联合共培养系统诱导骨髓间充质干细胞分化为视网膜色素上皮样细胞

目的 探讨添加脑源性神经生长因子（BDNF）、表皮生长因子（EGF）、碱性成纤维细胞生长因子（bFGF）的培养基联合人类视网膜色素上皮细胞（HRPECs）共培养对骨髓间充质干细胞（BMSCs）定向诱导分化的影响.方法 实验研究.实验分三组：HRPECs＋BDNF、EGF、bFGF＋BMSCs共培养组、BDNF、EGF、bFGF＋BMSCs共培养组和对照组（单独BMSCs）.第一组取第3代HRPECs接种在双层培养板的上层,将第3代人BMSCs接种于下层培养板中,在双层六孔板的每孔中加入混合有20 ng/mlbFGF、20 ng/ml EGF、20 ng/ml BDNF及10%胎牛血清（FBS）的DMEM-LG培养液（需做免疫细胞化学染色应同时在下层放入18 mm×18 mm盖玻片进行细胞爬片）.第二组取第3代人BMSCs接种在六孔培养板中,每孔中加入含20 ng/ml bFGF、20 ng/ml EGF、20 ng/ml BDNF及10%FBS的DMEM-LG培养液.第三组将第3代人BMSCs接种于六孔培养板中,加入10%FBS的DMEM-LG培养液.在倒置相差显微镜下观察细胞形态学变化.2周后停止培养,采用免疫细胞化学染色法和RT-PCR检测角蛋白18、RPE65蛋白存诱导细胞中的表达.数据采用Holm-Sidak法进行分析.结果 诱导2周后,第一组BMSCs细胞呈圆形、类圆形、不规则形、短棒状外观,细胞内有色素颗粒形成,其他两组没有类似改变.三组间免疫细胞化学染色法检测RPE65蛋白、角蛋白18,光密度值结果显示第一组和第二组间、第一组和第三组间差异有统计学意义（RPE65：t=37.416、36.236,P＜0.05;角蛋白18：t=38.611、37.532,P＜0.05）.而第二组和第三组间差异没有统计学意义（RPE65：t=1.180,P＞0.05;角蛋白18：t=1.079,P＞0.05）.RT-PCR检测相对mRNA表达量,结果显示第一组和第二组间、第一组和第三组间差异有统计学意义（RPE65/β-actin：t=176.110、174.820,P＜0.05;角蛋白18/β-actin：t=243.230、241.560,P＜0.05）.而第二组和第三组间差异无统计学意义（RPE65/β-actin：t=1.283.P＞0.05;角蛋白18/β-actin：t=1.670,P＞0.05）.结论 利用BDNF、EGF、bFGF联合HRPECs共培养可以使BMSCs分化为视网膜色素上皮样细胞.

Differentiation of bone marrow mesenchymal stem cells into retinal pigment epithelial-like cells by a systematic induction in vitro

Objective To investigate the differentiation features of bone marrow mesenchymal stem cells (BMSCs) cultivated with human retinal pigment epithelial cells (HRPECs), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) in vitro. Methods Experimental study. Three experimental groups were established based on the following criteria: HRPECs+EGF, BFGF, BDNF+BMSCs group (group Ⅰ), EGF, BFGF, BDNF+BMSCs group (group Ⅱ) and human BMSCs only (group Ⅲ). For group Ⅰ, HRPECs at passage 3were inoculated at the upper layer of a Transwell 6-well double layer culture plate, and human BMSCs at passage 3 were seeded at the lower layer of the culture plate; a DMEM-LG medium containing 20 ng/ml bFGF, 20 ng/ml EGF, 20 ng/ml BDNF and 10% fetal bovin serum (FBS) was added to each well (for immunocytochemical analysis, a 18 mm×18 mm cover slip was placed under the lower layer to allow cells to grow on it). For group Ⅱ, human BMSCs at passage 3 were seeded at the 6-well culture plate, and a DMEM-LG medium containing 20 ng/ml bFGF, 20 ng/ml EGF, 20 ng/ml BDNF and 10% FBS was added to each well. For group III, human BMSCs at passage 3 were seeded at the 6-well culture plate, and a DMEM-LG medium containing 10% FBS was added to each well. Cell morphology was monitored under an inverted microscope. After 2 weeks, cells were collected for immunocytochemistry and RT-PCR analysis. A Holm-Sidak test was used to analyze data. Results After induction, the cells in group Ⅰ presented a similar appearance to retinal pigment epithelial cells and intracellular pigment particles were visible. However, similar changes did not occur in group Ⅱ and group Ⅲ. Immunocytochemical analysis of RPE65 and keratin-18 showed the optical density value differences between group Ⅰ and group Ⅱ, and between group Ⅰ and group Ⅲwere statistically significant (RPE65: t=37.416, 36.236, P＜0.05; keratin-18: t=38.611,37.532, P＜0.05),while the difference between group Ⅱ and group Ⅲ was not statistically significant (RPE65: t=1.180,P＞0.05; keratin-18: t=1.079, P＞0.05). The expression of RPE65 and keratin-18 was examined by RT-PCR using cell extracted from the 6-well plate, and mRNA expression was calculated after a computer software analysis and a comparison to the internal control β-actin. The inter-group difference showed that the differences between group Ⅰ and group Ⅱ, and between group Ⅰand group Ⅲ were statistically significant (RPE65/β-actin: t=176.110, 174.820, P＜0.05; keratin-18/β-actin:t=243.230, 241.560, P＜0.05), but the difference between group Ⅱ and group Ⅲ was not statistically significant (RPE65/β-actin: t=1.283, P＞0.05; keratin-18/β-actin: t=1.670, P＞0.05). Conclusion BMSCs can be induced into retinal pigment epithelium-like cells when cocultured with HRPECs and BDNF, EGF, bFGF.