高糖与尼古丁对乳鼠心肌细胞凋亡的影响及机制研究

【作者】 周祥；

【导师】 孔祥清；

【作者基本信息】 南京医科大学 ， 内科学， 2010， 硕士

【摘要】 目的:研究高糖对乳鼠心肌细胞凋亡的影响及其机制。方法:分离培养SD乳鼠心肌细胞,分别用10-50 mmol/L终浓度的葡萄糖进行干预,MTT测定各组心肌细胞的生长活力;激光共聚焦显微镜、脱氧核糖核酸转移酶原位缺口末端标记法(TUNEL)和流式细胞术用于观察和检测心肌细胞凋亡;将心肌细胞分为3组:对照组、高糖组和抗氧化剂组,分别检测各组上清液中乳酸脱氢酶(LDH)、丙二醛(MDA)、超氧化物歧化酶(SOD)的含量以反映心肌细胞的氧化应激水平;荧光定量PCR检测高糖对凋亡相关基因P53、Pax3、Bmp4、Slug和Msx1表达的影响。结果:MTT结果显示高糖能浓度依赖性地抑制心肌细胞的生长活力;激光共聚焦显微术、TUNEL染色和流式细胞术均表明高糖能诱导心肌细胞凋亡;与对照组相比,高糖组LDH、MDA含量明显增高,SOD含量显著下降;与高糖组相比,抗氧化剂组LDH、MDA含量明显下降,SOD含量显著上升;抗氧化剂组LDH、MDA、SOD的含量较对照组无明显变化;流式细胞检测显示高糖组细胞凋亡率较对照组明显增加,而抗氧化剂组细胞凋亡率较高糖组则显著下降;荧光定量PCR结果表明高糖能上调P53、Msx1基因的表达,下调Pax3、Bmp4、Slug基因的表达。结论:高糖可能通过氧化应激和改变凋亡相关基因的表达从而诱导心肌细胞凋亡。目的:研究尼古丁对乳鼠心肌细胞凋亡的影响及其机制。方法:分离培养SD乳鼠心肌细胞,分别用0.1-100μM终浓度的尼古丁进行干预,MTT测定各组心肌细胞的生长活力;激光共聚焦显微镜、脱氧核糖核酸转移酶原位缺口末端标记法(TUNEL)和流式细胞术用于观察和检测心肌细胞凋亡;将心肌细胞分为3组:对照组、尼古丁组和抗氧化剂组,分别检测各组上清液中乳酸脱氢酶(LDH)、丙二醛(MDA)、超氧化物歧化酶(SOD)的含量以反映心肌细胞的氧化应激水平;荧光定量PCR检测尼古丁对凋亡相关基因P53、Bax、Bcl-2、Pax3、Bmp4、Slug和Msx1表达的影响。结果:MTT结果显示尼古丁能浓度依赖性地抑制心肌细胞的生长活力;激光共聚焦显微术、TUNEL染色和流式细胞术均表明尼古丁能诱导心肌细胞凋亡;与对照组相比,尼古丁组LDH、MDA含量明显增高,SOD含量显著下降;与尼古丁组相比,抗氧化剂组LDH、MDA含量明显下降,SOD含量显著上升;抗氧化剂组LDH、MDA、SOD的含量较对照组无明显变化;流式细胞检测显示尼古丁组细胞凋亡率较对照组明显增加,而抗氧化剂组细胞凋亡率较尼古丁组则显著下降;荧光定量PCR结果表明尼古丁能上调P53、Bax和Msx1基因的表达,下调Bcl-2、Pax3、Bmp4和Slug基因的表达。结论:尼古丁可能通过氧化应激和改变凋亡相关基因的表达诱导心肌细胞凋亡。更多还原

【Abstract】 Objective: To investigate the effects of high glucose on apoptosis in neonatal rat cardiomyocytes and to explore the potential mechanisms involved. Methods: The MTT assay was used to detect the viability of cultured cardiomyocytes exposed to different concentrations of glucose (10 to 50 mmol/L). Laser confocal microscopy, TUNEL assay and flow cytometry were utilized to detect cardiomyocyte apoptosis. Oxidative stress was evaluated by the levels of lactic dehydrogenase (LDH), malondialdehyde (MDA) and superoxide dismutase (SOD) in the supernatant of culture media. Real-time PCR was conducted to identify mRNA expression changes in apoptosis-related genes between the high glucose group and the control group. Results: High glucose was found to inhibit cardiomyocyte viability in a concentration-dependent manner. Our results demonstrated that high glucose can promote cardiomyocyte apoptosis and the antioxidant can protect cardiomyocytes from apoptosis via inhibition of high glucose-induced oxidative stress. Real-time PCR indicated that the expression of Pax3, Bmp4 and Slug were down-regulated in the high glucose group, while the expression of P53 and Msx1 were up-regulated. Conclusion: High glucose promotes cardiomyocyte apoptosis by inducing oxidative stress and disrupting apoptosis-related gene expression. Objective: To investigate the effects of nicotine on apoptosis in neonatal rat cardiomyocytes and to explore the potential mechanisms involved. Methods: The MTT assay was used to detect the viability of cultured cardiomyocytes exposed to different concentrations of nicotine (0.1 to 100μM). Laser confocal microscopy, TUNEL assay and flow cytometry were utilized to detect cardiomyocyte apoptosis. Oxidative stress was evaluated by the levels of lactic dehydrogenase (LDH), malondialdehyde (MDA) and superoxide dismutase (SOD) in the supernatant of culture media. Real-time PCR was conducted to identify mRNA expression changes in apoptosis-related genes between the nicotine and the control group. Results: Nicotine was found to inhibit cardiomyocyte viability in a concentration-dependent manner. Our results demonstrated that nicotine can promote cardiomyocyte apoptosis and antioxidant can protect cardiomyocytes from apoptosis via inhibition of nicotine-induced oxidative stress. Real-time PCR indicated that the expression of Bcl-2, Pax3, Bmp4 and Slug were down-regulated in the nicotine group, while the expression of P53, Bax and Msx1 were up-regulated. Conclusion: Nicotine promotes cardiomyocyte apoptosis by inducing oxidative stress and disrupting apoptosis-related gene expression.更多还原