| Ovarian cancer is the leading cause of mortality from gynecologic malignancies, as a result of the morbidity concealment, lacks the incipient symptoms, approximately 75% of patients are diagnosed in an advanced stage, thus the prognosis is very bad. Although the traditional treatment way consummated unceasingly in recent years, the five-year survival rate has not made distinct elevation, therefore, sought for the new method of treatment will have great significance for the ovarian cancer. Since Dr. Folkman established the relationship between angiogenesis and tumor growth in 1971 for the first time, the angiogenesis as well as the anti-angiogenesis therapy has been the hot spot of the tumor research area. It has already proved that like most of the solid tumors, the growth and metastasis of ovarian cancer has close relation with the new blood vessels formation . Thus, therapies targeting angiogenesis may bring new hope to the treatment of ovarian cancer. Although there are many angiogenesis inhibitors have entered clinical and have obtained promising therapeutic effect at present, the proper disadvantages of protein drugs such as short half-life , poor stability and high price are actually not allowed to be neglected. However,the anti-angiogenesis gene therapy can solve this problem to a great extent and attracts more and more attention accordingly.Human ribonuclease inhibitor (hRI) is a cytoplasmic acid protein with a molecular weight of 50 kDa. It lies in all the mammalian organs and tissues extensively,in which placental tissue contains most. RI can inhibit the activity of RNase A competitively by tight combination with it at a stoichiometric ratio 1:1, and then regulate the RNA level of cytoplasm. Angiogenin (Ang),a protein with angiogenic activity , has elevated expression in many tumor organizations. It is a member of the ribonuclease super family and has 35% identity with RNase A. The research indicated that the combining power between RI and Ang was much stronger and the Ang combining to RI lost its angiogenic activity, thus the tumor growth was reduced. Therefore , RI can inhibit the tumor growth by inhibiting the formation of its blood vessels, so it will be a potential anti-tumor drug. This study aimed to establish an ovarian cancer cell line SKOV3 stably expressing RI gene by the method of gene transfection and to investigate the effects of RI on the cell growth in vitro ,then hope to afford cell model and experimental evidence for the future study of RI gene in the therapy of ovarian cancer.Objective:To establish an ovarian cancer cell line SKOV3 stably expressing RI gene and to explore the effects of exogenous gene on the cell proliferation in vitro.Methods:We transfected the recombinant expression vector pLNCX-ri containing exogenous human RI cDNA into SKOV3 cell line by lipofectamine and classified the experimental cells into SKOV3 /RI, SKOV3 /Neo and SKOV3. The stable cell line was obtained by the selection of G418. The expression of RI gene was detected by reverse transcriptase- polymerase chain reaction and immnohistochemistry and the effects on the proliferation of SKOV3 cells were examined by MTT and FCM.Results:Exogenous RI gene had successfully been transferred into SKOV3 cells and obtained permanent expression. The growth speed of SKOV3 / RI was slower than the other two groups, there was significant difference between them ( P < 0.01). The SKOV3 / RI cells of G1 phase increased to 69.23% , which that of S phase decreased to 24.34% by analyzing cell cycle, there was significant difference compared with the other two groups (P < 0.01 ) . The cell apoptosis rate in SKOV3 /RI group(58.25 % )was significantly higher than those in SKOV3 group(17.97%,P<0.01)and SKOV3/Neo group(21.42%,P<0.01),but the apoptosis rate between the later two groups was similar(P>0.05).Conclusion: We can establish an ovarian cancer cell line stably expressing RI gene mediated by lipofactamine and the RI gene can inhibit the proliferation of SKOV3 cell in vitro. |
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