大鼠骨髓间充质干细胞诱导分化为视网膜光感受器样细胞的研究

发布时间：2018-11-02 15:46

【摘要】： 目的:探讨体外分离培养视网膜光感受器细胞以及体外培养的大鼠骨髓间充质干细胞(rat mesenchymal stem cells,rMSCs)诱导分化成视网膜光感受器样细胞的能力。方法:1、取SD大鼠眼球:①视网膜片行消化前后的HE染色。②分离光感受器细胞:形态学观察;视紫红质(Rhodopsin)免疫细胞化学染色行光感受器细胞的鉴定;同时收集光感受器细胞培养上清液。2、分离培养SD大鼠的骨髓间充质干细胞,取第三代rMSCs,采用光感受器细胞培养上清液诱导rMSC。诱导14d后采用免疫细胞化学染色和Rt-PCR观察rMSC视紫红质(Rhodopsin)、神经元特异性烯醇化酶(NSE),胶质纤维酸性蛋白(GFAP)的表达情况。结果:视网膜片的HE染色,消化前观察到完整的九层,消化后光感受器细胞大量缺失,而其它层完整,光感受器细胞免疫细胞化学鉴定阳性率达95%以上。诱导细胞14d后镜下观察到神经样的细胞形态,免疫细胞化学:实验组rMSC的Rhodopsin表达率为35.1%±6.2%,而对照组中无Rhodopsin阳性的细胞;实验组rMSC的NSE表达率为33.6%±4.0%,对照组为10.6%±5.0%,组间差异具有统计学意义(F =0.09,P 0.001);实验组rMSC的GFAP的表达率为9.6%±1.5%,对照组为:13.7%±3.0%,组间差异无统计学意义(F =1.726,P 0.05)。RT-PCR鉴定结果显示诱导组观察到Rhodopsin、NSE、GFAP表达,而对照组只有NSE、GFAP表达。结论:采用酶消化非离心的方法可以得到大量较纯且完整的光感受器细胞;体外培养的rMSC,经过视网膜光感受器细胞培养上清液诱导,可以分化为视网膜光感受器样细胞。
[Abstract]:Aim: to investigate the ability of retinal photoreceptor cells isolated and cultured in vitro and rat bone marrow mesenchymal stem cells (rat mesenchymal stem cells,rMSCs) to differentiate into retinal photoreceptor like cells. Methods: 1. The eyeballs of SD rats were taken out: 1 Retinal slices were stained with HE before and after digestion, 2 photoreceptor cells were isolated: morphological observation, rhodopsin (Rhodopsin) immunocytochemistry staining was used to identify photoreceptor cells; At the same time, the supernatant of photoreceptor cell culture was collected. 2. Bone marrow mesenchymal stem cells from SD rats were isolated and cultured. The third generation of rMSCs, was used to induce rMSC. by photoreceptor cell culture supernatant. Immunocytochemical staining and Rt-PCR were used to observe the expression of (NSE), glial fibrillary acidic protein (GFAP) in rMSC rhodopsin (Rhodopsin), neurons after 14 days of induction. Results: the complete nine layers were observed by HE staining before digestion. After digestion, a large number of photoreceptor cells were absent, while the other layers were intact. The positive rate of photoreceptor cell immunocytochemistry was over 95%. After 14 days of induction, the neuron-like cell morphology was observed under microscope. The immunocytochemistry showed that the Rhodopsin expression rate of rMSC was 35.1% 卤6.2 in the experimental group, but no Rhodopsin positive cells were found in the control group. The NSE expression rate of rMSC was 33.6% 卤4.0 in the experimental group and 10.6% 卤5.0 in the control group. The difference between the two groups was statistically significant (F = 0.09 P 0.001). The expression rate of rMSC GFAP in the experimental group was 9.6% 卤1.5 and that in the control group was 13.7% 卤3.0. There was no significant difference between the two groups (F = 1.726, P 0.05). The results of RT-PCR identification showed that Rhodopsin,NSE, was observed in the induced group. GFAP was expressed in the control group, but only NSE,GFAP in the control group. Conclusion: a large number of pure and complete photoreceptor cells can be obtained by non-centrifugation and rMSC, cultured in vitro can differentiate into retinal photoreceptor like cells induced by retinal photoreceptor cell culture supernatant.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R774.1

【参考文献】