anti-miR-28对鼠视网膜Müller细胞向光感受器细胞定向分化的调控作用

In our previous experiments, we have found out that some retinal stem cells derived from retinal Müller cells are able to differentiate to a tiny population of photoreceptors.Consequently, we intent to apply the lentiviral vector driving anti-miR-28 expression to up-regulate Crx expression and induce the differentiation of mouse retinal Müller cells to photoreceptors in vitro.Then in one group,NMDA and lentiviral vector driving anti-miR-28 expression are injected into the subretinal cavity of mouse model of photoreceptors light induced damage to dedifferentiate retinal stem cells from retinal Müller cells.In another group,the retinal stem cells derived from retinal Müller cells are transfected with lentiviral vector driving anti-miR-28 expression,and injected into the subretinal cavity of mouse model of photoreceptors light induced damage . ERG of the retinas of mouse is detected.The population of differentiated cells and photoreceptors are assessed by immunofluorescence, the expression of anti-miR-28,Crx, Rhodopsin,SWS1 and SWS2 are detected using quantitative PCR and Western blot examination . Additionally, the apoptosis of retinal cells are eventually detected with TUNEL analysis. Our research will demonstrate the role of anti-miR-28 on the expression of Crx to induce the differentiation of the retinal stem cells derived from retinal Müller cells to photoreceptors, thus providing a valuable tool for clinical gene therapy.

我们的前期研究发现鼠视网膜Müller细胞源性干细胞（MISC）可分化为少量光感受器细胞。在此研究基础上，本研究拟采用上调Crx基因的anti-miR-28慢病毒重组质粒，首先体外诱导小鼠视网膜MISC向光感受器细胞定向分化，并明确其调控机制。其次,一组将NMDA及anti-miR-28重组质粒注射入光感受器光损伤病变模型小鼠的视网膜下腔内；另一组视网膜下腔注射体外培养并转染了anti-miR-28重组质粒的鼠MISC, 采用ERG检测鼠视网膜功能的变化，免疫荧光染色检测视网膜细胞分化类型和光感受器细胞所占的比例，qPCR、Western blot方法分析视网膜anti-miR-28、Crx、Rhodopsin、SWS1和SWS2的表达差异。最后采用TUNEL技术分析视网膜细胞凋亡情况，从而明确外源性anti-miR-28对视网膜MISC向光感受器细胞定向分化的调控作用、机制及其副作用。

视网膜退行性疾病如遗传性视网膜色素变性、Leber’s先天性黑朦和视锥-视杆营养不良等是眼科严重的不可逆性致盲性眼病。虽然各种退行性视网膜疾病的发病机理不同，但它们具有共同的终点：视网膜光感受器细胞的损伤及凋亡。目前临床上药物和手术等治疗手段无法从根本上治愈视网膜退行性患者的视功能，如何减缓、阻止、逆转感光细胞的凋亡，甚至将损伤、凋亡细胞进行功能性地定位替换和整合，最终实现视网膜功能的重建，这将为此类患者的复明迎来曙光。Müller细胞是人和哺乳动物视网膜中最主要的神经胶质细胞，具有视网膜干细胞特性，能分化为各种视网膜神经元包括视网膜神经节细胞、双极细胞、无长突细胞和少量光感受器细胞；然而，由于缺乏定向诱导因子，分化为光感受器细胞的效率很低，仅为 7.51%，不足以补偿各种原因导致的光感受器细胞的损失。因此，Müller神经胶质细胞虽然是自体来源的理想的候选细胞，如何提高定向诱导分化效率是迫切亟需解决的问题。基于此，本课题对视网膜Müller细胞源性干细胞向光感受器样细胞定向分化的调控及其机制进行了深入研究。我们的研究结果显示，anti-miR-28能通过上调 CRX 基因诱导小鼠 Müller 细胞源性干细胞向光感受器方向定向分化，Otx2 基因可通过上调视锥视杆同源盒基因 Crx、视杆细胞发育转录因子 Nrl 和 Wnt 通路抑制因子 Dkk-1以及下调 Wnt通路关键因子totalβ-catenin、nuclear-catenin 促进视网膜 Müller 细胞源性干细胞定向分化为光感受器样细胞。在光感受器细胞损伤模型鼠视网膜下腔注射Otx2介导的Müller源性光感受器前体细胞，可促进大鼠视网膜形成新生的光感受器样细胞，并与相邻细胞存在突触连接，且视网膜功能有一定程度的恢复。从而明确了anti-miR-28、Otx2在对视网膜Müller细胞源性干细胞定向分化为光感受器样细胞的调控作用及其可能的信号通路，为后续的临床基因治疗提供坚实的理论和实践基础。