Apoptosis And Proliferation As Well As Regulation Mechanism Of Hela And Bovine Granulosa Cells Transfected With Eukaryotic Expression Plasmid Encoding Two Copies Of Inhibin A (1-32) Fragments

The objective of this study was to determine the effects of the inhibin a (1-32) fusion gene expression on proliferation, apoptosis, cell cycle, inhibin receptor, estradiol (E2) and progesterone (P4) of bovine granulosa cells (GCs) and cervical cancer cells (Hela) with a series of techniques such as gene cloning, plasmid extraction, transfection, RT-PCR, Real-time PCR, confocal microscopy, thiazolyl blue tetrazolium bromide (MTT), flow cytometry, Radio-Immunoassays (RIA), ELISA. We have three purposes in this study: the frist, detection the quality of inhibin plasmid pXAISI-EGFP, in order to prepare the basis for inhibin DNA vaccine; the second, clarify the regulatory mechanism of inhibin in follicular development, for promote development new technology of the breeding, improve animal reproduction; the third, explore the role of inhibin on the uterus, in order to improve the rate of embryonic development and prepare the theoretical foundation for the development of new technologies for anti-cancer diseases.1. Fragment ISI-EGFP obtained from the plasmid pEGISI kept in our laboratory by digestion with the restriction enzymes EcoR I and Not I was inserted into the vector pVAX-asd, resulting in an eukaryotic expression plasmid of inhibin pXAISI-EGFP, which encoding two copies of the inhibin a (1-32) fragments, hepatitis B surface antigen gene S (HBsAg-S) and green fluorescent protein gene (EGFP);2. Eukaryotic expression plasmid of inhibin pXAISI-EGFP transfected into GCs and Hela cells at 48h, the transcription process of ISI gene was confirmed by RT-PCR;3.The localization as well as expression of ISI-EGFP fusion protein was observed by confocal microscopy. The results showed that the inhibin fusion protein in Hela cells was localized only in the nucleus; inhibin fusion protein in GCs was localized only in the cytosol;4. Apoptosis (V-FITC/propidiumiodide) was analyzed by flow cytometry. The results showed that in the Hela cells, transfection can induce significant apoptosis, however inhibin can inhibtor apoptosis. After transfection for 48h, Hela cells in the control group, pXAISI-EGFP experimental group, pVAX-asd-EGFP negatie control apoptotic rates were:8.20±3.92%,24.86±5.38%,31.83±8.37%(p<0.05); After transfection for 48h, we proved that inhibin could induce the apoptosis of GCs by microscope;5. Proliferation was assessed using thiazolyl blue tetrazolium bromide (MTT). The results prove that proliferation of bovine granulosa cells and Hela cells were inhibited after transfection for 48h. The experimental group compared with control group and negative control group in Hela cells, inhibition rates were 74.35±1.51% and 38.64±3.60%; The experimental group compared with the control group and negative control group in GCs, inhibition rates were 41.86±1.24% and 26.31±0.78%;6.Cell cycle (propidium iodide) was analyzed by flow cytometry. The results showed that Inhibin was significant effectd on the cell cycle, both in Hela cells or in GCs, inhibin would increased the number of the cells in the S phase. GO/Gl phase,S phase and G2/M phase in blank control groups of GCs:64.92%±1.75,15.53%±1.98 and 19.55%±1.32; in pXAISI-EGFP groups:55.07%±1.55,25.50%±1.48 and 19.44%±1.71; in pVAX-asd-EGFP groups:62.71%±1.21,4.37%±1.37 and 32.91%±1.28. GO/G1 phase, S phase and G2/M phase in blank control groups of Hela cells:56.27±2.57%,30.24±1.36% and 13.49±3.94%; in pXAISI-EGFP groups 55.75±2.70%,39.41±2.71% and 4.84±0.04%; in pVAX-asd-EGFP groups 56.11±2.69%,36.20±1.48% and 7.67±1.25%.7.Four genes related apoptosis and inhibin receptor TGFβR3 were detected by Real-time PCR for further study. The results showed that levels expression of anti-apoptosis genes Bcl-2 and NFκB were down regulation, respectively as 0.62±0.02 and 0.38±0.01, pro-apoptosis gene Bax and wild type P53 up-regulation, respectively as 3.24±0.12 and 2.58±0.06 compared with controls in Hela cells; however, anti-apoptosis genes(Bcl-2, Bcl-xl) and pro-apoptosis gene(Bax, p53) all were up-regulation, respectively as 1.51±0.11,1.92±0.15,1.59±0.09,1.23±0.12 in GCs. Levels expression of inhibin receptor TGFβR3 in Hela cells and GCs, respectively16.15±0.14 (p<0.01) and 1.23±0.04 (p<0.05);8.Detection of estradiol, progesterone, and inhibin levels after transfection for 24h, 48h,72h,96h in medium of GCs by Radio-Immunoassays (RIA) and ELISA, The results showed that estradiol and estradiol/progesterone were increased after transfection for 24h, then lower; progesterone level was lower all the time compared with controls. However, inhibin level was increased all the time compared with controls, but lowest at 72h.These results indicate that inhibinα(1-32) fusion gene has antagonistic effect on cervical cancer cells and GCs.