CRX基因诱导Müller细胞源性干细胞定向分化为光感受器细胞的实验研究

摘要

目的 研究外源性视锥视杆细胞同源盒(CRX)基因是否能够诱导体外培养的视网膜Müller细胞源性干细胞向光感受器细胞定向分化.方法 实验研究.体外培养、扩增出生5～7 d昆明小鼠视网膜Müller细胞,通过无血清培养去分化为干细胞.采用免疫细胞化学法分别鉴定Müller细胞(GS和Vimentin)和干细胞(Nestin和Sox2)标志物的表达.取第2代祖细胞分为3个组:空白对照组、空载体组[采用只含绿色荧光蛋白(GFP)基因的慢病毒转染细胞]、CRX组(用含GFP+CRX基因的慢病毒转染细胞).诱导分化7 d后,采用q-PCR、免疫印迹法检测光感受器细胞标志CRX蛋白、视紫红质(Rhodopsin)、感光蛋白S-opsin的表达差异.组间均数的两两比较采用独立样本t检验.结果体外培养的Müller细胞96.03％±1.21％同时表达GS和Vimentin标志物,去分化后表达干细胞标志物Nestin和Sox2.CRX诱导分化7 d,CRX和Rhodopsin表达均明显增加,部分细胞发生神经元样改变.CRX组、空载体组及空白对照组CRX mRNA相对表达量分别为10.830±2.301、1.214±0.469、1.000±0.576,CRX组与空白对照组相比,差异有统计学意义(t=1.57,P=0.02),空载体组与空白对照组相比,差异无统计学意义(t=0.29,P=0.84).CRX组、空载体组及空白对照组Rhodopsin mRNA相对表达量分别为26.410±16.180、1.301±0.401、1.000±0.473,CRX组与空白对照组相比,差异有统计学意义(t=4.15,P=0.01),空载体组与空白对照组相比,差异无统计学意义(t=0.49,P=0.80).CRX组、空载体组、空白对照组CRX蛋白相对表达量分别为1.258±0.358,0.471±0.168,0.442±0.152.未检测到S-opsin的表达.结论 CRX基因能够诱导小鼠Müller细胞源性干细胞定向分化为视杆细胞,表明Müller细胞可以作为视网膜光感受器细胞替代治疗的内源性种子细胞.%Objective To investigate whether exogenous CRX gene would be able to induce Müller cells-derived progenitors to differentiate into photoreceptors. Methods Experimental study. Müller cells-derived progenitors resulted from primary Müller cells isolated from KunMing mice(5-7 days old) and cultured in free-serum media. Markers of Müller cells(glutamine synthetase, GS and Vimentin) and stem cells (Nestin and Sox2) were analysed by immnocytochemical assays. The secondary passage progenitors were divided into three groups: (1)the control group; (2)the empty vector group was transfected with lentivirus GFP;(3)the treated group was transfected with lentivirus GFP-CRX. After differentiation for 7 days, 7 days after differentiation, the expression of markers of photoreceptors were analyzed by q-PCR and Western blot assay. Results There were 96.03％± 1.21％of Müllerz cells cultured in vitro were immunoreactive to both GS and Vimentin. The dedifferentiation cells expressed Nestin and Sox2. After 7 days of induction, Exogenous CRX induced Müller cell-derived progenitors to differentiate into rod-like cells showed appearance like neuron morphology. q-PCR demonstrated that mRNAs of CRX and Rhodopsin were upregulated greatly. CRX mRNA were 9 times(P<0.05) and Rhodopsin mRNA were 20 times(P<0.05). The difference between the control group and the empty vector group was not statistically significant. Western blot showed that the expression of CRX was upregulated significantly, and was 2.7 times(P<0.05). But expression of Rhodopsin was weak and was nearly not detected in the control group and empty vector group. The expression of S-opsin was not detected. Conclusion CRX gene could induce the differentiation of Müller cell-derived progenitor into rod photoreceptors, indicating a new avenue to study müller cells as endogenous seed cells for retinal photoreceptor.