CREB蛋白与羊卵巢上皮细胞生长和凋亡关系的探讨

Investigation into CREB induced growth and apoptosis of ovine ovarian surface epithelial cells

目的 探讨cAMP反应序列结合蛋白 (CREB蛋白 )在羊卵巢上皮细胞生长和凋亡中的作用。方法 将CREB蛋白基因转入腺病毒载体PACsk 2CMV ,将野生型的CREB蛋白 (CREBWT)和主要的非活性形式的CREB蛋白 (CREBS13 3A)表达载体腺病毒转染羊卵巢上皮细胞 ,Westernblotting检测CREB蛋白的表达 ;应用CellTiter 96Aqueous细胞生长分析仪测定细胞数目 ;Caspase 3/ 7活性分析仪测定Caspase的活性。结果 Westernblotting显示CREB蛋白表达载体腺病毒转染羊卵巢上皮细胞后 ,细胞表达CREB蛋白 ,且随着病毒浓度的增加 ,CREB蛋白的表达增强 ;CellTiter 96Aqueous测试结果显示 :当病毒浓度为 10 7～ 10 9/ml时 ,细胞数目增多 ;当病毒浓度为 10 10 /ml时 ,细胞数目明显减少 ,CREBS13 3A表达载体的作用比CREBWT和大肠杆菌乳糖操纵子Z(LacZ)明显 (P <0 0 5 ) ;Caspase 3/ 7活性测定显示 :当病毒浓度为 10 10 /ml时 ,经LacZ表达载体转染的羊卵巢上皮细胞Caspase 3/ 7酶活性增加 2～ 3倍 ,CREBWT增加 5倍 ,CREBS13 3A 增加 11倍 (P <0 0 5 )。结论 CREB蛋白的表达水平及CREB蛋白的磷酸化程度对卵巢上皮细胞的存活是非常重要的 ,CREB蛋白及其相关因子在卵巢上皮细胞的生长和凋亡中具有重要的作用。

Objective: To investigate the role of cAMP Response Element Binding(CREB)in the regulation of ovine OSE cell growth and apoptosis. Methods: The CREB gene was introduced into the adenoviral vector pACsk.2CMV. Wild-type CREB and a dominant negative form of CREB (S133A) were introduced to ovine OSE cells by adenoviral-mediated expression. CREB protein was observed by Western blotting. Cell growth was measured by the Cell Titer 96 Aqueous cell proliferation assay. Caspase activity was measured by caspase 3/7 assay. Results: CREB protein was observed by Western blotting when ovarian surface epithelial (OSE) cells were infected with Ad CREB WT and CREB S133A . And the levels of the protein expression increased when OSE cells was infected with an increasing concentration of infecting virus. OSE cell number increased in the detection by the CellTiter 96 Aqueous when OSE cells were infected with 10 7 to 10 9 viral particles/ml of either Ad CREB WT or Ad CREB S133A . However, when cells were infected with 10 10 viral particles/ml, the expression of CREB S133A was reduced in cell number compared with OSE cells infected with either Ad CREB WT or Ad LacZ. The result of caspase 3/7 activity detection showed when cells were infected with 10 10 viral particles/ml, Ad LacZ induced caspase 3/7 activation increased by 2～3 folds above that of mock-infected cells and at the same viral concentration, however, caspase 3/7 activation increased by 5 folds in OSE cells when infected with Ad CREB WT and more significantly 11 folds in cells infected with Ad CREB S133A (P<0.05). Conclusion: The level and the phosphorylation of CREB protein are important to the cell survival. CREB and CREB associated factors play an essential role in the growth and apotosis of OSE cells.