Regulation Of Inhibin On Testosterone Secretion In The Leydig Cells Of The Rats And Consruction And Characterization Of PEGISI-CPG

In order to understand the regulating mechanism of Inhbin on testosterone secretion by the rat Leydig cells and to futher improve transfection efficiency of the Inhibin fusion expression plasmid, this experiment was conducted to explore the effect of inhibin on the experession of enzymes related to testosterone synthesis by transfecton of inhibin fusing expressing plasmid (pEGFP) in vitro or orally administering inhibin gene vaccine (pXAIS) in vivo. In the meanwhile, the newly constructed inhibin gene plasmid (pEGISI-CPG) was acquired by insertion of a new adjuvant CPG-OND into the restriction enzyme cutting site (SaiI) to improve the transfection efficiency,.The main research results were as follows:1 Primary culture and transfection of the Leydig cells of adlut rat testisEight-week male rats were randomly killed and the testises were isolated and the isolation purity reached 90%. What's more, the optimized conditions of transfection were screened by using different dosage of lipofectamine 3000 and pEGFP taking the Ledig cells of the rat testis as the model and the results showed that the highest transfection efficiency was 41.78±1.91% when using 0.75 ?L of lipofectamine 3000 and 0.75 ?g of pEGFP plasmid.2 Effect of pEGISI on the expression of mRNA expression levels of the differert key enzymes related to testosterone synthesismRNA expression levels of the different key enzymes related to testosterone synthesis(StAR, P450scc, CYP-17 and 3?-HSD) were detected and the results showed that:(1) The StAR mRNA expression levels of the pEGISI plasmid transfected cells were higher when compared to the transfected pEGFP plasmid (P<0.05); (2)The P450scc mRNA expression levels of the pEGISI plasmid transfected cells were remarkably higher in comparision with the pEGFP plasmid transfected cells (P<0.01); (3)The CYP-17 mRNA expression levels of the pEGISI plasmid transfected cells were obviously higher than in the pEGFP plamid (P<0.05); (4) The 3(3-HSD mRNA expression levels of the pEGISI plamid transfected cells were significantly higher in contrast to the pEGFP transfected plasmid (P<0.01). The above results demonstrate that pEGISI transfection can promote mRNA expression of the different testosterone synthetase in the Leydig cells of the rat testis invitro.3 Effect of pEGISI on the expression of protein expression levels of the different key enzymes related to testosterone synthesisProtein expression levels of the different key enzymes related to testosterone synthesis (3?-HSD, P450scc, StAR, cAMP and aromatizing enzyme) were detected by ELISA and the results showed that:(1) The 3?-HSD protein expression levels (106.76±3.93) of the pEGISIplamid transfected cells were higher than those in the pEGFP transfected plasmid (86.46±4.91) (P<0.05); (2) The P450scc protein expression levels (135.96±4.86) of pEGISI plamid transfected cells were higher in comparison with pEGFP plamid transfected cells (115.58±5.31) (P<0.05); (3) The StAR protein expression levesl (118.67±3.29) of the pEGISI plasmid transfected cells were higher when compared to the thansfected pEGFP plamid (97.37±4.68) (P<0.05); (4) The cAMP contents (6.35±0.49) of pEGISI plamid transfected cells were significantly higher in contrast to pEGFP transfected plasmid (4.43±0.74)·(P<0.05); The aromating enzyme expression level of transfection pEGISI plasmid (68.75±5.86) were higher compared to the pEGFP transfected plasmid (51.79±3.86) (P<0.05). These results illustrate that pEGISI transfection can promote protein expression of the different testosterone synthetase in the Leydig cells of the rat testis in vitro.4 Effect of orally administered inhibin gene vaccine (pXAIS) on the expression of mRNA expression levels of the different key enzymes related to testosterone synthesisRelative mRNA expression levels of the different testosterone synthetase (P450scc, StAR,3?-HSD and CYP-17) were determined on the 7th week after orally administering inhibin vaccine (pXAIS). The results showed that there was no obvious increase in the mRNA expression levels of the appointed key enzymes in the four tested groups compared with the control group (P>0.05).5 Effect of orally administered inhibin gene vaccine (pXAIS) on the expression of protein expression levels of the different key enzymes related to testosterone synthesisProtein expression levels of the different testosterone synthetase (P450scc, StAR and 3?-HSD) were determined on the 7th week after orally administering inhibin vaccine (pXAIS).The results showed that there was no obvious increase in the protein expression levels of the appointed key enzymes in the three tested groups compared with the control group (P>0.05).6 pEGISI-CPG construction and characterizationA 400 base pair band was exhibited after PCR and agarosegel electrophoresis, which was identical to the designed primer size and there were a large number of fluorescences after T293 cell transfection. These results reveal that the pEGISI-CPG was successfully constructed.The above results expound that key enzymes related to testosterone synthesis can be obviously expressed by Inhibin transfection in vitro, not affected by orally adminstrated Inhibin gene vaccine. In addition, pEGISI-CPG is successfully constructed after detection of transfection with T293 cells and PCR electrophoresis.