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目的探讨骨髓间充质干细胞(MSCs)移植对大鼠肝纤维化模型的作用与机制。方法分离大鼠MSCs培养传代至第4代。采用大鼠腹腔注射四氯化碳制造肝纤维模型。将造模成功SD大鼠60只,随机分为3组,每组20只:(1)对照组:鼠尾静脉注射等量的生理盐水;(2)MSCs组:鼠尾静脉注射MSCs悬液;(3)诱导组:鼠尾静脉注射经肝细胞生长因子(HGF)诱导14 d后的MSCs悬液。于移植后第1周、2周、3周、4周分别处死大鼠5只,检测大鼠血清透明质酸(HA)、层黏蛋白(LN)、Ⅳ型胶原水平;光镜下观察肝组织纤维化程度;分别用PCR法及Western检测大鼠肝脏Col-Ⅰ、RhoA、Cdc42、Rac1 mRNA基因及其蛋白质表达水平。结果(1)诱导组与MSCs组的纤维化程度评分随着时间延长逐渐下降,且在第4周诱导组评分明显低于MSCs组(P<0.05)。(2)移植3周后各组血清HA、LN、Ⅳ型胶原含量均显著下降,4周时诱导组和MSCs组各指标含量明显低于对照组,并且诱导组低于MSCs组(P<0.05)。(3)MSCs移植后诱导组、MSCs组大鼠肝脏组织Col-Ⅰ、RhoA、Cdc42、Rac1 mRNA和蛋白表达均随时间延长而下降,移植4周时诱导组各项指标明显低于MSCs组和对照组(P<0.05)。结论 MSCs移植可抑制肝纤维化大鼠肝组织中HA、LN、Ⅳ型胶原的分泌,并可下调肝纤维化大鼠肝组织Rho信号通路相关因子RhoA,Cdc42,Rac1 mRNA及蛋白表达。HGF诱导MSCs后抗大鼠肝纤维化作用显著增强。
Objective To investigate the effect and mechanism of bone marrow mesenchymal stem cells (MSCs) transplantation on rat liver fibrosis model. Methods Rat MSCs were isolated and passaged to passage 4. Rat models of hepatic fibrosis were made by intraperitoneal injection of carbon tetrachloride. Sixty Sprague-Dawley rats were randomly divided into 3 groups with 20 rats in each group: (1) control group: rats were injected intravenously with equal volume of normal saline; (2) MSCs group: MSCs were injected into caudal vein ; (3) Induction group: The MSCs suspension was induced by hepatocyte growth factor (HGF) for 14 days in the tail vein of rats. Five rats were sacrificed in the first week, the second week, the third week and the fourth week after transplantation. The levels of serum hyaluronic acid (HA), laminin (LN) and collagen Ⅳ in the rats were determined. The levels of Col-Ⅰ, RhoA, Cdc42, Rac1 mRNA and protein in rat liver were detected by PCR and Western blot respectively. Results (1) The scores of fibrosis degree in induction group and MSCs group decreased gradually with time, and the score of inducing group in 4th week was lower than that of MSCs group (P <0.05). (2) After 3 weeks of transplantation, the contents of HA, LN and type IV collagen in serum of all the groups were significantly decreased. At 4 weeks, the content of each index in induction group and MSCs group was significantly lower than that in control group (P <0.05) ). (3) The mRNA and protein expressions of Col-Ⅰ, RhoA, Cdc42 and Rac1 in liver tissue of MSCs transplantation group decreased with time after transplanted MSCs. At 4 weeks after transplanting, the indexes in induction group were significantly lower than those in MSCs group and Control group (P <0.05). Conclusion Transplantation of MSCs can inhibit the secretion of HA, LN and type Ⅳ collagen in liver tissue of rats with hepatic fibrosis and downregulate the expression of RhoA, Cdc42 and Rac1 mRNA and protein in liver tissue of liver fibrosis rats. HGF-induced MSCs anti-rat liver fibrosis significantly enhanced.