小分子水凝胶承载双基因修饰的MMSCs移植治疗心肌梗塞及其机制探讨

The use of stem cells to improve recovery of the injured heart after myocardial infarction (MI) is an important emerging therapeutic strategy. However, low rate of cells retention, survival and differentiation after transplantation are the major challenges for its development. Numbers of studies have found that hydrogels can mimic cardiac microenvironment and be used as a carrier of stem cells in three-dimensional environment. Moreover, transplantation of stem cells and hydrogels complex in the treatment of MI can significantly improve stem cells retention, survival and differentiation in the infarcted zone and thereby ameliorate cardiac function. We have synthesize series of small molecular hydrogels, which have shown a great prospect in three-dimensional cell culture, gene regulation of stem cells differentiation, liver tissue repair, wound healing as well as drug controlled delivery, however, its potential utility and mechanism in cardiac regeneration have yet to be tested. Besides, our previous works found that BCL2 and VEGF165 double gene-modified bone marrow mesenchymal stem cells(MMSCs) can dramatically promote the expression of cell survival and angiogenesis factors..In order to explore the roles of small molecular hydrogels in the treatment of MI, we will used electroactive small molecular hydrogels as three-dimensional cell culture scaffolds. In vitro, the adhesion, proliferation, differentiation of MMSCs cultured in this scaffolds will be characterized. Then, the small molecular hydrogels and/or BCL2 and VEGF165 double gene-modified MMSCs will be injected in the animal infarcted sites to explore its cardiac tissue repair potential. Our aims is to investigate the roles of small molecular hydrogels in preserving cardiac function,and clarify its underlying mechanism of cardiac tissue repair. This study, focused on the injectable cardiac tissue engineering strategy in the treatment of MI, is promising to lay a firm foundation for the biomaterials science.

干细胞治疗心肌梗死存在移植后细胞滞留率、存活率和分化率低的问题是目前制约其发展的瓶颈。水凝胶可模拟心肌内环境作为干细胞三维载体支架，经移植治疗心梗可明显提高细胞的滞留、存活和分化并改善心功能。我们前期研究制备了一系列小分子水凝胶，发现其在细胞三维培养、干细胞分化基因调控、创伤修复、肝组织修复及药物控释等领域显现了良好的应用前景，然而，小分子水凝胶在心脏修复中的应用尚未广泛开展；我们同时发现经BCL2和VEGF165双基因修饰的骨髓间充质干细胞（MMSCs）可提高细胞的存活并促进血管新生。为了进一步明确小分子水凝胶在心脏修复中的作用以期获得更好的疗效，本研究拟采用活性小分子水凝胶承载BCL2和VEGF165双基因修饰的MMSCs移植治疗心肌梗死，通过观察其对MMSCs粘附、增殖、分化的影响，评价其移植后的疗效，阐明其促进心肌修复的机制，为可注射性心肌组织工程治疗心梗奠定材料学基础。

干细胞移植治疗心肌梗死存在移植后细胞滞留率、存活率和分化率低的问题这是目前制约细胞治疗这种治疗手段向前发展的瓶颈。本研究中，通过结合生物材料、基因修饰和细胞治疗改善了骨髓间充质干细胞移植对心肌梗死的治疗效果，并对其机制进行了研究。首先我们成功构建了Bcl-2和VEGF双基因共表达慢病毒载体并构建能稳定表达目的基因的大鼠MSC稳转细胞株，构建的稳转细胞株拥有更快的增殖速度和对目的基因更高的表达。Bcl-2和VEGF双基因共表达的大鼠MSC在体外糖氧剥夺环境下发挥更好的自我保护作用，其机制为：减少凋亡，抑制自噬，增加旁分泌。我们开发了多肽DFEFKDFEFKYRGD小分子水凝胶，制备的水凝胶具有良好的细胞相容性、组织相容性及可生物降解性，可以满足心肌组织工程对可注射性支架材料的要求。我们探讨小分子水凝胶对MSC体外的存活、分化、粘附、增殖的作用，证实了小分子水凝胶具有促进细胞生长增殖、提高缺氧环境下MSC的存活及增强5-aza 诱导MSCs向心肌细胞分化的作用。最后我们使用小分子水凝胶承载Bcl-2和VEGF双基因修饰的大鼠MSC移植至大鼠梗死心肌局部，结果表明Bcl-2和VEGF双基因共表达的MSC在动物体内发挥了对心肌梗死更好的治疗作用，并且小分子水凝胶的辅助进一步改善了其对心肌梗死的治疗作用，其机制为更好的细胞滞留，减少凋亡，增强心肌再生，增强血管再生。本研究是生物材料、基因治疗和细胞治疗的联合应用，不仅为治疗心肌梗死也为心肌组织工程提供新的思路，为临床应用生物材料、基因治疗和细胞治疗心肌梗死提供有效的理论支持和保证。