【摘要】：本試驗通過對不同濃度蛋氨酸作用于熱處理后的體外培養(yǎng)奶牛乳腺上皮細(xì)胞進(jìn)行研究,測定其對乳腺上皮細(xì)胞增殖、抗氧化性、凋亡及相關(guān)基因與蛋白表達(dá)方面的影響,從細(xì)胞水平探討蛋氨酸對熱應(yīng)激下奶牛乳腺的保護(hù)作用。本試驗分為兩個部分:1、蛋氨酸對熱處理及非熱處理奶牛乳腺上皮細(xì)胞增殖、抗氧化性能的影響。用兩組含不同濃度(0、30、60mg/L)蛋氨酸的DMEM培養(yǎng)基培養(yǎng)細(xì)胞2d后,37℃組用37℃水浴0.5h,42℃組(熱處理組)42℃C水浴0.5h,恢復(fù)6h,通過MTT測定高溫?zé)崽幚韺w外培養(yǎng)乳腺上皮細(xì)胞增殖的影響,培養(yǎng)液中丙二醛(MDA)、一氧化氮(NO)、一氧化氮合酶(NOS)、乳酸脫氫酶(LDH)、超氧化物歧化酶(SOD)、過氧化氫酶(CAT)和谷胱甘肽過氧化物酶(GSH-Px)的活性以及細(xì)胞凋亡率。結(jié)果表明,與37℃組相比,42℃組MDA含量、NO含量、NOS活力、LDH活性均升高;細(xì)胞活力、SOD活性、CAT活性下降,GSH-Px酶活力升高。與Omg/L蛋氨酸組相比,添加60mg/L蛋氨酸組的細(xì)胞活力升高(P0.05);LDH活性顯著降低(P0.05);尤其 MDA、NO、NOS 含量極顯著降低(P0.01);添加 30mg/L、60mg/L蛋氨酸能增加熱處理下乳腺上皮細(xì)胞CAT活性(P0.05);SOD中,60mg/L蛋氨酸組含量最高(P0.05);培養(yǎng)液中GSH-Px活性,60mg/L蛋氨酸組最高(P0.01)。這說明42℃0.5h熱處理使細(xì)胞產(chǎn)生了熱應(yīng)激,細(xì)胞受到氧化應(yīng)激,有氧自由基含量上升。添加蛋氨酸可以有效緩解細(xì)胞熱應(yīng)激損傷,減少氧化應(yīng)激。2、蛋氨酸對熱處理及非熱處理乳腺上皮細(xì)胞凋亡、相關(guān)基因及蛋白表達(dá)的影響。用含不同濃度(0、30、60mg/L)蛋氨酸的DMEM培養(yǎng)基培養(yǎng)細(xì)胞2d后,42℃C水浴0.5h后立即放回37℃恢復(fù)培養(yǎng)6h,測定細(xì)胞凋亡、HSF-1mRNA、HSP70mRNA、Bax/Bc1-2mRNA的表達(dá)、HSP70蛋白的表達(dá)以及Caspase-3的活性。結(jié)果表明,流式細(xì)胞儀測定細(xì)胞凋亡,與0mg/L蛋氨酸組相比,添加30、60mg/L蛋氨酸能顯著降低細(xì)胞晚期凋亡/壞死率(P0.05),總死亡率差異極顯著(P0.01);60mg/L蛋氨酸組HSF-1mRNA、HSP70mRNA表達(dá)量、HSP70蛋白的表達(dá)顯著升高(P0.01);降低了 Bax/Bcl-2mRNA的表達(dá)量以及Caspase-3的活性(P0.01)。結(jié)論:培養(yǎng)基中添加60mg/L蛋氨酸能促進(jìn)高溫條件下乳腺上皮細(xì)胞增殖,起到抗凋亡的作用,提高HSF-1、HSP70的表達(dá),降低Bax/Bcl-2表達(dá)量以及Caspase-3的活性,對細(xì)胞起到保護(hù)效應(yīng)。
[Abstract]:In this study, the effects of methionine at different concentrations on the proliferation, antioxidation, apoptosis and the expression of related genes and proteins of bovine breast epithelial cells after heat treatment were studied, and the effects of methionine on the proliferation, antioxidation, apoptosis and the expression of related genes and proteins were measured. To investigate the protective effect of methionine on dairy cow mammary gland under heat stress at cell level. The experiment was divided into two parts: 1: 1. The effect of methionine on proliferation and antioxidation of mammary epithelial cells after heat treatment and non-heat treatment. The cells were cultured in DMEM medium containing different concentrations of methionine (0.30g / L) for 2 days. After 2 days, the cells were incubated in 37 鈩，

本文編號：2140010