摘要
目的:基于AngⅡ诱导心肌细胞凋亡模型的建立,探讨离体实验中中药复方的研究方法。方法:以原代心肌细胞、心肌细胞株H9c2为研究对象,含药血清为介质,AngⅡ为刺激因子,建立凋亡模型,采用MTT、流式、荧光定量PCR、DAPI染色检验凋亡率及凋亡相关基因的表达。结果:(1)以原代心肌细胞为研究对象,胎牛血清为介质,分为正常组和AngⅡ组(1×10-6mol/L),MTT及流式结果显示:与正常组比较,AngⅡ可以降低原代心肌细胞活性,诱导心肌细胞发生凋亡(P<0.05);荧光定量PCR结果显示:与正常组比较,AngⅡ可以促进促凋亡基因Bax、Caspase-3的表达,降低抗凋亡基因Bcl-2的表达(P<0.05)。(2)以心肌细胞株H9c2为研究对象,鼠血清为介质,分为正常组(10%正常鼠血清)、AngⅡ组(1×10-6mol/L+10%正常鼠血清)、中药组(1×10-6mol/L+10%中药鼠血清),MTT、流式及DAPI结果显示:与正常组比较,AngⅡ可降低H9c2细胞活性,诱导H9c2发生凋亡(P<0.05),加参方可干预AngⅡ作用(P<0.05)。(3)以心肌成纤维细胞为研究对象,分为无血清组、胎牛血清组、胎牛血清+Brdu组、鼠血清组,MTT检测心肌成纤维细胞活性,结果显示:与无血清组比较,胎牛血清、鼠血清均能促进心肌成纤维细胞增殖,鼠血清促进心肌成纤维细胞增殖更明显。结论:在建立凋亡模型、研究药物的抗凋亡作用的试验中,由于采用鼠血清培养原代心肌细胞,心肌成纤维细胞增殖明显,使原代心肌细胞的纯度明显降低,进而影响实验结果,含药血清并不适合作为中药媒介与原代心肌细胞相结合进行研究。
Objective: To explore the research method of traditional Chinese medicine in vitro experiment based on Ang Ⅱ induced cardiomyocyte apoptosis. Methods: The H9c2 cell line and primary cardiomyocytes were cultured and observed the effects of AngⅡ on cardiomyocyte apoptosis at different condition of culturing. MTT was used to study cell viability, while the induction of apoptosis and the expressions of apoptosis-related m RNA and protein were determined by the use of DAPI staining, flow cytometry and RT-PCR respectively. Results:(1)The primary cardiomyocytes were treated with the fetal bovine serum, and were divided into normal group and Ang Ⅱ group(1×10-6 mol/L). MTT and flow cytometry results showed that: Treatment with Ang Ⅱ for 24 h could reduce the cell viability of primary cardiomyocytes and induce the apoptosis of cardiomyocytes(P<0.05); The results of RT-PCR showed that Ang Ⅱ could promote the expressions of pro-apoptosis genes Bax and Caspase-3 and decrease the expression of anti-apoptotic gene Bcl-2(P<0.05).(2)H9c2 cell line were treated with rat serum, and were divided into rat serum group, AngⅡ group(1×10-6mol/L+10% rat serum) and Jiashen Formula group(1×10-6mol/L+10%Chineme medicine rat serum). MTT, flow cytometry and DAPI staining results showed that: AngⅡ could decrease the activity of H9c2 cells and induce the apoptosis of H9c2(P<0.05), and Jiashen Formula could interevent the effect of AngⅡ(P<0.05).(3)The myofibroblasts were divided into serum-free group, fetal bovine serum group, fetal bovine serum+Brdu group and rat serum group. MTT was used to assess the myocardial fibroblast activity. The results of MTT showed that compared with the serum-free group, fetal bovine serum and rat serum can promote the proliferation of cardiac fibroblasts, and the effect of rat serum was more obvious. Conclusion: In the establishment of apoptosis model and study on the anti-apoptotic effect of drugs, due to the use of rat serum to culture primary cardiomyocytes,cardiac fibroblasts were significantly increased, so that the purity of primary cardiomyocytes was significantly reduced, which affected the experimental results, and drug-containing serum is not suitable as a medium of traditional Chinese medicine and cardiomyocytes.
出处
《中华中医药杂志》
CAS
CSCD
北大核心
2017年第10期4699-4704,共6页
China Journal of Traditional Chinese Medicine and Pharmacy
基金
国家自然科学基金项目(No.81373853
No.81503419)
河南省高校科技创新团队支持计划项目(No.13IRTSTHN012)
河南中医学院科技创新团队支撑计划项目(No.2010XCXTD10)~~
关键词
心肌细胞
含药血清
AngⅡ
凋亡
Cardiomyocytes
Drug-containing serum
Ang Ⅱ
Apoptosis
