籽鹅卵泡颗粒细胞烯醇化酶过表达模型的建立及其对孕酮分泌的影响

Garnulosa cells model tansfected by ENO1 overexpression adenovirus vector and the effect of ENO1 overexpression on progesterone secretion

目的:建立α-烯醇化酶(ENO1)腺病毒过表达载体转染原代培养籽鹅卵泡颗粒细胞模型,探讨ENO1过表达对颗粒细胞孕酮分泌的影响。方法:采用ENO1腺病毒过表达载体以梯度感染复数值(MOI)100、250、350、400pfu/cell转染原代培养籽鹅卵泡颗粒细胞,于转染后24 h、48 h,荧光倒置显微镜下观察绿色荧光蛋白(GFP)表达。双抗体一步夹心法酶联免疫吸附试验(ELISA)研究ENO1过表达对颗粒细胞孕酮分泌的影响。结果:最佳转染条件是,当MOI为350 pfu/cell,转染48 h后,转染率达100%;通过荧光定量PCR与Western blot法,检测到ENO1 mRNA与蛋白均过表达(P<0.01);与培养液组和腺病毒空载体组相比较,ENO1过表达使颗粒细胞孕酮分泌量极显著增加(P<0.01)。结论:ENO1过表达会使体外原代培养的籽鹅卵泡颗粒细胞孕酮分泌量增加。

Objective： To construct primary cultured granulosa cells model of Zi Gooses tansfected by a-enolase （ENO1） overexpression adenovirus vector, and to detect the effect of ENO1 overexpression of granulose cells on progesterone secretion. Methods： Granulosa cells were infected with Ad-CMV-ENO1 in gradient multiplicity of infection（MOI）Ievels： 100, 250, 350 and 400 pfu/cell.Twenty four hours and 48 h af- ter infection, green fluorescent pr0tein（GDP）was respectively detected by fluorescence inverted microscopy. The effect of ENO1 overexpression of granulose cells on progesterone secretion was detected by the step double antibody sandwich enzyme-linked immunosorbent assay （ELISA）. Results：The optimal infection rate （100%）was achieved when MOI was 800 pfu/cell,4$h after infection. Real time RT-PCR and Western blot showed that the level of mRNA and protein expression EN01 were increased significantly after infection （P 〈 0.01） ; The granulosa cells pro- gesterone secretion of Ad-CMV-ENO1 group increased signigicantly （ P 〈 0.01 ）. Conclusion： ENO1 overexpression could make the primary culture follicle granulosa cells in vitro improve progesterone secretion.