骨髓间充质干细胞移植时机及移植时间长短对大鼠肝纤维化病变进程的影响

Effects of bone marrow mesenchymal stem cells transplantation at different time as well as the duration on hepatic fibrogenesis in rats

背景:研究发现肝纤维化环境是骨髓间充质干细胞适宜的分化环境,提示通过骨髓间充质干细胞移植治疗肝纤维化成为可能。目的:探讨骨髓间充质干细胞不同移植时期及移植时间长短对大鼠肝纤维化进程的影响。设计、时间及地点:细胞学体内实验,于2007-11/2008-08在福建医科大学及福建省科研所完成。材料:普通级五六周龄雄性SD大鼠5只用于制备骨髓间充质干细胞;清洁级成年雌性SD大鼠68只,分别于造模10周、12周后处死,前者分为对照组、造模第6周细胞移植组2个亚组,12只/组;后者分为对照组、造模第6周细胞移植组、造模第8周细胞移植组3个亚组,动物数量分别为16只,16只,12只。方法:各组大鼠均通过腹腔注射四氯化碳建立肝纤维化模型。取体外分离培养的第4代骨髓间充质干细胞悬液0.2mL(含5×106个细胞),分别于造模后第6,8周经尾静脉注入各细胞移植组大鼠体内,对照组同法注入L-DMEM培养基,各组于培养对应时间点处死。主要观察指标:大鼠一般情况及肝纤维化和炎症程度。结果:①造模10周后处死:与对照组比较,造模第6周细胞移植组肝纤维化评分均值明显降低(t=-2.45,P=0.023),炎症程度方面无明显差异(t=-0.60,P=0.554)。②造模12周后处死:对照组、造模第8周细胞移植组的肝纤维化及炎症程度评分均值均基本相似(t=-0.21,P=0.84;t=-0.18,P=0.86),2组均明显高于造模第6周细胞移植组(t=6.17,t=8.61,P<0.01)。③造模10周后处死与造模12周后处死比较:随细胞移植时间的延长,造模第6周细胞移植组肝纤维化及炎症程度评分均值均明显降低(t=-6.98,t=-9.78,P<0.01)。结论:早期移植骨髓间充质干细胞可以减轻CCl4引起的肝脏炎症程度,同时改善大鼠的肝纤维化程度,此作用与移植时间成正相关。

BACKGROUND： Research demonstrated that hepatic fibrosis is suitable for the differentiation of bone marrow mesenchymal sterr ceils （MSCs）, which suggests it is possible to treat hepatic fibrosis with MSCs transplantation. OBJECTIVE： To investigate the effects of MSCs transplantation at different Umes as well as the duration on hepatic fibrogenesis in rats. DESIGN, TIME AND SETTING： The in vivo cytology experiment was performed at the Fujian Medical University and Fujian Institute of Medical Sciences between November 2007 and August 2008. MATERIALS： Five male SD rats, with 5-6 weeks old were prepared for MSCs; 68 adult, male SD rats with clean grade were sacdficed at weeks 10 and 12 after model preparation. Rats killed at 10 weeks were divided into control and 6th weeks MSCs transplantation sub-groups, with 12 animals in each group. Rats killed at 12 weeks were divided into control, 6th weeks MSCs transplantation and 8th weeks MSCs transplantation sub-groups, with 16, 16 and 12 animals in each group. METHODS： Combined with stationary culture MSCs of male SD rats were isolated and purified by density gradient centrifugation. Totally 0.2 mL forth passage cells （5×10^6 mL）, expanded by subculture, were respectively injected into the homogeneous female rats via the caudalis vena at weeks 6 and 8 after the hepatic fibrosis model induced by carbon tetrachloride （CCL4） in rats. In the control group, L-DMEM was injected with the same method. MAIN OUTCOME MEASURES： The Scheuer inflammation activity score and Schmid M fibrosis grading score. RESULTS： （1）Rats killed at 10 weeks： The Scheuer inflammation score of the 6th weeks MSCs transplantation group was obvious decreased than that of control group （t=-2.45, P=-0.023）, while there was no difference in inflammation activity grade was found （t=- 0.60, P=0.554）, （2）Rats killed at 12 weeks： the Schmid M fibrosis score and Scheuer inflammation score were basically similar in control and 8th weeks MSCs transplantation groups （t=-0.21,P=-0.84; t=-0.18, P=0.86）, which was greater than the 6th weeks MSCs transplantation group （t=6.17, t=-8.61, P 〈 0.01）. （3）Compared between rats killed at 10 weeks and killed at 12 weeks after model preparation, the fibrosis score and inflammation score was dramatically decreased in the 6th weeks MSCs transplantation group as the increasing of transplantation duration （t=- 6.98, t=- 9.78, P 〈 0.01）. CONCLUSION： Eady MSCs transplantation can alleviate inflammation reaction and improve fibrosis stage in transplanted rats induced by CCL4. The improve degree of inflammation reaction and fibrosis stage has a positive correlation with time.