Bambance-bambance na nodules na huhu da aka gano ta hanyar ƙididdige hoto (CT) ya kasance ƙalubale a aikin asibiti.Anan, mun siffata metabolome na duniya na samfuran serum 480, gami da kulawar lafiya, nodules na huhu mara kyau, da adenocarcinoma na huhu.Adenocarcinomas suna nuna bayanan martaba na musamman na metabolomic, yayin da nodules marasa lafiya da masu lafiya suna da kamanceceniya a cikin bayanan metabolomic.A cikin ƙungiyar ganowa (n = 306), an gano saitin metabolites 27 don bambanta tsakanin nodules mara kyau da mara kyau.AUC na samfurin nuna bambanci a cikin ingantacciyar ciki (n = 104) da kuma tabbatarwa na waje (n = 111) ƙungiyoyi shine 0.915 da 0.945, bi da bi.Binciken hanya ya nuna karuwar glycolytic metabolites da ke hade da ragewar tryptophan a cikin kwayar cutar adenocarcinoma na huhu idan aka kwatanta da nodules mara kyau da kuma kula da lafiya, kuma ya ba da shawarar cewa shan tryptophan yana inganta glycolysis a cikin kwayoyin cutar ciwon huhu.Bincikenmu yana nuna darajar masu amfani da kwayoyin halitta a cikin tantance haɗarin nodules na huhu da CT ya gano.
Binciken farko yana da mahimmanci don inganta ƙimar rayuwa ga masu ciwon daji.Sakamako daga gwajin gwajin cutar daji na huhu na Amurka (NLST) da Nazarin NELSON na Turai sun nuna cewa yin gwaji tare da ƙananan ƙididdiga masu ƙima (LDCT) na iya rage yawan mutuwar cutar kansar huhu a cikin ƙungiyoyi masu haɗari1,2,3.Tun lokacin da aka yi amfani da LDCT da yawa don nazarin ciwon huhu na huhu, abubuwan da suka faru na binciken rediyo na gaggawa na nodules na huhu na asymptomatic ya ci gaba da karuwa 4.An ayyana nodules na huhu a matsayin ɓangarorin hangen nesa har zuwa 3 cm a diamita 5.Muna fuskantar matsaloli wajen tantance yiwuwar malignancy da kuma magance yawan adadin nodules na huhu da aka gano ba zato ba tsammani akan LDCT.Ƙayyadaddun CT na iya haifar da gwaje-gwaje akai-akai akai-akai da sakamako mai kyau na ƙarya, wanda zai haifar da shiga tsakani da ba dole ba da overtreatment6.Sabili da haka, akwai buƙatar haɓaka masu dogara da masu amfani da kwayoyin halitta don gano ainihin ciwon huhu a farkon matakai da kuma bambanta mafi yawan nodules a farkon ganowa 7.
Cikakken nazarin kwayoyin jini na jini (serum, plasma, na gefe na jini mononuclear sel), ciki har da genomics, proteomics ko DNA methylation8,9,10, ya haifar da haɓaka sha'awar gano masu gano kwayoyin halitta don ciwon huhu.A halin yanzu, hanyoyin metabolomics suna auna samfuran ƙarshen salon salula waɗanda ayyukan endogenous da na waje suka rinjayi don haka ana amfani da su don hasashen farawa da sakamako.Liquid chromatography-tandem mass spectrometry (LC-MS) hanya ce ta ko'ina da ake amfani da ita don nazarin metabolomics saboda girman hankalinta da babban kewayon kuzari, wanda zai iya rufe metabolites tare da kaddarorin physicochemical daban-daban11,12,13.Ko da yake an yi amfani da nazarin metabolomic na duniya na plasma/serum don gano alamomin halittu masu alaƙa da cutar sankara na huhu14,15,16,17 da ingancin magani, 18 masu rarraba ƙwayoyin metabolite don bambance tsakanin nodules na huhu mara kyau da m.- gagarumin bincike.
Adenocarcinoma da squamous cell carcinoma su ne manyan nau'o'in nau'i biyu na ciwon daji na huhu marasa ƙananan (NSCLC).Gwaje-gwaje daban-daban na CT sun nuna cewa adenocarcinoma shine nau'in ciwon daji na huhu da aka fi sani da shi1,19,20,21.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 sinadirai 695, gami da sarrafa lafiya, nodules na huhu, da CT-gano ≤3 cm.Nunawa ga Stage I huhu adenocarcinoma.Mun gano wani panel na serum metabolites wanda ke bambanta adenocarcinoma na huhu daga nodules mara kyau da kuma kula da lafiya.Binciken wadatar hanyar hanya ya nuna cewa rashin daidaituwa na tryptophan da glucose metabolism sune sauye-sauye na yau da kullun a cikin adenocarcinoma na huhu idan aka kwatanta da nodules mara kyau da kulawar lafiya.A ƙarshe, mun kafa kuma mun tabbatar da rarrabuwa na sinadarai na ƙwayar cuta tare da ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai da azanci don bambancewa tsakanin muggan ƙwayoyin cuta da nodules na huhu waɗanda LDCT suka gano, waɗanda zasu iya taimakawa a farkon ganewar asali da ƙimar haɗari.
A cikin binciken na yanzu, an tattara samfurori na jima'i- da shekarun da suka dace da jima'i daga matakan lafiya na 174, marasa lafiya 292 tare da nodules na huhu, da marasa lafiya 229 tare da mataki na huhu adenocarcinoma.Ana nuna halayen alƙaluma na batutuwa 695 a cikin ƙarin Tebu 1.
Kamar yadda aka nuna a cikin Hoto 1a, an tattara jimlar samfuran 480 na serum, ciki har da 174 lafiya kula da lafiya (HC), 170 benign nodules (BN), da 136 mataki I huhu adenocarcinoma (LA) samfurori, an tattara a Sun Yat-sen University Cancer Center.Ƙungiyar ganowa don bayanan metabolomic mara niyya ta amfani da ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS).Kamar yadda aka nuna a Ƙarin Hoto na 1, bambance-bambancen metabolites tsakanin LA da HC, LA da BN an gano su don kafa samfurin rarrabuwa da kuma ci gaba da binciken bincike na banbamci.Samfura 104 da Cibiyar Ciwon daji ta Jami'ar Sun Yat-sen ta tattara da samfuran 111 da wasu asibitoci biyu suka tattara an ba su ingantaccen ciki da waje, bi da bi.
Yawan Nazari a cikin ƙungiyar ganowa waɗanda aka gudanar da bincike na metabolomics na duniya ta hanyar amfani da ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS).b Aƙalla ƙananan murabba'ai bincike na wariya (PLS-DA) na jimlar metabolome na 480 samfuran serum daga ƙungiyar binciken, gami da kulawar lafiya (HC, n = 174), nodules mara kyau (BN, n = 170), da kuma adenocarcinoma na huhu. (Los Angeles, n = 136).+ ESI, ingantaccen yanayin ionization electrospray, -ESI, yanayin ionization mara kyau na electrospray.c–e Metabolites tare da ɗimbin yawa daban-daban a cikin ƙungiyoyi biyu da aka bayar (Wilcoxon mai wutsiya biyu ya sanya hannu kan gwajin daraja, ƙimar gano ƙarya da aka daidaita p ƙimar, FDR <0.05) ana nuna su cikin ja (canji ninki> 1.2) da shuɗi (canji ninki <0.83) .) wanda aka nuna akan hoton dutsen mai aman wuta.f Taswirar taswirar zafi mai tsari yana nuna bambance-bambance masu mahimmanci a cikin adadin bayanan metabolites tsakanin LA da BN.Ana ba da bayanan tushen ta hanyar fayilolin bayanan tushen.
An yi nazarin jimlar ƙwayar ƙwayar ƙwayar ƙwayar cuta ta 174 HC, 170 BN da 136 LA a cikin ƙungiyar ganowa ta amfani da bincike na UPLC-HRMS.Da farko mun nuna cewa samfuran sarrafa ingancin (QC) sun taru sosai a tsakiyar tsarin nazarin manyan abubuwan da ba a kula da su ba (PCA), yana mai tabbatar da daidaiton aikin binciken na yanzu (Ƙarin Hoto 2).
Kamar yadda aka nuna a cikin ƙananan ƙananan murabba'i-wariya bincike (PLS-DA) a cikin Hoto 1 b, mun gano cewa akwai bambance-bambance a fili tsakanin LA da BN, LA da HC a cikin tabbatacce (+ ESI) da kuma mummunan (-ESI) hanyoyin ionization electrospray .ware.Koyaya, ba a sami bambance-bambance masu mahimmanci tsakanin BN da HC a cikin + ESI da -ESI yanayi.
Mun sami nau'ikan nau'ikan nau'ikan nau'ikan 382 tsakanin LA da HC, nau'ikan nau'ikan nau'ikan 231 tsakanin LA da BN, da nau'ikan nau'ikan nau'ikan 95 tsakanin BN da HC (Wilcoxon ya sanya hannu kan gwajin daraja, FDR <0.05 da canji mai yawa> 1.2 ko <0.83) (Hoto .1c-e). )..An ƙara yin bayanin kololuwa (Ƙarin Bayanan 3) a kan ma'ajin bayanai (mzCloud/HMDB/Chemspider library) ta ƙimar m/z, lokacin riƙewa da rarrabuwa taro bakan binciken (cikakkun bayanai da aka bayyana a cikin Sashen Hanyoyi) 22 .A ƙarshe, an gano 33 da 38 bayanan metabolites tare da bambance-bambance masu yawa a cikin LA da BN (Hoto 1f da Ƙarin Teburin 2) da LA da HC (Ƙarin Hoto 3 da Ƙari na 2), bi da bi.Sabanin haka, kawai 3 metabolites tare da bambance-bambance masu yawa a cikin BN da HC (Ƙarin Teburin 2), daidai da haɗuwa tsakanin BN da HC a cikin PLS-DA.Waɗannan bambance-bambancen metabolites sun ƙunshi nau'ikan sinadarai masu yawa (Ƙarin Hoto 4).A hade tare, waɗannan sakamakon suna nuna gagarumin canje-canje a cikin ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar cuta wanda ke nuna mummunan canji na ciwon huhu na farko idan aka kwatanta da nodules na huhu ko kuma batutuwa masu lafiya.A halin yanzu, kamanni na metabolome na BN da HC yana nuna cewa nodules na huhu na huhu na iya raba halaye da yawa na halitta tare da mutane masu lafiya.Ganin cewa maye gurbi na haɓakar haɓakar epidermal (EGFR) ya zama ruwan dare gama gari a cikin huhu adenocarcinoma subtype 23, mun nemi sanin tasirin maye gurbi na direba akan ƙwayar ƙwayar ƙwayar cuta.Sa'an nan kuma muka bincika gabaɗayan bayanan metabolomic na lokuta 72 tare da matsayin EGFR a cikin ƙungiyar adenocarcinoma huhu.Abin sha'awa, mun sami kwatankwacin bayanan martaba tsakanin EGFR mutant marasa lafiya (n = 41) da EGFR-nau'in marasa lafiya (n = 31) a cikin nazarin PCA (Ƙarin Hoto 5a).Duk da haka, mun gano 7 metabolites wanda yawansu ya canza sosai a cikin marasa lafiya tare da maye gurbin EGFR idan aka kwatanta da marasa lafiya tare da nau'in EGFR na daji (t gwajin, p <0.05 da sauyin ninka> 1.2 ko <0.83) (Ƙarin Hoto 5b).Yawancin waɗannan metabolites (5 daga 7) sune acylcarnitines, waɗanda ke taka muhimmiyar rawa a cikin hanyoyin oxidation fatty acid.
Kamar yadda aka kwatanta a cikin aikin aiki da aka nuna a cikin Hoto 2 a, an samo alamun biomarkers don rarrabuwar nodule ta amfani da mafi ƙarancin ma'aikatan ragewa da zaɓi dangane da nau'ikan metabolites na 33 da aka gano a cikin LA (n = 136) da BN (n = 170).Mafi kyawun haɗin sauye-sauye (LASSO) - tsarin koma baya na binary logistic.An yi amfani da ingantaccen giciye sau goma don gwada amincin samfurin.Zaɓuɓɓuka masu canzawa da daidaita sigina ana daidaita su ta hanyar yuwuwar ƙarar hukunci tare da siga λ24.An ci gaba da yin nazarin metabolomics na duniya da kansa a cikin ingantacciyar ciki (n = 104) da ingantattun ƙungiyoyin waje (n = 111) don gwada aikin rarrabuwa na ƙirar wariya.A sakamakon haka, an gano 27 metabolites a cikin saitin ganowa a matsayin mafi kyawun samfurin nuna bambanci tare da mafi girman darajar AUC (Fig. 2b), daga cikinsu 9 ya kara yawan aiki kuma 18 ya rage yawan aiki a LA idan aka kwatanta da BN (Fig. 2c).
Gudun aiki don gina nau'in nodule na huhu, gami da zaɓar mafi kyawun panel na metabolites na serum a cikin saitin ganowa ta amfani da ƙirar juzu'i na binary ta hanyar tabbatar da giciye mai ninki goma da kimanta aikin tsinkaya a cikin saitin tabbatarwa na ciki da waje.b Ƙididdiga ta giciye na samfurin koma baya na LASSO don zaɓin ƙirar halitta.Lambobin da aka bayar a sama suna wakiltar matsakaicin adadin masu alamar halitta da aka zaɓa a wani λ.Layin jajayen dige-dige yana wakiltar matsakaicin ƙimar AUC a madaidaicin lambda.Sandunan kuskuren launin toka suna wakiltar mafi ƙaranci da matsakaicin ƙimar AUC.Layin dige-dige yana nuna mafi kyawun samfuri tare da 27 zaɓaɓɓun alamomin halittu.AUC, yanki a ƙarƙashin madaidaicin yanayin aiki mai karɓa (ROC).c ninka canje-canje na metabolites 27 da aka zaɓa a cikin ƙungiyar LA idan aka kwatanta da ƙungiyar BN a cikin ƙungiyar ganowa.Rukunin ja - kunnawa.Rukunin shuɗi yana raguwa.d–f Siffofin aiki na mai karɓa (ROC) suna nuna ƙarfin ƙirar ƙima dangane da haɗe-haɗe na metabolite guda 27 a cikin saitin tabbatarwa, ciki, da waje.Ana ba da bayanan tushen ta hanyar fayilolin bayanan tushen.
An ƙirƙiri samfurin tsinkaya dangane da ma'aunin koma baya na ma'auni na waɗannan metabolites 27 (Ƙarin Tebu 3).Binciken ROC dangane da waɗannan metabolites na 27 ya ba da wani yanki a ƙarƙashin ƙimar (AUC) na 0.933, ƙwarewar ƙungiyar ganowa shine 0.868, kuma takamaiman shine 0.859 (Fig. 2d).A halin yanzu, a cikin 38 annotate bambance-bambancen metabolites tsakanin LA da HC, saitin metabolites 16 sun sami AUC na 0.902 tare da ji na 0.801 da ƙayyadaddun 0.856 a cikin nuna wariya LA daga HC (Ƙarin Hoto 6a-c).Hakanan an kwatanta ƙimar AUC dangane da ninki daban-daban na canza kofa don bambance-bambancen metabolites.Mun gano cewa ƙirar rarrabuwa ta yi mafi kyau wajen nuna wariya tsakanin LA da BN (HC) lokacin da aka saita matakin canjin ninka zuwa 1.2 da 1.5 ko 2.0 (Ƙarin Hoto 7a,b).Samfurin rarrabuwa, bisa ƙungiyoyin metabolite 27, an ƙara inganta shi a cikin ƙungiyoyin ciki da waje.AUC ya kasance 0.915 (hankali 0.867, ƙayyadaddun 0.811) don tabbatarwa na ciki da 0.945 (ƙananan 0.810, ƙayyadaddun 0.979) don ingantaccen waje (Fig. 2e, f).Don tantance ingancin haɗin gwiwa, samfuran 40 daga ƙungiyar waje an bincika su a cikin dakin gwaje-gwaje na waje kamar yadda aka bayyana a cikin sashin hanyoyin.Daidaiton rarrabuwa ya sami AUC na 0.925 (Ƙarin Hoto 8).Saboda ciwon huhu na huhu (LUSC) shine nau'in nau'in ciwon daji na huhu na huhu (NSCLC) na biyu bayan adenocarcinoma na huhu (LUAD), mun kuma gwada ingantacciyar damar amfani da bayanan martaba.BN da 16 lokuta na LUSC.AUC na wariya tsakanin LUSC da BN ya kasance 0.776 (Ƙarin Hoto na 9), yana nuna rashin ƙarfi idan aka kwatanta da wariya tsakanin LUAD da BN.
Nazarin ya nuna cewa girman nodules akan hotuna na CT yana da alaƙa da alaƙa da yiwuwar malignancy kuma ya kasance babban mahimmancin maganin nodule25,26,27.Binciken bayanai daga babban rukuni na binciken binciken NELSON ya nuna cewa hadarin rashin lafiya a cikin batutuwa tare da nodes <5 mm ya kasance ma kama da abin da ke cikin batutuwa ba tare da nodes 28 ba.Sabili da haka, ƙananan girman da ake buƙatar saka idanu na CT na yau da kullum shine 5 mm, kamar yadda British Thoracic Society (BTS) ya ba da shawarar, da 6 mm, kamar yadda Fleischner Society 29 ya ba da shawarar.Duk da haka, nodules ya fi girma fiye da 6 mm kuma ba tare da siffofi masu kyau ba, wanda ake kira nodules na huhu (IPN), ya kasance babban kalubale a kimantawa da gudanarwa a aikin asibiti30,31.Mu na gaba mun bincika ko girman nodule ya rinjayi sa hannu na metabolomic ta amfani da samfuran da aka tattara daga ganowa da ƙungiyoyin tabbatarwa na ciki.Mayar da hankali kan ingantattun alamomin halittu guda 27, mun fara kwatanta bayanan PCA na HC da BN sub-6 mm metabolomes.Mun gano cewa yawancin bayanan bayanan HC da BN sun mamaye, yana nuna cewa matakan ƙwayar ƙwayar ƙwayar ƙwayar cuta sun kasance iri ɗaya a cikin ƙungiyoyi biyu (Fig. 3a).Taswirorin fasalin da ke cikin jeri daban-daban sun kasance ana kiyaye su a cikin BN da LA (Fig. 3b, c), yayin da aka lura da rabuwa tsakanin mummuna da nodules mara kyau a cikin kewayon 6-20 mm (Fig. 3d).Wannan ƙungiyar tana da AUC na 0.927, ƙayyadaddun 0.868, da kuma hankali na 0.820 don tsinkayar mummunan nodules mai auna 6 zuwa 20 mm (Fig. 3e, f).Sakamakonmu ya nuna cewa mai rarrabawa zai iya ɗaukar sauye-sauye na rayuwa wanda ya haifar da canji mara kyau na farko, ba tare da la'akari da girman nodule ba.
ad Kwatanta bayanan bayanan PCA tsakanin ƙayyadaddun ƙungiyoyi dangane da rabe-rabe na metabolites 27.CC da BN <6 mm.b BN <6 mm vs BN 6-20 mm.a cikin LA 6-20 mm 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 Mai karɓar sifa mai aiki (ROC) mai lanƙwasa yana nuna aikin ƙirar nuna bambanci don nodules 6-20 mm.f An ƙididdige ƙididdige ƙididdigewa bisa ga tsarin jujjuyawar dabaru don nodules masu auna 6-20 mm.Layin launin toka mai digo yana wakiltar mafi kyawun ƙimar yanke (0.455).Lambobin da ke sama suna wakiltar adadin adadin da aka tsara don Los Angeles.Yi amfani da gwajin ɗalibi mai wutsiya biyu.PCA, babban bincike na bangaren.Yankin AUC a ƙarƙashin lanƙwasa.Ana ba da bayanan tushen ta hanyar fayilolin bayanan tushen.
Samfurori huɗu (shekaru 44-61) tare da nau'ikan nodule na huhu iri ɗaya (7-9 mm) an ƙara zaɓe don kwatanta aikin ƙirar tsinkayar ɓarna (Fig. 4a, b).A farkon nunawa, Case 1 an gabatar da shi azaman ƙaƙƙarfan nodule tare da ƙididdigewa, fasalin da ke da alaƙa da benignity, yayin da Case 2 an gabatar da shi azaman ƙaƙƙarfan ƙaƙƙarfan nodule wanda ba shi da fa'ida mara kyau.Zagaye uku na CT scans na bin diddigin ya nuna cewa waɗannan lamuran sun kasance masu ƙarfi a tsawon shekaru 4 kuma saboda haka ana ɗaukar su nodules mara kyau (Fig. 4a).Idan aka kwatanta da kimantawa na asibiti na serial CT scans, bincike na metabolite na jini-harbi guda ɗaya tare da ƙirar ƙira na yanzu da sauri kuma an gano waɗannan nodules mara kyau daidai bisa ƙayyadaddun yuwuwar (Table 1).Hoto na 4b a yanayin 3 yana nuna nodule tare da alamun ja da baya, wanda galibi ana danganta shi da malignancy32.Case na 4 da aka gabatar a matsayin nodule mai ƙarfi mara ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙaƙƙarfan nodule ba tare da wata shaida ta dalili mara kyau ba.Duk waɗannan shari'o'in an yi annabta a matsayin m bisa ga ƙirar ƙira (Table 1).An nuna kima na adenocarcinoma na huhu ta hanyar nazarin ilimin lissafi bayan aikin tiyata na huhu (Fig. 4b).Don saitin ingantacciyar hanyar waje, mai rarraba rayuwa daidai ya annabta lokuta biyu na nodules na huhu mara iyaka wanda ya fi 6 mm (Ƙarin Hoto 10).
Hotunan CT na taga axial na huhu na lokuta biyu na nodules mara kyau.A cikin yanayin 1, CT scan bayan shekaru 4 ya nuna barga mai ƙarfi nodule mai auna 7 mm tare da ƙididdiga a cikin ƙananan lobe na dama.A cikin yanayin 2, CT scan bayan shekaru 5 ya bayyana wani barga, wani sashi mai ƙarfi nodule tare da diamita na 7 mm a cikin lobe na dama na dama.b Axial taga CT hotuna na huhu da kuma daidaitattun nazarin ilimin cututtuka na lokuta biyu na mataki na adenocarcinoma kafin huhu na huhu.Case 3 ya bayyana nodule tare da diamita na 8 mm a cikin babban lobe na dama tare da ja da baya.Case 4 ya bayyana wani ɓangaren ƙaƙƙarfan ƙaƙƙarfan nodule-gilashin ƙasa mai auna 9 mm a babban lobe na hagu.Hematoxylin da eosin (H&E) tabo na nama na huhu da aka yi wa resected (sikelin sikelin = 50 μm) yana nuna alamar ci gaban acinar na huhu adenocarcinoma.Kibau suna nuna nodules da aka gano akan hotunan CT.Hotunan H&E hotuna ne na wakilci na filaye da yawa (> 3) ƙananan ƙananan ƙwayoyin cuta wanda masanin ilimin cututtuka ya bincika.
A hade tare, sakamakonmu yana nuna yuwuwar kimar ma'aunin ƙwayoyin cuta na ƙwayoyin cuta a cikin bambance-bambancen ganewar ƙwayar nodules na huhu, waɗanda na iya haifar da ƙalubale yayin kimanta gwajin CT.
Dangane da ingantacciyar ɓangarorin metabolite na metabolite, mun nemi gano alaƙar halittu na manyan canje-canjen rayuwa.Binciken haɓaka hanyoyin KEGG ta MetaboAnalyst ya gano hanyoyin 6 na yau da kullun da aka canza tsakanin ƙungiyoyin da aka bayar (LA vs. HC da LA vs. BN, daidaita p ≤ 0.001, sakamako> 0.01).Wadannan canje-canje sun kasance da damuwa a cikin pyruvate metabolism, tryptophan metabolism, niacin da nicotinamide metabolism, glycolysis, da TCA sake zagayowar, da purine metabolism (Fig. 5a).Daga nan mun ƙara yin niyya metabolomics don tabbatar da manyan canje-canje ta amfani da cikakken ƙididdigewa.Ƙaddamar da metabolites na gama gari a cikin hanyoyin da aka saba canzawa ta hanyar sau uku quadrupole mass spectrometry (QQQ) ta amfani da ingantattun matakan metabolite.Halayen ƙididdiga na samfurin binciken binciken metabolomics an haɗa su a cikin Ƙarin Table 4. Daidai da sakamakon mu na duniya metabolomics, ƙididdigar ƙididdiga ya tabbatar da cewa hypoxanthine da xanthine, pyruvate, da lactate sun karu a LA idan aka kwatanta da BN da HC (Fig. 5b, c, p <0.05).Koyaya, ba a sami bambance-bambance masu mahimmanci a cikin waɗannan metabolites tsakanin BN da HC ba.
Binciken haɓaka hanyoyin KEGG na mahimman metabolites daban-daban a cikin rukunin LA idan aka kwatanta da ƙungiyoyin BN da HC.An yi amfani da Globaltest mai wutsiya biyu, kuma an daidaita ƙimar p ta amfani da hanyar Holm-Bonferroni (daidaita p ≤ 0.001 da girman tasirin> 0.01).b-d Shirye-shiryen Violin suna nuna hypoxanthine, xanthine, lactate, pyruvate, da matakan tryptophan a cikin maganin HC, BN, da LA wanda LC-MS/MS ya ƙaddara (n = 70 kowace ƙungiya).Layuka masu dige fari da baƙi suna nuna tsaka-tsaki da huɗu, bi da bi.e Makircin Violin yana nuna daidaitaccen Log2TPM (rubutun da miliyan) mRNA magana na SLC7A5 da QPRT a cikin adenocarcinoma huhu (n = 513) idan aka kwatanta da nama na huhu na yau da kullun (n = 59) a cikin bayanan LUAD-TCGA.Akwatin farin yana wakiltar kewayon tsaka-tsaki, layin baƙar fata a kwance a tsakiyar yana wakiltar tsaka-tsaki, kuma layin baƙar fata na tsaye wanda ya shimfiɗa daga akwatin yana wakiltar tazarar amincewa ta 95% (CI).f Pearson daidaita makirci na SLC7A5 da GAPDH magana a cikin huhu adenocarcinoma (n = 513) da kuma al'ada huhu nama (n = 59) a cikin TCGA dataset.Yankin launin toka yana wakiltar 95% CI.r, Pearson haɗin kai.g Matakan tryptophan na salula na yau da kullun a cikin sel A549 waɗanda aka canza su tare da sarrafa shRNA marasa takamaiman (NC) da shSLC7A5 (Sh1, Sh2) wanda LC-MS/MS ya ƙaddara.Ana gabatar da ƙididdigar ƙididdiga na samfurori masu zaman kansu guda biyar a cikin kowane rukuni.h Matakan salula na NADt (jimlar NAD, gami da NAD + da NADH) a cikin ƙwayoyin A549 (NC) da SLC7A5 ƙwanƙwasa sel A549 (Sh1, Sh2).Ana gabatar da ƙididdigar ƙididdiga na samfurori masu zaman kansu guda uku a cikin kowane rukuni.i Ayyukan Glycolytic na sel A549 kafin da kuma bayan SLC7A5 knockdown an auna ta ta hanyar extracellular acidification rate (ECAR) (n = 4 samfurori masu zaman kansu na halitta a kowace ƙungiya).2-DG,2-deoxy-D-glucose.An yi amfani da gwajin ɗalibi mai wutsiya biyu a cikin (b–h).A cikin (g-i), sandunan kuskure suna wakiltar ma'anar ± SD, kowane gwaji an yi shi sau uku da kansa kuma sakamakon ya kasance iri ɗaya.Ana ba da bayanan tushen ta hanyar fayilolin bayanan tushen.
Idan aka yi la'akari da gagarumin tasirin canjin canjin tryptophan a cikin rukunin LA, mun kuma tantance matakan maganin tryptophan a cikin ƙungiyoyin HC, BN, da LA ta amfani da QQQ.Mun gano cewa an rage maganin tryptophan a cikin LA idan aka kwatanta da HC ko BN (p <0.001, Hoto 5d), wanda ya yi daidai da binciken da aka yi a baya cewa yaduwa matakan tryptophan ya ragu a cikin marasa lafiya da ciwon huhu fiye da kulawar lafiya daga rukunin kulawa33,34 ,35.Wani binciken da aka yi amfani da PET/CT tracer 11C-methyl-L-tryptophan ya gano cewa lokacin riƙe siginar tryptophan a cikin ƙwayar cutar kansar huhu ya ƙaru sosai idan aka kwatanta da raunuka marasa kyau ko nama na al'ada36.Muna tsammanin raguwar tryptophan a cikin jini na LA na iya yin nuni da ɗaukar tryptophan mai aiki ta ƙwayoyin kansar huhu.
Hakanan an san cewa ƙarshen samfurin hanyar kynurenine na catabolism tryptophan shine NAD + 37,38, wanda shine muhimmin mahimmanci don amsawar glyceraldehyde-3-phosphate tare da 1,3-bisphosphoglycerate a cikin glycolysis39.Yayin da binciken da aka yi a baya ya mayar da hankali kan rawar da ake yi na catabolism na tryptophan a cikin ka'idojin rigakafi, mun nemi bayyana ma'amala tsakanin dysregulation na tryptophan da hanyoyin glycolytic da aka lura a cikin binciken na yanzu.Iyali mai jigilar solute 7 memba 5 (SLC7A5) an san shi azaman jigilar tryptophan43,44,45.Quinolinic acid phosphoribosyltransferase (QPRT) wani enzyme ne da ke ƙasa daga cikin hanyar kynurenine wanda ke canza quinolinic acid zuwa NAMN46.Binciken bayanan LUAD TCGA ya nuna cewa duka SLC7A5 da QPRT sun kasance masu mahimmanci a cikin ƙwayar ƙwayar cuta idan aka kwatanta da nama na al'ada (Fig. 5e).An lura da wannan karuwa a cikin matakai na I da II da kuma matakai na III da IV na adenocarcinoma na huhu (Ƙarin Hoto 11), yana nuna damuwa da wuri a cikin ƙwayar cuta ta tryptophan da ke hade da tumorigenesis.
Bugu da ƙari, bayanan LUAD-TCGA ya nuna kyakkyawar alaƙa tsakanin SLC7A5 da GAPDH mRNA magana a cikin samfuran masu cutar kansa (r = 0.45, p = 1.55E-26, Hoto 5f).Sabanin haka, ba a sami alaƙa mai mahimmanci tsakanin irin waɗannan sa hannu na kwayoyin halitta a cikin nama na huhu na yau da kullun (r = 0.25, p = 0.06, Hoto 5f).Knockdown na SLC7A5 (Ƙarin Hoto 12) a cikin ƙwayoyin A549 ya rage yawan matakan tryptophan na salula da NAD (H) (Figure 5g, h), wanda ya haifar da raguwar ayyukan glycolytic kamar yadda aka auna ta hanyar extracellular acidification rate (ECAR) (Hoto 1).5i).Don haka, dangane da canje-canjen rayuwa a cikin jini da gano in vitro, muna tsammanin cewa metabolism na tryptophan na iya haifar da NAD + ta hanyar kynurenine kuma yana taka muhimmiyar rawa wajen haɓaka glycolysis a cikin ciwon huhu.
Nazarin ya nuna cewa yawancin nodules na nodules marasa iyaka wanda LDCT ya gano zai iya haifar da buƙatar ƙarin gwaji kamar PET-CT, huhu biopsy, da kuma overtreatment saboda rashin kuskuren kuskuren kuskure.31 Kamar yadda aka nuna a cikin Hoto 6. Bincikenmu ya gano wani kwamiti na metabolites na jini tare da yuwuwar ƙimar bincike wanda zai iya inganta haɓakar haɗarin haɗari da kuma kula da nodules na huhu da aka gano ta hanyar CT.
Ana kimanta nodules na huhu ta amfani da ƙananan ƙididdiga na ƙididdiga (LDCT) tare da fasalulluka na hoto masu nuna alamun rashin lafiya ko mummuna.Sakamakon rashin tabbas na nodules zai iya haifar da ziyara mai zuwa akai-akai, abubuwan da ba dole ba, da kuma wuce gona da iri.Haɗin nau'ikan nau'ikan ƙwayoyin cuta na ƙwayar cuta tare da ƙimar bincike na iya haɓaka ƙimar haɗari da kuma sarrafa nodules na huhu na gaba.PET positron emission tomography.
Bayanai daga binciken NLST na Amurka da binciken NELSON na Turai sun nuna cewa tantance ƙungiyoyi masu haɗari tare da ƙananan ƙididdigar ƙididdiga (LDCT) na iya rage yawan mutuwar ciwon daji na huhu1,3.Koyaya, kimanta haɗarin haɗari da kulawar asibiti na adadi mai yawa na nodules na huhu na huhu wanda LDCT ya gano ya kasance mafi ƙalubale.Babban makasudin shine a inganta daidaitaccen rarrabuwar ka'idoji na tushen LDCT da ke akwai ta hanyar haɗa amintattun amintattun halittu.
An gano wasu alamomin kwayoyin halitta, irin su metabolites na jini, ta hanyar kwatanta kansar huhu tare da ingantattun kulawa15,17.A cikin binciken na yanzu, mun mayar da hankali kan aikace-aikacen bincike na metabolomics na serum don bambanta tsakanin nodules na huhu da marasa kyau da LDCT suka gano ba zato ba tsammani.Mun kwatanta da duniya serum metabolome na lafiya iko (HC), benign huhu nodules (BN), da mataki I huhu adenocarcinoma (LA) samfurori ta amfani da UPLC-HRMS bincike.Mun gano cewa HC da BN suna da bayanan martaba iri ɗaya, yayin da LA ya nuna manyan canje-canje idan aka kwatanta da HC da BN.Mun gano nau'ikan metabolites guda biyu waɗanda suka bambanta LA daga HC da BN.
Tsarin gano tushen tushen LDCT na yanzu don nodules mara kyau da mara kyau ya dogara ne akan girman, yawa, ilimin halittar jiki da ƙimar girma na nodules akan lokaci30.Binciken da aka yi a baya ya nuna cewa girman nodules yana da alaƙa da yiwuwar ciwon huhu.Ko da a cikin marasa lafiya masu haɗari, haɗarin malignancy a cikin nodes <6 mm shine <1%.Hadarin malignancy ga nodules auna 6 zuwa 20 mm jeri daga 8% zuwa 64%30.Saboda haka, Fleischner Society ya ba da shawarar yanke diamita na 6 mm don bin CT na yau da kullum.29 Duk da haka, ƙididdigar haɗari da kuma kula da nodules na huhu marasa iyaka (IPN) wanda ya fi girma fiye da 6 mm ba a yi daidai ba 31.Gudanar da cututtukan zuciya na yau da kullun yana dogara ne akan jira a hankali tare da saka idanu na CT akai-akai.
Dangane da ingantaccen metabolome, mun nuna a karon farko haɗe-haɗe na sa hannu na metabolomic tsakanin mutane masu lafiya da nodules mara kyau <6 mm.Kwatankwacin ilimin halitta ya yi daidai da binciken CT na baya cewa haɗarin malignancy ga nodules <6 mm yana da ƙasa kamar ga batutuwa ba tare da nodes ba. kamanceceniya a cikin bayanan martaba na metabolomic, yana ba da shawarar cewa ma'anar aiki na etiology mara kyau daidai yake ba tare da la'akari da girman nodule ba.Don haka, na'urorin gano ƙwayoyin cuta na zamani na iya samar da gwaji guda ɗaya azaman gwajin ƙa'ida lokacin da aka fara gano nodules akan CT kuma mai yuwuwar rage sa ido na serial.A lokaci guda kuma, guda panel na biomarkers na rayuwa sun bambanta m nodules ≥6 mm a girman daga nodules mara kyau kuma sun ba da cikakkun tsinkaya ga IPNs masu girman girman girman da siffofi masu banƙyama akan hotunan CT.Wannan rarrabuwa na metabolism na serum ya yi kyau sosai a cikin tsinkayar rashin lafiyar nodules ≥6 mm tare da AUC na 0.927.A hade tare, sakamakonmu yana nuna cewa sa hannu na musamman na maganin ƙwayar cuta na iya yin nunin sauye-sauyen ƙwayar cuta da wuri-wuri kuma suna da ƙima a matsayin masu hasashen haɗari, mai zaman kansa ba tare da girman nodule ba.
Musamman ma, adenocarcinoma na huhu (LUAD) da kuma squamous cell carcinoma (LUSC) sune manyan nau'in ciwon daji na huhu marasa kanana (NSCLC).Ganin cewa LUSC yana da alaƙa da amfani da taba47 kuma LUAD shine mafi yawan tarihin tarihin nodules na huhu da aka gano akan CT screening48, ƙirar ƙirar mu an gina ta musamman don samfuran adenocarcinoma na mataki na.Wang da abokan aikinsa sun kuma mai da hankali kan LUAD kuma sun gano sa hannun lipid tara ta amfani da lipidomics don bambance kansar huhu na farko daga mutane masu lafiya17.Mun gwada samfurin ƙira na yanzu akan lokuta 16 na mataki I LUSC da 74 nodules benign kuma mun lura da ƙarancin tsinkayar LUSC (AUC 0.776), yana ba da shawarar cewa LUAD da LUSC na iya samun sa hannun nasu metabolomic.Lallai, an nuna LUAD da LUSC sun bambanta a fannin ilimin etiology, asalin halitta da ɓarnawar kwayoyin halitta49.Don haka, ya kamata a haɗa wasu nau'ikan ilimin tarihi a cikin ƙirar horarwa don gano tushen yawan jama'a na ciwon huhu a cikin shirye-shiryen tantancewa.
Anan, mun gano hanyoyi shida da aka fi canzawa akai-akai a cikin adenocarcinoma na huhu idan aka kwatanta da kulawar lafiya da nodules mara kyau.Xanthine da hypoxanthine sune na kowa metabolites na hanyar rayuwa ta purine.Daidai da sakamakonmu, masu tsaka-tsakin da ke hade da purine metabolism sun karu sosai a cikin jini ko kyallen jikin marasa lafiya tare da adenocarcinoma na huhu idan aka kwatanta da kulawar lafiya ko marasa lafiya a mataki na gaba15,50.Ƙwararren ƙwayar xanthine da matakan hypoxanthine na iya yin la'akari da anabolism da ake buƙata ta ƙwayoyin ciwon daji masu saurin yaduwa.Dysregulation na glucose metabolism sanannen alama ce ta metabolism na ciwon daji51.Anan, mun lura da karuwa mai yawa a cikin pyruvate da lactate a cikin rukunin LA idan aka kwatanta da ƙungiyar HC da BN, wanda ya yi daidai da rahotannin da suka gabata game da rashin daidaituwar hanyar glycolytic a cikin bayanan martaba na metabolome na marasa lafiya marasa kan gado (NSCLC). lafiya controls.Sakamakon ya daidaita52,53.
Mahimmanci, mun lura da alaƙar da ba ta dace ba tsakanin pyruvate da tryptophan metabolism a cikin maganin adenocarcinomas na huhu.An rage matakan tryptophan na jini a cikin rukunin LA idan aka kwatanta da ƙungiyar HC ko BN.Abin sha'awa, wani babban binciken da aka yi a baya ta hanyar amfani da wata ƙungiya mai yiwuwa ya gano cewa ƙananan matakan da ke tattare da tryptophan sun haɗu da haɗarin ciwon huhu na huhu 54.Tryptophan shine muhimmin amino acid wanda muke samu gaba daya daga abinci.Mun ƙarasa da cewa raguwar ƙwayar cutar tryptophan a cikin adenocarcinoma na huhu na iya nuna saurin raguwar wannan metabolite.An san cewa ƙarshen samfurin tryptophan catabolism ta hanyar kynurenine shine tushen de novo NAD + kira.Saboda ana samar da NAD + da farko ta hanyar hanyar ceto, mahimmancin NAD + a cikin metabolism na tryptophan a cikin lafiya da cuta ya rage don tantance46.Binciken mu na bayanan TCGA ya nuna cewa furcin mai ɗaukar nauyin jigilar solute transporter 7A5 (SLC7A5) ya karu sosai a cikin adenocarcinoma na huhu idan aka kwatanta da sarrafawa na al'ada kuma an danganta shi da kyau tare da bayanin glycolytic enzyme GAPDH.Nazarin da suka gabata sun fi mayar da hankali kan rawar da ake yi na tryptophan catabolism a cikin murkushe martanin rigakafi na antitumor40,41,42.Anan mun nuna cewa hana ɗaukar tryptophan ta hanyar ƙwanƙwasa SLC7A5 a cikin ƙwayoyin cutar kansar huhu yana haifar da raguwa mai zuwa a cikin matakan NAD na salula da haɓakar ayyukan glycolytic.A taƙaice, bincikenmu yana ba da tushen ilimin halitta don canje-canje a cikin ƙwayar ƙwayar ƙwayar cuta mai alaƙa da mummunan canji na adenocarcinoma na huhu.
Sauye-sauyen EGFR sune mafi yawan maye gurbin direba a cikin marasa lafiya tare da NSCLC.A cikin bincikenmu, mun gano cewa marasa lafiya tare da maye gurbin EGFR (n = 41) suna da cikakkun bayanan martaba na metabolomic kama da marasa lafiya da nau'in EGFR na daji (n = 31), kodayake mun sami raguwar matakan jini na wasu marasa lafiya na EGFR a cikin marasa lafiya na acylcarnitine.Ayyukan da aka kafa na acylcarnitines shine jigilar kungiyoyin acyl daga cytoplasm zuwa cikin matrix na mitochondrial, wanda ke haifar da oxidation na fatty acid don samar da makamashi 55.Dangane da bincikenmu, wani binciken da aka yi kwanan nan ya kuma gano bayanan bayanan metabolome iri ɗaya tsakanin EGFR mutant da EGFR nau'in ciwace-ciwacen daji ta hanyar nazarin metabolome na duniya na 102 huhu adenocarcinoma nama samfurori50.Abin sha'awa, an kuma sami abun ciki na acylcarnitine a cikin rukunin mutant na EGFR.Sabili da haka, ko canje-canje a cikin matakan acylcarnitine suna nuna sauye-sauye na rayuwa na EGFR da kuma hanyoyin da ke cikin kwayoyin halitta na iya cancanci ƙarin bincike.
A ƙarshe, bincikenmu ya kafa wani nau'in nau'in ƙwayar cuta na ƙwayar cuta don ganewar asali na nodules na huhu kuma yana ba da shawarar aikin aiki wanda zai iya inganta ƙimar haɗari da sauƙaƙe gudanarwa na asibiti dangane da binciken CT scan.
Kwamitin Da'a na Asibitin Cancer na Jami'ar Sun Yat-sen, Asibitin Farko na Jami'ar Sun Yat-sen, da Kwamitin Da'a na Asibitin Ciwon daji na Jami'ar Zhengzhou ne suka amince da wannan binciken.A cikin binciken da ƙungiyoyin tabbatarwa na ciki, sera 174 daga mutane masu lafiya da 244 sera daga nodules masu kyau an tattara su daga mutanen da ke yin gwajin lafiya na shekara-shekara a Ma'aikatar Kula da Ciwon daji da Rigakafin Ciwon daji, Cibiyar Ciwon daji ta Jami'ar Sun Yat-sen, da 166 benign nodules.magani.Stage I huhu adenocarcinomas an tattara su daga Sun Yat-sen University Cancer Center.A cikin ƙungiyar tabbatarwa ta waje, akwai lokuta 48 na nodules mara kyau, lokuta 39 na mataki na huhu adenocarcinoma daga Asibitin Farko na Jami'ar Sun Yat-sen, da lokuta 24 na mataki na adenocarcinoma na huhu daga Asibitin Cancer na Zhengzhou.Cibiyar Ciwon daji ta Jami'ar Sun Yat-sen ta kuma tattara lokuta 16 na mataki na ciwon huhu na huhu na squamous don gwada ikon ganowa na kafaffen rabe-raben rayuwa (an nuna halayen marasa lafiya a cikin Ƙarin Table 5).An tattara samfurori daga ƙungiyar ganowa da ƙungiyar tabbatarwa ta ciki tsakanin Janairu 2018 da Mayu 2020. An tattara samfurori na ƙungiyar tabbatarwa ta waje tsakanin Agusta 2021 da Oktoba 2022. Don rage girman bambancin jinsi, an ba da kusan adadin adadin maza da mata ga kowannensu. ƙungiya.Tawagar Ganowa da Ƙungiyar Binciken Ciki.An ƙaddara jinsin mahalarta bisa ga rahoton kai.An samu izini na sanarwa daga duk mahalarta kuma ba a bayar da diyya ba.Abubuwan da ke da nodules masu kyau sune waɗanda ke da kwanciyar hankali na CT scan a cikin shekaru 2 zuwa 5 a lokacin bincike, sai dai na 1 daga samfurin tabbatarwa na waje, wanda aka tattara da farko kuma an gano shi ta hanyar histopathology.Banda ciwon sankara na kullum.An tattara shari'o'in adenocarcinoma na huhu kafin a cire huhu kuma an tabbatar da su ta hanyar ganewar cututtuka.An tattara samfuran jinin azumi a cikin bututun rabuwa na jini ba tare da wani maganin rigakafi ba.An toshe samfuran jini na sa'a 1 a zafin jiki na ɗaki sannan kuma a sanya su a 2851 × g na minti 10 a 4 ° C don tattara ruwan sama.An daskarar da aliquots na jini a -80 ° C har sai an fitar da metabolite.Sashen rigakafin cutar kansa da gwajin likitanci na Cibiyar Ciwon daji ta Jami’ar Sun Yat-sen ta tattara tarin ruwan magani daga masu ba da gudummawa lafiya 100, gami da daidai da adadin maza da mata masu shekaru 40 zuwa 55.Daidaitaccen kundin kowane samfurin mai ba da gudummawa an haɗa shi, an shayar da tafkin da aka samu kuma an adana shi a -80 ° C.An yi amfani da cakuda ruwan magani azaman abin tunani don sarrafa inganci da daidaita bayanai.
An narke ruwan magani da samfuran gwaji kuma an fitar da metabolites ta hanyar amfani da hanyar haɗewa (MTBE / methanol / ruwa) 56.A taƙaice, an haɗa 50 μl na ruwan magani da 225 μl na methanol mai sanyin ƙanƙara da 750 μl na methyl tert-butyl ether (MTBE).Haɗa cakuda kuma a saka a kan kankara na 1 hour.Sa'an nan kuma aka haxa samfurori da vortex gauraye da 188 μl na MS-grade ruwa dauke da ciki nagartacce (13C-lactate, 13C3-pyruvate, 13C-methionine, da 13C6-isoleucine, saya daga Cambridge Isotope Laboratories).An sanya cakuda cakuda a 15,000 × g na 10 min a 4 ° C, kuma an canza ƙaramin lokaci zuwa cikin bututu biyu (125 μL kowane) don nazarin LC-MS a cikin halaye masu kyau da mara kyau.A ƙarshe, samfurin ya ƙafe zuwa bushewa a cikin babban ma'auni mai sauri.
An sake gina busassun metabolites a cikin 120 μl na 80% acetonitrile, vortexed na 5 min, kuma an sanya su a 15,000 × g na 10 min a 4 ° C.An canza ma'auni a cikin gilashin amber tare da microinserts don nazarin metabolomics.Binciken metabolomics mara niyya akan dandamalin ultra-performance ruwa 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 ingantaccen yanayin ion, matakan wayar hannu sun kasance 95% (A) da 50% acetonitrile (B), kowanne yana ɗauke da 10 mmol/L ammonium acetate da 0.1% formic acid.A cikin yanayi mara kyau, sassan wayar hannu A da B sun ƙunshi 95% da 50% acetonitrile, bi da bi, duka matakan sun ƙunshi 10 mmol / L ammonium acetate, pH = 9. Tsarin gradient shine kamar haka: 0-0.5 min, 2% B;0.5-12 min, 2-50% B;Minti 12–14, 50–98% B;14–16 min, 98% B;16–16.1.min, 98-2% B;16.1-20 min, 2% B. An kiyaye shafi a 40 ° C da samfurin a 10 ° C a cikin autosampler.Yawan kwarara ya kasance 0.3 ml/min, ƙarar allurar shine 3 μl.Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) tare da tushen electrospray ionization (ESI) an sarrafa shi cikin cikakken yanayin dubawa kuma an haɗa shi tare da yanayin saka idanu na ddMS2 don tattara manyan bayanai.An saita sigogi na MS kamar haka: fesa ƙarfin lantarki + 3.8 kV/- 3.2 kV, zafin jiki na capillary 320 ° C, iskar gas 40 arb, iskar gas 10 arb, zazzabi mai bincike 350 ° C, kewayon dubawa 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 10 μL aliquots na supernatant daga kowane samfurin.An yi nazarin allurar sarrafa ingancin inganci guda shida a farkon jerin nazarin don tantance daidaiton tsarin UPLC-MS.Ana gabatar da samfuran sarrafa ingancin lokaci-lokaci a cikin tsari.Dukkan nau'ikan samfuran 11 na samfuran jini a cikin wannan binciken an bincika su ta LC-MS.An yi amfani da Aliquots na cakuda ruwan magani daga masu ba da gudummawa lafiya 100 azaman abin tunani a cikin batches daban-daban don saka idanu kan tsarin cirewa da daidaitawa don tasirin tsari-zuwa-tsari.Binciken metabolomics marasa niyya na ƙungiyar ganowa, ƙungiyar tabbatarwa ta ciki, da ƙungiyar tabbatarwa ta waje a Cibiyar Metabolomics na Jami'ar Sun Yat-sen.dakin gwaje-gwaje na waje na Cibiyar Nazarin Fasaha da Gwaji ta Jami'ar Guangdong ta kuma yi nazarin samfurori 40 daga ƙungiyar waje don gwada aikin ƙirar ƙira.
Bayan hakar da sake gyarawa, an auna cikakkiyar ƙididdigewa na metabolites na jini ta amfani da ultra-high performance liquid chromatography-tandem mass spectrometry (Agilent 6495 triple quadrupole) tare da tushen ionization na electrospray (ESI) a cikin yanayin saka idanu da yawa (MMRM).An yi amfani da ginshiƙin ACQUITY BEH Amide (2.1 × 100 mm, 1.7 μm, Ruwa) don raba metabolites.Tsarin wayar hannu ya ƙunshi 90% (A) da 5% acetonitrile (B) tare da 10 mmol/L ammonium acetate da 0.1% ammonia bayani.Shirin gradient 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 40 ° C kuma samfurin a 10 ° C a cikin autosampler.Adadin kwarara shine 0.3 ml/min kuma ƙarar allurar shine 1 μL.An saita sigogi na MS kamar haka: ƙarfin lantarki na capillary ± 3.5 kV, matsa lamba nebulizer 35 psi, kwararar gas ɗin sheath 12 L / min, zazzabi gas mai zafi 350 ° C, bushewar gas zafin jiki 250 ° C, da bushewar iskar gas 14 l / min.Abubuwan MRM na tryptophan, pyruvate, lactate, hypoxanthine da xanthine sune 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).Don samfuran magani, tryptophan, pyruvate, lactate, hypoxanthine, da xanthine an ƙididdige su ta amfani da madaidaicin madaidaicin mafita na cakuda.Don samfuran tantanin halitta, abun ciki na tryptophan an daidaita shi zuwa ma'auni na ciki da yawan furotin tantanin halitta.
An yi aikin cirewa kololuwa (m/z da lokacin riƙewa (RT)) ta amfani da Compound Discovery 3.1 da TraceFinder 4.0 (Thermo Fisher Scientific).Don kawar da yuwuwar bambance-bambance a tsakanin batches, kowane ƙimar sifa na samfurin gwajin an raba ta hanyar ƙimar mafi girman abin da ake tunani daga wannan tsari don samun yalwar dangi.An nuna ma'auni na ma'auni na ma'auni na ciki kafin da bayan daidaitawa a cikin Ƙarin Table 6. Bambance-bambancen da ke tsakanin ƙungiyoyin biyu an kwatanta su da ƙimar gano ƙarya (FDR <0.05, Wilcoxon ya sanya hannu kan gwajin daraja) da ninka canji (> 1.2 ko <0.83).Bayanan MS masu asali na fasalulluka da aka fitar da kuma bayanan da aka gyara na maganin magani ana nuna su a cikin Ƙarin Bayanai 1 da Ƙarin Bayanai 2, bi da bi.An yi bayanin kololuwa bisa ƙayyadaddun matakan tantancewa guda huɗu, gami da abubuwan da aka gano na metabolites, abubuwan da aka sanya su, da azuzuwan da ba a san su ba, da abubuwan da ba a sani ba 22.Dangane da binciken bayanai a cikin Compound Discovery 3.1 (mzCloud, HMDB, Chemspider), mahaɗan halittu tare da MS/MS da suka dace da ingantattun ma'auni ko ainihin bayanan wasa a cikin mzCloud (maki> 85) ko Chemspider a ƙarshe an zaɓi su azaman tsaka-tsaki tsakanin metabolome mai bambanta.An haɗa bayanin kololuwa ga kowane fasali a cikin Ƙarin Bayanai 3. MetaboAnalyst 5.0 An yi amfani da shi don nazarin nau'in nau'in nau'in nau'in nau'i na nau'i-nau'i na yau da kullum.MetaboAnalyst 5.0 kuma ya kimanta nazarin haɓaka hanyoyin KEGG dangane da mabanbantan metabolites.Babban nazarin abubuwan da suka shafi mahimmanci (PCA) da ƙananan ƙananan murabba'ai (PLS-DA) an yi nazarin su ta amfani da fakitin software na ropls (v.1.26.4) tare da daidaitawa tari da sarrafa kai.Mafi kyawun ƙirar ƙirar halitta don tsinkayar cutar nodule an ƙirƙira ta ta amfani da koma bayan logistic na binary tare da ƙarancin cikakkar raguwa da ma'aikacin zaɓi (LASSO, R fakitin v.4.1-3).Ayyukan samfurin nuna bambanci a cikin ganowa da saitunan tabbatarwa an kwatanta su ta hanyar kimantawa AUC bisa nazarin ROC bisa ga kunshin pROC (v.1.18.0.).An sami mafi kyawun yanke yuwuwar bisa ga matsakaicin ƙimar Youden na ƙirar (hankali + takamaiman - 1).Samfurori masu ƙimar ƙasa ko mafi girma fiye da bakin kofa za a yi hasashen su azaman nodules mara kyau da adenocarcinoma huhu, bi da bi.
Kwayoyin A549 (#CCL-185, Tarin Al'adun Nau'in Amurka) an girma a cikin F-12K matsakaici mai ɗauke da 10% FBS.Gajerun jerin gajerun gashin gashi na RNA (shRNA) masu niyya SLC7A5 da kuma kulawar da ba ta da niyya (NC) an saka su a cikin vector plKO.1-puro.Jerin antisense na shSLC7A5 sune kamar haka: Sh1 (5′-GGAGAAACCTGATGAACAGTT-3′), Sh2 (5′-GCCGTGGACTTCGGAACTAT-3′).Antibodies zuwa SLC7A5 (#5347) da tubulin (#2148) an siyi su daga Fasahar Siginar salula.An yi amfani da ƙwayoyin rigakafi zuwa SLC7A5 da tubulin a dilution na 1:1000 don nazarin ɓangarorin Yamma.
The Seahorse XF Glycolytic Stress Test yana auna matakan acidification na waje (ECAR).A cikin binciken, an gudanar da glucose, oligomycin A, da 2-DG a jere don gwada ƙarfin glycolytic ta salula kamar yadda aka auna ta ECAR.
Kwayoyin A549 da aka canza su tare da kulawar marasa niyya (NC) da shSLC7A5 (Sh1, Sh2) an yi su cikin dare a cikin jita-jita na diamita 10 cm.An fitar da metabolites na sel tare da 1 ml na ƙanƙara-sanyi 80% methanol mai ruwa.Kwayoyin da ke cikin maganin methanol an goge su, an tattara su a cikin sabon bututu, kuma an sanya su a 15,000 × g na 15 min a 4 ° C.Tattara 800 µl na ruwa mai ƙarfi da bushewa ta amfani da na'ura mai saurin sauri.Sannan an yi nazarin busassun ƙwayoyin metabolite don matakan tryptophan ta amfani da LC-MS/MS kamar yadda aka bayyana a sama.Matakan NAD(H) na salula a cikin sel A549 (NC da shSLC7A5) an auna su ta amfani da kit ɗin launi mai ƙididdigewa NAD+/NADH (#K337, BioVision) bisa ga umarnin masana'anta.An auna matakan furotin don kowane samfurin don daidaita adadin metabolites.
Ba a yi amfani da hanyoyin ƙididdiga don tantance girman samfurin da farko ba.Nazarin metabolomics na baya da ke nufin ganowar biomarker15,18 an yi la'akari da su azaman ma'auni don ƙayyade girman, kuma idan aka kwatanta da waɗannan rahotanni, samfurin mu ya isa.Ba a cire samfurori daga ƙungiyar binciken ba.Samfurori na jini an sanya su ba da gangan ba ga ƙungiyar ganowa (la'o'i 306, 74.6%) da ƙungiyar tabbatarwa ta ciki (sharuɗɗan 104, 25.4%) don nazarin metabolomics da ba a yi niyya ba.Mun kuma zaɓi shari'o'i 70 ba da gangan ba daga kowace ƙungiya daga saitin ganowa don nazarin metabolomics da aka yi niyya.An makantar da masu binciken zuwa aikin rukuni yayin tattara bayanai da bincike na LC-MS.Ana siffanta ƙididdigar ƙididdiga na bayanan metabolomics da gwaje-gwajen tantanin halitta a cikin sassan Sakamako, Tatsuniya, da Sassan Hanyoyi.An yi ƙididdige ƙimar tryptophan ta salula, NADT, da ayyukan glycolytic sau uku da kansu tare da sakamako iri ɗaya.
Don ƙarin bayani game da ƙirar binciken, duba Abstract Rahoton Fayil na Halitta mai alaƙa da wannan labarin.
Danyen bayanan MS na abubuwan da aka fitar da kuma daidaitattun bayanan MS na maganin magani ana nuna su a Ƙarin Bayanai 1 da Ƙarin Bayanai 2, bi da bi.An gabatar da bayanin kololuwa don fasalulluka daban-daban a cikin Ƙarin Bayanai 3. Za a iya sauke bayanan LUAD TCGA daga https://portal.gdc.cancer.gov/.Ana ba da bayanan shigarwa don tsara jadawalin a cikin bayanan tushen.An bayar da bayanan tushen don wannan labarin.
Rukunin Nazarin Binciken Huhu na Ƙasa, da dai sauransu. Rage yawan mace-macen cutar kansar huhu tare da ƙananan ƙididdiga masu ƙididdiga.Arewacin Ingila.J. Med.365, 395-409 (2011).
Kramer, BS, Berg, KD, Aberle, DR da Annabi, PC huhu ciwon daji screening ta amfani da low-kashi helical CT: sakamakon daga National Lung Screening Study (NLST).J. Med.Allon 18, 109-111 (2011).
De Koning, HJ, et al.Rage mace-macen cutar kansar huhu tare da gwajin CT volumetric a cikin gwajin da bazuwar.Arewacin Ingila.J. Med.382, 503-513 (2020).
Lokacin aikawa: Satumba 18-2023