【摘要】： 目的:探討體外分離培養(yǎng)視網(wǎng)膜光感受器細(xì)胞以及體外培養(yǎng)的大鼠骨髓間充質(zhì)干細(xì)胞(rat mesenchymal stem cells,rMSCs)誘導(dǎo)分化成視網(wǎng)膜光感受器樣細(xì)胞的能力。 方法:1、取SD大鼠眼球:①視網(wǎng)膜片行消化前后的HE染色。②分離光感受器細(xì)胞:形態(tài)學(xué)觀察;視紫紅質(zhì)(Rhodopsin)免疫細(xì)胞化學(xué)染色行光感受器細(xì)胞的鑒定;同時(shí)收集光感受器細(xì)胞培養(yǎng)上清液。2、分離培養(yǎng)SD大鼠的骨髓間充質(zhì)干細(xì)胞,取第三代rMSCs,采用光感受器細(xì)胞培養(yǎng)上清液誘導(dǎo)rMSC。誘導(dǎo)14d后采用免疫細(xì)胞化學(xué)染色和Rt-PCR觀察rMSC視紫紅質(zhì)(Rhodopsin)、神經(jīng)元特異性烯醇化酶(NSE),膠質(zhì)纖維酸性蛋白(GFAP)的表達(dá)情況。 結(jié)果:視網(wǎng)膜片的HE染色,消化前觀察到完整的九層,消化后光感受器細(xì)胞大量缺失,而其它層完整,光感受器細(xì)胞免疫細(xì)胞化學(xué)鑒定陽性率達(dá)95%以上。誘導(dǎo)細(xì)胞14d后鏡下觀察到神經(jīng)樣的細(xì)胞形態(tài),免疫細(xì)胞化學(xué):實(shí)驗(yàn)組rMSC的Rhodopsin表達(dá)率為35.1%±6.2%,而對(duì)照組中無Rhodopsin陽性的細(xì)胞;實(shí)驗(yàn)組rMSC的NSE表達(dá)率為33.6%±4.0%,對(duì)照組為10.6%±5.0%,組間差異具有統(tǒng)計(jì)學(xué)意義(F =0.09,P 0.001);實(shí)驗(yàn)組rMSC的GFAP的表達(dá)率為9.6%±1.5%,對(duì)照組為:13.7%±3.0%,組間差異無統(tǒng)計(jì)學(xué)意義(F =1.726,P 0.05)。RT-PCR鑒定結(jié)果顯示誘導(dǎo)組觀察到Rhodopsin、NSE、GFAP表達(dá),而對(duì)照組只有NSE、GFAP表達(dá)。 結(jié)論:采用酶消化非離心的方法可以得到大量較純且完整的光感受器細(xì)胞;體外培養(yǎng)的rMSC,經(jīng)過視網(wǎng)膜光感受器細(xì)胞培養(yǎng)上清液誘導(dǎo),可以分化為視網(wǎng)膜光感受器樣細(xì)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% 鹵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% 鹵4.0 in the experimental group and 10.6% 鹵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% 鹵1.5 and that in the control group was 13.7% 鹵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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R774.1

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本文編號(hào)：2306227