Yuav Ua Li Cas Hloov Kev Loj Hlob Factor B1 (TGF 1) cuam tshuam rau raum Fibrosis
Mar 18, 2022
Yog xav paub ntxiv:ali.ma@wecistanche.com
Inhibition ntawm kev hloov pauv kev loj hlob b1 signaling nyob rau hauv interstitial hlwb attenuates profibrotic gene qhia thiab khaws cia erythropoietin ntau lawm thaum sim raum fibrosis hauv nas.
Michaela AA Fuchs, Katharina AE Broeker & et al.
Lub raum fibrosisNws yog tus cwj pwm los ntawm kev loj hlob ntawm myofibroblasts los ntawm cov neeg nyob hauv lub raum thiab cov hlwb nkag. Myofibroblast tsim thiab extracellular matrix ntau lawm thaum lub raum puas yog tshwm sim los ntawm ntau yam cytokines. Ntawm cov no,transforming kev loj hlob factor B1(TGF 1) suav hais tias yog lub hauv paus ua rauraum fibrosis. Peb pom ib qho kev nthuav qhia siab heev ntawm TGF 1(transforming kev loj hlob factor B1)thiab TGF receptor 2 (TGF -R2) mRNAs hauv lub raum interstitial hlwb hauv kev simfibrosis. Ntawm no, peb tau tshawb xyuas qhov kev koom tes ntawm TGF 1 (transforming kev loj hlob factor B1) signaling nyob rau hauv lub raum interstitial hlwb rau organ fibrosis siv cov qauv ntawm adenine-induced nephropathy thiab unilateral ureteral occlusion nyob rau hauv nas. Rau lub hom phiaj no TGFB1 signaling tau cuam tshuam los ntawm inducible deletion ntawm TGF -R2 noob nyob rau hauv interstitial hlwb qhia cov fibroblast marker platelet-derived kev loj hlob yam receptor-B. Kev nthuav qhia ntawm cov noob profibrotic tau txo qis mus txog 50 feem pua hauv lub raum tsis muaj TGF -R2 hauv cov hlwb zoo rau platelet-derived kev loj hlob ntawm receptor-B. Tsis tas li ntawd, kev tshem tawm ntawm TGF -R2 tiv thaiv kev poob ntawm erythropoietin ntau lawm hauv ureter-ligated ob lub raum. Tshwj xeeb tshaj yog, fibrosis-txoj kev qhia ntawm x-smooth nqaij actin raws li ib tug myofibroblast marker thiab deposits ntawm extracellular collagens tsis tau hloov nyob rau hauv nas nrog lub hom phiaj deletion ntawm TGF -R2. Yog li, peb qhov kev tshawb pom pom tias muaj txiaj ntsig zoo ntawm TGF 1(transforming kev loj hlob factor B1)Kev taw qhia nyob rau hauv cov hlwb interstitial uas pab txhawb rau profibrotic gene qhia thiab downregulation ntawm erythropoietin ntau lawm tab sis tsis mus rau kev loj hlob ntawm myofibroblasts thaum lub sij hawm.raum fibrosis.
Nyem rau cistanche hmoov cov txiaj ntsig kev noj qab haus huv rau kab mob raum
Kev loj hlob thiab kev loj hlob ntawmraum fibrosisyog ib qho cim ntawm kab mob raum mob. Nws yog feem ntau xav tias yog qhov tshwm sim ntawm kev tsim khoom ntau ntxiv thiab tso tawm ntawm cov khoom siv hluav taws xob ntxiv (ECM) xws li collagens, fibronectin, lossis tenascins. Tsis tu ncuaraum fibrosisua rau muaj kev nce zuj zus ntawm glomerular thiab tubular muaj nuj nqi thiab tseem txwv cov endocrine kev ua haujlwm ntawm ob lub raum uas ua rau cov khoom tsim tsis txaus ntawm erythropoietin (EPO) ua rau mob ntshav qab zib.-y
Ntawm cov cytokines thiab signaling yam uas muaj feem xyuam rau cov kab mob raum thiabfibrosiskev vam meej,transforming kev loj hlob factor 1(TGF 1) tau kawm nyob rau hauv ntau yam qauv.6.0-1 TGF 1(transforming kev loj hlob factor B1)khi rau TGF receptor 2 (TGF -R2). Kev tsim ntawm heteromeric complex nrog TGF receptor 1 yog qhov tseem ceeb rau kev ua kom lub cev ntawm SMAD signaling cascade. i.1-18 Nyob rau hauv lub raum puas, nthuav qhia ntawm TGF 1(transforming kev loj hlob factor B1)mRNA nyob rau hauv ntau lub raum cell hom tau raug tshaj tawm.1517,19-2lAnimportant roleofTGF 1 nyob rau hauvraumfibrosiskuj tau piav qhia los ntawm kev tshawb pom tias kev tswj hwm ntawm exogenous TGF 1(transforming kev loj hlob factor B1)tuaj yeem txhawb nqaraum fibrosisthiab TGF 1(transforming kev loj hlob factor B1)Cov tshuaj tiv thaiv tuaj yeem ua rau muaj kev sim ua kom muaj zogfibrosis,10.fibrosis.1,1
Qhov kev qhia ntawm TGF -R2 tau raug tshaj tawm nyob rau hauv ntau hom cell, yog li txwv cov ntaub ntawv muaj txiaj ntsig ntawm kev tshawb pom tau nrog kev cuam tshuam thoob ntiaj teb TGF 1.(transforming kev loj hlob factor B1)teeb liab. Qhov no kuj tseem yuav piav qhia qee qhov kev tsis sib haum xeeb hauv xyoo tas los no.1.1.2 TGF 1(transforming kev loj hlob factor B1)kuj tau tshaj tawm los txhawbraum fibrosislos ntawm kev txhim kho cov infiltration ntawm macrophages, uas yuav transdifferentiate rau hauv myofibroblasts.
Txawm li cas los xij, lub luag haujlwm tseem ceeb ntawm TGF 1(transforming kev loj hlob factor B1)hauvraumfibrosistsis ntev los no tau sib tw los ntawm qhov pom tiasraumfibrosistsis txo los ntawm thaiv TGF 1(transforming kev loj hlob factor B1)signaling nyob rau hauv lub hlwb nquag tsim matrix proteins. Nws yog qhov tsim nyog tias kev tsim cov matrix protein ntau ntxiv yog tsim los ntawm myofibroblasts, uas tuaj yeem hloov pauv los ntawm ntau hom cell xws li fibroblasts, monocytes, pericytes, thiab tubular thiab endothelial cells.6,10,2-32 Cov txheeb ze koom nrog. Cov hom cell no rau cov neeg myofibroblast tuaj yeem sib txawv nyob ntawm qhov kev sim ua qauv. Txawm li cas los xij, qhov kev pom zoo loj hlob tuaj yog tias cov neeg nyob hauv fibroblasts, pericytes, thiab cov pob txha pob txha-derived hlwb yog lub ntsiab precursors ntawm myofibroblasts nyob rau hauv lub raum thiab pab txhawb rau lub cev.raumfibrosisnyob rau hauv ib qho piv txwv zoo sib xws. Ib txoj kev tshawb fawb tsis ntev los no kuj muaj ntau hom cell hauv lub raum. paub tseeb tias PDGFR-f hlwb ua lub hauv paus tseem ceeb rau myofibroblasts hauv tib neeg lub raum. Cov PDGFR-f cov hlwb no kuj suav nrog txhua lub raum hlwb tuaj yeem tsim EpOs,-9
Lub raum fibrosisyog txuam nrog txo qis peev xwm ntawm lub raum los tsim EPO.-,3,0 Txawm hais tias cov laj thawj rau qhov no tsis tau ua kom pom tseeb, nws tau xav tias kev hloov pauv ntawm interstitial PDGFR-ntxiv cov hlwb rau hauv myofibroblasts mus. koom tes nrog kev poob lawv lub peev xwm los tsim EPO. Nyob rau hauv kev pom ntawm lub luag hauj lwm tseem ceeb ntawm interstitial PDGFR-B hlwb raws li haiv neeg EPO cov neeg tsim khoom thiab ua ntej ntawm myofibroblasts, peb xav kom txhais lub luag haujlwm ntawm TGF 1.(transforming kev loj hlob factor B1)signaling nyob rau hauv lub cell cov neeg.
Rau lub hom phiaj no, peb tsim tus qauv nas nrog tamoxifen-inducible tshem tawm ntawm TGF -R2 hauv PDGFR- ntxiv cov hlwb (PDGFR- CeERT7 ntxiv rau TGF -R2 /, tom qab ntawd hu ua TGF -R2ko nas). Tom qab tshem tawm ntawm TGF(transforming kev loj hlob yam)-R2 hauv PDGFR- ntxiv rau cov hlwb hauv cov nas laus, tsiaj txhu tau kawm hauv 2 qauv ntawm kev simraumfibrosis: unilateral ureteral occlusion (UUO) thiab adenine-induced nephropathy (AN). Adenine-induced nephropathy yog ib qho qauv kev puas tsuaj uas muaj zog sib npaug rau tib neeg siv lead ua-induced pathologies, whereas UUO yog tus qauv kev puas tsuaj rau kev ntxhov siab.2933,42- Siv cov RNAscope tshwj xeeb hauv situ hybridization txoj kev tso cai rau colocalization ntawm phiaj thiab cim mRNAs nqes mus rau ib leeg-cell daws teeb meem. Hauv cov qauv pathological no, cov teebmeem ntawm TGFB-R2 tshem tawm hauv PDGFR- fcells ntawm kev qhia ntawm d-smooth leeg actin (a-SMA) ua tus cim rau kev tsim myofibroblast. Tsis tas li ntawd, peb tau tshawb xyuas cov noob profibrotic thiab tso tawm ntawm ECM. Lub peev xwm ntawm lub raum los tsim EPO hauv cov xeev ntawm kev simraum fibrosistau soj ntsuam los ntawm kev ntsuas ntawm plasma EPO qib thiab tus naj npawb thiab thaj chaw ntawm EPO mRNA-qhia cov hlwb hauv lub raum puas.
Txoj kev
Tsiaj
TGF -R2lo nas tau tsim los ntawm kev sib tw PDGFR- cexr2 nas (tamoxifen-inducible Cre recombinase qhia nyob rau hauv kev tswj hwm ntawm PDGFR- txhawb nqa; JAX Tshuag #029684) thiab mic nrog loxP-franked TGF6- R2 alleles (IAX stock #012603) in a mixed C57BJ/6 and 129S1/SvmJ background. JAX Tshuag #007676) tau bred rau hauv TGF -R2ko nas. Genotyping tau ua siv cov primers teev nyob rau hauv Table 1. Littermates tsis zoo rau Cre tau siv raws li cov tsiaj tswj, Tsiaj txhu tau khaws cia ntawm tus qauv nas chow (0.6 feem pua NaCl; Ssniff) nrog dawb Tag nrho cov kev sim tsiaj tau ua raws li Phau Ntawv Qhia Txog Kev Saib Xyuas thiab Kev Siv Cov Tsiaj Txhaum Cai uas tau luam tawm los ntawm US National Institutes of Health thiab pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees hauv zos.
Kev kho mob tamoxifen
Cre recombination tau tshwm sim los ntawm kev noj zaub mov uas muaj 400 mg ntawm tamoxifen citrate ib kilogram (A115T00404, Sniff) rau 4 lub lis piam tom qab los ntawm 3- lub lim tiam nrog cov qauv chow. Kev txiav tawm ntawm floxed TGF-R2 noob tau lees paub los ntawm polymerase saw cov tshuaj tiv thaiv,
Adenine-induced nephropathy
Adenine-inducedfibrosistau tsim nyob rau hauv cov nas laus. Tom qab kev kho tamoxifen thiab lub sijhawm so hauv qab no, txiv neej nas tau noj cov zaub mov uas muaj adenine (0.2 feem pua ) txuas ntxiv rau 3 lub lis piam. Kev sim tau ua tiav tom qab 3 lub lis piam (3-lub lim tiam adenine).
Hauv qhov chaw hybridization ntawm RNAscope
Localization ntawm mRNA qhia tau kawm nrog RNAscope Multiplex Fluorescent v2 cov khoom siv (Advanced Cell Diagnostics ACD) raws li cov chaw tsim khoom cov lus qhia. Lub raum tau kho nrog 10 feem pua ntxhiab tsw buffered formalin tov, lub cev qhuav dej hauv ethanol series, thiab muab tso rau hauv paraffin. Hybridization signals tau kuaj pom ntawm 5-μm cov ntaub so ntswg seditions siv TSA Plus fluorophores Cy3 thiab Cy5 (PerkinElmer). Slices tau mounted nrog ProLong Kub Antifade Mountant (Thermo Fisher Scientific, Waltham, MA) thiab saib nrog Axio Observer Z1 microscope (Zeiss). Kev tswj qhov zoo thiab qhov tsis zoo tau raug kaw txhua hnub. RNAsape probes tau teev nyob rau hauv Table 2
Table 1|Primer sequences siv rau genotyping ntawm nas
TSEEM CEEB
TGF 1(transforming kev loj hlob factor B1)thiab TGF(transforming kev loj hlob yam)-R2 qhia nyob rau hauv cov hlwb interstitial yog txhim kho thaum lub sij hawm sim kab mob raum
Localization ntawm TGFB1 thiab TGFB-R2 mRNA qhia nyob rau hauv lub raum nyob rau hauv basal tej yam kev mob thiab thaum lub sij hawm simfibrosistau ua los ntawm RNAscope hauv situ hybridization. Nyob rau hauv basal tej yam kev mob, TGFB-R2 mRNA qhia tau pom nyob rau hauv tubulointerstitial PDGFR-B hlwb nyob rau hauv tag nrho cov me nyuam mos zones thiab nyob rau hauv glomerular mesangial hlwb (Daim duab 1, sab laug). TGF -R2 kuj tau kuaj pom hauv cov tubules ze ze; qhia nyob rau hauv lwm yam tubular ntu yog qis dua (Cov duab ntxiv S1 thiab S2).
Table 2|RNAscope probes siv rau hauv situ hybridization
TGFb 1(transforming kev loj hlob factor B1)mRNA tau nyob rau hauv interstitial PDGFR-bþ hlwb thoob plaws txhua lub raum zones thiab hauv glomerular mesangial hlwb (Daim duab 1). Tubules tshwm tsis zoo rau TGFb1 mRNA. Qhov no cuam tshuam PDGFR-ntxiv cov hlwb ua lub ntsiab lus qhia rau TGF 1(transforming kev loj hlob factor B1)nyob rau hauv lub raum nyob rau hauv basal mob.
Table 3|Primer sequences siv rau ib tug ntau npaum li cas polymerase saw cov tshuaj tiv thaiv
Tom qab 3 lub lis piam ntawm kev noj zaub mov adenine (AN) lossis 5-hnub UUO, raum TGF 1(transforming kev loj hlob factor B1)thiab TGF -R2 mRNA qhia theem tau nce siab hauv cov nas tswj (Daim duab 2).
Kev nthuav qhia ntau ntxiv ntawm TGF 1(transforming kev loj hlob factor B1)thiab TGF -R2 tom qab AN lossis UUO tau nyob hauv RNAscope. Los ntawm colocalization ntawm cov cim tshwj xeeb ntawm tes, peb tuaj yeem txheeb xyuas lub raum cell hom uas qhia txog kev txhim kho ntawm TGF -R2 thaum lub raum puas (Daim duab 3; Cov duab ntxiv S2 thiab S3). Hauv AN, TGF -R2 mRNA qhia tau nce siab hauv interstitial PDGFR- ntxiv rau cov hlwb hauv thaj chaw fibrotic (Daim duab 3a). Cov tubules puas nrog siab TGF -R2 qhia tau tshwm sim nrog flattened epithelial thiab tau ncig los ntawm PDGFR- ntxiv cov hlwb nrog TGF -R2 thiab TGF 1.(transforming kev loj hlob factor B1)mRNA signals (Daim duab 3a thiab c, xub; Ntxiv daim duab S2). Tubules nrog nce TGF -R2 cov cim qhia pom ntawm novo TGF 1(transforming kev loj hlob factor B1)mRNA qhia (Daim duab 3c; Ntxiv daim duab S3).
Hauv UUO ob lub raum, cov teeb liab nce ntxiv rau TGF -R2 mRNA tau kuaj pom nyob rau hauv cov hlwb interstitial ntawm cov seem sab nrauv medulla (Daim duab 3b) thiab cortex (Cov Duab Ntxiv S2). Cov PDGFR- ntxiv rau cov hlwb no kuj tau pom cov cim qhia ntxiv rau TGF 1(transforming kev loj hlob factor B1)(Daim duab 3d).
Hauv cov ntsiab lus, cov kev tshawb pom no tau pom tias muaj kev ua kom muaj zog ntawm TGF 1(transforming kev loj hlob factor B1)Kev taw qhia ntawm interstitial PDGFR- ntxiv rau cov hlwb thiab cov tubules puas thaum lub raum puas tsuaj.
Daim duab 1|Basal qhia ntawm (sab laug) transforming growth factor b receptor 2 (TGFb-R2) and (right) transforming growth factor b1 (TGFb1) mRNA on the obmile sections of wild-type nass.
Inducible deletion ntawm TGF -R2 nyob rau hauv PDGFR- ntxiv rau cov hlwb attenuates profibrotic gene qhia thaum lub sij hawm sim kab mob.
Txhawm rau txheeb xyuas qhov tseeb ntawm TGF 1(transforming kev loj hlob factor B1)Kev taw qhia rau kev ua haujlwm ntawm PDGFR- ntxiv rau cov hlwb, peb tau tshuaj xyuas cov neeg laus TGF -R2ko nas. Basal qhia ntawm TGF 1(transforming kev loj hlob factor B1)tsis hloov tom qab tshem tawm ntawm TGF -R2 nyob rau hauv interstitial hlwb ntawm lub raum (Daim duab 2). Kev nthuav qhia ntawm TGF -R2 mRNA tau txo qis hauv cov tsiaj txhu (Figure2). Qhov seem seem yog vim tubular qhia ntawm TGF -R2 (Daim duab 1). Lub raum morphology tsis txawv hauv TGF -R2ko nas mus txog 8 lub lis piam tom qab tshem tawm ntawm TGF -R2.
Qhov pom kev nce hauv TGF -R2 qhia thaum lub sij hawm AN thiab UUO tau txo qis hauv TGF -R2ko ob lub raum piv nrog raum tswj (Daim duab 2). TGF 1(transforming kev loj hlob factor B1)kev qhia tau txo qis tsuas yog tom qab AN.
Ib txoj kev tshawb fawb histological ntawm phosphorylated SMAD3 pom tau tias txo qhov kev hloov pauv ntawm qhov hloov pauv mus rau hauv lub nucleus ntawm a-SMAþ interstitial hlwb hauv TGFb-R2ko tsiaj, qhia tias cuam tshuam TGFb1 signaling tom qab ua tiav Cre mediated deletion ntawm TGFb-R2 (Daim duab 4).
Daim duab 2|Kev hloov pauv kev loj hlob b1 (TGFb1) thiab TGFb receptor 2 (TGFb-R2) mRNA abundance nyob rau hauv tswj lub raum thiab ob lub raum nrog tamoxifen-inducible deletion ntawm TGFb-R2 nyob rau hauv platelet-derived kev loj hlob factor receptor b-positive hlwb (TGFb-R2ko) nyob rau hauv basal hlwb thiab cov kab mob pathological.
Inducible deletion ntawm TGFb-R2 nyob rau hauv PDGFR-bD hlwb attenuates profibrotic noob qhia thaum lub sij hawm sim kab mob.
Thaumfibrosis, zus tau tej cov matrix Cheebtsam yog txhim khu kev qha, ua rau nws kim heev deposition ntawm ECM thiab caws pliav tsim. Txhawm rau ntsuas lub luag haujlwm ua tau ntawm TGF 1(transforming kev loj hlob factor B1)signaling nyob rau hauv cov neeg nyob hauv PDGFR-Bf hlwb rau cov matrix ntau lawm, peb piv cov noob qhia ntawm fibrotic cim ntawm kev tswj thiab TGF -R2ko nas.
Adenine pub mis thiab UUO coj mus rau -25-fold nce ntawm Collal mRNA hauv lub raum tswj. Whereas hauv TGF -R2ko ob lub raum, Collar mRNA nce tau txo los ntawm -50 feem pua piv rau cov tswj (Daim duab 5). Adenine pub thiab UUO kuj tau ua rau pom qhov nce ntawm collagen 3al thiab fibronectin mRNAs. Qhov nce ntawm ob qho tib si mRNAs kuj tau txo qis hauv TGF -R2ko ob lub raum.
NYEEM NO PART Ⅱ
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Nco tseg: cov saum toj no tsis yog daim ntawv teev npe tag nrho
