c-Kit+Lin-细胞体外扩增及体内分化的实验研究

Study of the expansion of c-kit+Lin-cells and the potential of differentiation into liver cell in mouse

关键词:  骨髓衍生肝干细胞  免疫磁珠分选  体外扩增培养  干细胞移植  c-Kit+Lin-细胞

Key words:  [Key words]　bone marrow derived liver stem cells  MACS  expand and culture in vitro  stem cell transplantation  c-Kit+Lin- cells

摘要:

目的　研究移植刚分选及扩增后雄性小鼠的骨髓c-Kit+Lin-细胞到酒精性肝纤维化雌性模型小鼠体内后，模型小鼠的肝脏形态改变、肝功能变化和移植细胞的分布、分化及归巢情况。　方法　　采用酒精灌胃法建立酒精性肝纤维化雌性小鼠模型，造模成功后随机选取8只模型小鼠（模型组）与正常小鼠8只（对照组）比较两组肝功能变化。剩余模型小鼠再随机选取32只，分为4组处理，并再随机选取8只正常小鼠（第1组）与其对照。模型小鼠的4组分别为：移植前取血处死组（第2组），移植新鲜分选的c-Kit+ Lin-细胞组（第3组），移植扩增后的c-Kit+ Lin-细胞组（第4组），仅予以注射Buffer组（第5组）。采用免疫磁珠法分选雄性小鼠骨髓c-Kit+Lin-细胞；利用SCF+ HGF+ FL+LIF +TPO+ IL-3因子组合对其进行体外扩增；将刚分选及扩增后c-Kit+Lin-细胞移植入酒精性肝纤维化雌性模型小鼠体内，检测两组肝功能改变、肝脏纤维化改善、含雄性小鼠Y染色体性别决定基因cDNA(Sry)的移植细胞在受体肝内定居分化情况。　结果　　（1） 两移植组在移植30天后，较移植前肝脏纤维化改善明显；（2） 两移植组在移植30天后AST、ALT、ALB与移植前比较差异有显著性意义（P<0.05）；（3）两移植组均可见含雄性小鼠Y染色体性别决定基因cDNA(Sry)的移植细胞，并同时表达ALB mRNA，计数细胞比例，比较差异无显著性意义（P>0.05）。　结论　　移植c-Kit+ Lin-细胞后能在受体肝内定居并转化成有功能新生肝细胞；移植c-Kit+ Lin-细胞后能明显改善肝损伤小鼠的肝功能及肝脏纤维化；扩增对c-Kit+Lin-细胞的归巢、在体内分化没有明显影响。

Abstract:

[Abstract] Objective　To elucidate the liver function recovery and the translated stem cells’location and differentiation of the hepatic fibrosis female rats induced by alcohol in the group of translated expanded and fresh isolated c-Kit+Lin- cells.　Methods　We used alcohol to establish the female rats hepatic fibrosis model. After the modeling randomly selected eight mice (model group) compared with normal mice (control group) liver function. The remaining mice were randomly selected 32 divided into 4 groups.Randomly selected eight normal mice (group 1)as a control group.C-kit+Lin- cells were isolated from mouse bone marrow by using a high-gradient magnetic cell sorting system (MACS),and expanded with combinations of SCF+ HGF+ FL+LIF +TPO+ IL-3 for 7days in a liquid culture system. We translated the expanding c-kit+Lin- cells into the normal female rats and hepatic fibrosis female rats induced by alcohol. Observe the translated stem cells'location and differentiation in the rats body and count the cells which expressed both gene of Sry, and compare the two group. 　Results (1) After 30 days,HE and VG histological examination showed that the translated groups were less fibrosis and the control group obviously. (2) After 30 days,AST,ALT,ALB after transplantation compared with pretrans plant significant difference (P<0.05). (3) Results of hybridization of Y-chromosome determined cDNA and albumin mRNA indicated that in the translated group there existed hepatic cells which derived from stem cells in the liver. The difference was not statistically significant (P>0.05).　Conclusion In this experiment condition,expanding c-Kit+Lin- cell did not effect the stem cells location and differentiation. The liver function recovery in the group translated and fresh isolated c-Kit+Lin- cells ，reverse the liver fibrosis and there existed hepatic cells which derived from stem cells in the liver.