本文選題：乳酸閾 + 心肌細(xì)胞　； 參考：《華中科技大學(xué)》2011年碩士論文

【摘要】：目的: 通過建立乳酸孵育模型觀察原代心肌細(xì)胞和成纖維細(xì)胞的細(xì)胞活力并計(jì)算細(xì)胞存活率,探討大鼠心肌細(xì)胞與成纖維細(xì)胞的乳酸閾。并提取細(xì)胞蛋白,通過Western blot方法行蛋白分析,觀察大鼠心肌細(xì)胞與成纖維細(xì)胞單羧酸轉(zhuǎn)運(yùn)蛋白的表達(dá)情況。旨在為進(jìn)一步研究心肌細(xì)胞與非心肌細(xì)胞之間的乳酸穿梭奠定基礎(chǔ)。 方法: 體外培養(yǎng)原代大鼠心肌細(xì)胞和成纖維細(xì)胞,添加pH值相同、濃度不同的乳酸(1mmol/L,2mmol/L,4mmol/L,8mmol/L,16mmol/L,32mmol/L)孵育,建立乳酸孵育模型,通過測(cè)定OD值觀察細(xì)胞的活力并計(jì)算細(xì)胞存活率。提取原代心肌細(xì)胞和成纖維細(xì)胞的蛋白,并用Western blot方法進(jìn)行蛋白分析,觀察MCT1及MCT4在這兩種細(xì)胞上的表達(dá)情況。 結(jié)果: (1)原代心肌細(xì)胞在乳酸濃度為2mmol/L和4mmol/L時(shí),其OD值最大,細(xì)胞存活率最高,與其他組之間比較有顯著差異,P㩳0.05。這兩組之間無差異,P㧐0.05。(2)原代成纖維細(xì)胞在乳酸濃度為4mmol/L和8mmol/L時(shí),其OD值最大,細(xì)胞存活率最高,與其他組之間比較有顯著差異,P㩳0.05。這兩組之間無差異,P㧐0.05。(3)兩種細(xì)胞MCT1的表達(dá)量均比MCT4的表達(dá)高,P㩳0.05。而且心肌細(xì)胞的MCT1,MCT4表達(dá)量均比成纖維細(xì)胞高,P㩳0.05。 討論: 原代心肌細(xì)胞在乳酸濃度為2mmol/L和4mmol/L時(shí),細(xì)胞活力最高,細(xì)胞存活率最高,說明在此濃度時(shí)細(xì)胞吸收乳酸最多,給細(xì)胞提供了相對(duì)充足的能量,故細(xì)胞存活率較其它組高。由此推斷心肌細(xì)胞的乳酸閾為2mmol/L-4mmol/L。然而隨濃度更進(jìn)一步增高,細(xì)胞活力下降,可能是因?yàn)槿樗釢舛冗^高,細(xì)胞由以攝取乳酸為主轉(zhuǎn)為以排出乳酸為主,故細(xì)胞能量供應(yīng)減少,細(xì)胞存活率下降。同樣成纖維細(xì)胞在濃度為4mmol/L和8mmol/L時(shí),細(xì)胞活力最高,細(xì)胞存活率最高。由此推斷成纖維細(xì)胞的乳酸閾為4mmol/L-8mmol/L。 MCT1和MCT4在原代心肌細(xì)胞和成纖維細(xì)胞均有表達(dá),說明這兩種細(xì)胞均具備乳酸穿梭的轉(zhuǎn)運(yùn)體,可以將乳酸轉(zhuǎn)入或轉(zhuǎn)出細(xì)胞。而且負(fù)責(zé)轉(zhuǎn)入的MCT1高表達(dá),負(fù)責(zé)轉(zhuǎn)出的MCT4低表達(dá)表明兩種細(xì)胞均以乳酸轉(zhuǎn)入及氧化利用為主。MCT4的低表達(dá)可能是因?yàn)镸CT4需要某種刺激表達(dá)才會(huì)上調(diào)。同時(shí)心肌細(xì)胞上MCT1與MCT4的表達(dá)均比成纖維細(xì)胞上的高,提示心肌細(xì)胞乳酸代謝更活躍。 結(jié)論: 原代心肌細(xì)胞和成纖維細(xì)胞的乳酸閾的不同提示兩種細(xì)胞乳酸代謝方式不同,這是二者間可能存在乳酸穿梭的生化基礎(chǔ)。心肌細(xì)胞和成纖維細(xì)胞表達(dá)的單羧酸轉(zhuǎn)運(yùn)蛋白種類及豐度不同,這是二者之間可能存在乳酸穿梭的結(jié)構(gòu)基礎(chǔ)。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R341

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