Binciken bambance-bambancen ƙwayoyin cuta na huhu da aka gano ta hanyar amfani da kwamfuta (CT) ya ci gaba da zama ƙalubale a aikin asibiti. A nan, mun kwatanta yanayin duniya na samfuran jini 480, waɗanda suka haɗa da kula da lafiya, ƙwayoyin cuta na huhu masu kyau, da kuma ciwon huhu na mataki na I. Ciwon daji na Adenocarcinoma yana nuna yanayin rayuwa na musamman, yayin da ƙwayoyin cuta masu kyau da mutane masu lafiya suna da kamanceceniya sosai a cikin bayanan metabolomic. A cikin ƙungiyar gano (n = 306), an gano saitin ƙwayoyin cuta 27 don bambance tsakanin ƙwayoyin cuta masu kyau da marasa kyau. AUC na samfurin bambance-bambance a cikin ƙungiyoyin tabbatarwa na ciki (n = 104) da tabbatarwa na waje (n = 111) sun kasance 0.915 da 0.945, bi da bi. Binciken hanya ya nuna ƙaruwar ƙwayoyin cuta masu glycolytic da ke da alaƙa da raguwar tryptophan a cikin jinin adenocarcinoma na huhu idan aka kwatanta da ƙwayoyin cuta masu kyau da kuma kula da lafiya, kuma ya nuna cewa shan tryptophan yana haɓaka glycolysis a cikin ƙwayoyin cutar kansa ta huhu. Bincikenmu ya nuna muhimmancin sinadaran metabolite na jini wajen tantance haɗarin ƙwayoyin huhu da CT ta gano.
Gano cutar kansa da wuri yana da matuƙar muhimmanci don inganta rayuwar masu fama da cutar kansa. Sakamakon gwajin gwajin cutar kansar huhu na ƙasa na Amurka (NLST) da kuma binciken NELSON na Turai ya nuna cewa tantancewa da ƙaramin adadin tomography mai ƙididdigewa (LDCT) na iya rage mace-macen cutar kansar huhu a cikin ƙungiyoyi masu haɗari1,2,3. Tun lokacin da aka yi amfani da LDCT sosai don tantance cutar kansar huhu, yawan binciken da aka yi ta hanyar rediyo na ƙwayoyin huhu marasa alamun cutar ya ci gaba da ƙaruwa. 4. An bayyana ƙwayoyin huhu a matsayin masu hangen nesa har zuwa 3 cm a diamita. 5. Muna fuskantar matsaloli wajen tantance yuwuwar kamuwa da cutar kansa da kuma magance yawan ƙwayoyin huhu da aka gano ba zato ba tsammani akan LDCT. Iyakan CT na iya haifar da gwaje-gwaje akai-akai da sakamakon ƙarya, wanda ke haifar da shiga tsakani da magani fiye da kima. Saboda haka, akwai buƙatar haɓaka ingantattun alamun biomarkers masu amfani don gano cutar kansar huhu daidai a farkon matakai da kuma bambance yawancin ƙwayoyin cuta marasa lahani a farkon gano cutar 7.
Cikakken bincike na kwayoyin halitta na jini (magani, plasma, ƙwayoyin mononuclear na jini na gefe), gami da genomics, proteomics ko DNA methylation8,9,10, ya haifar da ƙaruwar sha'awar gano alamun cutar kansar huhu. A halin yanzu, hanyoyin metabolomics suna auna samfuran ƙarshen ƙwayoyin halitta waɗanda ke da tasiri ta hanyar ayyukan endogenous da exogenous kuma saboda haka ana amfani da su don annabta farkon cutar da sakamakonta. Liquid chromatography-tandem mass spectrometry (LC-MS) hanya ce da ake amfani da ita sosai don nazarin metabolomics saboda babban ƙarfinsa da babban kewayon kuzari, wanda zai iya rufe metabolites tare da halaye daban-daban na physicochemical11,12,13. Kodayake an yi amfani da nazarin metabolomic na duniya na plasma/serum don gano alamun biomarkers da ke da alaƙa da gano cutar kansar huhu14,15,16,17 da ingancin magani, har yanzu ana ci gaba da yin nazari sosai kan rarrabuwar metabolites na jini18 don bambance tsakanin nodules na huhu marasa kyau da marasa kyau. - babban bincike.
Ciwon daji na huhu na Adenocarcinoma da squamous cell carcinoma su ne manyan nau'ikan ciwon daji na huhu na ƙananan ƙwayoyin halitta (NSCLC). Gwaje-gwaje daban-daban na CT sun nuna cewa adenocarcinoma shine nau'in ciwon daji na huhu da aka fi sani da histological 1,19,20,21. A cikin wannan binciken, mun yi amfani da ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) don yin nazarin metabolomics akan jimillar samfuran jini 695, gami da kula da lafiya, nodules na huhu masu kyau, da kuma CT-gano ≤3 cm. Binciken cutar kansar huhu na Stage I. Mun gano wani kwamiti na metabolites na jini wanda ke bambanta adenocarcinoma na huhu daga nodules masu kyau da kula da lafiya. Binciken wadatar hanyoyin ya nuna cewa rashin daidaituwar tryptophan da metabolism na glucose canje-canje ne na yau da kullun a adenocarcinoma na huhu idan aka kwatanta da nodules masu kyau da kula da lafiya. A ƙarshe, mun kafa kuma mun tabbatar da wani nau'in rarrabawa na metabolism na jini tare da takamaiman ƙwarewa da ƙwarewa don bambance tsakanin ƙwayoyin huhu masu cutarwa da marasa lahani waɗanda LDCT ta gano, wanda zai iya taimakawa wajen gano bambance-bambancen farko da kimanta haɗari.
A cikin wannan binciken, an tattara samfuran jini masu dacewa da jinsi da shekaru daga marasa lafiya 174, marasa lafiya 292 da ke da ƙwayoyin cuta marasa lafiya na huhu, da kuma marasa lafiya 229 da ke da cutar sankarar huhu ta mataki na I. An nuna halayen alƙaluma na mutane 695 a cikin Tebur na Ƙarin Bayani na 1.
Kamar yadda aka nuna a Hoto na 1a, an tattara jimillar samfuran jini guda 480, gami da samfuran kula da lafiya guda 174 (HC), ƙwayoyin cuta marasa kyau guda 170 (BN), da kuma samfuran adenocarcinoma na huhu guda 136 (LA) a Cibiyar Ciwon daji ta Jami'ar Sun Yat-sen. Ƙungiyar gano ƙwayoyin cuta don tantance yanayin metabolomic mara niyya ta amfani da ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). Kamar yadda aka nuna a cikin Hoto na Ƙarin 1, an gano bambance-bambancen metabolites tsakanin LA da HC, LA da BN don kafa samfurin rarrabuwa da kuma ƙarin bincika nazarin hanyoyin daban-daban. An tattara samfura 104 da Cibiyar Ciwon daji ta Jami'ar Sun Yat-sen ta tattara da samfuran 111 da wasu asibitoci biyu suka tattara don tabbatarwa ta ciki da waje, bi da bi.
a Nazarin yawan jama'a a cikin ƙungiyar binciken da aka gudanar da binciken metabolomics na jini ta amfani da ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). b Binciken bambance-bambancen murabba'i na ƙananan murabba'i (PLS-DA) na jimlar metabolome na samfuran jini 480 daga ƙungiyar binciken, gami da kula da lafiya (HC, n = 174), nodules marasa kyau (BN, n = 170), da kuma adenocarcinoma na huhu na mataki na I (Los Angeles, n = 136). +ESI, yanayin ionization na electrospray mai kyau, -ESI, yanayin ionization na electrospray mara kyau. c–e Metabolites tare da yalwar abubuwa daban-daban a cikin ƙungiyoyi biyu da aka bayar (gwajin matsayi na Wilcoxon mai wutsiya biyu, ƙimar gano ƙarya da aka daidaita ƙimar p, FDR <0.05) an nuna su da ja (canjin ninkawa > 1.2) da shuɗi (canjin ninkawa < 0.83). ) da aka nuna akan hoton dutsen mai aman wuta. f Taswirar zafi ta rukuni mai tsari wanda ke nuna bambance-bambance masu mahimmanci a cikin adadin metabolites da aka ambata tsakanin LA da BN. Ana bayar da bayanan tushe ta hanyar fayilolin bayanai na tushe.
An yi nazarin jimillar metabolome na 174 HC, 170 BN da 136 LA a cikin rukunin binciken ta amfani da nazarin UPLC-HRMS. Da farko mun nuna cewa samfuran kula da inganci (QC) sun taru sosai a tsakiyar samfurin nazarin manyan sassan (PCA) wanda ba a kula da shi ba, yana tabbatar da daidaiton aikin binciken na yanzu (Hoto na Ƙari na 2).
Kamar yadda aka nuna a cikin nazarin bambance-bambancen da ba su da bambanci tsakanin ɓangarorin biyu (PLS-DA) a cikin Hoto na 1 b, mun gano cewa akwai bambance-bambance bayyanannu tsakanin LA da BN, LA da HC a cikin yanayin ionization na electrospray mai kyau (+ESI) da mara kyau (−ESI). Duk da haka, ba a sami wani muhimmin bambanci tsakanin BN da HC a cikin yanayin +ESI da -ESI ba.
Mun sami siffofi daban-daban guda 382 tsakanin LA da HC, siffofi daban-daban guda 231 tsakanin LA da BN, da siffofi daban-daban guda 95 tsakanin BN da HC (gwajin matsayi na Wilcoxon da aka sanya hannu, FDR <0.05 da sauyi mai yawa >1.2 ko <0.83) (Hoto na .1c-e). . An ƙara yin bayanin kololuwa (Bayanan Ƙarin 3) akan rumbun bayanai (ɗakin karatu na mzCloud/HMDB/Chemspider) ta hanyar ƙimar m/z, lokacin riƙewa da binciken taro na rarrabuwa (cikakkun bayanai da aka bayyana a cikin sashin Hanyoyi) 22. A ƙarshe, an gano metabolites masu ba da bayani guda 33 da 38 tare da bambance-bambance masu mahimmanci a cikin yawa don LA idan aka kwatanta da BN (Hoto na 1f da Tebur na Ƙarin 2) da LA idan aka kwatanta da HC (Hoto na Ƙarin 3 da Tebur Na Ƙarin 2), bi da bi. Sabanin haka, an gano metabolites guda 3 ne kawai tare da bambance-bambance masu yawa a cikin BN da HC (Jadawali na Ƙarin 2), daidai da haɗuwa tsakanin BN da HC a cikin PLS-DA. Waɗannan metabolites daban-daban suna rufe nau'ikan biochemicals iri-iri (Hoto na Ƙarin 4). Idan aka haɗa su, waɗannan sakamakon suna nuna manyan canje-canje a cikin metabolome na jini wanda ke nuna canjin cutar kansar huhu a matakin farko idan aka kwatanta da nodules na huhu marasa kyau ko marasa lafiya. A halin yanzu, kamanceceniya tsakanin metabolome na jini na BN da HC yana nuna cewa nodules na huhu marasa kyau na iya raba halaye da yawa na halitta tare da mutane masu lafiya. Ganin cewa maye gurbi na kwayar halittar epidermal growth factor receptor (EGFR) ya zama ruwan dare a cikin nau'in adenocarcinoma na huhu na 23, mun nemi tantance tasirin maye gurbi na direba akan metabolome na jini. Sannan muka bincika cikakken bayanin metabolomic na shari'o'i 72 tare da matsayin EGFR a cikin ƙungiyar adenocarcinoma na huhu. Abin sha'awa, mun sami bayanai iri ɗaya tsakanin marasa lafiya da suka canza EGFR (n = 41) da marasa lafiya da suka canza EGFR (n = 31) a cikin nazarin PCA (Hoto na Ƙarin 5a). Duk da haka, mun gano metabolites guda 7 waɗanda yawansu ya canza sosai a cikin marasa lafiya da suka canza EGFR idan aka kwatanta da marasa lafiya da ke da EGFR na daji (gwajin t, p < 0.05 da canjin ninkawa > 1.2 ko < 0.83) (Hoto na Ƙarin 5b). Yawancin waɗannan metabolites (5 cikin 7) acylcarnitines ne, waɗanda ke taka muhimmiyar rawa a cikin hanyoyin iskar shaka mai kitse.
Kamar yadda aka nuna a cikin aikin da aka nuna a Hoto na 2 a, an sami alamun alamun don rarrabuwar ƙwayoyin cuta ta amfani da masu sarrafa raguwar ƙarancin cikakku da zaɓi bisa ga metabolites 33 daban-daban da aka gano a cikin LA (n = 136) da BN (n = 170). Mafi kyawun haɗin masu canji (LASSO) - samfurin binary logistic regression model. An yi amfani da ingantaccen giciye-naɗi mai ninki goma don gwada amincin samfurin. Ana daidaita zaɓin masu canzawa da daidaita sigogi ta hanyar hukuncin haɓaka yuwuwar tare da siga λ24. An ƙara yin nazarin metabolomics na duniya daban-daban a cikin ƙungiyoyin tantancewa na ciki (n = 104) da na tabbatarwa na waje (n = 111) don gwada aikin rarrabuwa na samfurin rarrabewa. Sakamakon haka, an gano metabolites 27 a cikin saitin gano a matsayin mafi kyawun samfurin rarrabewa tare da mafi girman matsakaicin ƙimar AUC (Hoto na 2b), daga cikinsu 9 suna da ƙaruwar aiki da raguwar aiki 18 a cikin LA idan aka kwatanta da BN (Hoto na 2c).
Tsarin aiki don gina na'urar rarraba nodule ta huhu, gami da zaɓar mafi kyawun kwamitin metabolites na jini a cikin saitin ganowa ta amfani da samfurin komawar binary logistic ta hanyar tantancewa sau goma da kuma kimanta aikin hasashen a cikin saitin tabbatarwa na ciki da waje. b Ƙididdigar tantancewa ta giciye na samfurin komawar LASSO don zaɓin alamar biomarker na metabolism. Lambobin da aka bayar a sama suna wakiltar matsakaicin adadin alamun biomarker da aka zaɓa a wani λ da aka bayar. Layin ja mai dige-dige yana wakiltar matsakaicin ƙimar AUC a lambda mai dacewa. Sandunan kuskuren launin toka suna wakiltar mafi ƙarancin da matsakaicin ƙimar AUC. Layin dige-dige yana nuna mafi kyawun samfurin tare da alamun biomarker 27 da aka zaɓa. AUC, yanki a ƙarƙashin lanƙwasa halayyar aiki mai karɓar (ROC). c Canje-canje na metabolites 27 da aka zaɓa a cikin ƙungiyar LA idan aka kwatanta da ƙungiyar BN a cikin ƙungiyar ganowa. Ja ginshiƙi ja - kunnawa. Ginshiƙin shuɗi raguwa ne. d–f Lanƙwasa halayyar aiki mai karɓar karɓa (ROC) yana nuna ikon samfurin bambance-bambance bisa ga haɗuwar metabolite 27 a cikin saitin tabbatarwa na ganowa, na ciki, da na waje. Ana bayar da bayanan tushe a cikin nau'in fayilolin bayanai na tushe.
An ƙirƙiri samfurin hasashen bisa ga ma'aunin ma'aunin komawa baya na waɗannan metabolites 27 (Jadawali na Ƙarin 3). Binciken ROC bisa ga waɗannan metabolites 27 ya samar da yanki a ƙarƙashin ƙimar lanƙwasa (AUC) na 0.933, ƙwarewar ƙungiyar gano shine 0.868, kuma takamaiman shine 0.859 (Hoto na 2d). A halin yanzu, daga cikin metabolites 38 da aka yi bayani dalla-dalla tsakanin LA da HC, saitin metabolites 16 ya cimma AUC na 0.902 tare da ƙarfin hali na 0.801 da takamaiman hali na 0.856 wajen bambance LA daga HC (Hoto na Ƙarin 6a-c). An kuma kwatanta ƙimar AUC bisa ga ma'aunin canjin ninkaya daban-daban don metabolites daban-daban. Mun gano cewa samfurin rarrabuwa ya yi mafi kyau wajen bambancewa tsakanin LA da BN (HC) lokacin da aka saita matakin canjin ninkaya zuwa 1.2 akan 1.5 ko 2.0 (Hoto na Ƙarin 7a,b). An ƙara tabbatar da samfurin rarrabuwa, wanda ya dogara da ƙungiyoyin metabolite guda 27, a cikin ƙungiyoyin ciki da waje. AUC shine 0.915 (jijiya 0.867, takamaiman 0.811) don tabbatarwa ta ciki da 0.945 (jijiya 0.810, takamaiman 0.979) don tabbatarwa ta waje (Hoto na 2e, f). Don tantance ingancin dakin gwaje-gwaje, an yi nazarin samfura 40 daga ƙungiyar waje a cikin dakin gwaje-gwaje na waje kamar yadda aka bayyana a cikin sashin Hanyoyi. Daidaiton rarrabuwa ya cimma AUC na 0.925 (Hoto na Ƙarin 8). Saboda cutar kansa ta ƙwayar huhu (LUSC) ita ce nau'i na biyu mafi yawan kamuwa da cutar kansar huhu ba ƙaramin ƙwayar halitta ba (NSCLC) bayan cutar kansar huhu (LUAD), mun kuma gwada ingantaccen amfani da bayanan metabolism. BN da shari'o'i 16 na LUSC. AUC na nuna wariya tsakanin LUSC da BN ya kasance 0.776 (Karin Hoto na 9), wanda ke nuna ƙarancin iyawa idan aka kwatanta da wariya tsakanin LUAD da BN.
Nazarin ya nuna cewa girman ƙurajen da ke kan hotunan CT yana da alaƙa mai kyau da yuwuwar kamuwa da cutar kansa kuma ya kasance babban abin da ke tantance maganin ƙurajen25,26,27. Binciken bayanai daga babban rukunin binciken tantance NELSON ya nuna cewa haɗarin kamuwa da cutar kansa a cikin mutanen da ke da ƙurajen <5 mm ma yayi kama da na mutanen da ba su da ƙurajen 28. Saboda haka, mafi ƙarancin girman da ke buƙatar sa ido kan CT na yau da kullun shine 5 mm, kamar yadda ƙungiyar Thoracic ta Burtaniya (BTS) ta ba da shawara, da 6 mm, kamar yadda Fleischner Society ta ba da shawara 29. Duk da haka, ƙurajen da suka fi girma fiye da 6 mm kuma ba tare da siffofi marasa lahani ba, waɗanda ake kira ƙurajen huhu marasa tabbas (IPN), sun kasance babban ƙalubale a cikin kimantawa da gudanarwa a cikin aikin asibiti30,31. Na gaba mun bincika ko girman ƙurajen ya shafi sa hannun metabolomic ta amfani da samfuran da aka tattara daga binciken da kuma haɗin gwiwar tabbatarwa na ciki. Mun mai da hankali kan alamun biomarkers 27 da aka tabbatar, da farko mun kwatanta bayanan PCA na ƙwayoyin cuta na HC da BN sub-6 mm. Mun gano cewa yawancin bayanan da aka tattara don HC da BN sun yi karo, wanda ke nuna cewa matakan metabolite na jini iri ɗaya ne a cikin ƙungiyoyi biyu (Hoto na 3a). Taswirorin fasali a cikin kewayon girma daban-daban sun kasance an kiyaye su a cikin BN da LA (Hoto na 3b, c), yayin da aka lura da rabuwa tsakanin ƙwayoyin cuta masu haɗari da marasa kyau a cikin kewayon 6-20 mm (Hoto na 3d). Wannan rukunin yana da AUC na 0.927, takamaiman 0.868, da kuma jin daɗin 0.820 don annabta mummunan ƙwayoyin cuta masu auna 6 zuwa 20 mm (Hoto na 3e, f). Sakamakonmu ya nuna cewa mai rarrabawa zai iya kama canje-canjen metabolism da ke faruwa sakamakon canjin cutar da wuri, ba tare da la'akari da girman ƙwayoyin cuta ba.
Talla Kwatanta bayanan PCA tsakanin ƙungiyoyi da aka ƙayyade bisa ga rarrabuwar metabolism na metabolites 27. CC da BN < 6 mm. b BN < 6 mm vs BN 6-20 mm. a cikin LA 6-20 mm idan aka kwatanta da LA 20-30 mm. g BN 6-20 mm da LA 6-20 mm. GC, n = 174; BN < 6 mm, n = 153; BN 6-20 mm, n = 91; LA 6-20 mm, n = 89; LA 20-30 mm, n = 77. e Lanƙwasa halayyar aiki mai karɓa (ROC) yana nuna aikin samfurin bambance-bambance ga nodules 6-20 mm. f An ƙididdige ƙimar yuwuwar bisa ga samfurin komawar logistic don nodules waɗanda ke auna 6-20 mm. Layin launin toka mai dige-dige yana wakiltar ƙimar yankewa mafi kyau (0.455). Lambobin da ke sama suna wakiltar kashi na shari'o'in da aka yi hasashen za su faru a Los Angeles. Yi amfani da gwajin t na ɗalibi mai wutsiya biyu. PCA, babban nazarin sassan. Yankin AUC a ƙarƙashin lanƙwasa. Ana bayar da bayanan tushe a cikin nau'in fayilolin bayanai na tushe.
An ƙara zaɓar samfura huɗu (masu shekaru 44-61) waɗanda ke da irin wannan girman ƙwayoyin huhu (7-9 mm) don nuna aikin samfurin hasashen cutar kansa da aka gabatar (Hoto na 4a, b). A gwajin farko, Shari'a ta 1 ta gabatar a matsayin ƙwayar cuta mai ƙarfi tare da calcification, wata alama da ke da alaƙa da rashin lafiya, yayin da Shari'a ta 2 ta gabatar a matsayin ƙwayar cuta mai ƙarfi wadda ba ta da wata alama ta rashin lafiya. Zagaye uku na binciken CT na biye sun nuna cewa waɗannan shari'o'in sun kasance masu karko tsawon shekaru 4 kuma saboda haka an ɗauke su a matsayin ƙwayoyin cuta marasa lafiya (Hoto na 4a). Idan aka kwatanta da kimantawa ta asibiti na gwajin CT na serial, nazarin metabolite na jini guda ɗaya tare da samfurin rarrabuwa na yanzu da sauri kuma ya gano waɗannan ƙwayoyin cuta marasa lafiya daidai bisa ga ƙuntatawa na yuwuwar (Tebur na 1). Hoto na 4b a cikin shari'a ta 3 yana nuna ƙwayar cuta mai alamun ja da baya daga pleural, wanda galibi ana danganta shi da cutar kansa32. Shari'a ta 4 ta gabatar a matsayin ƙwayar cuta mara ƙarfi wacce ba ta da wata shaida ta dalili mai kyau. An yi hasashen cewa duk waɗannan lamuran za su iya zama masu illa bisa ga samfurin rarrabuwa (Tebur 1). An nuna kimantawar cutar adenocarcinoma ta huhu ta hanyar binciken histopathology bayan tiyatar cire huhu (Hoto na 4b). Don saitin tabbatarwa na waje, mai rarraba metabolism ya yi hasashen daidai shari'o'i biyu na ƙwayoyin huhu marasa tabbas waɗanda suka fi 6 mm girma (Hoto na Ƙari na 10).
Hotunan CT na tagar axial na huhu na lokuta biyu na nodules marasa lahani. A yanayin 1, CT scan bayan shekaru 4 ya nuna wani santsi mai ƙarfi wanda yake auna 7 mm tare da calcification a cikin ƙananan lobe na dama. A yanayin 2, CT scan bayan shekaru 5 ya nuna wani santsi mai ƙarfi wanda yake da diamita na 7 mm a cikin saman lobe na dama. b Hotunan CT na taga axial na huhu da kuma nazarin cututtuka masu dacewa na lokuta biyu na adenocarcinoma na mataki na I kafin a cire huhu. Halin 3 ya nuna santsi mai diamita na 8 mm a cikin saman lobe na dama tare da ja da baya daga pleural. Halin 4 ya nuna wani santsi mai ƙarfi na gilashin ƙasa wanda yake auna 9 mm a cikin saman lobe na hagu. Tabon Hematoxylin da eosin (H&E) na kyallen huhu da aka cire (ma'aunin sikelin = 50 μm) yana nuna yanayin girma na acinar na adenocarcinoma na huhu. Kibiyoyi suna nuna santsi da aka gano akan hotunan CT. Hotunan H&E wakilci ne na filayen microscopic da yawa (>3) da masanin ilimin cututtuka ya bincika.
Idan aka haɗa su wuri ɗaya, sakamakonmu yana nuna yuwuwar ƙimar biomarkers na metabolite a cikin ganewar asali na bambance-bambancen ƙwayoyin huhu, wanda zai iya haifar da ƙalubale yayin kimanta gwajin CT.
Dangane da wani kwamitin metabolite mai bambanci da aka tabbatar, mun nemi gano alaƙar halittu da manyan canje-canjen rayuwa. Binciken haɓaka hanyoyin KEGG ta MetaboAnalyst ya gano hanyoyi 6 da aka saba canzawa sosai tsakanin ƙungiyoyi biyu da aka bayar (LA vs. HC da LA vs. BN, an daidaita p ≤ 0.001, sakamako > 0.01). Waɗannan canje-canjen an siffanta su da rikice-rikice a cikin metabolism na pyruvate, metabolism na tryptophan, metabolism na niacin da nicotinamide, glycolysis, zagayowar TCA, da metabolism na purine (Hoto na 5a). Daga nan muka ci gaba da yin metabolomics da aka yi niyya don tabbatar da manyan canje-canje ta amfani da cikakken ƙididdigewa. Tabbatar da metabolites na yau da kullun a cikin hanyoyin da aka saba canzawa ta hanyar spectrometry na mass triple quadrupole (QQQ) ta amfani da ingantattun ƙa'idodin metabolite. An haɗa halayen alƙaluma na samfurin da aka yi niyya na nazarin metabolomics a cikin Tebur na Ƙarin 4. Daidai da sakamakon metabolomics na duniya, nazarin adadi ya tabbatar da cewa hypoxanthine da xanthine, pyruvate, da lactate sun ƙaru a cikin LA idan aka kwatanta da BN da HC (Hoto na 5b, c, p <0.05). Duk da haka, babu wani muhimmin bambanci a cikin waɗannan metabolomics tsakanin BN da HC.
Binciken hanyoyin haɓaka hanyoyin KEGG na metabolites daban-daban a cikin ƙungiyar LA idan aka kwatanta da ƙungiyoyin BN da HC. An yi amfani da gwajin Global mai wutsiya biyu, kuma an daidaita ƙimar p ta amfani da hanyar Holm-Bonferroni (an daidaita p ≤ 0.001 da girman tasirin > 0.01). b–d Hotunan Violin suna nuna matakan hypoxanthine, xanthine, lactate, pyruvate, da tryptophan a cikin jini HC, BN, da LA wanda LC-MS/MS ta ƙaddara (n = 70 a kowace rukuni). Layukan fari da baƙi masu dige-dige suna nuna matsakaicin da quartile, bi da bi. e Hotunan Violin suna nuna daidaitaccen Log2TPM (kwafi a kowace miliyan) mRNA na SLC7A5 da QPRT a cikin adenocarcinoma na huhu (n = 513) idan aka kwatanta da nama na huhu na yau da kullun (n = 59) a cikin bayanan LUAD-TCGA. Akwatin fari yana wakiltar kewayon interquartile, layin baƙi mai kwance a tsakiya yana wakiltar matsakaici, kuma layin baƙi mai tsaye wanda ya fito daga akwatin yana wakiltar tazara ta amincewa ta 95% (CI). f Tsarin haɗin Pearson na bayyanar SLC7A5 da GAPDH a cikin adenocarcinoma na huhu (n = 513) da kyallen huhu na yau da kullun (n = 59) a cikin bayanan TCGA. Yankin launin toka yana wakiltar CI 95%. r, Pearson correlation coefficient. g Matakan tryptophan na ƙwayoyin halitta masu daidaituwa a cikin ƙwayoyin A549 waɗanda aka yi musu transfected tare da kulawar shRNA mara takamaiman (NC) da shSLC7A5 (Sh1, Sh2) waɗanda LC-MS/MS ta ƙaddara. An gabatar da nazarin ƙididdiga na samfuran biyar masu zaman kansu na halitta a cikin kowane rukuni. h Matakan ƙwayoyin halitta na NADt (jimillar NAD, gami da NAD+ da NADH) a cikin ƙwayoyin A549 (NC) da SLC7A5 knockdown A549 (Sh1, Sh2). An gabatar da nazarin ƙididdiga na samfuran uku masu zaman kansu na halitta a cikin kowane rukuni. i An auna aikin Glycolytic na ƙwayoyin A549 kafin da kuma bayan ƙwanƙwasa SLC7A5 ta hanyar ƙimar acidification na extracellular (ECAR) (n = samfuran 4 masu zaman kansu na halitta a kowace ƙungiya). 2-DG, 2-deoxy-D-glucose. An yi amfani da gwajin t na ɗalibi mai wutsiya biyu a cikin (b–h). A cikin (g–i), sandunan kuskure suna wakiltar matsakaicin ± SD, kowane gwaji an yi shi sau uku daban-daban kuma sakamakon ya yi kama da juna. Ana bayar da bayanan tushe a cikin nau'in fayilolin bayanai na tushe.
Idan aka yi la'akari da tasirin da canjin metabolism na tryptophan a cikin ƙungiyar LA ya yi, mun kuma tantance matakan tryptophan na jini a cikin ƙungiyoyin HC, BN, da LA ta amfani da QQQ. Mun gano cewa an rage tryptophan na jini a cikin LA idan aka kwatanta da HC ko BN (p < 0.001, Hoto na 5d), wanda ya yi daidai da binciken da aka yi a baya cewa matakan tryptophan da ke yawo a jiki sun yi ƙasa a cikin marasa lafiya da ke fama da cutar kansar huhu fiye da na lafiya daga ƙungiyar kulawa33,34,35. Wani bincike da aka yi amfani da PET/CT tracer 11C-methyl-L-tryptophan ya gano cewa lokacin riƙe siginar tryptophan a cikin kyallen kansar huhu ya ƙaru sosai idan aka kwatanta da raunuka marasa kyau ko kyallen jiki na yau da kullun36. Mun yi hasashen cewa raguwar tryptophan a cikin jini na LA na iya nuna yawan shan tryptophan mai aiki ta ƙwayoyin cutar kansar huhu.
An kuma san cewa ƙarshen hanyar kynurenine ta catabolism ta tryptophan shine NAD+37,38, wanda shine muhimmin abu don amsawar glyceraldehyde-3-phosphate tare da 1,3-bisphosphoglycerate a cikin glycolysis39. Duk da cewa binciken da aka yi a baya ya mayar da hankali kan rawar da catabolism na tryptophan ke takawa a cikin daidaita garkuwar jiki, mun yi ƙoƙarin bayyana alaƙar da ke tsakanin rashin daidaituwar tryptophan da hanyoyin glycolytic da aka lura a cikin binciken na yanzu. An san cewa dangin mai jigilar Solute 7 memba 5 (SLC7A5) shine mai jigilar tryptophan43,44,45. Quinolinic acid phosphoribosyltransferase (QPRT) wani enzyme ne da ke ƙasa da hanyar kynurenine wanda ke canza quinolinic acid zuwa NAMN46. Binciken bayanan LUAD TCGA ya nuna cewa duka SLC7A5 da QPRT sun karu sosai a cikin kyallen ƙari idan aka kwatanta da kyallen halitta (Hoto na 5e). An lura da wannan ƙaruwa a matakai na I da II da kuma matakai na III da IV na adenocarcinoma na huhu (Karin Hoto na 11), wanda ke nuna rikice-rikicen farko a cikin metabolism na tryptophan da ke da alaƙa da cutar tumorigenesis.
Bugu da ƙari, bayanan LUAD-TCGA sun nuna kyakkyawar alaƙa tsakanin bayyanar SLC7A5 da GAPDH mRNA a cikin samfuran marasa lafiya na ciwon daji (r = 0.45, p = 1.55E-26, Hoto na 5f). Sabanin haka, ba a sami wata muhimmiyar alaƙa tsakanin irin waɗannan alamun kwayoyin halitta a cikin kyallen huhu na yau da kullun ba (r = 0.25, p = 0.06, Hoto na 5f). Rushewar SLC7A5 (Hoto na Ƙarin 12) a cikin ƙwayoyin A549 ya rage matakan tryptophan da NAD(H) na ƙwayoyin halitta sosai (Hoto na 5g,h), wanda ya haifar da raguwar aikin glycolytic kamar yadda aka auna ta hanyar ƙimar acidification na extracellular (ECAR) (Hoto na 1). 5i). Don haka, bisa ga canje-canjen rayuwa a cikin jini da gano a cikin vitro, mun yi hasashen cewa metabolism na tryptophan na iya samar da NAD+ ta hanyar hanyar kynurenine kuma yana taka muhimmiyar rawa wajen haɓaka glycolysis a cikin ciwon huhu.
Nazarin ya nuna cewa adadi mai yawa na ƙwayoyin huhu marasa tabbas da LDCT ta gano na iya haifar da buƙatar ƙarin gwaji kamar PET-CT, biopsy na huhu, da kuma fiye da kima saboda ganewar cutar kansa ta ƙarya.31 Kamar yadda aka nuna a Hoto na 6, bincikenmu ya gano wani rukunin ƙwayoyin halitta na jini tare da ƙimar ganewar asali wanda zai iya inganta rarrabuwar haɗari da kuma kula da ƙwayoyin huhu ta hanyar CT.
Ana kimanta ƙwayoyin huhu ta amfani da ƙaramin adadin kwamfuta (LDCT) tare da fasalulluka na hoto waɗanda ke nuna dalilai marasa lahani ko masu cutarwa. Sakamakon rashin tabbas na ƙwayoyin cuta na iya haifar da ziyarar bibiya akai-akai, shiga tsakani marasa amfani, da kuma fiye da kima. Haɗa masu rarraba ƙwayoyin halitta na jini tare da ƙimar ganewar asali na iya inganta kimanta haɗari da kuma kula da ƙwayoyin huhu na gaba. PET positron emission tomography.
Bayanai daga binciken NLST na Amurka da kuma binciken NELSON na Turai sun nuna cewa tantance ƙungiyoyi masu haɗari masu yawa tare da ƙaramin adadin kwamfuta (LDCT) na iya rage mace-macen cutar kansar huhu1,3. Duk da haka, kimanta haɗari da kuma kula da asibiti na adadi mai yawa na ƙwayoyin huhu da LDCT ta gano sun kasance mafi ƙalubale. Babban burin shine inganta daidaitaccen rarrabuwa na ka'idojin LDCT da ke akwai ta hanyar haɗa alamun biomarkers masu inganci.
An gano wasu kwayoyin halitta masu nuna alama, kamar su metabolites na jini, ta hanyar kwatanta ciwon daji na huhu da hanyoyin da suka dace da lafiya15,17. A cikin wannan binciken, mun mayar da hankali kan amfani da nazarin metabolites na jini don bambance tsakanin nodules na huhu marasa kyau da marasa kyau waɗanda LDCT ta gano ba zato ba tsammani. Mun kwatanta metabolome na jini na duniya na kula da lafiya (HC), nodules na huhu marasa kyau (BN), da samfuran adenocarcinoma na huhu na mataki na I ta amfani da nazarin UPLC-HRMS. Mun gano cewa HC da BN suna da irin wannan bayanin metabolism, yayin da LA ta nuna manyan canje-canje idan aka kwatanta da HC da BN. Mun gano saitin metabolites na jini guda biyu waɗanda ke bambanta LA daga HC da BN.
Tsarin gano ƙwayoyin cuta marasa lahani da masu cutarwa na yanzu wanda aka yi bisa ga LDCT ya dogara ne akan girma, yawa, yanayin jiki da kuma yawan girma na ƙwayoyin cuta a tsawon lokaci30. Nazarin da aka yi a baya ya nuna cewa girman ƙwayoyin cuta yana da alaƙa da yuwuwar kamuwa da cutar kansar huhu. Ko da a cikin marasa lafiya masu haɗari, haɗarin kamuwa da cutar kansa a cikin ƙwayoyin cuta <6 mm shine <1%. Haɗarin kamuwa da cutar kansa ga ƙwayoyin cuta masu girman 6 zuwa 20 mm ya kama daga 8% zuwa 64%30. Saboda haka, ƙungiyar Fleischner ta ba da shawarar a yi diamita na yankewa na 6 mm don bin diddigin CT na yau da kullun. 29 Duk da haka, ba a yi cikakken bincike game da haɗari da kuma kula da ƙwayoyin cuta marasa tabbas na huhu (IPN) waɗanda suka fi 6 mm girma ba 31. Kula da cututtukan zuciya na haihuwa a halin yanzu yawanci ya dogara ne akan jira mai lura tare da sa ido akai-akai na CT.
Dangane da ingantaccen metabolome, mun nuna karo na farko na haɗuwar sa hannun metabolomic tsakanin mutane masu lafiya da nodules marasa lafiya <6 mm. Kamanceceniyar halittu ta yi daidai da binciken CT na baya cewa haɗarin cutar kansa ga nodules <6 mm yayi ƙasa da na mutanen da ba su da nodules.30 Ya kamata a lura cewa sakamakonmu ya kuma nuna cewa nodules marasa lafiya <6 mm da ≥6 mm suna da babban kamanceceniya a cikin bayanan metabolomic, yana nuna cewa ma'anar aikin etiology marasa lafiya ya yi daidai ba tare da la'akari da girman nodules ba. Don haka, allunan metabolite na zamani na jini na iya samar da gwaji ɗaya a matsayin gwajin cirewa lokacin da aka fara gano nodules akan CT kuma yana iya rage sa ido kan serial. A lokaci guda, allunan biomarkers iri ɗaya sun bambanta nodules masu cutarwa ≥6 mm daga nodules marasa lafiya kuma sun ba da hasashen daidai ga IPNs masu girman iri ɗaya da fasalulluka marasa tabbas akan hotunan CT. Wannan mai rarraba metabolism na jini yayi aiki sosai wajen annabta cutar kansa na nodules ≥6 mm tare da AUC na 0.927. Idan aka haɗa su wuri ɗaya, sakamakonmu ya nuna cewa alamun metabolism na musamman na jini na iya nuna canje-canjen metabolism da suka faru da wuri ta hanyar ƙari kuma suna da ƙima mai yuwuwa a matsayin masu hasashen haɗari, ba tare da la'akari da girman nodule ba.
Abin lura shi ne, ciwon huhu na adenocarcinoma (LUAD) da ciwon huhu na squamous cell carcinoma (LUSC) su ne manyan nau'ikan ciwon huhu na ƙananan ƙwayoyin halitta (NSCLC). Ganin cewa LUSC yana da alaƙa sosai da amfani da taba47 kuma LUAD shine mafi yawan tarihin ƙwayoyin huhu na bazata da aka gano a gwajin CT48, an gina samfurin rarrabuwarmu musamman don samfuran adenocarcinoma na mataki na I. Wang da abokan aikinsa sun kuma mai da hankali kan LUAD kuma sun gano alamun lipid guda tara ta amfani da lipidomics don bambance ciwon huhu na mataki na farko daga mutanen da ke da lafiya17. Mun gwada samfurin rarrabuwa na yanzu akan shari'o'i 16 na LUSC na mataki na I da ƙananan ƙwayoyin cuta 74 kuma mun lura da ƙarancin daidaiton hasashen LUSC (AUC 0.776), yana nuna cewa LUAD da LUSC na iya samun nasu sa hannun metabolomic. Hakika, an nuna cewa LUAD da LUSC sun bambanta a cikin etiology, asalin halitta da canje-canjen kwayoyin halitta49. Saboda haka, ya kamata a haɗa wasu nau'ikan histology a cikin samfuran horo don gano cutar kansar huhu bisa ga yawan jama'a a cikin shirye-shiryen tantancewa.
A nan, mun gano hanyoyi shida da aka fi canzawa a cikin adenocarcinoma na huhu idan aka kwatanta da lafiyayyun sarrafawa da kuma nodules marasa lahani. Xanthine da hypoxanthine sune abubuwan da aka saba samu a cikin hanyar metabolism ta purine. Daidai da sakamakonmu, tsaka-tsakin da ke da alaƙa da metabolism na purine sun ƙaru sosai a cikin jini ko kyallen jikin marasa lafiya da adenocarcinoma na huhu idan aka kwatanta da lafiyayyun sarrafawa ko marasa lafiya a matakin kafin shiga cikin jini15,50. Matakan xanthine da hypoxanthine na jini masu ƙaruwa na iya nuna anabolism da ake buƙata ta hanyar yaɗuwar ƙwayoyin cutar kansa cikin sauri. Rashin daidaita metabolism na glucose sanannen alama ce ta metabolism na ciwon daji51. A nan, mun lura da ƙaruwa mai yawa a cikin pyruvate da lactate a cikin ƙungiyar LA idan aka kwatanta da ƙungiyar HC da BN, wanda ya yi daidai da rahotannin da suka gabata na rashin daidaituwar hanyar glycolytic a cikin bayanan metabolome na marasa lafiya da ba ƙaramin ƙwayar cutar kansar huhu ba (NSCLC) da kuma kula da lafiyayyun sarrafawa. Sakamakon ya kasance daidai52,53.
Abu mai mahimmanci, mun lura da alaƙar da ke tsakanin pyruvate da metabolism na tryptophan a cikin jini na adenocarcinomas na huhu. Matakan tryptophan na jini sun ragu a cikin ƙungiyar LA idan aka kwatanta da ƙungiyar HC ko BN. Abin sha'awa, wani babban bincike da aka yi a baya ta amfani da ƙungiyar masu zuwa ya gano cewa ƙarancin matakan tryptophan da ke yawo suna da alaƙa da ƙaruwar haɗarin kamuwa da cutar kansar huhu 54. Tryptophan muhimmin amino acid ne da muke samu gaba ɗaya daga abinci. Mun kammala da cewa raguwar tryptophan a cikin jini a cikin adenocarcinoma na huhu na iya nuna raguwar wannan metabolite cikin sauri. An san cewa ƙarshen samfurin catabolism na tryptophan ta hanyar hanyar kynurenine shine tushen haɗin de novo NAD+. Saboda ana samar da NAD+ galibi ta hanyar hanyar ceto, har yanzu ba a tantance mahimmancin NAD+ a cikin metabolism na tryptophan a cikin lafiya da cuta ba46. Bincikenmu na bayanan TCGA ya nuna cewa bayyanar tryptophan transporter solute transporter 7A5 (SLC7A5) ya ƙaru sosai a cikin adenocarcinoma na huhu idan aka kwatanta da sarrafawa na yau da kullun kuma yana da alaƙa mai kyau da bayyanar glycolytic enzyme GAPDH. Nazarin da suka gabata sun fi mayar da hankali kan rawar da tryptophan catabolism ke takawa wajen danne martanin rigakafin cutar kansa40,41,42. A nan mun nuna cewa hana shan tryptophan ta hanyar rage SLC7A5 a cikin ƙwayoyin cutar kansar huhu yana haifar da raguwar matakan NAD na ƙwayoyin halitta da kuma raguwar ayyukan glycolytic tare. A taƙaice, bincikenmu yana ba da tushen halitta don canje-canje a cikin metabolism na jini da ke da alaƙa da canjin cutar kansar huhu mai haɗari.
Sauye-sauyen EGFR sune abubuwan da suka fi haifar da maye gurbi a cikin marasa lafiya da ke fama da NSCLC. A cikin bincikenmu, mun gano cewa marasa lafiya da ke fama da maye gurbi na EGFR (n = 41) suna da cikakkun bayanan metabolomic kamar marasa lafiya da ke fama da EGFR na daji (n = 31), kodayake mun gano raguwar matakan jini na wasu marasa lafiya da ke fama da maye gurbi na EGFR a cikin marasa lafiya da ke fama da acylcarnitine. Aikin da aka kafa na acylcarnitine shine jigilar ƙungiyoyin acyl daga cytoplasm zuwa cikin matrix na mitochondrial, wanda ke haifar da iskar shaka na fatty acids don samar da kuzari 55. Daidai da bincikenmu, wani bincike na baya-bayan nan ya kuma gano irin wannan bayanin metabolome tsakanin maye gurbi na EGFR da ciwon daji na EGFR ta hanyar nazarin metabolome na duniya na samfuran nama na adenocarcinoma na huhu 10250. Abin sha'awa, an kuma sami abubuwan da ke cikin acylcarnitine a cikin ƙungiyar maye gurbi na EGFR. Saboda haka, ko canje-canje a cikin matakan acylcarnitine suna nuna canje-canjen metabolism da EGFR ya haifar da kuma hanyoyin kwayoyin halitta na iya cancanci ƙarin bincike.
A ƙarshe, bincikenmu ya kafa wani nau'in rarraba metabolism na jini don bambance-bambancen ganewar asali na nodules na huhu kuma yana ba da shawarar tsarin aiki wanda zai iya inganta kimanta haɗari da sauƙaƙe gudanarwa na asibiti bisa ga gwajin CT scan.
Kwamitin Ɗabi'a na Asibitin Ciwon Daji na Jami'ar Sun Yat-sen, Asibitin Farko da ke Jami'ar Sun Yat-sen, da Kwamitin Ɗabi'a na Asibitin Ciwon Daji na Jami'ar Zhengzhou sun amince da wannan binciken. A cikin ƙungiyoyin bincike da tantancewa na ciki, an tattara sera 174 daga mutane masu lafiya da sera 244 daga nodules marasa lahani daga mutanen da ake yi musu gwajin lafiya na shekara-shekara a Sashen Kula da Cututtukan Daji da Rigakafi, Cibiyar Ciwon Daji ta Jami'ar Sun Yat-sen, da sera 166 marasa lahani. An tattara adenocarcinomas na huhu na Mataki na I daga Cibiyar Ciwon Daji ta Jami'ar Sun Yat-sen. A cikin ƙungiyar tantancewa ta waje, akwai lokuta 48 na nodules marasa lahani, lokuta 39 na adenocarcinoma na huhu na mataki na I daga Asibitin Farko da ke Jami'ar Sun Yat-sen, da lokuta 24 na adenocarcinoma na huhu na mataki na I daga Asibitin Cutar Daji na Zhengzhou. Cibiyar Ciwon Daji ta Jami'ar Sun Yat-sen ta kuma tattara lokuta 16 na ciwon daji na huhu na matakin squamous cell don gwada ikon gano cutar da aka kafa ta hanyar rarraba metabolism (an nuna halayen marasa lafiya a cikin Tebur na Ƙarin Bayani na 5). An tattara samfura daga ƙungiyar binciken da ƙungiyar tantancewa ta ciki tsakanin Janairu 2018 da Mayu 2020. An tattara samfura don ƙungiyar tantancewa ta waje tsakanin Agusta 2021 da Oktoba 2022. Don rage wariya tsakanin jinsi, an ba da adadin maza da mata daidai gwargwado ga kowace ƙungiya. Ƙungiyar Bincike da Ƙungiyar Bita ta Cikin Gida. An ƙayyade jinsi na mahalarta bisa ga rahoton kansu. An sami izini daga duk mahalarta kuma ba a bayar da diyya ba. Wadanda ke da ƙwayoyin cuta marasa kyau su ne waɗanda ke da ƙimar CT scan mai ƙarfi a shekaru 2 zuwa 5 a lokacin binciken, sai dai mutum 1 daga samfurin tantancewa na waje, wanda aka tattara kafin tiyata kuma aka gano shi ta hanyar histopathology. Banda ciwon huhu na yau da kullun. An tattara shari'o'in adenocarcinoma na huhu kafin a cire huhu kuma an tabbatar da su ta hanyar ganewar asali. An tattara samfuran jinin azumi a cikin bututun rabuwa na jini ba tare da wani maganin hana zubar jini ba. An sanya samfuran jini na tsawon awa 1 a zafin ɗaki sannan a sanya su a centrifuge a 2851 × g na minti 10 a 4°C don tattara ruwan jini. An daskare ƙwayoyin magani a -80°C har sai an cire sinadarin metabolites. Sashen Rigakafin Ciwon daji da Binciken Lafiya na Cibiyar Ciwon daji ta Jami'ar Sun Yat-sen ya tattara tarin maganin daga masu ba da gudummawa 100 masu lafiya, ciki har da adadin maza da mata masu shekaru 40 zuwa 55 daidai. An haɗa daidai adadin kowanne samfurin mai ba da gudummawa, an raba wurin da aka samo shi kuma an adana shi a -80°C. An yi amfani da cakuda maganin a matsayin kayan aiki don kula da inganci da daidaita bayanai.
An narke samfuran magani na asali da na gwaji sannan aka cire metabolites ta amfani da hanyar cirewa ta haɗin gwiwa (MTBE/methanol/water) 56. A takaice, an haɗa 50 μl na magani da 225 μl na methanol mai sanyi da 750 μl na methyl tert-butyl ether (MTBE). A juya cakuda a saka a kan kankara na tsawon awa 1. Daga nan aka haɗa samfuran kuma aka haɗa su da 188 μl na ruwan MS mai ɗauke da ma'aunin ciki (13C-lactate, 13C3-pyruvate, 13C-methionine, da 13C6-isoleucine, waɗanda aka saya daga Cambridge Isotope Laboratories). Daga nan aka sanya cakuda a centrifuge a 15,000 × g na tsawon minti 10 a zafin jiki na 4 °C, sannan aka canza matakin ƙasa zuwa bututu biyu (125 μL kowannensu) don nazarin LC-MS a cikin yanayi mai kyau da mara kyau. A ƙarshe, an fitar da samfurin zuwa busasshe a cikin injin tattara iska mai saurin gudu.
An sake haɗa busassun metabolites a cikin 120 μl na acetonitrile 80%, an yi tururi na minti 5, sannan a sanya su a centrifuge a 15,000 × g na minti 10 a 4°C. An canza supernatants zuwa cikin kwalban amber tare da microinserts don nazarin metabolomics. Binciken metabolomics mara niyya akan dandamalin ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). An raba metabolites ta amfani da tsarin Dionex Ultimate 3000 UPLC da ginshiƙin ACQUITY BEH Amide (2.1 × 100 mm, 1.7 μm, Waters). A cikin yanayin ion mai kyau, matakan wayar hannu sune 95% (A) da 50% acetonitrile (B), kowannensu yana ɗauke da 10 mmol/L ammonium acetate da 0.1% formic acid. A yanayin rashin kyau, matakan motsi na A da B sun ƙunshi 95% da 50% acetonitrile, bi da bi, duka matakan sun ƙunshi 10 mmol/L ammonium acetate, pH = 9. Shirin gradient ɗin ya kasance kamar haka: minti 0–0.5, 2% B; minti 0.5–12, 2–50% B; minti 12–14, 50–98% B; minti 14–16, 98% B; minti 16–16.1., 98 –2% B; minti 16.1–20, 2% B. An kiyaye ginshiƙin a 40°C kuma samfurin a 10°C a cikin samfurin atomatik. Yawan kwararar ya kasance 0.3 ml/min, girman allurar shine 3 μl. An yi amfani da na'urar auna ma'aunin Q-Exactive Orbitrap (Thermo Fisher Scientific) tare da tushen ionization na electrospray (ESI) a cikin cikakken yanayin scanning kuma an haɗa shi da yanayin sa ido na ddMS2 don tattara manyan bayanai. An saita sigogin MS kamar haka: ƙarfin feshi +3.8 kV/- 3.2 kV, zafin capillary 320°C, iskar kariya 40 arb, iskar gas mai taimako 10 arb, zafin zafi na bincike 350°C, kewayon scanning 70–1050 m/h, ƙuduri. 70 000. An samo bayanai ta amfani da Xcalibur 4.1 (Thermo Fisher Scientific).
Don tantance ingancin bayanai, an samar da samfuran kula da inganci (QC) ta hanyar cire ƙananan ƙwayoyin cuta 10 μL daga kowane samfurin. An yi nazarin allurar rigakafin inganci guda shida a farkon jerin nazarin don tantance daidaiton tsarin UPLC-MS. Daga nan ana gabatar da samfuran kula da inganci lokaci-lokaci a cikin rukunin. LC-MS ta yi nazarin dukkan rukunin samfuran jini guda 11 a cikin wannan binciken. An yi amfani da ƙananan ƙwayoyin cuta na cakuda wurin wanka na jini daga masu ba da gudummawa 100 masu lafiya a matsayin kayan aiki na tunani a cikin rukunin daban-daban don sa ido kan tsarin cirewa da kuma daidaita tasirin rukuni-zuwa-rukuni. An gudanar da nazarin metabolomics mara niyya na ƙungiyar ganowa, ƙungiyar tabbatarwa ta ciki, da ƙungiyar tabbatarwa ta waje a Cibiyar Metabolomics ta Jami'ar Sun Yat-sen. Dakin gwaje-gwaje na waje na Cibiyar Nazarin Fasaha da Gwaji ta Jami'ar Guangdong shi ma ya yi nazarin samfura 40 daga rukunin waje don gwada aikin samfurin rarrabuwa.
Bayan cirewa da sake haɗa su, an auna cikakken adadin metabolites na jini ta amfani da spectrometry na ruwa mai ƙarfi-mai aiki (Agilent 6495 triple quadrupole) tare da tushen ionization na electrospray (ESI) a cikin yanayin sa ido kan amsawa da yawa (MRM). An yi amfani da ginshiƙin ACQUITY BEH Amide (2.1 × 100 mm, 1.7 μm, Ruwa) don raba metabolites. Matakin wayar hannu ya ƙunshi 90% (A) da 5% acetonitrile (B) tare da 10 mmol/L ammonium acetate da 0.1% maganin ammonia. Shirin gradient ɗin ya kasance kamar haka: 0–1.5 min, 0% B; 1.5–6.5 min, 0–15% B; 6.5–8 min, 15% B; 8–8.5 min, 15%–0% B; 8.5–11.5 min, 0% B. An kiyaye ginshiƙin a zafin 40 °C da kuma samfurin a zafin 10 °C a cikin na'urar daukar hoton atomatik. Yawan kwararar ya kasance 0.3 mL/min kuma girman allurar shine 1 μL. An saita sigogin MS kamar haka: ƙarfin lantarki na capillary ±3.5 kV, matsin lamba na nebulizer 35 psi, kwararar iskar sheath 12 L/min, zafin iskar sheath 350 °C, zafin iskar busasshe 250 °C, da kuma kwararar iskar busasshe 14 l/min. Juyawar MRM na tryptophan, pyruvate, lactate, hypoxanthine da xanthine sun kasance 205.0–187.9, 87.0–43.4, 89.0–43.3, 135.0–92.3 da 151.0–107. 9 bi da bi. An tattara bayanai ta amfani da Mass Hunter B.07.00 (Agilent Technologies). Ga samfuran jini, an auna tryptophan, pyruvate, lactate, hypoxanthine, da xanthine ta amfani da lanƙwasa na daidaita ma'aunin maganin cakuda na yau da kullun. Ga samfuran tantanin halitta, an daidaita yawan tryptophan zuwa ma'aunin ciki da kuma yawan furotin na tantanin halitta.
An yi amfani da Compound Discovery 3.1 da TraceFinder 4.0 (Thermo Fisher Scientific). Don kawar da bambance-bambance masu yuwuwa tsakanin rukuni-rukuni, kowane kololuwar halayyar samfurin gwajin an raba shi da kololuwar halayyar kayan tunani daga rukuni ɗaya don samun yalwar dangi. An nuna bambance-bambancen daidaitattun ka'idojin ciki kafin da bayan daidaitawa a cikin Tebur na Ƙarin 6. Bambance-bambance tsakanin ƙungiyoyin biyu an siffanta su da ƙimar gano ƙarya (FDR <0.05, gwajin matsayi na Wilcoxon da aka sanya hannu) da canjin ninkawa (>1.2 ko <0.83). Bayanan MS marasa inganci na fasalulluka da aka cire da bayanan MS da aka gyara a cikin jini an nuna su a cikin Ƙarin Bayanai 1 da Bayanan Ƙarin Bayani 2, bi da bi. An yi bayanin kololuwar bisa ga matakai huɗu da aka ƙayyade na ganewa, gami da metabolites da aka gano, mahaɗan da aka yi bayanin su a zahiri, azuzuwan mahaɗan da aka siffanta a zahiri, da mahaɗan da ba a sani ba 22. Dangane da binciken bayanai a cikin Compound Discovery 3.1 (mzCloud, HMDB, Chemspider), an zaɓi mahaɗan halittu tare da daidaitattun daidaitattun MS/MS ko kuma cikakkun bayanai daidai a cikin mzCloud (maki > 85) ko Chemspider a ƙarshe a matsayin tsaka-tsaki tsakanin bambancin metabolome. An haɗa bayanan mafi girma ga kowane fasali a cikin Ƙarin Bayanan 3. An yi amfani da MetaboAnalyst 5.0 don nazarin univariate na yawan metabolites da aka daidaita. MetaboAnalyst 5.0 kuma ya kimanta nazarin haɓaka hanyar KEGG bisa ga metabolites daban-daban. An bincika babban nazarin sassan (PCA) da nazarin bambance-bambancen murabba'i na ɓangaren ƙananan murabba'i (PLS-DA) ta amfani da fakitin software na ropls (v.1.26.4) tare da daidaita tari da kuma daidaita atomatik. An samar da mafi kyawun samfurin biomarker na metabolite don annabta cutar kansa ta hanyar amfani da binary logistic regression tare da ƙarancin raguwa da mai aiki na zaɓi (LASSO, R package v.4.1-3). An siffanta aikin samfurin bambance-bambancen a cikin saitin ganowa da tabbatarwa ta hanyar kimanta AUC bisa ga nazarin ROC bisa ga kunshin pROC (v.1.18.0.). An sami mafi kyawun yankewar yiwuwar dangane da matsakaicin ma'aunin Youden na samfurin (ƙwaƙwalwa + takamaiman - 1). Za a yi hasashen samfuran da ƙimarsu ƙasa da ko sama da matakin a matsayin nodules marasa lahani da adenocarcinoma na huhu, bi da bi.
An shuka ƙwayoyin A549 (#CCL-185, American Type Culture Collection) a cikin matsakaicin F-12K wanda ke ɗauke da 10% FBS. An saka jerin gajerun RNA (shRNA) waɗanda ke nufin SLC7A5 da kuma mai sarrafa abin da ba a ta'allaka ba (NC) a cikin vector lentiviral pLKO.1-puro. Jerin maganin hana ji na shSLC7A5 sune kamar haka: Sh1 (5′-GGAGAAACCTGAACAGTT-3′), Sh2 (5′-GCCGTGGACTTCGGAACTTAT-3′). An sayi ƙwayoyin rigakafi zuwa SLC7A5 (#5347) da tubulin (#2148) daga Fasahar Siginar Kwayoyin Halitta. An yi amfani da ƙwayoyin rigakafi zuwa SLC7A5 da tubulin a cikin narkewar 1:1000 don nazarin Western blot.
Gwajin Damuwa na Seahorse XF Glycolytic yana auna matakan acidification na extracellular (ECAR). A cikin gwajin, an ba da glucose, oligomycin A, da 2-DG a jere don gwada ƙarfin glycolytic na ƙwayoyin halitta kamar yadda ECAR ta auna.
An yi wa ƙwayoyin A549 da aka yi musu allurar da ba ta da wani tasiri (NC) da kuma shSLC7A5 (Sh1, Sh2) fenti na dare a cikin kwano mai diamita 10 cm. An cire ƙwayoyin halitta da 1 ml na methanol mai sanyi 80% na ruwa mai sanyi. An goge ƙwayoyin da ke cikin maganin methanol, an tattara su cikin sabon bututu, sannan aka sanya su a centrifuge a 15,000 × g na tsawon minti 15 a 4°C. A tattara 800 µl na supernatant a bushe ta amfani da injin tattara iska mai sauri. Daga nan aka yi nazarin ƙwayoyin metabolite da aka busar don matakan tryptophan ta amfani da LC-MS/MS kamar yadda aka bayyana a sama. An auna matakan NAD(H) na ƙwayoyin halitta a cikin ƙwayoyin A549 (NC da shSLC7A5) ta amfani da kayan aikin launi na NAD+/NADH (#K337, BioVision) bisa ga umarnin masana'anta. An auna matakan furotin ga kowane samfurin don daidaita adadin metabolites.
Ba a yi amfani da hanyoyin kididdiga ba don tantance girman samfurin da farko. An yi la'akari da nazarin metabolomics na baya da aka yi niyya ga gano alamun halittu15,18 a matsayin ma'auni don tantance girman, kuma idan aka kwatanta da waɗannan rahotannin, samfurinmu ya isa. Babu wani samfuri da aka cire daga ƙungiyar binciken. An sanya samfuran jini bazuwar zuwa ga ƙungiyar gano (lamurra 306, 74.6%) da ƙungiyar tabbatarwa ta ciki (lamurra 104, 25.4%) don nazarin metabolomics marasa niyya. Mun kuma zaɓi shari'o'i 70 bazuwar daga kowace ƙungiya daga saitin gano don nazarin metabolomics da aka yi niyya. Masu binciken sun makance ga aikin rukuni yayin tattara bayanai da nazarin LC-MS. An bayyana nazarin ƙididdiga na bayanan metabolomics da gwaje-gwajen tantanin halitta a cikin sassan Sakamako, Tatsuniyoyi, da Hanyoyi. An yi ƙididdige ayyukan tryptophan tantanin halitta, NADT, da glycolytic sau uku daban-daban tare da sakamako iri ɗaya.
Don ƙarin bayani game da tsarin binciken, duba Takaitaccen Rahoton Fayil na Halitta wanda ke da alaƙa da wannan labarin.
An nuna bayanan MS na ainihin siffofin da aka cire da kuma bayanan MS da aka daidaita na maganin ma'auni a cikin Ƙarin Bayanai na 1 da Ƙarin Bayanai na 2, bi da bi. An gabatar da mafi girman bayanin fasali na bambance-bambance a cikin Ƙarin Bayanai na 3. Ana iya sauke bayanan LUAD TCGA daga https://portal.gdc.cancer.gov/. Bayanan shigarwa don tsara jadawalin an bayar da su a cikin bayanan tushe. An bayar da bayanan tushe don wannan labarin.
Ƙungiyar Nazarin Duban Huhu ta Ƙasa, da sauransu. Rage mace-macen cutar kansar huhu ta hanyar amfani da ƙaramin adadin tomography na kwamfuta. Arewacin Ingila. J. Med. 365, 395–409 (2011).
Kramer, BS, Berg, KD, Aberle, DR da Annabi, PC Gwajin cutar kansar huhu ta amfani da ƙaramin allurar CT mai maganin helical: sakamakon Nazarin Binciken Huhu na Ƙasa (NLST). J. Med. Allon 18, 109–111 (2011).
De Koning, HJ, da sauransu. Rage mace-macen cutar kansar huhu ta hanyar gwajin CT mai girma a cikin gwaji da aka yi bazuwar. Arewacin Ingila. J. Med. 382, 503–513 (2020).
Lokacin Saƙo: Satumba-18-2023