Insm1促进视网膜祖细胞向光感受器分化的作用及其机制研究

The low efficiency of photoreceptor cell differentiation is one of the important reasons of limiting photoreceptor cell transplantation for the treatment of retinal degeneration. In recent years, it has been found that Insulinoma associated-1 (INSM-1) can promote retinal progenitor cell (RPC) differentiation into photoreceptor cells, inhibit RPC differentiation into glial cells. The role of Insm1 in the differentiation of retinal progenitor cells to photoreceptors and its mechanism is not clear, We put forward the hypothesis:.Insm1 is a regulatory factor for the differentiation of retinal progenitor cells into photoreceptor cells, which may promote the differentiation of retinal progenitor cells into photoreceptor cells by inhibiting Wnt signaling pathways. To verify this hypothesis, we will use the flow cytometry, real-time quantitative polymerase chain reaction, protein immunoblotting and other technical means to detect regulation of the retinal progenitor cells to photoreceptor cell differentiation by Insm1, and the mechanism of Insm1 promoted retina progenitor cells differentiated into photoreceptors. In this study, Insm1 was used to promote RPC differentiated into photoreceptor cells as the entry point, which provided a new idea for RPC efficient differentiation into photoreceptors. It is unclear as to why PRP not given prior to AGV implantation. please mention the reasons.

光感受器细胞分化效率低是限制光感受器细胞移植治疗视网膜变性的重要原因之一。近年研究发现胰岛素瘤相关蛋白1（Insulinoma associated-1, INSM-1）可以促进视网膜祖细胞（retinal progenitor cell, RPC）向光感受器细胞分化，抑制RPC向胶质细胞分化，但Insm1对视网膜祖细胞向光感受器分化的作用及其机制仍知之甚少，为此，我们提出假说：.Insm1是RPC向光感受器细胞分化的调控因子，其可能通过抑制Wnt信号通路进而促进RPC向光感受器细胞分化。为验证这一假说，我们将采用流式细胞术、实时定量聚合酶链式反应、蛋白免疫印迹法等技术手段，检测Insm1对RPC向光感受器细胞分化的调控作用，研究Insm1在促进RPC向光感受器分化的调控机制，本课题以Insm1促进RPC向光感受器细胞分化为切入点，为RPC高效定向分化为光感受器细胞的研究提供新思路。

背景及研究内容：胰岛素瘤相关蛋白1（Insulinoma associated-1, Insm-1）可以促进视网膜祖细胞（retinal progenitor cell, RPC）向光感受器细胞分化，但Insm1对视网膜祖细胞向光感受器分化的作用及其机制仍知之甚少，因此，本项目通过对Insm1 在不同发育阶段小鼠视网膜及光感受器细胞的表达的检测，Insm1 的表达对光感受器细胞分化的影响，探讨Insm1调控视网膜祖细胞向光感受器细胞分化的作用及机制。.结果：1.通过免疫组化及流式细胞学检查，在胚胎12.5天小鼠视网膜检测到Insm1表达，Insm1阳性细胞的分布与视网膜光感受器分布一致，并且Insm1阳性细胞表达视网膜光感受器前体细胞的标志物Otx2。在小鼠出生后14天，Insm1的表达逐渐消失。2.Insm1对RPC向光感受分化的影响：视网膜祖细胞过表达Insm1后，在诱导分化14天时，视杆细胞标志物Rhodopsin表达比例（2.21%±0.67%）较空载体组（2.02%±0.74%）无明显增加（P=0.75），而在诱导分化20天和25天，空载体组Rhodopsin表达比例分别为13.43%±1.22%，24.41%±0.70%，Insm1过表达组Rhodopsin表达比例分别为21.96%±1.29%，35.42%±0.77%，均高于空载体组（P<0.05）。在诱导分化第14、20、25天，Insm1过表达组胶质细胞标志物谷氨酰胺合成酶 （glutamine synthetase，GS）的表达比例较空载体组均显著降低（P<0.05）。对照组与空载体组不同时间Rhodospin和GS表达比例差异无统计学意义。3.本研究发现对于Insm1过表达的视网膜祖细胞，SHH和Hes1表达增强，S-opsin，Rhodopsin的表达提高，并使GS和GFAP表达降低。然而，通过抑制SHH或Hes1表达，可消除Insm1过表达对S-opsin,Rhodopsin,GS和GFAP表达水平的影响。.科学意义：本研究显示Insm1是视网膜发育过程中感光细胞分化所必需的。Insm1通过上调SHH和Hes1促进RPC分化为发育中视网膜的感光细胞。