唐國(guó)華 ,屈麗華
(1.南華大學(xué)生命科學(xué)研究中心,湖南衡陽421001;2.南華大學(xué)醫(yī)學(xué)院組織學(xué)與胚胎學(xué)教研室)
腎臟是人體重要的器官,不僅能分泌尿液、排泄代謝廢物,還具有調(diào)節(jié)血壓、體液平衡,以及骨骼密度的功能。全球有10%的慢性腎臟疾病患者進(jìn)展為慢性腎功能不全、尿毒癥,是導(dǎo)致人類死亡的主要原因之一[1-2]。國(guó)內(nèi)外學(xué)者們一直致力于研究腎臟發(fā)育的分子機(jī)制,并試圖利用干細(xì)胞移植,修復(fù)腎臟損傷,重建終末期腎病患者腎臟的功能。
胚胎后腎間充質(zhì)細(xì)胞是一種多潛能干細(xì)胞,研究表明它能分化形成腎上皮細(xì)胞[3-4]。因此,利用后腎間充質(zhì)細(xì)胞移植治療很可能成為改善急性腎小管壞死預(yù)后的重要方法[4-5]。白血病抑制因子(leukemia inhibitory factor,LIF)是一類細(xì)胞因子,最早發(fā)現(xiàn)于小鼠KrebsⅡ腹水瘤細(xì)胞條件培養(yǎng)液中,能誘導(dǎo)白血病細(xì)胞株M1細(xì)胞向正常細(xì)胞分化[6]。已有研究報(bào)道LIF能誘導(dǎo)神經(jīng)前體細(xì)胞分化為星形膠質(zhì)細(xì)胞、少突神經(jīng)膠質(zhì)細(xì)胞及感覺神經(jīng)元[7-8];JAKSTAT信號(hào)介導(dǎo)的LIF受體激活是形成上皮小管所必需[9-10],提示LIF在細(xì)胞分化過程中具有重要作用。本文分離并鑒定后腎間充質(zhì)細(xì)胞,利用體外細(xì)胞培養(yǎng)技術(shù),在培養(yǎng)液中加入細(xì)胞因子LIF進(jìn)行誘導(dǎo),用免疫熒光染色和RT-PCR檢測(cè)后腎間充質(zhì)細(xì)胞分化形成腎上皮細(xì)胞過程中的形態(tài)學(xué)和基因表達(dá)變化,旨在闡明后腎間充質(zhì)細(xì)胞分化形成上皮細(xì)胞過程中的生物學(xué)特性變化,理解腎臟發(fā)生和發(fā)育過程,為腎臟組織工程學(xué)研究奠定理論基礎(chǔ)。
Balb/c小鼠(湖南斯萊克景達(dá)實(shí)驗(yàn)動(dòng)物有限公司);兔多克隆抗體Pax2(1:250,Abcam),laminin α5(1:250,Abcam),anti-rabbit FITC(1:250,Santa Cruz);DMEM-LG培養(yǎng)基(Gibco);胎牛血清FBS(Gibco);胰酶(Amersco);轉(zhuǎn)化生長(zhǎng)因子(transforming growth factor alpha,TGF-α)(10 ng/mL,Boehringer Mannheim),成纖維細(xì)胞生長(zhǎng)因子(fibroblast growth factor,FGF 2)(50 ng/mL,Boehringer Mannheim)和白血病抑制因子LIF(10 units/mL,Chemicon);RNeasy Mini Kit(Qiagen);SupercriptⅡRNase H-Reverse Transcriptase(Invitrogen);引物(Invitrogen);VECTASHIELD Fluorescent Mounting Media(Vector Laboratories)。
1.2.1 后腎間充質(zhì)細(xì)胞的分離和培養(yǎng) 取孕11.5天Balb/c雌鼠(以發(fā)現(xiàn)陰栓當(dāng)日為受孕0.5天),斷頸處死,在解剖顯微鏡下剝離胎盤,獲取E11.5小鼠胚胎。未分化的后腎間充質(zhì)細(xì)胞位于胚胎后肢的后1/3處,即輸尿管的背側(cè),輸尿管芽向背部延伸處[11]。在小鼠前肢下方橫向剪斷,沿胚胎神經(jīng)管縱向剪開,截?cái)辔膊?根據(jù)后腎位置及其形狀分離得到胚胎后腎間充質(zhì)組織,去除輸尿管芽 (圖1);將解剖分離后組織置于4℃預(yù)冷的DMEM清洗備用。
圖1 分離后腎間充質(zhì)組織解剖示意圖 A:沿虛線,即從胚胎前肢的下方橫向剪斷;B:沿神經(jīng)管縱向剪開,并截去尾部,使輸尿管暴露于視野下;C:未分化的后腎間充質(zhì)細(xì)胞(MM)位于胚胎后肢的后1/3處,即輸尿管芽向背部延伸處
1.2.2 后腎間充質(zhì)細(xì)胞的培養(yǎng)和分化誘導(dǎo) 將解剖分離的后腎組織在膠原酶溶液中(DMEM+10%FBS+0.2%膠原酶 +50 IU/mL DNase)37℃孵育15 min;殘存后腎組織以0.25%胰酶/0.02%EDTA(1:1)室溫消化至組織塊消失;加入含10%FBS的DMEM,1 000 r/min離心5 min;棄上清,含10%FBS的DMEM重懸細(xì)胞,細(xì)胞濃度調(diào)整至5×104/cm2,放入37℃、含5%CO2、飽和濕度的培養(yǎng)箱中培養(yǎng)。分別加入生長(zhǎng)刺激因子 TGF-α(10 ng/mL)、FGF 2(50 ng/mL)和LIF(10 units/mL),以誘導(dǎo)后腎間充質(zhì)細(xì)胞分化形成腎上皮細(xì)胞[3,12-14]。
1.2.3 后腎間充質(zhì)細(xì)胞的鑒定 將解剖分離好的后腎間充質(zhì)組織或者涂有后腎間充質(zhì)細(xì)胞的玻片放在4%多聚甲醛固定20 min;PBST(0.2%的Triton X-100)處理10 min,增加膜的通透性;含1%BSA的PBS封閉15 min;加入一抗Pax2(1:250,Abcam)或laminin α5(1:250,Abcam)4℃孵育過夜;PBS洗 3次,每次5 min;加入熒光素標(biāo)記的二抗anti-rabbit FITC(1:250,Santa Cruz)室溫下避光孵育1 h;PBS洗3次,每次5min;防熒光淬滅封片劑(VECTASHIELD)固定,熒光顯微鏡下觀察。
1.2.4 RT-PCR分析 RNeasy Mini Kit試劑盒提取分離好的小鼠后腎間充質(zhì)細(xì)胞RNA(見試劑盒抽提方法);分光光度計(jì)(Nanodrop)檢測(cè)RNA的濃度和質(zhì)量;SupercriptⅡRNase H-Reverse Transcriptase合成cDNA(500ngRNA每個(gè)反應(yīng))。引物序列如下:
利用后腎間充質(zhì)細(xì)胞標(biāo)記基因Pax-2抗體,對(duì)解剖分離得到的后腎組織進(jìn)行染色,免疫組化分析表明小鼠后腎間充質(zhì)組織定位正確(圖2)。
圖2 Pax2抗體鑒定后腎間充質(zhì)組織 A:普通光學(xué)顯微鏡觀察;B:熒光顯微鏡剝離E12.5胚胎中的后腎間充質(zhì)組織,兔多克隆抗體Pax2染色,表達(dá)陽性
分離的后腎間充質(zhì)組織經(jīng)膠原酶和胰酶消化形成單一、懸浮的后腎間充質(zhì)細(xì)胞。用免疫熒光染色檢測(cè)上皮細(xì)胞基底膜極性蛋白laminin α5的表達(dá)情況。結(jié)果顯示:FGF2/TGF-α培養(yǎng)48 h,基底膜極性蛋白laminin α5不表達(dá),細(xì)胞無極性且排列紊亂,表明細(xì)胞還未被誘導(dǎo)分化為上皮細(xì)胞(圖3A,3D)。加入LIF培養(yǎng)48 h,laminin α5蛋白開始表達(dá),細(xì)胞成簇,具有頂端膜等上皮細(xì)胞的特點(diǎn),細(xì)胞由原來的無規(guī)則排列增殖形成“S”或“C”型小體樣的鏈狀結(jié)構(gòu) (圖3B,3E)。LIF誘導(dǎo)1周后,細(xì)胞簇進(jìn)一步發(fā)育形成具有連續(xù)基底膜包圍的腎小管或者腎小球結(jié)構(gòu)(圖3C,3F)。
RT-PCR結(jié)果顯示:加入FGF2、TGF-a培養(yǎng)48 h后,Wt1、Pax2和Wnt4等與細(xì)胞聚集形成相關(guān)的蛋白在后腎間充質(zhì)細(xì)胞中有表達(dá),早期的上皮膠原蛋白CollagenⅣ在這個(gè)時(shí)期也有表達(dá),而特異性上皮細(xì)胞標(biāo)記蛋白E-cadherin,laminin α5以及足細(xì)胞標(biāo)記蛋白podocalyxin不表達(dá);加入LIF誘導(dǎo)培養(yǎng)48 h后,細(xì)胞粘性蛋白 E-cadherin以及基質(zhì)蛋白lamininα5開始表達(dá),而足細(xì)胞的標(biāo)志蛋白podocalyxin則在誘導(dǎo)1周后,才有表達(dá),標(biāo)志著腎小球開始形成(圖4)。
圖3 LIF誘導(dǎo)后腎間充質(zhì)細(xì)胞分化為上皮細(xì)胞的生物形態(tài)學(xué)變化 A-C:普通光學(xué)顯微鏡觀察;D-F:熒光顯微鏡.A,D:FGF2/TGF-a處理培養(yǎng)后腎間充質(zhì)細(xì)胞48 h,細(xì)胞呈不規(guī)則排列,laminin α5不表達(dá);B,E:FGF2/TGF-a/LIF處理培養(yǎng)后腎間充質(zhì)細(xì)胞48 h,細(xì)胞呈鏈狀排列,伴隨著基質(zhì)膜蛋白laminin α5的表達(dá);C,F:由基質(zhì)膜蛋白laminin α5包圍,細(xì)胞開始形成管狀和早期的腎小球結(jié)構(gòu)
后腎間充質(zhì)細(xì)胞是腎臟的多能干細(xì)胞,經(jīng)過間充質(zhì)-上皮細(xì)胞的分化過程,形成極性細(xì)胞,最終能分化為腎單位[3,15-16]。腎單位是腎臟的結(jié)構(gòu)和功能單位。因此,利用后腎間充質(zhì)細(xì)胞治療腎臟疾病成為國(guó)際研究焦點(diǎn)。
圖4 RT-PCR檢測(cè)后腎間充質(zhì)細(xì)胞分化形成上皮細(xì)胞過程中的基因表達(dá)變化
腎臟發(fā)育過程中,腎單位上皮細(xì)胞的形成是受各種不同細(xì)胞之間信號(hào)相互作用。該相互作用包括輸尿管芽、后腎間充質(zhì)細(xì)胞和腎基質(zhì)三者之間的網(wǎng)絡(luò)調(diào)控[16-21]。本實(shí)驗(yàn)分離了上皮始祖細(xì)胞(即后腎間充質(zhì)細(xì)胞),直接采用上皮細(xì)胞發(fā)育所需的生長(zhǎng)因子處理,使得復(fù)雜的調(diào)控網(wǎng)絡(luò)簡(jiǎn)單化。轉(zhuǎn)化生長(zhǎng)因子(TGF-a)和成纖維細(xì)胞生長(zhǎng)因子(FGF)是保持細(xì)胞正常生長(zhǎng)和增殖,并保證細(xì)胞分化為上皮細(xì)胞所必需的刺激因子。若無上述刺激因子,后腎間充質(zhì)細(xì)胞會(huì)出現(xiàn)凋亡現(xiàn)象[22];加入FGF2/TGF-a處理,后腎間充質(zhì)細(xì)胞至少能存活1周以上,但并不能誘導(dǎo)分化形成上皮細(xì)胞[3,23]。而白血病抑制因子(LIF)是后腎間充質(zhì)細(xì)胞分化為腎上皮細(xì)胞所必需的因子[12,14]。本實(shí)驗(yàn)中生長(zhǎng)刺激因子作用對(duì)象為單一的上皮始祖細(xì)胞,排除了輸尿管芽或者腎基質(zhì)對(duì)后腎間充質(zhì)作用的干擾,研究了轉(zhuǎn)錄調(diào)控子(Pax-2,WT-1),細(xì)胞基質(zhì)蛋白 (E-cadherin,lamininα5,CollagenⅣ),糖蛋白(Podocalyxinn),信號(hào)分子(Wnt4)與腎上皮細(xì)胞各個(gè)發(fā)育階段之間的直接關(guān)系,解決了細(xì)胞示蹤技術(shù)不成熟這一難題。
本實(shí)驗(yàn)利用LIF誘導(dǎo)單一后腎間充質(zhì)細(xì)胞的模型,對(duì)后腎間充質(zhì)分化為上皮細(xì)胞的生物學(xué)特性進(jìn)行了研究。laminin α5免疫熒光染色結(jié)果顯示,無LIF處理,后腎間充質(zhì)細(xì)胞不能分化形成上皮細(xì)胞;加入LIF刺激,無規(guī)則排列的細(xì)胞增殖聚集 (圖3A,3D),形成“S”或“C”型小體樣的鏈狀結(jié)構(gòu) (圖3B,3E),進(jìn)一步發(fā)育形成連續(xù)基底膜包圍的腎小管或者腎小球結(jié)構(gòu) (圖3C,3F)。本實(shí)驗(yàn)還發(fā)現(xiàn),在TGF-a和 FGF誘導(dǎo)下,后腎間充質(zhì)細(xì)胞高表達(dá)Pax2、WT-1、Wnt4和CollagenⅣ,使得后腎間充質(zhì)細(xì)胞區(qū)別于其他間質(zhì)細(xì)胞[24-26]。CollagenⅣ的高表達(dá)標(biāo)志著后腎間充質(zhì)細(xì)胞已經(jīng)在向上皮細(xì)胞轉(zhuǎn)化[24]。但是E-cadherin,lamininα5,podocalyxin等細(xì)胞極性標(biāo)記分子只有在加入刺激因子LIF后才開始表達(dá),細(xì)胞極性標(biāo)記分子的表達(dá)標(biāo)志著腎小球的形成(圖4)[27-29]。綜上所述,本課題組成功分離了后腎間充質(zhì)細(xì)胞,證實(shí)了LIF能誘導(dǎo)小鼠后腎間充質(zhì)細(xì)胞分化為腎小囊和腎小管上皮細(xì)胞,LIF誘導(dǎo)細(xì)胞分化過程中伴隨細(xì)胞極性標(biāo)記分子基因的高表達(dá),本研究為闡明腎臟發(fā)生發(fā)育的分子機(jī)制及慢性腎臟疾病的細(xì)胞治療法提供了理論依據(jù)。
[1]Hadaya K,de Rham C,Bandelier C,et al.Natural killer cell receptor repertoire and their ligands,and the risk of CMV infection after kidney transplantation[J].Am J Transplant,2008,8(12):2674-2683.
[2]Coresh J,Levey AS,Levin A,et al.A stable definition of chronic kidney disease improves knowledge and patient care[J].BMJ,2013,347(11):5553.
[3]Karavanova ID,Dove LF,Resau JH,et al.Conditioned medium from a rat ureteric bud cell line in combination with bFGF induces complete differentiation of isolated metanephric mesenchyme[J].Development,1996,122(12):4159-4167.
[4]Villanueva S,Ewertz E,Carrion F,et al.Mesenchymal stem cell injection ameliorates chronic renal failure in a rat model[J].Clin Sci(Lond),2011,121(11):489-499.
[5]Yokote S,Yokoo T.Organogenesis for kidney regeneration[J].Curr Opin Organ Transplant,2013,18(2):186-190.
[6]Metcalf D,Hilton D,Nicola N.Clonal analysis of the actions of the murine leukemia inhibitory factor on leukemic and normal murine hemopoietic cells[M].Leukemia,1988,2(4):216.
[7]Bonni A,Sun Y,Nadal-Vicens M,et al.Regulation of gliogenesis in the central nervous system by the JAKSTAT signaling pathway[J].Science,1997,278(5337):477-483.
[8]Murphy M,Reid K,Hilton DJ,et al.Generation of sensory neurons is stimulated by leukemia inhibitory factor[J].PNAS,1991,88(8):3498-3501.
[9]Boccaccio C,Andò M,Tamagnone L,et al.Induction of epithelial tubules by growth factor HGF depends on the STAT pathway[J].Nature,1998,391(6664):285-288.
[10]Sierra-Honigmann MRo,Nath AK,Murakami C,et al.Biological action of leptin as an angiogenic factor[J].Science,1998,281(5383):1683-1686.
[11]Challen GA,Martinez G,Davis MJ,et al.Identifying the molecular phenotype of renal progenitor cells[J].J Am Soc Nephrol,2004,15(9):2344-2357.
[12]Barasch J,Yang J,Ware CB,et al.Mesenchymal to epithelial conversion in rat metanephros is induced by LIF[J].Cell,1999,99(4):377-386.
[13]Oliver JA,Barasch J,Yang J,et al.Metanephric mesenchyme contains embryonic renal stem cells[J].Am J Physiol Renal Physiol,2002,283(4):799-809.
[14]Plisov SY,Yoshino K,Dove LF,et al.TGF beta 2,LIF and FGF2 cooperate to induce nephrogenesis[J].Development,2001,128(7):1045-1057.
[15]Yang J,Blum A,Novak T,et al.An epithelial precursor is regulated by the ureteric bud and by the renal stroma[J].Dev Biol,2002,246(2):296-310.
[16]Chai OH,Song CH,Park SK,et al.Molecular regulation of kidney development[J].Anat Cell Biol,2013,46(1):19-31.
[17]Barasch J,Qiao J,McWilliams G,et al.Ureteric bud cells secrete multiple factors,including bFGF,which rescue renal progenitors from apoptosis[J].Am J Physiol Renal Physiol,1997,273(5):757-767.
[18]Grobstein C.Inductive interaction in the development of the mouse metanephros[J].J Exp Zool,1955,130(2):319-339.
[19]Schuchardt A,D'Agati V,Larsson-Blomberg L,et al.Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret[J].Nature,1994,273(5):380-383.
[20]Barasch J,Yang J,Qiao J,et al.Tissue inhibitor of metalloproteinase-2 stimulates mesenchymal growth and regulates epithelial branching during morphogenesis of the rat metanephros[J].J Clin Inves,1999,103(9):1299-1307.
[21]Barasch J.Genes and proteins involved in mesenchymal to epithelial transition[J].Curr Opin Nephrol Hypertens,2001,10(3):429.
[22]Koseki C,Herzlinger D,al-Awqati Q.Apoptosis in metanephric development[J].J Cell Biol,1992,119(5):1327-1333.
[23]Barasch J,Pressler L,Connor J,et al.A ureteric bud cell line induces nephrogenesis in two steps by two distinct signals[J].Am J Physiol Renal Physiol,1996,271(1):50-61.
[24]Dudley AT,Godin RE,Robertson EJ.Interaction between FGF and BMP signaling pathways regulates development of metanephric mesenchyme[J].Genes Dev,1999,13(12):1601-1613.
[25]Zhou TB.Signaling pathways of PAX2 and its role in renal interstitial fibrosis and glomerulosclerosis[J].J Recept Signal Transduct Res,2012,32(6):298-303.
[26]Hou XM,Chen X,Wang YL.The role of Pax2 in regulation of kidney development and kidney disease[J].Yi Chuan,2011,33(9):931-938.
[27]Mori K,Yang J,Barasch J.Ureteric bud controls multiple steps in the conversion of mesenchyme to epithelia[J].Semin Cell Dev Biol,2003,14(4):209-216.
[28]Miner JH,Li C.Defective glomerulogenesis in the absence of laminin α5 demonstrates a developmental role for the kidney glomerular basement membrane[J].Dev Biol,2000,217(2):278-289.
[29]Piepenhagen PA,Nelson WJ.Biogenesis of polarized epithelial cells during kidney development in situ:roles of E-cadherin-mediated cell-cell adhesion and membrane cytoskeleton organization[J].Mol Biology Cell,1998,9(11):3161-3177.