鸡Bcl-2基因的克隆及其对卵泡颗粒细胞凋亡的影响

Chicken Bcl-2 Gene Cloning and It′s Effects on Growth and Apoptosis of Ovarian Granular Cells in Laying Hens

将鸡的 Bcl- 2基因克隆到真核表达载体 JL V中 ,构建了重组质粒 JL VB。 0 .2μg/孔 JL VB重组质粒经脂质体介导转染卵泡颗粒细胞 ,应用流式细胞术等方法分析了转染 Bcl- 2基因后原代细胞和传代细胞的生长与凋亡情况。与对照组相比 ,转染后的传代细胞分裂速度较快 (P<0 .0 1) ,凋亡比率较低 ,G2 / M+S期细胞比例极显著高于对照组 (P<0 .0 1)。这些结果表明 ,Bcl- 2基因具有促进细胞分裂、抑制细胞凋亡、延长细胞寿命的作用 ,这种作用是直接的。在转染时间为 12 h、DNA量为 0 .3μg/孔的条件下 ,较好的脂质体介导用量为 1.2 μL/孔。

Chicken Bcl 2 gene was recombinant with JLV plasmid and the recombinant expression plasmid JLVB was induced to chicken granulosa cells with liposome.The granulosa cells were isolated from follicles of laying Romen hens and incubated in 2 mL medium 199 supplementing with 5% calf serum for 3 days,and then were transferred by JLVB with 0 0,0 6,1 2,2 4 and 4 8 μL of liposome in 24 well plates.The growth and apoptosis of primary cells and subcultured cells were investigated by flow cytometry and other methods.The results showed that Bcl 2 gene transferred cells grew more quickly( P <0 01),with lower ratio of apoptosis and higher ratio of G 2/M+S phase( P <0 01),in comparison with the control cells during the subculture period.These results further indicated that Bcl 2 gene could play a direct roles in control of growth,a development and apoptosis of ovarian granular cells in hens.The plasmid JLVB transferred had a better result as 1 2 μL per well liposome was used.