本文選題：視黃酸 + 心肌細(xì)胞��； 參考：《桂林醫(yī)學(xué)院》2012年碩士論文

【摘要】：目的：研究外源性視黃酸(Retinoic acid, RA)對(duì)乳鼠心肌細(xì)胞凋亡的影響,并研究乳鼠心肌細(xì)胞中Fas,Fas1, Casepase-3,Pax-8細(xì)胞調(diào)控因子的表達(dá)水平,分析RA誘導(dǎo)心肌損傷的機(jī)制。方法：1.體外培養(yǎng)乳鼠原代心肌細(xì)胞以及明確心肌細(xì)胞干預(yù)的最佳時(shí)間：在無(wú)菌條件下取出Balb/c乳鼠心臟,采用胰酶-差速貼壁法分離并純化獲得乳鼠心肌細(xì)胞,并進(jìn)行原代心肌細(xì)胞培養(yǎng)。2.RA干預(yù)心肌細(xì)胞后進(jìn)行指標(biāo)的檢測(cè)：設(shè)置的空白對(duì)照組為選用含20%胎牛血清的DMEM高糖培養(yǎng)基培養(yǎng)心肌細(xì)胞；實(shí)驗(yàn)組分別使用含不同濃度視黃酸的培養(yǎng)基干預(yù)心肌細(xì)胞,在用藥干預(yù)后的0h、12h、24h、48h使用倒置顯微鏡觀(guān)察心肌細(xì)胞形態(tài)并動(dòng)態(tài)記錄細(xì)胞搏動(dòng)力；用MTT法來(lái)檢測(cè)心肌細(xì)胞的抑制率；流式細(xì)胞儀檢測(cè)心肌細(xì)胞凋亡率；3.RA干預(yù)心肌細(xì)胞后Fas、Fas1、Caspase-3、Pax-8調(diào)控因子檢測(cè)：Western blot測(cè)定Fas、Fas1、Caspase-3、Pax-8蛋白的表達(dá)水平。RT-PCR檢測(cè)Fas、Fasl mRNA表達(dá)水平。結(jié)果：1.成功培養(yǎng)Balb/c乳鼠心肌原代細(xì)胞。培養(yǎng)72h的心肌細(xì)胞生長(zhǎng)旺盛,表現(xiàn)為搏動(dòng)力強(qiáng),心肌細(xì)胞增殖逐漸進(jìn)入平臺(tái)期,為最佳的細(xì)胞干預(yù)期。2.視黃酸濃度≥2umo1/L時(shí)能顯著抑制正常Balb/c乳鼠心肌細(xì)胞的生長(zhǎng),使細(xì)胞的搏動(dòng)力減弱、凋亡增加,同時(shí)干預(yù)的藥物濃度越高、時(shí)間越長(zhǎng),抑制越明顯。3.與對(duì)照組相比,2umol/L RA組隨作用時(shí)間的延長(zhǎng),Fas、Fas1、Caspase-3蛋白的表達(dá)逐漸增強(qiáng)(P0.05)；與之相對(duì)應(yīng)的是Pax-8蛋白的表達(dá)則逐漸降低(P0.05)；Fas、Fas1mRNA表達(dá)逐漸增強(qiáng)(P0.05)。結(jié)論：1.采用胰酶-差速貼壁法及化學(xué)藥物抑制法能獲取存活率高、純度高的心肌原代細(xì)胞；心肌細(xì)胞培養(yǎng)72h為最佳的心肌細(xì)胞干預(yù)時(shí)間點(diǎn)。2.高濃度RA能抑制心肌細(xì)胞生長(zhǎng),使細(xì)胞搏動(dòng)力減弱或消失,并誘導(dǎo)心肌細(xì)胞凋亡。3.RA通過(guò)調(diào)節(jié)Fas、Fas1、Caspase-3、Pax-8的表達(dá),抑制正常心肌細(xì)胞的增殖,促進(jìn)細(xì)胞的凋亡。
[Abstract]:Aim: to study the effect of Retinoic acid (RA) on cardiomyocyte apoptosis and the expression of Fas-Fas1, Casepase-3 Pax-8 in neonatal rat cardiomyocytes, and to analyze the mechanism of myocardial injury induced by RA. Method 1: 1. The optimal time for primary culture of neonatal rat cardiomyocytes in vitro and the intervention of cardiomyocytes were as follows: the hearts of Balb/c rats were removed under aseptic conditions and isolated and purified by trypsin differential adherent method. The primary cardiomyocyte culture. 2. The indexes were detected after RA intervention. The blank control group was cultured on DMEM medium containing 20% fetal bovine serum. The experimental groups were treated with different concentrations of retinoic acid to intervene cardiomyocytes. The morphology of cardiomyocytes was observed and the pulsatility of cardiomyocytes was recorded dynamically by inverted microscope at 0 h, 12 h, 24 h and 48 h after intervention, and the inhibition rate of cardiomyocytes was detected by MTT method. Apoptosis rate of cardiomyocytes was measured by flow cytometry. After RA intervention, the expression of Fas-Fas1Caspase-3 and Pax-8 was detected by Western blot. The expression level of Fas-Fas1Caspase-3 and Pax-8 protein was detected by RT-PCR. The expression of Fas-Fasl mRNA in Fas-Fas1Caspase-3 was detected by reverse transcription-polymerase chain reaction (RT-PCR). The result is 1: 1. Primary myocardial cells were successfully cultured in neonatal Balb/c mice. After 72 hours of culture, cardiomyocytes grew vigorously, showing strong pulsatility, and the proliferation of cardiomyocytes gradually entered the plateau phase, which was the best cell dry expectation. 2. When the concentration of retinoic acid 鈮，

本文編號(hào)：1965643