| Backgrounds: Human ribonuclease inhibitor (hRI) is ubiquitous in the cytoplasm of human cell with molecular weight of about 50 KD and its gene locates in the No.11 chromosome (11p15.5). hRI consists of 15 leucine-rich repeat( LRP). Its tertiary structure is symmetrical horseshoe-shape. Moreover, according to its primary structure, there are 32 highly conserved cysteine residues in its molecule;this results in great sensitivity to oxidants. And at least 30 cysteine residues should exist with reduced thiol groups which is necessary for hRI to maintain its biological activities. RI can inhibit the activity of ribonuclease A (RNase A) by tight combination with it (ki=4.0×10-14M). The amino acid sequence of angiogenin (Ang) is high conservative compared with RNase A. Ang is a member of RNase superfamily. Ang is produced and secreted by tumor cells and normal cells and its molecular mass is 14.4 kDa. It has a robust capacity to induce blood vessel formation in vivo and in vitro. It is expressed widely in many kinds of cell and tissue, such as cultured human colon cancer cells and hepatic cells, gastric cancer, colonic cancer, mammary cancer, carcinoma of corpus uteri and so on. It also exits in normal tissues and cells, such as plasm, renal cells, epithelial cells, smooth muscle cells, blood vessle epithelial cells, fibroblasts and so on. The wide expression of Ang suggests that it is restrictively controlled in the body. On the basis of analysis of cDNA sequence, Ang is 35% identical with human pancreatic RNase. Ang forms more restricted complex (Ki=7.1×10-16 M) with RI than that formed by RI and RNase A. X-ray analysis showed that Ang had a very similar space structure with RNase A.They can both form tight complex with RI. Because RI can form the tight complex with Ang, so it can inhibit the activity of Ang,RI may inhibit angiogenesis through blocking on b-FGF pass way too.Objective: In order to observe the influence of GST-RI on cerebral ischemia-reperfusioninjury in rats, the experimental model was established in rats. The other aim is to evaluate the inhibiting effect of fusion protein GST-RI on tumor growth in the animal body.Methods: In this experiment, the fusion expressive plasmid pGEX-6p-1-hri was transfected into the bacterium Rosetta. The fusion protein GST-hRI was induced at 16 degree Celsius with 0.5mmol IPTG for 10 hrs. After hypersound, the supernatant including fusion proteins was collected and purified through GST affinity chromatography. Concentrated by dialysis against 20% PEG-6000, purified GST-hRI was ready for administration to peritoneum of the mice. Low GST-hRI, middle GST-hRI, high GST-hRI and GST were pre-administered to the mice. Seven days later, ligature both sides of common carotid artery for 30 minutes followed by reperfusing for 24 hours in mice. Decollate them to die. The protective antioxidation effects of GST-hRI fusion protein was evaluated by determining the level of SOD (Superoxide Dismutase),MDA (malondialdehyde) and GSH-Px (Glutathione peroxidase) .Pathological analysis on H&E-stained slides was carried out to observe morphological change of the hippocamp of brain.The mice inoculated B16 melonoma were hypodermicaly treated with physiologic saline,low GST-RI,high GST-RI,and Cyclophosphamide , respectively, to know whether RI can inhibit the tumor growth. Results:1. The data showed that GST-RI significantly decreased level of MDA and (had) obviously increased (protective effect on) activity of SOD, GSH-Px in the hippocamp of brain that underwent ischemia following reperfusion in mice.2. The mice carrying B16 melonoma were treated through hypodermic injection with the GST-RI, there's the linearity relation for the dose of GST-RI and the effecting to inhibit the tumor. When the dose is 5000U(0.52mg/kg) ,the inhibition rate is 34.62%.When the dose is up to10000U(1.04mg/kg), the inhibition rate is 46.32% and the average inhibition rate is 40.50% .Our date shows that the group treated by GST-RI has slower tumor growth rate, lighter average tumor weight in contrast to other four groups. Conclusion: The results suggested GST-RI have protective effect on cerebral ischemia reperfusion injury and its mechanism might be related to its anti-oxidation. Our research shows that GST-RI can inhibit the neovascularization powerfully in the B16 melonoma by hypodermic inoculating in mice. Our results will supply valuable data and clue for clinical apply of RI in the treatment of the diseases relating oxidativestress and anti-tumor.
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