Effect Of Inhibin Gene Transfection On Proliferation, Apotosis, Steroidogenesis Of Rat Leydig Cells

The objective is to determine the effects of an inhibin a (1-32) fragment gene transfection on proliferation, apoptosis, and steroidogenesis of rat Leydig cells. Leydig cell is isolated from adult rat testes, and purifyied, identified before used to be transfected with inhibin gene. Transfection conditions of Leydig cell are optimized while lipofectamine2000and lipofectamine LTX are used for transfection. Proliferation, apoptosis and testosterone secretion of Leydig cell are detected after transfection with pEGISI.Results:1. The viability and purity of the isolated Leydig cells were96.33%and91.9%respectively. Transfection efficiency of lipofectamine LTX was higher than lipofectamine2000. The transfection efficiency was highest (33.68±1.67)%, when the ratio of lipofectamine LTX and pEGISI was2μ:1.0μg.2. Transfection inhibited Leydig cell proliferation compared to control group (P<0.05), but compared to pEGFP group with no significant effect (P>0.05). Apoptosis was higher (P<0.01) than in control,but compared to pEGFP group with no significant effect (P>0.05). Transfection with pEGISI groups and controls increased testosterone synthesis after culturing for48h, with suppression (P<0.01) in transfected Leydig cells after96h. Transfection reduced (P<0.01) testosterone secretion in Leydig cell compared to controls after48h and96h, and increased (P<0.01) testosterone secretion in Leydig cell compared to pEGFP groups after96h。Conclusion:The application of a low concentration of collagenase for differential digestion allows isolation of large quantities of viable Leydig cells for the study of Leydig cell function. The ratio of lipofectamine LTX and pEGISI which was2μL:1.0μg were the best transfection reagent and condition. Transfection inhibited Leydig cell proliferation and apoptosis,but increased testosterone secretion compared to pEGFP group.