大鼠骨髓间充质干细胞的标记跟踪及自体移植对骨折愈合的影响

Experimental study on the effect of rats bone marrow stromalstem cell autologous transplantation on fracture healing

目的:体外分离培养大鼠骨髓间充质干细胞(BMSCs),分别采用BrdU、DAPI对BMSCs进行体外染色标记,同时自体移植BrdU标记的BMSCs,观察其对大鼠胫骨骨折愈合的影响。方法:BMSCs取自SD大鼠的股骨和胫骨,分离培养BMSCs至第三代时,采用流式细胞仪对其表面抗原CD34、CD44和CD45进行测定;同时分别用BrdU、DAPI对BMSCs进行染色标记。选取3月龄雌性SD大鼠24只,随机分为三组(n=8),A组为左胫骨骨折组,B组为左胫骨骨折+DMEM组,C组为左胫骨骨折+骨髓间充质干细胞移植组。术前两周C组取自体BMSCs,采用BrdU标记,术后2天将标记好的细胞以局部注射的方法移植到骨折断端,同时B组局部注射等量DMEM。各组动物分别于术后1、2、3、4周拍摄左胫骨DR,测量骨痂宽度,评价骨折愈合情况。结果:流式细胞仪检测证实:实验成功分离培养出了高纯度的BMSCs(免疫表型CD44阳性,CD34、CD45阴性);采用BrdU(浓度10μmol/L,时间48hours)和DAPI(浓度20μg/ml,时间30 min)染色标记BMSCs安全有效;BrdU标记的BMSCs在移植后第3周时仍然存在荧光标记。术后1、2、3、4周各胫骨骨痂宽度测量:术后第1、2周,三组实验动物骨痂宽度无明显差异(P>0.05),术后第3、4周时C组骨痂宽度较A、B两组差异有统计学意义(P<0.05)。结论:采用黏附贴壁分离培养法能够有效获取高纯度的BMSCs;运用Brdu、DAPI方法标记BMSCs效果良好,方法可靠;大鼠BMSCs的自体移植能够促进骨折愈合。

Objective:In vitro isolation and purification of rat bone marrow mesenchymal stem cells(BMSCs)were explored;in vitro staining of BMSCs was performed by Brd U、DAPI,and autologous transplantation of BMSCs to observe its effect on the healing of tibial fracture in rats. Methods:BMSCs were taken from the femur and tibia of SD rats,and when the BMSCs were isolated to the third generation,their surface antigen CD34、CD44 and CD45 was determined by flow cytometry,while BMSCs were stained with Brd U and DAPI respectively.Twenty-four female SD rats were randomly divided into three groups(n=8):Group A was left tibial fracture group,Group B was left tibial fracture+DMEM,and Group C was left tibial fracture +BMSCs.Two days post-surgery,autologous cells labeled with Brd U were transplanted to the fracture of group C,and Group B injected with equal amounts of DMEM simultaneously.Taking the left tibial DR at 1,2,3 and 4 weeks after surgery,the callus was measured and the fracture healing was evaluated. Results:Flow cytometry confirmed that BMSCs were expressed CD44 positive,CD34,CD45 negative;Brd U(10μmol/L,time 48 hours)and DAPI(20μg/m L,time 30 min)stained were safe and effective;Brd U-labeled BMSCs were still fluorescent at week 3 after transplantation.The callus of each tibia was measured at 1,2,3 and 4 weeks after surgery,there was no significant difference of callus in the three group at first and second weeks after surgery(P>0.05),but the callus of group C was statistically significant at the third and fourth weeks after surgery compared with groups A and B(P<0.05). Conclusion:High-purifiedBMSCs can be obtained by adherent method;the ways of labeling BMSCsby Brd U and DAPIis stable andreliable;autologous transplantation of BMSCs can promote tibia fracture healing.