补肾化瘀生新方影响间充质干细胞存活率和分化率在防治心梗后心肌重塑中的作用

With the deepening of researches into the treatment of acute myocardialinfarction (AMI) with stem cells, people realize that the ischemic-hypoxia microenvironment formed after AMI is the major cause of the low survival rate of bone mesenchymal stem cells (BMSCs). The microenvironment, as the soil of stem cells’ growth, plays an important role in the survival and proliferation. Traditional Chinese medicine can correct the imbalance of yin and yang in the internal environment, improve the ischemic-hypoxia microenvironment after AMI, making it more suitable microenvironment for the survival of stem cells. This is undoubtedly a new idea that TCM interferes myocardial remodeling after AMI..On the basis of the study of myocardial remodeling after the treatment of AMI with the method of promoting blood circulation to remove blood stasis and mobilizing BMSCs under the support of the National Natural Science Foundation（81072798／H2708), using Bushen Huayu Shengxin Decoction as intervention means and the models of myocardial ischemic-hypoxia microenvironment and MicroRNA (miRNA) microarray technology, the author explored the influence of Bushen Huayu Shengxin Decoction on the miRNAsexpression, survival rate of BMSCs and the survival and transdifferentiation rate of transplanted BMSCs after AMI. The study reveals that Bushen Huayu Shengxin Decoction can improve the micro-environment of the myocardial infarction area, increase BMSCs’survival rate and improve the myocardial remodeling after the treatment of AMI, which reveals the scientific nature of traditional Chinese medicine in improving the survival rate and differentiation rate of BMSCs，and provide a new pattern for the prevention and treatment of myocardial remodeling.

随着干细胞治疗心肌梗死（AMI）研究深入，已经认识到AMI后形成的缺血缺氧微环境是导致间充质干细胞（BMSCs）生存率低主要原因。微环境是干细胞发育的土壤，在干细胞存活、增殖分化中扮演着重要作用。中医药通过体内用药能纠正阴阳失调的机体内环境，也应该能改善梗死区缺血缺氧微环境，使之成为更适合干细胞生存的微环境。这无疑是中医药干预AMI后心肌重塑新思路。.拟在国家自然基金（81072798／H2708）活血化瘀法动员BMSCs防治心梗后心肌重塑研究基础上，以名老中医经验方-补肾化瘀生新方为干预手段，利用心肌缺血缺氧微环境模型和miRNA微阵列等技术，研究其对miRNAs表达、BMSCs存活率以及对静脉移植BMSCs在心梗区分化率的影响，揭示补肾化瘀生新方改良梗死区微环境提高BMSCs存活率和分化率防治心梗后心肌重塑机制，阐明中医药提高BMSCs存活率和分化率科学性，为心肌重塑的防治提供新模式。

目的：探讨补肾化瘀生新方改良心肌梗死区微环境提高骨髓间充质干细胞增殖分化率在防治心梗后心肌重塑中的作用；.方法：体外研究：提取鉴定纯化培养BMSCs；制备补肾化瘀生新方和生理盐水含药血清；建立缺血缺氧微环境细胞模型。CD105+-BMSCs随机分为：正常组：常规培养；模型组：缺氧无血清培养40h；补肾化瘀生新组：加入补肾化瘀生新方的含药血清预处理30min，进行缺氧无血清处理40h。荧光显微镜观察凋亡细胞形态；AnnexinV/PI双染流式细胞仪检测细胞凋亡；免疫荧光技术检测Vimentin、MHC、Cx43、GATA-4和cTnT表达；RT-PCR技术检测microRNA-124a、Survivn、GATA-4和cTnTmRNA表达。在体研究：采用 Olivette法建立急性心肌梗死模型，随机分为补肾化瘀组（按2.728g/ml灌胃），模型组和正常组生理盐水灌胃，2次/日，连续15d。同时补肾化瘀组尾静脉注射3.0×106／ml的BMSCs，模型组和正常组给予同等剂量PBS注射，1次/周。免疫荧光测定梗死边缘区SDF-1、MHC、Vimentin、CX-43和CXCR4蛋白表达；RT-PCR技术检测Beclin1和p-mTOR/mTOR-mRNA、Western-blot法检测LC3B, Beclinl蛋白表达；光镜和电镜观察心肌梗死边缘区病理和超微结构：结果：1、随着代次的增加细胞凋亡逐渐增多，与模型组比补肾化瘀组的G0/G1期明显降低，S期增高P<0.05；2、与模型组比补肾化瘀组CTNT、GATA-4、Vimentin、MHC、microRNA-124、Survivn量增加明显P<0.05。3、补肾化瘀生新方联合静脉移植BMSCs研究发现：与模型组相比补肾化瘀组cTnT、Vimentin、GATA4、CX-43和SDF-1阳性细胞数P<0.05。另发现抑制自噬体过度形成可减轻心肌缺血再灌注损伤，与模型组比较，补肾化瘀组beclin1、mTOR mRNA和蛋白表达量升高P<0.05；结论：1、补肾化瘀生新方干预缺血缺氧微环境提升BMSCs在缺血缺氧微环境下增殖分化率，进一步验证改良梗死区微环境提高 BMSCs 存活率防治心梗后心肌重塑的可行性；2、从细胞学与分子生物学水平阐释干细胞与微环境关系，为中医药改良微环境提高干细胞增殖分化率提供实验依据。