DDR1基因过表达对水牛乳腺上皮细胞的影响

为揭示盘状结构域受体1（discoidin domain receptor1，DDR1）基因对水牛泌乳性能的影响，本研究构建了水牛DDR1基因真核表达载体，并对其最佳转染时间进行摸索，同时分析DDR1基因过表达对水牛乳腺上皮细胞的影响。琼脂糖凝胶电泳检测结果显示，载体片段大小与目标载体片段大小一致，均为8.8 kb，测序结果显示其与目的片段序列匹配率为100%。细胞转染试验结果显示，DDR1基因过表达最佳转染时间为48 h。细胞增殖检测结果显示，DDR1基因过表达组与对照组细胞相对荧光值差异不显著（P>0.05）。细胞凋亡检测结果显示，DDR1基因过表达对水牛乳腺上皮细胞的晚期凋亡率（19.87% VS 17.49%）无影响（P>0.05），但极显著增加了水牛乳腺上皮细胞早期凋亡率（6.48% VS 1.35%，P<0.01）。同时，DDR1基因过表达上调了水牛乳腺上皮细胞中抑凋亡基因BCL-2和XIAP的表达，而下调了促凋亡基因P53的表达。此外，DDR1基因过表达显著提高了水牛乳腺上皮细胞的迁移率（66.26% VS 58.76%，P<0.05）。综上，试验成功构建了水牛DDR1基因真核表达载体并证明了DDR1基因过表达促进了水牛乳腺上皮细胞的早期凋亡和迁移，为今后进一步研究水牛乳腺发育和泌乳性能提供了一定理论依据。

Effect of DDR1 Gene over Expression on Buffalo Mammary Epithelial Cells

In order to reveal the effect of discoidin domain receptor 1 (DDR1) gene on the lactation performance of buffalo,in this study,the eukaryotic expression vector of buffalo DDR1 gene was constructed,and its optimal transfection time was groped.The effects of DDR1 gene expression on buffalo mammary epithelial cells were also analyzed.Results of agarose gel electrophoresis showed that the fragment size of vetor was 8.8 kb,it was the same as the target carrier fragment size,and the sequencing results showed that the match rate with the target fragment sequence was 100%.Results of cell transfection test showed that the optimal transfection time of DDR1 gene over expression was 48 h.Results of cell proliferation test showed that there was no significant difference in the relative fluorescence value between the DDR1 gene over expression group and the control group (P>0.05).Apoptosis test results showed that the late apoptosis rate was not affected (19.87% VS 17.49%) by the DDR1 gene over expression (P>0.05),but the early apoptosis rate of buffalo mammary epithelial cells (6.48% VS 1.35%) was extremely significantly increased (P<0.01).Moreover,DDR1 gene over expression increased the expression of apoptosis-inhibiting genes BCL-2 and XIAP in buffalo mammary epithelial cells,and reduced the expression of apoptosis-promoting gene P53.In addition,over expression of the DDR1 gene significantly improved the migration rate of buffalo mammary epithelial cells (66.26% VS 58.76%,P<0.05).In conclusion,the research successfully constructed the eukaryotic expression vector of buffalo DDR1 gene and proved that over expression of DDR1 gene promoted the early apoptosis and migration of buffalo mammary epithelial cells,and laid a foundation for the future study of the development and lactation performance of buffalo mammary gland.