用Canstatin-N抑制大鼠视网膜血管增生的研究

发布时间：2018-03-05 06:10

本文选题：Canstatin-N　切入点：糖尿病　出处：《海南大学》2012年硕士论文　论文类型：学位论文

【摘要】：糖尿病视网膜病变是糖尿病严重的并发症之一,是致盲的主要原因之一,严重地影响着人们的生存质量。糖尿病视网膜血管增生将最终致盲,Canstatin-N是一种分子量小而活性高的血管生成抑制因子。本论文研究应用Canstatin-N蛋白治疗糖尿病视网膜血管增生。应用PCR的方法克隆Canstatin-N基因,将Canstatin-N基因重组于毕赤酵母表达载体pGAP9K的多克隆位点,构建毕赤酵母组成型分泌型表达载体pGAP9K-canstatin-N。通过电转法将pGAP9K-canstatin-N转化于毕赤酵母GS115菌种,用基因特异引物PCR验证重组子,用G418抗性筛选出高拷贝的重组子作为工程菌。在发酵罐中进行高密度发酵GS115(pGAP9K-canstatin-N)工程菌分泌表达重组Canstatin-N。用SP-Sepharose Fast Flow阳离子交换纯化重组蛋白。经过纯化的重组Canstatin-N蛋白具有抑制鸡胚绒毛尿囊膜血管生成的生物学活性。应用DeadEndTM Fluorometric TUNEL System (Promega)分析证明浓度为0.12μg/mL的Canstatin-N蛋白诱发81%的血管内皮细胞凋亡(P0.01)。成功构建用链脲佐菌素诱发SD大鼠糖尿病视网膜血管增生模型。分别应用滴眼法和皮下注射法治疗糖尿病SD鼠模型的视网膜血管增生。用药两月后,注射生理盐水对照组10个视野(40×)167条血管,注射Canstatin-N蛋白组10个视野(40×)110条血管(P0.05)；滴生理盐水对照组10个视野(40×)177条血管,滴Canstatin-N蛋白眼药水组10个视野(40×)41条血管(P0.01)；对注射法与滴眼法治疗后视网膜血管数量统计分析表明两组之间呈显著性差异(P0.01)。以上结果表明Canstatin-N的注射给药法和滴眼给药都能有效地治疗SD大鼠糖尿病视网膜血管增生,但是滴眼法的疗效显著高于注射给药法。制备眼药水法简易易行,并且疗效显著,具有深入研究和开发Canstatin-N蛋白眼药水作为治疗人眼睛血管增生药物的前景。
[Abstract]:Diabetic retinopathy is one of the serious complications of diabetes mellitus and one of the main causes of blindness. Diabetic retinal vascular hyperplasia will eventually cause blindness. Canstatin-N is a low molecular weight and high activity angiogenesis inhibitor. In this study, Canstatin-N protein was used to treat diabetic retinal vascular hyperplasia. The Canstatin-N gene was cloned by PCR, and the Canstatin-N gene was recombined into the polyclonal site of Pichia pastoris expression vector pGAP9K. The secretory expression vector pGAP9K-canstatin-Nwas constructed. PGAP9K-canstatin-N was transformed into GS115 strain by electroporation. The recombinant plasmid was verified by gene specific primer PCR. High copy recombinant bacteria were screened by G418 resistance. Recombinant Canstatin-Ns were secreted and expressed by GS115pGAP9K-canstatin-N in fermenter. The recombinant protein was purified by SP-Sepharose Fast Flow cation exchange. The purified recombinant Canstatin-N protein was obtained. The results of DeadEndTM Fluorometric TUNEL System analysis showed that Canstatin-N protein (0.12 渭 g / mL) could induce apoptosis of vascular endothelial cells at 81% 渭 g / mL (P0.01A) and could inhibit the angiogenesis of chorioallantoic membrane in chorioallantoic chorioallantoic chorioallantoic cells. The diabetic retinal vascular hyperplasia model of SD rats induced by streptozotocin was successfully constructed. The retinal vascular hyperplasia of diabetic SD rats was treated by eye drops and subcutaneous injection respectively. There were 10 visual fields in the control group, 40 脳 10 in the Canstatin-N protein group, 40 脳 10 in the Canstatin-N protein group and 40 脳 10 in the normal saline group, respectively, and 40 脳 10 vessels in the 10 visual fields of the control group were injected with normal saline, and 40 脳 10 vessels in the 10 visual fields of the control group were injected with Canstatin-N protein. In the Canstatin-N protein eye drops group, there were 10 visual fields and 40 脳 10 vessels in the eye drops group (P 0.01), and the statistical analysis of retinal blood vessels after injection and eye drop showed that there was a significant difference between the two groups in the number of retinal blood vessels (P 0.01), and there was a significant difference between the two groups in the number of retinal blood vessels (P 0.01). The above results show that both Canstatin-N injection and eye drip can effectively treat diabetic retinal vascular hyperplasia in SD rats, but the effect of eye drop is significantly higher than that of injection, and the preparation of eye drops is easy and effective. It has the prospect of further research and development of Canstatin-N protein eye drops as a drug for the treatment of human eye angiogenesis.
【学位授予单位】：海南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R774.1

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