体外定向诱导成年大鼠骨髓间充质干细胞向许旺细胞样细胞分化的研究

Induction of Schwann cell-like differentiation in bone mesenchymal stem cell of adult rat in vitro

目的探讨体外定向诱导成年大鼠骨髓间充质干细胞(BMSC)向许旺细胞样细胞转化的有效方法。方法分别进行成年大鼠许旺细胞原代培养和BMSC分离培养。根据诱导方法不同,分为化学诱导组和共培养诱导组。采用显微镜观察许旺细胞和BMSC的生长情况,分别采用免疫荧光化学染色法[检测神经胶质纤维酸性蛋白(GFAP)抗体和S-100抗体等许旺细胞标志蛋白]和流式细胞术鉴定两种细胞;显微镜下动态观察两组细胞的形态和生长情况;共培养诱导组共培养3 d后,化学诱导组诱导作用4 h、1 d后分别采用免疫荧光化学染色的方法评估两组细胞的诱导分化情况。结果化学诱导组诱导后的细胞发生了明显的许旺细胞样细胞形态改变,预诱导4 h后即出现GFAP抗体表达阳性,维持诱导1 d后,GFAP抗体阳性表达率为(80.9±3.5)%,S-100抗体阳性表达率为(59.0±1.1)%。诱导2 d后分化细胞开始出现脱落死亡,3 d后大部分分化细胞脱落死亡。共培养诱导组共培养3 d后,被诱导的BMSC未出现类似化学诱导组明显的细胞形态改变,GFAP抗体阳性表达率为(89.8±2.4)%,S-100抗体阳性表达率为(80.9±1.7)%。共培养诱导组S-100、GFAP抗体阳性表达率均高于化学诱导组,差异均有统计学意义(均为P<0.01)。结论共培养诱导法不仅对BMSC向许旺细胞样细胞定向分化具有明确的效果,而且对BMSC的存活和增殖均有促进作用,因此该法相对于化学诱导法更加安全和有效。

Objective To explore the effective method of induction of Schwann cell-like differentiation in bone mesenehymal stem cell （BMSC） of adult rat in vitro. Methods Primary culture of Schwann cell and isolated culture of BMSC were separately conducted. According to different induction methods, the cells were divided into chemical induction group and co-culture induction group. The growth of Schwann cell and BMSC was observed under light microscope. These two kinds of cells were identified by immunofluoreseenee staining [ detecting Sehwann cell marker proteins ： glial fibrillary acidic protein （GFAP） antibody and S-100 antibody ] and flow cytometry. The shape and growth of cells in two groups were dynamically observed by light microscope. The induced differentiation was evaluated with immunofluoreseence staining at 3rd day after co-culture induction in the co-culture induction group and at 4 h and 1st day after chemical induction in the chemical induction group. Results In the chemical induction group, the BMSC appeared typical Schwann cell-like morphology. The positive expression of GFAP antibody appeared at 4 h after preliminary induction. Meanwhile, the positive expression rate of GFAP and S-100 antibody was （80. 9± 3.5）% and （59.0 ± 1.1）% at 1st day after ind,lction. The induced BMSC began to die at 2na day after chemical induction and most of the induced BMSC had died at 3rd day after chemical induction. At 3rd day after co-culture induction, few induced BMSC showed obvious morphological changes like those in chemical induction group. The positive expression rate of GFAP and S-100 antibody was （ 89. 8 ± 2. 4 ） % and （ 80. 9 ± 1.7 ） %. The positive expression rate of GFAP and S-100 antibody in the co-culture induction group was higher than those in the chemical induction group and the difference had statistical significance （all in P 〈 0. 01 ）. Conclusions The co-culture induction not only has obvious effect on Schwann cell-like differentiation in BMSC, but also promotes the survival and proliferation of BMSC. Thus, co-culture induction is more safe and effective than chemical induction.