| Human Ribonuclease Inhibitor (hRI), an acidic protein (pI~4.7), is ubiquitous in the cytoplasm of human cell with molecular weight of about 50kDa, and its gene locates in No.11 chromosome (11p15.5). Its three-dimensional structure is charactered by alternating units ofα-helix andβ-strand that form a striking horseshoe shape. The activity of ribonuclease A (RNase A) can been inhibited by hRI through tight combination with it (Ki=4.0×10-14M). Angiogenin (Ang) is a member of RNase A superfamily. Its amino acid sequence is highly conservative compared with RNase A. Based on the analysis of cDNA sequence, Ang is 35% identical with human pancreatic RNase A. Ang forms more restricted complex (Ki=7.1×10-16 M) with hRI than that formed by hRI and RNase A. Ang hold a robust capacity to induce blood vessel formation in vivo and in vitro. Because hRI can form the tight complex with Ang, so it can inhibit the activity of Ang. It has previously been shown that the placental ribonulease inhibitor can effectively block angiogenin-induced angiogenesis in the chick chorioallantoic membrane (CAM) assay. Our investigation showed that exogenous ribonuclease inhibitor can inhibit the tumor growth in mice, including Ca761, S180, H22 solid tumor. Neovascular glaucoma is a disease which can not been cured easily, resulted from retinal vein blocking, diabetic retinal ischemia or inflammation. They lead to neovascularization at anterior chamber angle and the surface of iris, and then anterior chamber angle adhesions. Consequently, neovascular glaucoma formed. The principal therapy of neovascular glaucoma is to relieve obstruction by trabeculectomy, followed by antivascularization drugs. Because hRI can inhibit Ang, it may be become a potential antivascularization drug. In this experiment, fusion protein GST-hRI was applied treatment New Zealand rabbits performed trabeculectomy to observe its antinovascularization.On the other hand, hRI consists a high content of cyeteine. There are 32 highlyconserved cysteine residues in every molecule, which leads hRI to great sensitivity to oxidants. At least 30 cysteine residues should exist with reduced thiol groups which is necessary for hRI to maintain its biological activities. High content of cyeteine maybe enable hRI to antioxidation. Our laboratory had performed experiments in vivo and in vitro about the antioxidative effect of hRI. We found that rat glial cell line C6 transfected with hRI cDNA can resist more injuries with H2O2 than the control C6 cells. The resistance to the oxidation stress of cells is monitored by morphological observation and assays of LDH, MDA, GSH level. hRI have protective effect on mice damaged by CCl4 through improve the activity of SOD and decreasing the content of MDA. A large quantity of free radical produced in the process of reduced monosaccaride oxidating damages proteins and membrane of lens epithelial cell. Degenerated proteins and damaged cells lead to genesis and development of diabetic cataract. In this experiment, GST-hRI was applid treatment diabetic cataract animal model of Wistar Rat to observe its protective effect on diabetic cataract.In order to save cost, simply processes, increase soluble yields, make purification easy, our laboratory constructed fusion expressive plasmid pGEX-6p-1-hri. Culture E.coli Rosetta with fusion expressive plasmid pGEX-6p-1-ri, collect cell pept, broke cells, centrifuge and then transfer supernatant to loading on Glutathione-Sepharose 4B affinity chromatography column to extract and purify fusion protein GST-hRI . After identified, it was cleaved with Prescission Protease, to evaluate the activity difference between fusion GST-hRI and purified hRI. Then GST-hRI was used to animal model.In this experiment, I found the activety of GST-hRI is superior to hRI at the aspect of inhibiting RNase A degenerating yeast RNA. GST-hRI have the protective effect on delaying the development of diabetic cataract and on antineovaccularization in a dose-dependent way.
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