Stucture Modification Of Endostatin With PEG And Its Experimental Study Of Structure And Ffect

ObjectiveReseachrs of Harward University found a new endogenous angionesis inhabitor-endostatin (ES) for the first time in world in 1977. Experiments showed endostatin had good effect in inhabiting neocascularization, tumour growth and metastasis. The rssults shocked the world. Recent clinical study also showed certain effect. It is regarded as a most promising drug for tumours.Pathologic neovscularization formation plays important role in many eye diseases like corneal CNV after alkli burn, neovasculaar glaucoma,diabetic retinopathy. Prevention and treatment of ocular neocascularization has always been the focus of reseach. Although many anti-neocascularization drugs are constantly developed in recent years, the clinical effect is far from satisfaction. So far there is no ideal clinical medicine for ocular neovascularization. Many reserch reveled exciting results of endostatin in the inhabition of ocular experimental neovascularzation.Since endostatin is a protein product. It has the disadvantage of short half time, unstable and poor bioavailability. If we can modify its structure chemically, to improve its bioavailability, elongate half time, reduce dosage or delay administration intervals. We can greatly improve the treatment effect of endostain, and promote its clinical use.To improve the treatment effect of endostain, we used PEG to modify its structure. Because PEG so far is the best and most popular modifier forproteins. Compared with other modifiers, it has less toxity, no a&Btigenity, better binding effect to proteins and better biocompatibility (approved by FDA). The purpose of modification is to improve its bioavailability, increase stability and elongate half time. We also try to study the bioactivity of PEG-endostatin and its relationship of structure and bioactivity, for the purpose of obtaining an ideal endostatin modification. Materials and Methods1. Purification of endostatinThe culture suernatant of Pichia yeast containing human endostatin geng on the fourth day was purified through CM-II ion exchange chromatography column and Sephadex G-50 column.The resulting of endostatin exhibited one protein band on SDS-PAGE at the position of 20 KD. We found that purified enodstatin could effiectively inhibit the growth and multiplication of human umbilical vein endothelial cell (HUECV) through MTT colorimetry.2. Chemical modification of endostatin by PEG-6000PEG-6000 was activated by Cyanuric chiliride. The activated PEG modified endostatin in PH 8.0, lOmmol/L borax buffer. The process of modification was monitored by SDS-PAGE. We compared degree of free amino group modification, percentage of activity retention and result of electrophoresis, we assigned that the reaction time of modification by PEG was 24h.We purified PEG-ES through Sephadex G-50 gelfiltration column after pretreatment of the terminated solution of modification and identified by electrophoresis. Then we study the heat stability of ES and PEG-ES in 25 °C and 37°C water bath.3. Study of biological activity of ES and PEG-ESThe inhibitive activity to angiogenesis of ES and PEG-ES was studied by chicken chorioallantoic membrane (CAM) model. We observed inhibitive effect of endostatin and modified endostatin on CAM angiogenesis.Cornea CNV animal models was prepared with alkaline burn, then acted ESand PEG-ES were applied on rabbit ##rnea: Inhabition effect of endstatin and modified endostatin on CNV of cornea was observed. In the mean time, the expressions of VEGF were detected by immunohistochemical assay, the quantity of microvessels was observed under microscope. Results1. We for the first time successfully purified endostatin with ion exchange chromatography and gelfiltration chromatograph and obtained endostatin which had very high purity. With this method, it could decrease the cost of purify of endostatin.2. The process of endostatin modification with PEG was optimized, PEG-ES was successfully separated by column chromatography, and the ideal modified product PEG-ES under present conditions was obtained.3. We observed the inhibitive effect on HEUCV, CAM and CNV.3.1 We found that endostatin and its modification product could significantly inhibit the multiplication of HUECV.3.2 The results showed that purified endostain could significantly inhibit angiogenesis of CAM induced by bFGF. Compared with the bFGF group, blood vessel of CAM in endostatin group were significantly reduced (PO.05). The inhibitive effect enhanced, offset and density of blood vessel reduced when concentration of endostatin augmented. When concentration of endostatin was enhanced to a degree, blood vessel of CAM did not growth and embryo died. PEG-ES could also significantly inhibit angiogenesis of CAM winduced by bFGF (P<0.05), but the inhibitive activity was as much as ES.3.3 The results showed that PEG-endostatin treatment group and recombinant endostatin treatment group showed shorter length of blood vessels and smaller area of blood vessels than normal saline group (p<0.05) . PEG-endostatin treatment group showed shorter length of blood vessels and smaller area of blood vessels than recombinant endostatin treatment group (p<0.05) .Theexpressiohcof VEGF and microvessel quantity in PEG-endostatin treatment group and recombinant endostatin treatment group were also significantly lower than those in normal saline group (p<0.05) .The expression of VEGF and microvessel quantity in PEG-endostatin treatment group were lower than those in recombinant endostatin treatment group (p<0.05) .Conclusion1. For the first time in the world we successfully modifiy endostatin with PEG and PEG-ES is successfully separated and purified. We obtain a stable modified endostatin.2. PEG-ES and endostatin both can significantly inhibit the proliferation of HUECV, angiogenesis of CAM and rabbit corneal CNV induced by alkli burn. PEG-ES has better inhabition effect than endostatin.