Studies On The Inhibitory Action Of Ribonuclease Inhibitor (RI) Gene On Xenografted Tumor Of Ovarian Cancer In Nude Mice

Ribonuclease inhibitor (RI) is an acidic cytoplasmic glycoprotein with a 50 kDa estimated molecular weight. RI can inhibit the activities of Ribonuclease A (RNase A). RI is mainly constructed by seven leucine-rich repeats and every repeat contains 57 amino acid residues. The regular circular permutation of leucine-rich repeats makes the spatial structure of N-end approach to C-end. Every repeating unit also contains two A and B leucine repeats. RI is rich in leucine and cysteine. RI can combine with RNase A (Ki=4×10﹣14mol/L)and inhibits the activities of RNase A. Angiogenin (Ang)forms more restricted complex with RI than that formed by RI and RNase A. Ang is a protein with molecular weight of 14.4KDa .It is secreted by tumor cells and normal cells and can induce the angiogenesis of tumor remarkably. Ang is highly identical with RNase A and they have a very similar spatial structure. They can both form tight complex with RI. So RI can inhibit angiogenesis and will be a promising drug of anti- angiogenesis of tumor.Malignant tumors detriment the health of mankind severely. Ovarian cancer is one of three main malignant tumors of the female reproductive system. Owing to its multi-category of tissue and lack of effective diagnostic strategies of early stage, the 5-year survival rate for patients with ovarian cancer is still low. Moreover, the curative effects of surgery, irradiation and chemotherapy are not excellent. It severely threatens the life of patient with gynecological tumor. Neovascularization plays an important role in the growth and metabasis of tumor. Neovascularization is the process of persistent growth of blood vessels stimulated by tumor and supply thetumor with oxygen and nutrients. Clinical trials and experiments indicated that the tumor will stop grow, if the tumor achieves 1~2mm3 without new blood vessel formation. Furthermore, Neovascularization is the premise of infiltration and metabasis of tumor. So determinate goals of treatment can be achieved through inhibiting angiogenesis of tumor.The essence of gene therapy is to recover the structure and function of tissues and cells through delivering a normal gene into abnormal cells in place of mutational or absent gene or knocking out the mutational gene. The gene therapy of anti-tumor vessel is prominent with regard to the latest curative study of ovarian cancer. This strategy has gain determinate curative effects in animal experiments and some clinical patients with stageⅠ,ⅡandⅢ. It will be a new effective curative method after operation, irradiation and chemotherapy of ovarian cancer.RI can inhibit the activities of Ang obviously. Ang is secreted many kind of cells besides tumor cells and can promote proliferation of vascular endothelial cells. So RI has the effect of anti-tumor. Animal experiments indicated that RI can inhibit the growth of some transplantation tumor of animal in vivo. Previous experiments showed that RI gene powerfully inhibited the growth of C6 glioma cells. In addition, RI could inhibit some kings of transplant tumor growth in mice, including melanoma, S180 sarcoma, and breast solid tumor.This study expands further the research of the inhibitory action of RI on ovarian cancer on the basis of finished study. The purpose of this dissertation is to extract and purify RI gene and to study the effect of RI on gene therapy of anti-tumor vessel of ovarian cancer. The main work is as following:Firstly, the extraction, purification and identification of PLNCX-RiPLNCX-Ri transformed competent DH5α-bacteria. PLNCX-Ri was prepared through alkaline lysis of SDS after transformation. PLNCX-Ri was purified by precipitation method of PEG. PLNCX was conducted by the same method above. The purity quotient and concentration of RI gene were identified by agarose electrophoresis and UV-754 ultraviolet spectrophotometer. The results showed that the bands of PLNCX-Ri and PLNCX appeared. The purity quotient and concentration of DNA meted theexperimental demand.Secondly, the cell culture of ovarian cancer (Skov3), the establishment of models of xenografted tumor of ovarian cancer in nude mice and curative analysis of RI gene.The cell line Skov3 of ovarian cancer was selected and cultured. The nude mice were injected hypodermically the tumor cells of logarithmic growth so as to establish the models of xenografted tumor of ovarian cancer. Injected nude mice by PLNCX-Ri and PLNCX were used respectively as experimental group and control group. The incidence rate of tumor, inhibiting rate of tumor, latent periods and tumor weight were observed by contrast. The different expressions of RI gene in two groups were analyzed by immunohistochemistry. Microvessel density was observed through HE dyeing. The results showed injected PLNCX-Ri group showed lower incidence rate of tumor, longer latent periods, lighter tumor weight, higher expression of RI gene and lower microvessel density than those in the control group. The result suggested the inhibition of tumor is related to reduction of angiopoiesis.The above-mentioned finding indicated RI gene inhibited the growth of xenografted tumor of ovarian cancer. The mechanism relates to that RI inhibited angiogenesis of tumor by inhibiting the activities of Ang. This experimental data could provide valuable evidence for further studies of RI gene and clinical trials.