骨髓基质干细胞旁分泌作用促进大鼠损伤脊髓的血管新生

Paracrine of bone marrow mesenchymal stem cells promotes angiogenesis in rats with spinal cord injury

目的 观察移植骨髓基质干细胞（BMSCs）旁分泌作用对大鼠损伤脊髓血管新生的促进作用. 方法 用密度梯度离心及贴壁法相结合的方法分离、纯化、培养大鼠BMSCs,应用流式细胞技术检测BMSCs表面细胞标志CD34、CD45、CD29、CD90;根据改良Allen法制备大鼠脊髓损伤模型,54只清洁型SD大鼠分为假手术组、对照组和实验组（n=1 8）,假手术组和实验组经尾静脉注射BMSCs,对照组经尾静脉注射PBS.移植BMSCs后l、7、14d应用免疫荧光标记观察羧基荧光素乙酰乙酸标记的BMSCs表达血管内皮细胞生长因子（VEGF）和碱性成纤维细胞生长因子（bFGF）的情况;应用免疫组化检测移植BMSCs对损伤脊髓第Ⅷ因子相关抗原表达的影响;应用逆转录-聚合酶链反应法检测移植BMSCs对损伤脊髓VEGF和bFGF mRNA表达的影响. 结果 激光共聚焦显微镜显示移植的BMSCs可在脊髓损伤处及损伤附近区域聚集、存活且表达VEGF和bFGF.实验组大鼠脊髓内第Ⅷ因子相关抗原的灰度值、微血管密度以及VEGF mRNA和bFGF mRNA的表达量明显高于对照组和假手术组,差异均有统计学意义（P＜0.05）;而且实验组14d微血管密度、VEGF mRNA、bFGF mRNA的表达量较1、7d明显升高,差异均有统计学意义（P＜0.05）. 结论 BMSCs在损伤脊髓内可以通过旁分泌VEGF和bFGF增加微血管密度,促进损伤脊髓的结构和功能恢复.

Objective To explore the effects of paracrine of the transplanted bone marrow mnesenchymal stem cells （BMSCs） on angiogenesis in rats with spinal cord injury （SCI）.Methods BMSCs were isolated,purified and cultured in vitro by Percoll density gradient centrifugation combined with adherent method.The specific cellular surface mnarker CD34,CD45,CD29 and CD90 of BMSCs were analyzed with a flow cytometer.The rat SCI model was prepared according to the Allen method.54 healthy adult Sprague-dawely （SD） male rats were randomly divided into sham-operation group,control group and experimental group.BMSCs were injected 3 days after injury in sham-operation and experimental groups via the caudal vein,and PBS was injected in control group.Expression of VEGF and bFGF in transplanted BMSCs labeled with CFSE was observed at 1,7 and 14 days after transplantation by immunofluorescence staining under laser confocal scanning microscopy.Influence of transplanted BMSCs on expression of factor Ⅷ related antigen in injured spinal cord was detected by immunohistochemical method.Influence of transplanted BMSCs on expression of VEGF and bFGF mRNA in injured spinal cord was detected by RT-PCR method.Results Under laser confocal scanning microscopy,the transplanted BMSCs could gather together and live in injured spinal cord and express VEGF and bFGF.Compared with control rats,the gray value of factor VⅧ related antigen in the injured spinal cord of experimental rats was significantly higher,the micro-vessel density was significantly greater,and the expression of VEGF and bFGF mRNA was significantly higher （P 〈 0.05）.The micro-vessel density and the expression of VEGF and bFGF mRNA on day 14 after transplantation in the experimental group were significantly higher than on day 1 and day 7 （P 〈 0.05）.Conclusion The transplanted BMSCs may promote structural and functional recovery of the injured spinal cord by paracrining VEGF and bFGF and increasing micro-vessel density in the injured spinal cord after SCI in rats.