鹿红方激活心脏干细胞促进心肌细胞再生的机制研究

Cardiac stem cells transplantation can promote the cardiomyocytes regeneration and repair damaged cardiac muscle,thus creates a new outlook for the treatment of myocardial infarction.Currently the therapeutic target of promoting cardiomyocytes regeneration is gradually moving from inducing cardiac stem cells to differentiate into cardiomyocytes to promoting the division and proliferation of cardiomyocytes by cardiac stem cells.Based on our early study and the literature reports,Luhong formula not only can promote cardiomyocytes regeneration by enhancing the paracrine effect of cardiac stem cells to cardiomyocytes,but may also by enhancing the cell-cell contact effect of cardiac stem cells to cardiomyocytes.At the same time, cardiac stem cells can promote cardiomyocytes divide and proliferate through activiating the PI3K/Akt signaling pathway.Hence we proposed that Luhong formula can promote cardiomyocytes regeneration based on enhancing the cell-cell contact effect and the paracrine effect of cardiac stem cells to cardiomyocytes by activating the PI3K/Akt signaling pathway in the damaged cardiomyocytes after myocardial infarction.In order to test our hypothesis,we created a murine acute myocardial infarction model with a specific marker on cardiomyocytes and co-cultured cardiac stem cells and cardiomyocytes in a transwell contact-free system.This study can provide evidence and strategy for using Chinese medicine to promoting cardiomyocytes regeneration.

心脏干细胞移植促进心肌细胞再生、修复受损心肌，为心肌梗死开创了新的治疗前景。目前促进心肌细胞再生的治疗靶标，正逐渐从诱导心脏干细胞分化，向心脏干细胞促进心肌细胞分裂再生转移。基于课题组前期研究及文献报道，鹿红方既可通过增强心脏干细胞对心肌细胞的旁分泌作用，也可能通过增强心脏干细胞对心肌细胞的直接接触作用，促进心肌细胞再生，而PI3K/Akt通路为心脏干细胞促进心肌细胞分裂再生的下游信号通路。根据以上内容提出：鹿红方可通过增强心脏干细胞对心肌细胞的直接接触作用和旁分泌作用，激活心肌细胞PI3K/Akt通路，促进心肌细胞分裂再生。本研究拟制备特殊标记心肌细胞的小鼠心梗模型、心脏干细胞-心肌细胞穿透小室联合培养模型，探讨鹿红方激活心脏干细胞促进心肌细胞再生的机制，为中医药辅助干细胞疗法促进心肌再生提供依据和思路。

背景：干细胞移植修复受损心肌，为心肌梗死开创了新的治疗前景。基于课题组前期研究及文献报道，鹿红方既可增强Sca-1阳性细胞对心肌细胞的旁分泌作用，也可增强Sca-1阳性细胞对心肌细胞的直接接触作用，保护受损心肌，而PI3K/Akt通路是调控细胞代谢、生长、增殖与凋亡的关键通路。本项目提出：鹿红方可通过增强Sca-1阳性细胞对心肌细胞的直接接触作用和旁分泌作用，激活PI3K/Akt通路，保护受损心肌。.方法：结扎冠状动脉前降支制备心肌梗死小鼠模型，并分离提取乳鼠Sca-1+细胞。利用Sca-1+细胞-心肌细胞穿透小室联合培养模型，评价Sca-1+细胞对心肌细胞的间接作用。组织学检查评价心肌梗死面积和心肌损伤程度，心脏彩超评价小鼠心脏功能。分别使用western blot和RT-PCR检测PI3K/AKT通路蛋白、基因表达情况，免疫组化和免疫荧光检测Sca-1阳性细胞在体内的存活分化情况。ELISA检测血清或细胞上清液中VEGF、HGF、IL6的表达情况。.结果：细胞实验结果显示，无论是Sca-1+细胞还是Sca-1+细胞培养基干预后的乳鼠心肌细胞，PI3K、AKT的表达均上调，Ki67、H3P、Brdu阳性的乳鼠心肌细胞比例升高；当鹿红方与Sca-1+细胞同时干预乳鼠心肌细胞时，Ki67、H3P、Brdu阳性的乳鼠心肌细胞比例更高。动物实验结果显示，鹿红方协同Sca-1+细胞或Sca-1+细胞培养基注射后的心梗小鼠，PI3K、AKT蛋白表达上调，心肌梗死面积减小，心功能提高，但未发现Ki67阳性的心肌细胞，进一步研究发现鹿红方与Sca-1+细胞干预后的心梗小鼠微血管数量增加。.结论：①鹿红方增强心脏Sca-1+细胞对心肌细胞的直接接触作用，激活β-整合素/PI3K/Akt通路，促进乳鼠心肌细胞生长。②鹿红方增强心脏Sca-1+细胞对心肌细胞的旁分泌作用，激活PI3K/Akt通路，促进乳鼠心肌细胞生长。③鹿红方增强Sca-1+细胞对心肌细胞的直接接触作用和旁分泌作用，激活心脏组织中的PI3K/Akt通路，保护受损心肌，提高心梗小鼠的心功能，其机制可能与促进微血管新生有关。