胚胎肝前体细胞与转化生长因子β1信号介导的肝星状细胞联合移植治疗急性肝损伤

Intrasplenic co-transplantation of fetal hepatic progenitor cells and transforming growth factor beta 1 induced hepatic stellate cells ameliorates acute liver injury

背景:研究表明,移植的胚胎肝前体细胞在受体内大量增殖及长期存活困难,与转化生长因子β1(transforming growth factorβ1,TGF-β1)介导的肝星状细胞联合移植有望解决这一科学难题。目的:观察TGF-β1介导的小鼠肝星状细胞与小鼠胚胎肝前体细胞联合移植对急性肝损伤的治疗作用。方法:①建立慢病毒介导的稳定过表达TGF-β1基因的小鼠肝星状细胞株,通过细胞免疫荧光、qRT-PCR、western blotting法检测肝星状细胞转染目的基因情况;②体外培养小鼠胚胎肝前体细胞株mHPCs-E_(14.5)并以细胞免疫荧光染色法鉴定;③通过CCl4腹腔注射联合2/3肝切除构建急性肝损伤小鼠模型,然后进行mHPCs-E_(14.5)单独移植(mHPCs-E_(14.5)移植组),mHPCs-E_(14.5)与mHSCs-pHBLV-CMVIE-TGF-β1联合移植(过表达TGF-β1联合移植组),mHPCs-E_(14.5)与mHSCs-pHBLV-CMVIE-GFP联合移植(对照联合移植组);④在肝切除后14 d,共聚焦免疫荧光检测各组小鼠脾实质内ALB、CK19、a-SMA阳性表达,全自动生化分析仪检测小鼠血清谷丙转移酶、谷草转移酶活性。结果与结论:①成功建立稳定过表达TGF-β1基因的小鼠肝星状细胞株,与空载对照组相比,慢病毒mHSCs-pHBLV-CMVIE-TGF-β1组目的基因TGF-β1、α-SMA表达明显增高(P<0.01);②mHPCs-E_(14.5)细胞株大量表达AFP,微弱表达ALB和CK19,提示该细胞株为胚胎肝前体细胞;③mHPCs-E_(14.5)移植组脾实质内存在少量CK19阳性细胞和ALB阳性细胞;对照联合移植组脾实质内存在少量CK19阳性细胞、α-SMA阳性细胞及较多的ALB阳性细胞,而过表达TGF-β1联合移植组存在大量CK19阳性细胞、α-SMA阳性细胞,少量ALB阳性细胞;④细胞移植后血清谷丙转移酶及谷草转移酶有所下降,过表达TGF-β1联合移植组下降更明显(P<0.05); ⑤结果提示,移植的胚胎肝前体细胞在脾实质内定植并分化为肝细胞和胆管细胞,TGF-β1信号介导的肝星状细胞诱导胚胎肝前体细胞向胆管细胞分化而且能有效促进小鼠急性肝损伤的修复过程。

BACKGROUND:Difficulty in long-time survival and continuous proliferation is the main problem for transplanted fetal hepatic progenitor cells and co-transplantation with transforming growth factor beta 1(TGF-β1)-induced hepatic stellate cells may be a promising way to solve this scientific obstacle.OBJECTIVE:To explore the therapeutic effects of co-transplantation of fetal hepatic progenitor cells and TGF-β1-induced hepatic stellate cells on acute liver injury in mice.METHODS:Over-expression vector pHBLV-CMVIE-TGF-β1 was infected to mouse hepatic stellate cells and transfection efficiency was detected by immunocytochemistory,western blot and qRT-PCR.Hepatic progenitor cells,mHPCs-E14.5,were cultured and identified by immunofluorescence in vitro.The mouse model of acute liver injury was established by intraperitoneal injection of CCl4 in combination with 2/3 partial hepatectomy,followed by mHPCs-E14.5 transplantation,co-transplantation of mHPCs-E14.5 and mHSCs-pHBLV-CMVIE-TGF-β1(experimental co-transplantation group)or co-transplantation of mHPCs-E14.5 and mHSCs-pHBLV-CMVIE-GFP(control co-transplantation group)for cell transplantation assay.Confocal immunofluorescence staining against CK19,ALB,a-SMA was performed to analyze the engraftment and differentiation of transplanted cells in the splenic parenchyma 14 days post-transplantation;serum alanine transferase and aspartate transferase levels were monitored using an automatic biochemistry analyzer.RESULTS AND CONCLUSION:(1)A hepatic stellate cell line that over-expressing TGF-β1 was successfully established and expression levels of TGF-β1 andα-smooth muscle actin were efficiently up-regulated in the over-expression group(P<0.01).(2)mHPCs-E14.5 expressed massive AFP and low levels of ALB and CK19,confirming that this cell line was in complete conformity with fetal hepatic progenitor cells in vitro.(3)CK19 and ALB positive cells existed in the splenic parenchyma in mHPCs-E14.5 transplantation group.Highly expressed ALB but less expressedα-SMA and CK19 were observed in the control co-transplantation group,while massive CK19 and a-SMA positive cells as well as less level of ALB positive cells existed in the experimental co-transplantation group.Serum alanine transferase and aspartate transferase levels were decreased remarkably after cell transplantation,and moreover,the decrease was more obvious in the experimental co-transplantation group(P<0.05).Overall,transplanted fetal hepatic progenitor cells engraft and differentiate into hepatocytes and cholangiocytes in the splenic parechyma successfully in vivo.Importantly,hepatic stellate cells induced by TGF-β1 promote the differentiation of fetal hepatic progenitor cells into cholangiocytes and accelerate recovery from CCl4/partial hepatectomy induced acute liver injury.