TGF-β_1对奶牛乳腺上皮细胞增殖、凋亡及HSP70蛋白表达的影响  

Effects of TGF-β_1 on Proliferation, Apoptosis and HSP70 Expression of Mammary Epithelial Cells of Dairy Cow

作　　者：狄和双[1] 杜娟[1] 张中霞[1] 黄静燕[1] 崔群维[1] 程凯宁[1] 王根林[1] 

机构地区：[1]南京农业大学动物科技学院,南京210095

出　　处：《中国农业科学》2008年第10期3249-3255,共7页Scientia Agricultura Sinica

基　　金：国家科技部奶业支撑计划(2006BAD04A12;2006BAD04A01)

摘　　要：【目的】探讨TGF-β1对奶牛乳腺上皮细胞增殖、凋亡和HSP70蛋白表达的影响。【方法】TGF-β1作用乳腺上皮细胞不同时间,收集细胞及培养液,应用MTT检测细胞增殖、流式细胞术检测细胞凋亡率、比色法检测LDH活性、琼脂糖凝胶电泳检测DNA完整性、Western bolt技术分析HSP70蛋白的表达量。【结果】1、5、10ng·ml-1的TGF-β1均能抑制乳腺上皮细胞的增殖,且随浓度增加抑制作用加大,呈现剂量依赖性,其中10ng·ml-1TGF-β1组与对照组差异显著(P<0.05);10ng·ml-1TGF-β1作用于乳腺上皮细胞,随着作用时间的延长,细胞凋亡率逐渐升高,其中6、12和24h与对照组相比差异极显著(P<0.01);LDH活力随作用时间延长而升高,12、24h与对照组差异极显著(P<0.01);DNA完整性随TGF-β1作用时间的延长发生不同程度降解;TGF-β1作用2～6h细胞大量表达HSP70,6h达到高峰,而后下降,2～12h的表达量均极显著高于对照组(P<0.01)。【结论】TGF-β1可以抑制奶牛乳腺上皮细胞的增殖,具有浓度依赖性。10ng·ml-1TGF-β1可以显著增加细胞凋亡率,提高细胞培养液中LDH活力,使细胞DNA发生不同程度的降解,同时HSP70蛋白随TGF-β1作用时间发生显著的表达变化。本试验结果表明,TGF-β1可以以促进上皮细胞凋亡的方式,促进奶牛乳腺发生退化。[Objective] The aims of present study were to investigate the effects of TGF-β1 on proliferation, apoptosis and HSP70 expression of the mammary epithelial cells of dairy cow. [Method] Bovine mammary epithelial cells were cultured in DMEM/F12＋10%CS medium with TGF-β1 and cells and supernatant were collected. The cell proliferation and apoptosis, activity of LDH, integrity of DNA, and expression of heat shock protein 70 （HSP70） were detected by MTT, How cytometry, lactate dehydrogenase （LDH） kit, agarose gel electrophoresis and Western bolt, respectively. [Result] The results showed that cells proliferations were inhibited after treated with TGF-β1 in dose dependent way. The rate of apoptosis cells significantly increased after treatment with TGF-β1 for 6, 12, 24 hours （P〈0.01） .The activity of LDH was significantly higher in the treated groups than that in the control group（P〈0.01 ）. The nucleotide of DNA was degradated gradually as treatment time increased. The expression of HSP70 increased after TGF-β1 treatment for more than 2 hours and it reaclaed the highest level when treated for 6 h, then decreased. [Conculsion] TGF-β1 could inhibit the proliferation of bovine mammary epithelial cells in a dose-dependent way. At a dose of 10 ng·ml^-1, TGF-β1 could significantly stimulate cell apoptosis and increase LDH activity, which were resulted in degradation of DNA. Long-term culture of bovine mammary epithelial cells with TGF-β1 could lead to the up-regulation of the expression of HSP70. It is suggested that TGF-β1 could promote the apoptosis pathway of bovine mammary epithelial cells, and accelerate the involution of bovine mammary.

关 键 词：TGF-β1 乳腺上皮细胞 细胞凋亡 HSP70 

分 类 号：S823[农业科学—畜牧学]