大鼠心肌细胞和成纤维细胞乳酸阈及单羧酸转运蛋白表达情况的研究

发布时间：2018-05-19 13:31

本文选题：乳酸阈 + 心肌细胞　；参考：《华中科技大学》2011年硕士论文

【摘要】：目的: 通过建立乳酸孵育模型观察原代心肌细胞和成纤维细胞的细胞活力并计算细胞存活率,探讨大鼠心肌细胞与成纤维细胞的乳酸阈。并提取细胞蛋白,通过Western blot方法行蛋白分析,观察大鼠心肌细胞与成纤维细胞单羧酸转运蛋白的表达情况。旨在为进一步研究心肌细胞与非心肌细胞之间的乳酸穿梭奠定基础。方法: 体外培养原代大鼠心肌细胞和成纤维细胞,添加pH值相同、浓度不同的乳酸(1mmol/L,2mmol/L,4mmol/L,8mmol/L,16mmol/L,32mmol/L)孵育,建立乳酸孵育模型,通过测定OD值观察细胞的活力并计算细胞存活率。提取原代心肌细胞和成纤维细胞的蛋白,并用Western blot方法进行蛋白分析,观察MCT1及MCT4在这两种细胞上的表达情况。结果: (1)原代心肌细胞在乳酸浓度为2mmol/L和4mmol/L时,其OD值最大,细胞存活率最高,与其他组之间比较有显著差异,P㩳0.05。这两组之间无差异,P㧐0.05。(2)原代成纤维细胞在乳酸浓度为4mmol/L和8mmol/L时,其OD值最大,细胞存活率最高,与其他组之间比较有显著差异,P㩳0.05。这两组之间无差异,P㧐0.05。(3)两种细胞MCT1的表达量均比MCT4的表达高,P㩳0.05。而且心肌细胞的MCT1,MCT4表达量均比成纤维细胞高,P㩳0.05。讨论: 原代心肌细胞在乳酸浓度为2mmol/L和4mmol/L时,细胞活力最高,细胞存活率最高,说明在此浓度时细胞吸收乳酸最多,给细胞提供了相对充足的能量,故细胞存活率较其它组高。由此推断心肌细胞的乳酸阈为2mmol/L-4mmol/L。然而随浓度更进一步增高,细胞活力下降,可能是因为乳酸浓度过高,细胞由以摄取乳酸为主转为以排出乳酸为主,故细胞能量供应减少,细胞存活率下降。同样成纤维细胞在浓度为4mmol/L和8mmol/L时,细胞活力最高,细胞存活率最高。由此推断成纤维细胞的乳酸阈为4mmol/L-8mmol/L。 MCT1和MCT4在原代心肌细胞和成纤维细胞均有表达,说明这两种细胞均具备乳酸穿梭的转运体,可以将乳酸转入或转出细胞。而且负责转入的MCT1高表达,负责转出的MCT4低表达表明两种细胞均以乳酸转入及氧化利用为主。MCT4的低表达可能是因为MCT4需要某种刺激表达才会上调。同时心肌细胞上MCT1与MCT4的表达均比成纤维细胞上的高,提示心肌细胞乳酸代谢更活跃。结论: 原代心肌细胞和成纤维细胞的乳酸阈的不同提示两种细胞乳酸代谢方式不同,这是二者间可能存在乳酸穿梭的生化基础。心肌细胞和成纤维细胞表达的单羧酸转运蛋白种类及丰度不同,这是二者之间可能存在乳酸穿梭的结构基础。
[Abstract]:Objective: The lactic acid threshold of rat cardiomyocytes and fibroblasts was studied by establishing lactate incubation model to observe the viability of primary cardiomyocytes and fibroblasts and to calculate the cell survival rate. The expression of monocarboxylic acid transporter in rat cardiomyocytes and fibroblasts was observed by Western blot assay. The aim is to lay a foundation for further study of lactate shuttling between cardiomyocytes and non-cardiomyocytes. Methods: Primary rat cardiomyocytes and fibroblasts were cultured in vitro and incubated with lactic acid of 1 mmol / L ~ (2) mol / L ~ (2) mol / L ~ (4) mol / L ~ (8) mmol / L ~ (16) mmol / L ~ (32) mmol 路L ~ (-1) 路L ~ (-1). The cell viability was observed and cell survival rate was calculated by measuring OD value. The proteins of primary cardiomyocytes and fibroblasts were extracted and analyzed by Western blot to observe the expression of MCT1 and MCT4 in these two kinds of cells. Results: 1) when the lactate concentration was 2mmol/L and 4mmol/L, the OD value of primary cardiomyocytes was the highest and the cell survival rate was the highest, and there was a significant difference between the two groups. There was no difference between the two groups (P < 0.05. 2) when the lactate concentration was 4mmol/L and 8mmol/L, the OD value of the primary fibroblasts was the highest and the cell survival rate was the highest, and there was significant difference between the two groups. There was no difference between the two groups in the expression of MCT1. The expression of MCT1 in the two groups was higher than that in MCT4. Moreover, the expression of MCT1 and MCT4 in cardiac myocytes was higher than that in fibroblasts. Discussion: When the concentration of lactic acid was 2mmol/L and 4mmol/L, the primary cardiomyocytes had the highest cell viability and the highest survival rate, which indicated that the cells absorbed the most lactic acid at this concentration, which provided the cells with relatively sufficient energy, so the cell survival rate was higher than that of the other groups. The lactate threshold of cardiomyocytes was estimated to be 2 mmol / L-4 mmol / L. However, with the further increase of the concentration, the cell viability decreased, possibly because the lactic acid concentration was too high, the cells changed from lactic acid intake to lactic acid excretion, so the cell energy supply decreased and the cell survival rate decreased. The same fibroblasts had the highest cell viability and the highest cell survival rate at the concentrations of 4mmol/L and 8mmol/L. It was deduced that the lactate threshold of fibroblasts was 4 mmol / L-8 mmol / L. Both MCT1 and MCT4 were expressed in primary cardiomyocytes and fibroblasts, indicating that both cells had lactate shuttle transporters, which could transfer lactic acid into or out of cells. Furthermore, the high expression of MCT1 and the low expression of MCT4, which were responsible for the transfer, suggested that the low expression of Lactic acid in both cells was probably due to the need for some stimulation expression of MCT4 in order to be up-regulated. At the same time, the expression of MCT1 and MCT4 in cardiomyocytes was higher than that in fibroblasts, suggesting that lactate metabolism was more active in cardiomyocytes. Conclusion: The difference of lactate threshold between primary cardiomyocytes and fibroblasts suggests that the lactate metabolic patterns of the two cells are different, which may be the biochemical basis of lactate shuttle between the two cells. The species and abundance of monocarboxylic acid transporter expressed in cardiomyocytes and fibroblasts are different, which may be the structural basis of lactate shuttle between them.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R341

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