关键转录因子诱导间充质干细胞分化为视网膜色素上皮细胞的机制及功能研究

Multi-differentiation capacities and paracrine functions make mesenchymal stem cells (MSCs) a candidate for treatment of retinal degenerative disease (RD). However, MSCs possess lower retinal cell lineage differentiation capacities, and pathological niche in part of RD disease, especially wet aged-related macular degeneration, can yield epithelial-mesenchymal transition (EMT) inducers, which reduce the therapeutic functions of MSCs. Based on this scenario, we transfected human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) with transcriptional factors c-Myc、Nr2e1、Six6、Lhx2 and Crx. hUC-MSCs was successfully transformed into retinal pigment epithelial (RPE) cells, which is termed induced RPE (iRPE) cells. iRPE cells expressed RPE specific markers and exhibited phagocytic functions by engulfing POS. When subjected to TGFβ1 or TGFβ2 treatment in vitro, mimicing the transplanted cells under EMT induction in RD, the EMT markers in iRPE cells were not upregulated, and phosphorylation of smad2/3 and notch signaling pathway were not activated, which indicated that iRPE cells had anti-EMT functions. When transplanted into sodium iodate induced RD rat model, iRPE cells showed stronger vision rescue function compared with hUC-MSCs. Furthermore, the safety of iRPE cells were confirmed in immunodeficiency mice. These results prompt us to further induce adipose tissue derived stem cells into iRPE cells by transcriptional factors. Furthermore, the RPE functional and EMT related target genes of transcriptional factors will be clarified by CHIP-SEQ and Luciferase reporting system, and their therapeutic functions and safety will be evaluated in RD rat model and immunodeficiency mice respectively. This study will not only clarify the mechanism of induction of MSCs into RPE cells, but also provide more functional seed cells for treatment of RD disease.

多向分化和旁分泌特性赋予间充质干细胞（MSCs）治疗视网膜退行性疾病（RD）的潜能。但MSCs较低的视网膜细胞分化能力及部分RD病变微环境中存在的上皮间质转化（EMT）诱导作用，降低了MSCs的治疗功能。基于此，我们预实验采用转录因子c-Myc、Nr2e1、Six6、Lhx2和Crx成功诱导人脐带间充质干细胞（hUC-MSCs）分化为视网膜色素上皮细胞（induced RPE,iRPE），在TGFβ诱导下，iRPE中SMAD2/3磷酸化和Notch3信号通路不被激活，证实其抗EMT功能。iRPE治疗作用及安全性分别在RD大鼠模型和裸鼠中得到验证。在此基础上，本课题将进一步诱导人脂肪干细胞分化为iRPE，在两种MSCs来源的iRPE中通过确定关键转录因子直接调控的RPE功能和EMT相关靶基因，阐明iRPE抗EMT的分子机制，并验证其视网膜保护作用和安全性，以期为临床治疗RD提供优质种子细胞。

视网膜退行性疾病（Retinal degenerative disease, RD）包括多种疾病。其中，以年龄相关性黄斑变性（Age-related macular degeneration, AMD）和视网膜色素变性（Retinitis pigmentosa, RP）两类最具代表性。我国流行病学调查显示，50岁以上人群中，AMD的发病率高达4.7%－9.5%，RP的发病率也在1/1000左右。RD往往会导致严重的视力损害，最终会致盲，从而严重地影响患者的生活质量，并成为家庭和社会的问题。间充质干细胞（Mesenchymal stem cells，MSCs）是存在于多种组织中具有自我更新和分化能力的一小部分细胞，目前已发现多种组织来源的MSCs，如骨髓、脐带华氏胶及脂肪来源的MSCs。脐带间充质干细胞和脂肪干细胞易获得，增殖能力强，因而越来越受到重视。我们前期研究在RCS大鼠中证明多种MSCs视网膜下腔移植能够明显抑制感光细胞的凋亡,延缓视力的损失，并进一步证明不同的MSCs亚群具有不同的治疗作用。但有限的分化能力削弱了MSCs的治疗效果。为了克服这一限制，我们用视网膜发育相关转录因子（TFs）的组合将hUCMSCs和hADSCs转化为诱导RPE（iRPE）细胞,获得一下主要的研究结果：a).确定能转分化MSCs为iRPE细胞的关键转录因子组合；b).iRPE细胞表现出与诱导多能干细胞衍生的RPE细胞相当的特性，并具有EMT抗性；c) iRPE细胞移植到碘酸钠诱导的大鼠AMD模型的视网膜下腔间隙后，显示出比MSCs更好的治疗功能；d).iRPE细胞中高表达的PTPN13通过使Syntenin去磷酸化，进而促进TGF-βR内吞和变性，赋予细胞EMT抵抗能力；e)iRPE细胞在裸鼠皮下显示出一定的安全性，没有肿瘤的产生。该课题的实施将解决移植MSCs体内分化为RPE细胞能力有限的问题，同时iRPE细胞的抗EMT功能将克服疾病微环境对移植细胞的EMT诱导作用。iRPE细胞有望成为临床治疗RD疾病的优质种子细胞，同时为治疗其它EMT相关疾病提供新的思路。课题实施期间，课题资助发表文章7篇，在投一篇，申请专利一项，资助毕业博士和硕士各一名。