转基因骨髓源性肝干细胞移植对肝纤维化大鼠细胞外基质积聚的影响

Effects of gene-transfected bone marrow-derived liver stem cell transplantation on accumulation of extracellular matrix in rats with liver fibrosis

目的探讨尿激酶型纤溶酶原激活物（uPA）基因修饰骨髓源性肝干细胞（BDLSC）移植对四氯化碳（CCl4）诱导的肝纤维化大鼠肝脏细胞外基质（ECM）积聚的影响。方法体外将携带人uPA基因的腺病毒（AduPA）转染BDLSC。采用皮下注射CCl4方法建立大鼠肝纤维化模型。将纯系Fisher大鼠随机分为正常组、模型组、BDLSC组（尾静脉注入2×10^6BDLSC）和BDLSC—uPA组（尾静脉注入2×10^6AduPA转染的BDLSC），各9只。观察各组大鼠肝功能和血清ECM水平的变化；采用半定量逆转录（RT）-PCR方法检测大鼠肝组织Ⅰ、Ⅲ型胶原（COLⅠ、COLⅢ）、基质金属蛋白酶（MMP）-2、3、9及金属蛋白酶组织抑制物（TIMP）-1、2mRNA的表达。结果BDLSC—uPA组大鼠肝功能明显改善；血清ECM水平和羟脯氨酸含量明显低于模型组和BDLSC组；肝组织COLⅠmRNA（38．9±2．7）、COLⅢ mRNA（8．5±1．6）表达均明显低于模型组和BDLSC组（均P〈0．05），MMP-2 mRNA（157．5±32．6）、MMP-3 mRNA（105．5±14．6）、MMP-9mRNA（187．5±22．8）表达均高于模型组和BDLSC组（均P〈0．05），而TIMP-1、2mRNA的表达则差异无统计学意义（P〉0．05）。结论uPA基因修饰BDLSC移植可能通过上调MMP的表达。促进ECM的降解，从而改善纤维化大鼠的肝功能，抑制大鼠肝纤维化。

Objective To explore the effects of urokinase-type plasminogen activator （uPA） gene-modified bone marrow-derived stem cell （BDLSC） transplantation on accumulation of extracellular matrix （ECM） in hepatic tissue in liver fibrosis. Methods BDLSCs obtained from 10 male Fisher344 rats were transfected by adenovirus-mediated human uPA （AduPA） in vitro. Twenty-seven female rats were randomly divided into 3 equal groups to undergo subcutaneous injection of carbon tetraehloride to establish liver fibrosis models and then randomly divided into 3 equal groups： model group injected with normal saline via caudal vein, BDLSC group injected with 2 × 10^6 BDLSCs via caudal vein, and BDLSC-uPA group injected with 2 × 10^6 AduPA-transfected BDLSCs. Eight weeks later the serum levels of alanine transaminase （ALT）, aspartate aminotransferase （AST）, total bilirubin （TBIL）, albumin （ALB）, and ECM levels, i. e., hyaluronic acid, laminin （LN） , and proeollagen Ⅲ （ PC Ⅲ） , were detected. Then the rats were killed with their livers taken out. The hydroxyproline （Hyp） content of the liver was detected by alkaline hydrolysis. RT-PCR was used to examine the expression of collagen Ⅰ and Ⅲ （ COL Ⅰ and COL Ⅲ ） , matrix metalloproteinases-2, 3, and 9 （MMP-2, 3, and 9）, and tissue inhibitor of metalloproteinase-1 and2 （TIMP-1 and 2）. Results Compared with those of the model group the levels of ALT, AST, and TBIL of the BDLSC-uPA group were all significantly lower, and the ALB level was higher （ all P 〈 0.05 ）. The ECM levels of BDLSC-uPA group were all significantly lower than those of the model group or BDLSC group too （ all P 〈 0.05 ）. Hyp content of the liver decreased dramatically. The mRNA expression levels of COL Ⅰ and COLⅢ of the liver of the BDLSC-uPA group were significantly lower （38.9 ± 2.7,8.5 ± 1.6）, and the mRNA＇expression levels of MMP-2, -3, and MMP-9 mRNA （ 157.5 ± 32.6,105.5 ± 14.6,187.5 ± 22.8） were significantly higher than those of the model group or BDLSC group （ all P 〈 0.05 ）, but no significant differences were observed in the mRNA expression of TIMP-1 and 2 mRNA between the 3 groups （ all P 〉 0.05）. Conclusion uPA gene-modified BDISC transplantation improves the liver function and suppresses the hepatic fibrosis in liver cirrhosis through up-regulating the expression of MMPs and promoting the degradation of ECM.