DDR1对奶牛乳腺上皮细胞增殖与凋亡的调控作用

Effects of Interference and Overexpression of DDR1 on Proliferation and Apoptosis of Mammary Epithelial Cells in Dairy Cows

旨在通过干扰或过表达盘状蛋白结构域受体1(DDR1)基因,探讨其对奶牛乳腺上皮细胞增殖、凋亡及周期的影响。本研究将DDR1基因小干扰RNA片段和过表达载体pcDNA3.1-DDR1转染奶牛乳腺上皮细胞,采用qRT-PCR和Western blot检测细胞中DDR1的干扰和过表达效果;利用CCK-8法检测细胞增殖能力;流式细胞术检测细胞周期和细胞凋亡(早凋、晚凋)的变化;利用qRT-PCR检测增殖与凋亡相关基因的表达情况。结果,在奶牛乳腺上皮细胞中干扰DDR1基因后,其mRNA和蛋白表达分别下调94%和30%,而过表达DDR1基因后,其mRNA和蛋白表达分别上调68.24和1.38倍;干扰DDR1极显著抑制细胞的增殖(P<0.01),细胞早凋与晚凋比例极显著上升(P<0.01);干扰组G1期细胞比例极显著上升(P<0.01),S期细胞比例极显著下降(P<0.01),G2期细胞比例显著下降(P<0.05),提示细胞阻滞在G0/G1期。相反,过表达DDR1能显著促进细胞增殖(P<0.05),显著抑制细胞晚期凋亡(P<0.05),G1期细胞比例极显著下降(P<0.01),S期细胞比例极显著上升(P<0.01),促进细胞由G1期向S期的转换。qRT-PCR检测结果显示,干扰DDR1后显著上调BAX、Caspase9基因的表达(P<0.05),极显著上调P53、FAS基因的表达(P<0.01),且显著下调PCNA、CyclinB1基因的表达(P<0.05);过表达DDR1后,分别显著和极显著下调Cytc与BAX基因的表达(P<0.05,P<0.01),并极显著上调CyclinB1基因的表达(P<0.01)。综上可见,DDR1能够调控奶牛乳腺上皮细胞的增殖、凋亡和周期,可为阐明DDR1参与奶牛乳腺上皮细胞生长发育的分子机制提供参考。

The aim of this study was to investigate the effect of discoidin domain receptor 1(DDR1)gene on the proliferation,apoptosis and cell cycle of dairy cow mammary epithelial cells through interfering and overexpressing DDR1.The small RNA interference fragment of DDR1 gene and overexpression vector pcDNA3.1-DDR1 were transfected into dairy cow mammary epithelial cells,qRT-PCR and Western blot were used to detect the interference and overexpression efficiency of DDR1;CCK-8 was employed to detect cell proliferation;Flow cytometry was performed to detect the changes of cell cycle and apoptosis(early apoptosis and late apoptosis).qRT-PCR was used to detect the expression of genes related to proliferation and apoptosis.After interfering DDR1 gene in dairy cow mammary epithelial cells,mRNA and protein expression levels of DDR1 were down-regulated by 94%and 30%,respectively;While after overexpressing DDR1 gene,its mRNA and protein expression were up-regulated 68.24 and 1.38 times,respectively.Interfering DDR1 extremely significantly inhibited the proliferation of cells(P<0.01),the ratio of early and late apoptotic cells was extremely significantly increased(P<0.01);the ratio of G1 phase cells in the interference group was extremely significantly increased(P<0.01),and the proportions of S and G2 phase cells were significantly decreased(P<0.01 and P<0.05),which suggested that the cells were blocked in G0/G1 phase.Conversely,overexpression of DDR1 could significantly promote cell proliferation(P<0.05),significantly inhibit the late apoptosis of cells(P<0.05),the proportion of G1 phase cells was extremely significantly decreased(P<0.01),and the proportion of S phase cells was extremely significantly increased(P<0.01),suggesting the enhancement of transition from G1 to S phase.The results of qRT-PCR detection showed that after interfering DDR1,the expressions of BAX and Caspase9 genes were significantly up-regulated(P<0.05),and the expressions of P53 and FAS genes were extremely significantly up-regulated(P<0.01),while the expressions of PCNA and CyclinB1 genes were significantly down-regulated(P<0.05).After overexpression of DDR1,the expressions of Cytc and BAX genes were significantly(P<0.05)and extremely significantly down-regulated(P<0.01),respectively,and the expression of CyclinB1 gene was extremely significantly up-regulated(P<0.01).In summary,DDR1 can regulate the proliferation,apoptosis and cycle of dairy cow mammary epithelial cells,this result will provide a reference for elucidating the molecular mechanism of DDR1 involved in the growth and development of dairy cow mammary epithelial cells.