应用CRISPR/dCas9技术介导miR-145调控间充质干细胞定向分化治疗老年ED的实验研究

Erectile dysfunction (ED) in the elderly is a common disease nowadays. It is essential to find new therapeutic strategies for prevention and treatment of ED with aged in the field of andrology. Our previous studies have found that bone marrow mesenchymal stem cell (BMSC)transplantation could improve the erectile function in the aged rats, but it can't last longer due to the lack of local recruitment and oriented differentiation of BMSC in vivo. How to promote the local recruitment in vivo and oriented differentiation of BMSC? Recent studies have demonstrated that miR-145 might regulate the oriented differentiation of stem cell into smooth muscle cell (SMC). So we assume that the oriented differentiation of BMSC could be regulated using miR-145 modification. Our further studies have showed that miR-145 mimics could promote the oriented differentiation of BMSC into SMC. This study attempts to explore the effect of BMSC genetically modified with the miR-145 knockin reporter line using CRISPR/dCas9 system on improving erectile function in the aged rats. It is envisaged that gene modified BMSC could recruit and orientedly differentiate into corpus cavernous smooth muscle cell (CCSMC) in vivo after transplantation. Additionally, its mechanism would be elucidated. The purpose of this study is to provide potential therapeutic strategy in improving ED with aged using transplantation of gene modified BMSC.

当前人口老龄化，如何防治老年ED是男科学研究的重要课题。课题组前期研究发现：间充质干细胞(BMSC)移植可改善老龄ED大鼠阴茎勃起功能，但作用时间不持久，究其原因是BMSC移植于体内难以富集和定向分化。那么如何促使BMSC在体内局部滞留并定向转化？最近有文献报道miR-145可调控干细胞定向分化为平滑肌细胞。因此我们设想是否可应用miR-145修饰BMSC以调控其定向转化？进一步预实验研究证实导入miR-145模拟物可促使BMSC定向分化为平滑肌细胞。本项目拟应用CRISPR/dCas9技术筛选靶向miR-145高表达的SgRNA并构建载体转染BMSC，然后分别在细胞和动物实验水平探讨miR-145对BMSC定向分化和勃起功能的影响，以期基因修饰BMSC移植在体内局部富集并快速定向分化修复老龄大鼠变性纤维化的阴茎海绵体窦，同时阐明其分子机制，旨在为BMSC移植长效治疗老年ED提供科学依据。

在该基金资助下，我们成功建立了老龄ED动物模型，并在BMSCs作为细胞载体进行基因治疗ED的系列实验研究中得到了应用。本项目通过构建慢病毒载体LV-miR-145-GFP，转染SD大鼠BMSCs并注射移植于老龄ED大鼠阴茎海绵体内，7d及14d后评估勃起功能；随后留取各组阴茎海绵体组织标本研究miR-145基因修饰BMSC移植对老年ED大鼠阴茎海绵体形态学、蛋白表达及TGFβ/Smad信号通路可能的作用，并通过TargetScan及双荧光素酶实验探索miR-145可能的靶点。之后，采用EdU标记干细胞，通过体内及体外实验探究miR-145对BMSC增殖、迁徙、凋亡及分化的影响及干细胞的旁分泌作用。结果表明在注射移植后，miR-145基因修饰BMSC治疗组的老龄大鼠勃起功能得到显著改善，且效果优于单纯BMSC治疗组；miR-145基因修饰BMSC注射移植后可提高阴茎海绵体组织中平滑肌细胞/胶原比例及平滑肌标志分子表达，并降低纤维化相关标志分子及p-Smad2表达。以上作用可能与miR-145抑制BMSC迁徙及凋亡，促进BMSC海绵体内滞留，从而增强旁分泌作用靶向下调KLF4及TGFBR2表达有关。本项目在阐明miR-145基因修饰BMSC移植改善老龄ED大鼠勃起功能及组织形态学病理变化的同时，也通过分子生物学技术揭示了miR-145基因修饰增强BMSC作用的机制。该项目系列研究成果详见结题正文成果部分。