神经导向因子Slit2修饰骨髓间充质干细胞的表达研究  

Research of axon guidance cues Slit2 expression in gene modified bone marrow mesenchymal stem cells

作　　者：柴源[1] 蒋莱[2] 周玉云[1] 王晓鹤[1] 马学玲[1] 

机构地区：[1]哈尔滨医科大学第四临床医学院,黑龙江哈尔滨150000 [2]齐齐哈尔医学院附属第三医院,黑龙江齐齐哈尔161000

出　　处：《中风与神经疾病杂志》2015年第12期1084-1088,共5页Journal of Apoplexy and Nervous Diseases

基　　金：国家自然科学基金(No.81441039);教育部高等学校博士学科点专项科研基金新教师类(No.20122307120031)

摘　　要：目的基因修饰骨髓间充质干细胞,使其神经导向因子Slit2蛋白表达稳定增多,从而使骨髓间充质干细胞更好地促进脑缺血后血管生成,显著改善神经功能恢复。方法采用密度梯度离心法分离骨髓间充质干细胞,P3代细胞进行流式细胞仪鉴定,通过激光共聚焦显微镜观察其Slit2蛋白分布情况。利用脂质体转染技术,将p Sec Tag B/h Slit2-C-myc重组质粒转入骨髓间充质干细胞,经过Zeocin稳定筛选后,通过RT-PCR及Western blot检测骨髓间充质干细胞中重组Slit2蛋白的表达情况。结果 (1)成功分离培养大鼠骨髓间充质干细胞并通过流式细胞仪鉴定;(2)通过激光共聚焦显微镜发现Slit2蛋白在骨髓间充质干细胞的细胞膜及细胞质中均有表达;(3)成功将p Sec Tag B/h Slit2-C-myc重组质粒转染骨髓间充质干细胞;(4)RT-PCR及Western blot证实无论在基因水平或者蛋白水平,Slit2在转染后骨髓间充质干细胞中表达均增多。结论成功通过基因转染的方法实现骨髓间充质干细胞中重组Slit2蛋白表达量稳定增加,推动了进一步体内移植探讨其与血管生成机制的研究。Objective To stably up-regulate the expression of axon guidance cues Slit2 by gene modify bone marrow mesenchymal stem cells, promote it more effectively regulate angiogenesis, significantly improve functional recovery after cerebral ischemia. Methods Using density gradient centrifugation method to separate and culture rats bone marrow mesenchymal stem cells, then select the third generation cells for flow eytometry identified. Using confocal laser scanning microscope to observe Slit2 expression of rat bone marrow mesenchymal stem cells. Transfect the pSeeTagB/hSlit2-C-myc recombinant plasmid into rat bone marrow mesenchymal stem cells by liposome, after Zeocin screen, reverse transeription-PCR and estern blot were used to detect Slit2 expression in bone marrow mesenchymal stem cells. Results （ 1 ） Bone marrow mesenchymal stem cells were successfully cultured and identified by flow cytometry. （2）The confocal laser scanning microscope showed that the Slit2 proteins were expressed both in cell membrane and cytoplasm. （3） Successfully transfected the pSeeTagB/ hSlit2-C-myc recombinant plasmid into the cells. （4）Reverse transcription-PCR and western blot results showed that both in gene and protein level the expressions of Slit2 were all increased. Conclusion The expression of Slit2 was stably up-regulated in bone marrow mesenchymal stem cells, facilitate further study about the mechanism of bone marrow mesenchymal stem cells promote angiogenesis after cerebral infarction.

关 键 词：SLIT2 骨髓间充质干细胞 基因修饰 血管生成 

分 类 号：R743[医药卫生—神经病学与精神病学]