Proliferation And Apoptosis Effect Of Hepatocellular Carcinoma Cells And The Expression Of The Gene And Protein Of Survivin, Fas,TNF-a By Combining Somatostatin And Oxaliplatin To Intervene On HepG2

Objective : The experiment combines Somatostatin (SST) and Oxaliplatin (L-OHP)to intervene the cultured out-of-human body HepG2. The proliferation and apoptosis effect of HepG2 are checked through the method of MTT,DAPI and DNA-Ladder. The expression of the gene and protein of survivin, fas ,TNF-a are checked through PT-PCR and Western Blot method.This experiment tries to explore the mechanism of the combination effect of the two drugs, and provide the laboratory basis of liver cancer pathogenesis and treatment.Methods: We establish cell models with HepG2 and set up scientific test groups according to medical statistics adopting random samples. There are 3 small comparison groups and 5 small experimental groups.6 parallel cases for each small group and altogether 48 cases. We use Oh, 0 concentration as the blank comparison group(no drug group), we use Somatostatin (SST) 1250 ng/L or Oxaliplatin (L-OHP) 5mg/L as one drug to intervene the other two comparison groups respectively. The 5 experimental groups are divided according to different dose of first drug intervention(Somatostatin 125ng/L or 1250ng/L or 2500ng/L) and different time interval of second drug intervention(add Oxaliplatin after 24h or 48h or 72h respectively),ref figure 1. We apply MTT and DAPI to check the cancer cell proliferation and apoptosis; apply DNA Ladder to check the splitting condition among Nucleosomes ; apply RT-PCR and Western-blot to check the expression of the gene and protein of survivin,fas and TNF-a. P<0.05 is statistically significant after we analyze the data with the SPSS13.0 statistic soft ware. Results:1) HepG2 cells, intervened by the combination of oxaliplatin and somatostatin, can be observed a growth inhibition under the inverse phase microscope. With the increase of concentration and the passage of time, the number of apoptotic cells gradually raises.2) MTT indicates an apparent decline of cell survival rate in the Somatostatin and Oxaliplatin combination experimental groups than in the blank comparison group and the one drug intervention groups.; 3) DAPI shows a gradual intensification of nucleus cracking. At the earlier stage, nucleus swelling and deformation appear; at the later stage, nucleus cracking and the formation of small apoptotic bodies appear. 4) DNA Ladder shows the formation of apoptotic ladder, especially apparent in the Somatostatin and Oxaliplatin combination experimental groups. 5)PT-PCR electrophoresis indicates a gradual decrease of survivin mRNA and a gradual increase of fas mRNA and Tnf–a mRNA(P<0.05). 6) Western blot electronphoresis indicates a gradual decrease of survivin protein and a gradual increase of fas protein and Tnf-a ptotein. The combining use of Somatostatin and Oxaliplatin can weaken the expression of survivin mRNA and protein and enhance the expression of fas &Tnf–a mRNA and protein.Conclusion 1)Survivin, fas, TNF-a may mediated liver cancer apoptosis. 2)Oxaliplatin and somatostatin can reduce the growth speed of Hepatocellular carcinoma cell ,can induce the Apoptosis of Hepatocellular carcinoma cell. 3)Combined use of somatostatin and oxaliplatin can significantly induce apoptosis in human hepatoma cells. This effect may be related to reduced survivin mRNA and protein, increased fas &TNF-αmRNA and protein expression. Yet it remains to be a question for further research if Surivin, which plays the roll as the upstream acting element of fas, Tnf-a, acts as the promoter.