Studying On Using Canstatin-N Protein To Treat Of Retinal Vascular Proliferation

Diabetic retinopathy is one of the serious complications of diabetes, it serious impacts on people’s quality of life in the reason of diabetic retinopathy would final blinding. Canstatin-N is an angiogenesis inhibitor with the characteristics of smaller molecular weight and higher biological activity. Here, we studying on using Canstatin-N protein to treat of retinal vascular proliferation.For obtaining of recombinant Canstatin-N by Pichia pastoris, the sequence of canstatin-N was amplified by PCR from the canstatin-N gene containing vector of pET-CN. The sequence of canstatin-N was cloned to the MCS of pGAP9K to generate of pGAP9K-CN which was transformed into Pichia pastoris GS115by electroporation. The recombinants containing of this gene were proved by PCR with gene specific primers and the higher copied recombinants selected as by G418was used engineering strain of GS115(pGAP9K-CN). The recombinant Canstatin-N was secreted expression in the process of high-density cell culture of GS115(pGAP9K-CN) in bioreactor and the purified recombinant Canstatin-N was obtained by SP-Sepharose FF ion-exchange chromatography.The purified recombinant Canstatin-N behaves the function of suppression of new blood vessel growth on CAM and induced an average of81%ECV304cell apoptosis analyzed DeadEndTM Fluorometric TUNEL System (P<0.01).Retina vascular hyperplasia model of SD mouse have been successful constructed, which was induced by Streptozotocin. Treatment of retina vascular hyperplasia model of SD mice were proceeded by two ways, one is eye drops and another is subcutaneous injection. The treatments were maintained two months. It’s167blood vessels on the retina of10eyeshot (40×) of the injection control group (physiological saline) but only110blood vessel of the injection treatment group (Canstatin-N)(P<0.05). For the eye drops treatments, there are177blood vessels on the retina of10eyeshot (40×)of the injection eye drops group (physiological saline) but only41blood vessels of the eye drops treatment group (P<0.01).And also, the eye drops treatments is significantly better than the injection treatment (P<0.01) The results above indicated that the method of using Canstatin-N to treat of retina vascular hyperplasia model of SD mice by eye drops and by subcutaneous injection are all effective, but the curative effect by eye drops method is significantly higher than the method of subcutaneous injection. Preparation eye drops is so simple that it’s easy to implement but it’s curative effect is so notable that Canstatin-N protein behaves the future of depth research and development as the drug of Canstatin-N eye drops to treat of human retina vascular hyperplasia.