Effect Of Several Plasmids Of SS And MSTN Genes On The Cell Growth And Proliferation As Well As Appoptosis Of Hela Cells

The objective of this study was to determine the effects of SS and MSTN gene expression on proliferation, apoptosis, cell cycle, SSTR of cervical cancer cells(Hela) with a series of techniques such as cell culture, plasmid transfection, RT-PCR, Real-time PCR, confocal microscopy, MTT, flow cytometry. The purposes of present study to:(1) Comparise of the effect of growth and proliferation in SS and MSTN gene plasmid transfected in Hela cells in order to select the best group in promoting apoptosis of Hela cells; (2) estimate the pathway involved in anti-tumor effects of SS and MSTN in order to provide available way for gene therapy of tumors; (3) Compare the expression abundance of the plasmid selected to provide a theory basis for exploring SS and MSTN of DNA vaccine at cellular level.1. SS and MSTN gene plasmid were transfected in Hela cells with LipofectamineTM2000 for 48h. SS and MSTN mRNA were subsequently detected by electrophoresis after reverse transcription, which inditates that SS and MSTN gene plasmid can be transcripted normally in Hela Cells.2. After 48 hous'transfection of plamid in Hela cells, the Hela cells was treated with DAPI dark staining. Based on the confocal microscopy method, it was found that SS fusion proteins was observed only in cellular nuleus of transfect Hela cell, but MSTN fusion protein was observed in both cytoplasm and neculus.3. Proliferation was assessed using MTT assay in Hela cells transfected with SS or MSTN gene for 24h,48h,72h and 96h, respectively. It was shown that both SS and MSTN transfection suppressed cell proliferation (P<0.05) as compared to the control.. Among the treatments, pEGMS transfection of 72h showed the lowest cell proliferation rate.4. By emptying flow cytometry, apoptosis (Annexin V-APC/7-AAD) assay was conducted on the Hela cell with 48 hours'transfection of SS or MSTN gene. It was shown that R5 zone cells (early apoptosis cells) in treated cells were significantly increased (P<0.05) comparing to that of control. The highest rate of apoptosis was observed in pEGMS treatment about 27.47±2.86%, which was 8.15% higher than the control. It was implied that SS and MSTN gene plasmid can promote apoptosis of Hela cells.5. By using flow cytometry, cell cycle assay was conducted in Hela cell transfected with SS or MSTN gene. It was found that the number of cells in G0-G1 phase was decreased by gene trasnfection (P<0.05), while the number of cells in S phase and G2-M phase were increased by gene trasnfection (P<0.05). The results indicated that SS and MSTN plasmids arrested Hela cells mainly in S phase.6. SSTR-1, SSTR-2, SSTR-3, SSTR-4 and SSTR-5 receptors cDNA could be detected by reverse transcription PCR in Hela cells transfected with plasmid SS and MSTN for 48h, which indicated that the receptors of the 5 kinds participated in the regulattion of SS expression.7. MRNA expression of three genes related to apoptosis (Bax, Bcl-2 and p53) were detected by real time PCR. It was shown that the mRNA expression abundance of Bax and p53 were up-regulated by gene transfection (P<0.05), while the mRNA expression of Bcl-2 was down-regulated by gene transfection (p<0.05).These results of present study indicate that SS and MSTN gene plasmids inhibited cell proliferation. Additionally, pEGS2SS-V and pGS/2SS-IL6-asd showed the more significant effect on inhibiting cell proliferation in SS gene plasmids and pEGMS showed the most significant effect on inhibiting cell proliferation while pEGS/2SS+ pEGMS and pEGS2SS-V + pEGMS were not more significant than others. Somatostatin-induced and Myostatin-induced cell growth inhibition includes induction of cell cycle arrest and apoptosis. SS and MSTN plasmids arrested Hela cell mainly in S phase and promoted apoptosis of Hela cells as indicated by increased bax expression and decreased bcl-2 expression. From the perspective of transcription and expression, pEGS2SS-V and pGS/2SS-IL6-asd gene plasmids could be expressed better in Hela cells which were indirected that pEGS2SS-V and pGS/2SS-IL6-asd gene plasmids have a better effect on SS of DNA vaccine at cellular level.