钙敏感受体对胚胎干细胞移植治疗心肌梗死心功能的影响及其机制研究

Myocardial infarction (MI) is one of the leading causes of morbidity and mortality in the world. Embryonic stem cells( ESCs) are pluripotent stem cells, which have potential of multiple differentiation. ESC-derived cardiomyocytes (ESC-CMs) have the potential to replenish the loss of myocardium that occurs in myocardial infarction and other cardiac diseases. The calcium-sensing receptor (CASR) is crucial to the regulation of calcium metabolism and cell proliferation, differentiation and so on. Our team previously found that CaSR is functionally expressed in ESCs, and the activated CaSR contributes to intracellular calcium increment through Gq-PLC-inositol triphosphate (IP3) pathway, can promote the cell proliferation.But effects of calcium sensing receptor on embryonic stem cells transplantation for the cardiac function of myocardial infarction has not been reported. We speculated that the inhibition of CaSR activity is possibly more beneficial to the improve heart function and heart systolic function of myocardialinfarction after embryonic stem cells transplantation. To confirm this hypothesis, we will determine the effects of CaSR agonist，inhibitor, siRNA on mESCs differentiation, and will observe the the effects of gene silencing and over-expression of CaSR on the embryonic stem cells transplantation for the cardiac function of myocardial infarction in both of the MI of rat animals and cells, and indicate the effect and molecular mechanism of CaSR , and will provide a new thinking and new targets for the treatment of the of myocardial infarction.

心肌梗死（MI）是世界上发病率和死亡率较高的一类疾病。胚胎干细胞是多能干细胞，具有多向分化潜能。胚胎心肌细胞可以补充心肌梗死等心脏疾病损失的心肌细胞。钙敏感受体（CaSR）是调节钙代谢的重要因素，还参与细胞增殖、分化等的调节。本课题组首次证明胚胎干细胞有CaSR 表达，用Ca2+和新霉素激活CaSR，可通过G-PLC-IP3 途径使[Ca2+]i 升高，并促进细胞增殖，而CaSR对胚胎干细胞移植治疗心肌梗死后心功能的影响国内外尚无报导。我们推测"抑制梗死心肌CaSR 活性有利于改善胚胎干细胞移植后心室重构和心脏功能的恢复"。为证实该假说，本文拟观察CaSR基因沉默、激动剂和抑制剂对胚胎干细胞分化的影响，通过复制大鼠在体和细胞MI模型，观察CaSR沉默或过表达(激动剂或抑制剂)对胚胎干胞移植治疗心肌梗死后心功能的影响，阐明CaSR在其中的作用及机制，以便为心肌梗死的治疗提供新思路

目的：探讨钙敏感受体（calcium-sensing receptor ，CaSR）活性改变对小鼠胚胎干细胞移植治疗心肌梗死后心功能和细胞凋亡的影响，及其相关通路。.方法：制备大鼠心肌梗死动物和细胞模型：（1）动物模型组分为：A.正常对照组，B. AMI组，C. mESCs移植组，D. mESCs移植+CaSR激动剂组和E. mESCs移植+CaSR抑制剂组。（2）细胞模型组分为：1.正常对照组，2.单纯MI模型组，3.模型＋mESCs　4.模型＋mESCs+CaSR激动剂组，5. 模型＋mESCs移植+CaSR抑制剂组，6.模型＋mESCs移植+CaSRsiRNA组。形态学观察及TTC染色检测动物模型心肌梗死状态，超声心动图检测血流动力学指标的改变；TUNEL染色观察心肌细胞凋亡情况；Real-Time PCR观察大鼠心肌CaSR RNA、Western blot检测CaSR、Bax、Bcl- 2、cleaved-caspase- 3、cleaved-caspase-9、cyt-C、p-ERK、p-JNK和p-p38 的蛋白表达；紫外分光法检测乳酸脱氢酶( LDH ) 、肌酸激酶( CK )活性，超氧化物岐化酶(SOD)的活性和丙二醛(MDA)含量，ELISA法检测肌钙蛋白( cTnT) 水平。结果：动物模型组移植后与B组比较： C、E组梗死面积显著减少( P < 0.05)，LVSP和+ dp / dtmax、-dp/dtmax明显增大( P < 0.05)，而LVEDP 明显小于B组( P < 0. 01)；C和E组细胞凋亡指数、CaSR蛋白和RNA及p-ERK、p-JNK 、p-P38、Bax、cleavde caspase-3和cleavde caspase-9蛋白明显减少( P < 0. 05)，CK和 LDH活性、 cTnT水平减少( P < 0. 05)；心肌细胞超微结构改善明显；其中动物模型以E组各相指标改变更明显，心功能改善更显著。结论：抑制CaSR联合ESCs移植能明显改善心功能，其效果优于单独ESCs移植，其机制可能与抑制MAPK通路减少细胞凋亡有关。