Notch信号通路在脐带间充质干细胞治疗扩张型心肌病鼠中的作用

发布时间：2018-09-13 15:43

【摘要】：目的探究Notch通路在人脐带间充质干细胞治疗扩张型心肌病鼠中的作用。方法1、人脐带间充质干细胞对γ-分泌酶抑制剂诱导的扩张型心肌病叙利亚金黄地鼠疾病模型的治疗作用雄性健康叙利亚金黄地鼠(简称地鼠)60只,随机分为正常组(15只)、实验组(分为DAPT组、DAPT对照组、干细胞组,各15只)。其中实验组地鼠每天进行腹腔注射γ-分泌酶抑制剂(DAPT)构建扩张型心肌病(DCM)模型,同时每周分别向DAPT对照组(即DMEM组)和干细胞组肌肉注射干细胞重悬培养液DMEM和人脐带间充质干细胞(h HUCMSCs),应用超声心动图和血浆BNP水平评估地鼠心功能情况,采用HE染色和Masson染色观察心肌组织变化情况。2、检测人脐带间充质干细胞治疗扩张型心肌病大鼠后心肌组织Notch信号通路的活性110只SPF级健康、雄性SD大鼠随机分为正常组(20只)、扩张型心肌病组(简称DCM组,90只)。DCM组大鼠持续8周、每周1次经腹腔注射阿霉素构建扩张型心肌病模型,将建模成功后的60只DCM组大鼠随机分为DCM对照组(简称DMEM组)、h HUCMSCs低剂量组(简称低剂量组)、h HUCMSCs高剂量组(简称高剂量组),分别经肌肉注射DMEM、低剂量干细胞和高剂量干细胞治疗。h HUCMSC治疗2周及4周后,应用超声心动图和血浆BNP水平评估地鼠心功能情况,采用Western Blot检测心肌组织NICD(或者N1ICD,Notch信号通路游离的胞内段)和Notch信号通路下游因子Hes1的蛋白含量。结果1、人脐带间充质干细胞对γ-分泌酶抑制剂诱导的扩张型心肌病叙利亚金黄地鼠的治疗作用超声心动图结果,注射药物前各组差异无统计学意义(P0.05);注射药物后DAPT组、DMEM组、干细胞组与正常组相比差异有统计学意义(P0.01),DAPT组、DMEM组与干细胞组相比差异有统计学意义(P0.05),而DAPT组和DMEM组无显著差异(P0.05)。血浆BNP水平,注射药物后DAPT组、DMEM组与正常组、干细胞组相比差异有统计学意义(P0.01),而干细胞组较正常组无显著差异(P0.05)。HE染色显示,与DAPT组和DMEM组相比,正常组和干细胞组心肌细胞水肿减轻,炎性细胞浸润减少,心肌纤维排列较整齐;Masson染色显示干细胞组心肌间蓝染的胶原纤维沉积减少。2、人脐带间充质干细胞治疗扩张型心肌病鼠心肌组织中Notch信号通路活性正常组大鼠治疗前、治疗2周及4周后超声心动图结果无显著差异(P0.05);h HUCMSCs移植2周后DMEM组、低剂量组及高剂量组治疗前后超声心动图超声结果无显著差异(P0.05);h HUCMSCs移植4周后DMEM组、低剂量组及高剂量组治疗前后LVEDD、LVESD无显著差异,而LVEF、LVFS差异显著,且DMEM组LVEF、LVFS低于低剂量组和高剂量组(P0.05)。Western Blot检测结果显示治疗4周后各组之间心肌组织中NICD、Hes1蛋白含量差异无统计学意义(P0.05)。结论1、h UCMSCs可有效改善Notch信号通路被抑制后引起的扩张型心肌病地鼠的心功能;2、阿霉素诱导的扩张型心肌病大鼠Notch信号通路活性较正常组无显著改变,肌肉注射h UCMSCs可明显改善心功能,但对心肌组织Notch信号通路的活性无显著影响。以上结果提示h UCMSCs对扩张型心肌病的修复作用不依赖于对Notch信号通路的调节。
[Abstract]:Objective To explore the role of Notch pathway in human umbilical cord mesenchymal stem cells (HUCMSCs) in the treatment of dilated cardiomyopathy (DCM) in mice. Methods 1. Human umbilical cord mesenchymal stem cells (HUCMSCs) were used to treat DCM induced by gamma-secretase inhibitors in Syrian golden hamsters. Sixty healthy male Syrian hamsters (hamsters) were randomly divided into normal control group and control group. The hamsters in the experimental group were injected intraperitoneally with gamma-secretase inhibitor (DAPT) every day to establish dilated cardiomyopathy (DCM) model. At the same time, the hamsters in the experimental group were injected intramuscularly with DMEM and human umbilical cord mesenchymal filling (HUCM) in the DAPT control group (DMEM) and the stem cell group respectively weekly. Cardiac function of hamsters was assessed by echocardiography and plasma BNP levels. The changes of myocardial tissue were observed by HE staining and Mason staining. 2. The Notch signaling pathway activity of myocardial tissue in dilated cardiomyopathy rats treated with human umbilical cord mesenchymal stem cells (HUCMSCs) was detected in 110 SPF healthy male SD rats. DCM rats were injected intraperitoneally with adriamycin once a week for 8 weeks to establish dilated cardiomyopathy model. 60 DCM rats were randomly divided into DCM control group (DMEM group), low dose group of hUCMSCs (low dose group) and high dose group of hUCMSCs (abbreviated as high dose group). After 2 and 4 weeks of treatment with HUCMSC, echocardiography and plasma BNP levels were used to assess the cardiac function of hamsters. Western Blot was used to detect NICD (or intracellular segment of N1ICD, Notch signaling pathway) and downstream Notch signaling pathway in myocardium. Results 1. The therapeutic effect of human umbilical cord mesenchymal stem cells on dilated cardiomyopathy induced by gamma-secretase inhibitor in Syrian golden hamsters showed no significant difference between the groups before injection (P 0.05); there was statistical difference between DAPT group, DMEM group and normal group after injection of drugs. Significance (P 0.01), DAPT group, DMEM group and stem cell group were significantly different (P 0.05), but there was no significant difference between DAPT group and DMEM group (P 0.05). Compared with DAPT group and DMEM group, myocardial edema, inflammatory cell infiltration and myocardial fiber arrangement were reduced in normal group and stem cell group. Masson staining showed that the deposition of blue-stained collagen fibers was decreased in stem cell group. 2. Notch signaling pathway was activated in human umbilical cord mesenchymal stem cells treated dilated cardiomyopathy rats. There was no significant difference in echocardiographic results between two and four weeks before and after treatment in normal rats (P 0.05); there was no significant difference in echocardiographic results between two weeks after transplantation in DMEM group, low dose group and high dose group (P 0.05); there was no significant difference in LVEDD and LVESD between four weeks after transplantation in DMEM group, low dose group and high dose group. There were significant differences in LVEF and LVFS, and LVEF and LVFS in DMEM group were lower than those in low-dose group and high-dose group (P 0.05). Western Blot test results showed that there was no significant difference in the contents of NICD and Hes1 protein between the groups after 4 weeks of treatment (P 0.05). 2. Notch signaling pathway activity of adriamycin-induced dilated cardiomyopathy rats was not significantly changed compared with the normal group. Intramuscular injection of H UCMSCs could significantly improve cardiac function, but had no significant effect on Notch signaling pathway activity of myocardial tissue. Regulation of Notch signaling pathway.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R725.4

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