The Effects of RMP on Biological Property of Hepatoma Cells SMMC-7721
|Keywords||RMP polyclonal antibody hepatoma cell cell proliferation cell apoptosis|
Objective:To further study the effects of RMP (RPB5-mediating protein) on biological property we prepare polyclonal antibody against,and estab investigate RMP functionin hepatoma cells.Methods:1. Preparation of RMP polyclonal antibody2. Western blot was used to detect the specificity of RMP polyclonal antibody3. Establish RMP function feedback system,RT-PCR and Western blot was used to detect the expressin of mRNA and protein.4. MTT assay was used to detect proliferation of each cell line.5. MTT assay was used to detect capability of cell adhesion .6. Scarification assay was used to detect capability of cell migration.7. Transwell was used to detect attack of each cell line.8. From the irradiation of the 60Coγ-ray, flow cytometry was applied to measure the effect of RMP on cell cycle and apoptosis of SMMC-7721.9. RT-PCR was used to detect the expression of related genes of cell cycle and apoptosis.Results:1. Purified GST-RMP protein was used to immunize rabbits to prepare polyclonal antibody. Western blot indicated that the polyclonal antibody had high antigenicity.2. RMP function feedback cell lines were established. Result of RT-PCR and Western blot showed that RMP gene could feedback expression in SMMC-7721 cells.3. MTT assay showed that RMP promoted the growth of SMMC-7721 cells. The adhesion capability of cell lines was decreased.4. Scarification assay showed that the migration capability was enhanced compared with the negative control.5. The attact of cell line was strengthened.6. From the irradiation of the 60Coγ-ray , the results of flow cytometry demonstrated that the rate of G2 phase arrest and apoptosis was reduced with time.7. RT-PCR showed that the expression of bax,Caspase 3 was down-regulated and the expression of Cyclin B,CDK1,p21 and Bcl-2 was up-regulated.Conclusion:GST-RMP protein was expressed in BL21 (DE3) inducing by IPTG, prepare and obtain RMP polyclonal antibody. RMP could promote the growth of SMMC-7721 cells. At the same time,the adhesion capability of stable cell line was degraded but the migration capability was increased. The attact of cell line was strengthened. In the case of 60Coγ-ray irradiation,reply expression the RMP gene leaded to G2 phase arrest and apoptosis reduction. These experimental results indicated that RMP plays an important role on anti-radiation and anti-apoptosis in the process of apoptosis of human hepatoma cells line SMMC-7721 induced by 60Coγ-ray.