联合应用骨髓间充质干细胞与促肝细胞生长素治疗大鼠急性肝衰竭

Combination of mesenchymal stem cells and promoting hepatocyte growth factor for the treatment of acute liver failure in rats

目的评价骨髓间充质干细胞(MSCs)和促肝细胞生长素(pHGF)联合治疗大鼠急性肝衰竭(ALF)的可行性。方法将ALF造模成功的37只SD大鼠分为4组:CCl4组(A组,9只),CCl4/MSCs(B组,9只),CCl4/pHGF组(C组,9只)和CCl4/pHGF+MSCs组(D组,10只)。用携带hrGFP基因的慢病毒感染MSCs(hrGFP-MSCs),并以hrGFP示踪。对C、D组大鼠给予CCl4,同时腹腔注射pHGF;B组和D组造模12h后肝内注射hrGFP-MSCs悬液。对各组大鼠分别在造模后24h、72h、7天检测血清丙氨酸转氨酶(ALT)水平,造模后72h检测血清IL-6、IL-10、TNF-α炎症因子水平,造模后24h取肝组织行HE染色,造模72h行PCNA免疫组化染色检测肝细胞增殖情况。造模后72h后取注射部位肝组织行冰冻切片观察移植hrGFP-MSCs肝内分布情况,对非注射部位肝组织进行实时荧光定量RT-PCR检测hrGFP基因含量。结果联合治疗后,ALF大鼠外周血ALT水平降低,IL-6、IL-10、TNF-α炎症因子水平下调,肝脏病理组织学损害减轻,肝细胞增殖率增高。造模后72h,B组与D组注射部位肝组织内可见移植的hrGFP-MSCs,非注射部位肝组织未见hrGFP荧光阳性细胞,但用实时荧光定量RT-PCR技术可在非注射部位肝组织内检测到hrGFP基因,且D组含量高于B组。结论 pHGF与MSCs联合治疗大鼠ALF的疗效优于MSCs或pHGF单独治疗,可能与pHGF促使其肝内定植MSCs增多有关。

Objective To explore the feasibility of combination treatment acute liver failure （ALF） rats with bone marrow mesenchymal stem cells （MSCs） and promoting hepatocyte growth factor （pHGF）. Methods After preparation of ALF models, 37 SD rats were randomly divided into 4 groups： CC14 group （group A, n=9）, CC14/MSCs group （group B, n= 9）, CC14/pHGF group （group C, n=9） and CC14/pHGF＋MSCs group （group D, n=10）. MSCs had been infected with hrGFP-lentivius, in order to trace the transplantation of MSCs into liver. Rats of groups C and D were injected pHGF when given CC14. Rats of groups B and D were injected hrGFP-MSCs suspension into liver within 12 h of modeling. Serum ALT level 24 h, 72 h and 7 days after modeling, as well as IL-6, IL-10, TNF-α level at 72 h after modeling were detected. HE staining of liver tissue at 24 h after modeling and PCNA staining at 72 h were observed. The distribution of hrGFP-MSCs in the injection site of liver tissue were observed with cryosections, while the quantity of hrGFP gene in the non-injection of liver tissue were measured by RT-PCR at 72h. Results Combination of MSCs and pHGF therapy of ALF rats inhibited ALT levels, down-regulated IL-6, IL-10 and TNF-α inflammation factor, improved liver injury and enhanced liver regener- ation. There was a lot of transplanted cells in the injection site, while no specific fluorescence-positive cells were observed in non-injection site of liver tissue in group B and D at 72 h. However, the quantity of hrGFP gene was detected by RT- PCR in non-injection site of liver tissue. Conclusion Combination therapy of MSCs and pHGF for ALF rats is more effec- tive than MSCs or pHGF alone, for pHGF may increase the quantity of transplanted MSCs into ALF rat liver.