肼致心肌成纤维细胞凋亡机制及NADH防治作用的研究

发布时间：2018-06-04 00:49

本文选题：肼 + 心肌成纤维细胞　；参考：《武汉大学》2005年硕士论文

【摘要】：目的:探讨肼致心肌成纤维细胞凋亡的作用及机制;观察外源性NADH的抗肼毒作用。方法:体外培养S-D大鼠乳鼠(1-3天)心肌成纤维细胞,细胞分四组:正常组(对照组)、肼损伤组、正常+NADH组(NADH组)和肼损伤+NADH组。肼损伤分别采用1、2、4、8、16mM(mmol/l)等5个不同浓度,外源性NADH治疗采用400umol/L浓度。各组细胞分别处理培养72h后,通过用MTT法检测肼的细胞毒作用、光学倒置相差显微镜和Hochest33258染色后荧光显微镜观察细胞处理前后的形态学变化、Annexin V/PI双染流式细胞仪检测细胞凋亡率和坏死率确定肼诱导心肌成纤维细胞凋亡的浓度;选用凋亡率最高的肼损伤组,通过Rodamine 123荧光染色流式细胞仪检测细胞内线粒体膜电位的变化;Western-blot检测蛋白Bax、Bcl-2、Caspase-3表达的变化探讨肼致心肌成纤维细胞凋亡的机制和NADH的抗肼毒作用。结果:(一)肼对心肌成纤维细胞的作用: 1.MTT检测显示,肼可以抑制细胞增殖,其作用随肼浓度增高而增强,与正常组比较差异具有显著性(p0.01)。 2.荧光染色和流式细胞仪检测显示,肼能导致心肌成纤维细胞凋亡,当肼浓度为2mM时凋亡细胞最多,随浓度增大凋亡细胞逐渐减少,坏死细胞逐渐增多。 (二)肼致心肌成纤维细胞凋亡的作用机制和NADH的防治作用: 1.MTT检测显示,肼可以抑制细胞增殖,其作用随肼浓度增高而增强,与正常组比较差异具有显著性(p0.01);用400umol/L浓度NADH治疗后细胞抑制减弱,与肼损伤组比较差异具有显著性(p0.01)。 2.光学倒置相差显微镜和Hochest33258荧光染色观察显示,肼损伤组与正常组相比,细胞生长、增殖受到抑制,细胞数明显少于正常组,细胞出现凋亡核浓缩、核边集形态;肼+NADH组细胞生长、增殖较肼损伤组有明显改善,细胞凋亡的核浓缩、核边集减少。 3.流式细胞仪检测显示,肼损伤组细胞凋亡率和坏死率均高于正常组,差异均有显著性(P0.05),肼+NADH组凋亡率和坏死率均低于肼损伤组(P0.05)。
[Abstract]:Aim: to investigate the effect and mechanism of hydrazine on myocardial fibroblast apoptosis and to observe the anti-hydrazine toxicity of exogenous NADH. Methods: myocardial fibroblasts of S-D rats were cultured in vitro for 1-3 days. The cells were divided into four groups: normal group (control group, hydrazine injury group, normal NADH group, nadh group) and hydrazine injured NADH group. Five different concentrations of hydrazine were used in the treatment of hydrazine injury, including 816 mmol / L of 816 mmol / L, respectively. Exogenous NADH was used in the treatment of 400umol/L. After 72 hours of culture, the cytotoxicity of hydrazine was detected by MTT assay. Morphological changes before and after cell treatment were observed by optical inverted phase contrast microscope and Hochest33258 staining. Apoptosis rate and necrosis rate were detected by Annexin V/PI double staining flow cytometry to determine the concentration of hydrazine induced apoptosis of myocardial fibroblasts. In the hydrazine injury group with the highest apoptosis rate, the changes of mitochondrial membrane potential in the cells were detected by Rodamine 123 fluorescence staining flow cytometry. The changes of the expression of Baxan Bcl-2Caspase-3 protein were detected by Western-blot. The mechanism of hydrazine induced apoptosis of myocardial fibroblasts and the anti-hydrazine toxicity of NADH were investigated. Results the effects of 1% hydrazine on myocardial fibroblasts were as follows: The results of 1.MTT showed that hydrazine could inhibit cell proliferation, and its effect was enhanced with the increase of hydrazine concentration, which was significantly different from that of normal group (P 0.01). 2. Fluorescence staining and flow cytometry showed that hydrazine could induce apoptosis of cardiac fibroblasts. When the concentration of hydrazine was 2mM, the number of apoptotic cells was the most, and the number of necrotic cells increased with the increase of concentration of hydrazine. (2) the mechanism of apoptosis of myocardial fibroblasts induced by hydrazine and the preventive and therapeutic effects of NADH: The results of 1.MTT showed that hydrazine could inhibit cell proliferation, and the effect of hydrazine increased with the increase of hydrazine concentration, which was significantly different from that of normal group (P 0.01), and decreased after NADH treatment with 400umol/L concentration, which was significantly different from that of hydrazine injury group (P 0.01). 2. Optical inverted phase contrast microscope and Hochest33258 fluorescence staining showed that compared with the normal group, the cell growth and proliferation were inhibited in the hydrazine injury group, and the number of cells was significantly lower than that in the normal group. The cell growth and proliferation in hydrazine NADH group were significantly improved compared with those in hydrazine injury group. 3. The results of flow cytometry showed that the apoptosis rate and necrosis rate of hydrazine injury group were higher than that of normal group, and the difference was significant (P 0.05). The apoptosis rate and necrosis rate of hydrazine NADH group were lower than that of hydrazine injury group (P 0.05).
【学位授予单位】：武汉大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R363

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