本文選題：肼 + 心肌成纖維細胞��； 參考：《武漢大學》2005年碩士論文

【摘要】：目的:探討肼致心肌成纖維細胞凋亡的作用及機制;觀察外源性NADH的抗肼毒作用。 方法:體外培養(yǎng)S-D大鼠乳鼠(1-3天)心肌成纖維細胞,細胞分四組:正常組(對照組)、肼?lián)p傷組、正常+NADH組(NADH組)和肼?lián)p傷+NADH組。肼?lián)p傷分別采用1、2、4、8、16mM(mmol/l)等5個不同濃度,外源性NADH治療采用400umol/L濃度。各組細胞分別處理培養(yǎng)72h后,通過用MTT法檢測肼的細胞毒作用、光學倒置相差顯微鏡和Hochest33258染色后熒光顯微鏡觀察細胞處理前后的形態(tài)學變化、Annexin V/PI雙染流式細胞儀檢測細胞凋亡率和壞死率確定肼誘導心肌成纖維細胞凋亡的濃度;選用凋亡率最高的肼?lián)p傷組,通過Rodamine 123熒光染色流式細胞儀檢測細胞內(nèi)線粒體膜電位的變化;Western-blot檢測蛋白Bax、Bcl-2、Caspase-3表達的變化探討肼致心肌成纖維細胞凋亡的機制和NADH的抗肼毒作用。 結(jié)果:(一)肼對心肌成纖維細胞的作用: 1.MTT檢測顯示,肼可以抑制細胞增殖,其作用隨肼濃度增高而增強,與正常組比較差異具有顯著性(p0.01)。 2.熒光染色和流式細胞儀檢測顯示,肼能導致心肌成纖維細胞凋亡,當肼濃度為2mM時凋亡細胞最多,隨濃度增大凋亡細胞逐漸減少,壞死細胞逐漸增多。 (二)肼致心肌成纖維細胞凋亡的作用機制和NADH的防治作用: 1.MTT檢測顯示,肼可以抑制細胞增殖,其作用隨肼濃度增高而增強,與正常組比較差異具有顯著性(p0.01);用400umol/L濃度NADH治療后細胞抑制減弱,與肼?lián)p傷組比較差異具有顯著性(p0.01)。 2.光學倒置相差顯微鏡和Hochest33258熒光染色觀察顯示,肼?lián)p傷組與正常組相比,細胞生長、增殖受到抑制,細胞數(shù)明顯少于正常組,細胞出現(xiàn)凋亡核濃縮、核邊集形態(tài);肼+NADH組細胞生長、增殖較肼?lián)p傷組有明顯改善,細胞凋亡的核濃縮、核邊集減少。 3.流式細胞儀檢測顯示,肼?lián)p傷組細胞凋亡率和壞死率均高于正常組,差異均有顯著性(P0.05),肼+NADH組凋亡率和壞死率均低于肼?lián)p傷組(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).
【學位授予單位】：武漢大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R363

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