本文選題：Canstatin-N　切入點(diǎn)：糖尿病　出處：《海南大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：糖尿病視網(wǎng)膜病變是糖尿病嚴(yán)重的并發(fā)癥之一,是致盲的主要原因之一,嚴(yán)重地影響著人們的生存質(zhì)量。糖尿病視網(wǎng)膜血管增生將最終致盲,Canstatin-N是一種分子量小而活性高的血管生成抑制因子。本論文研究應(yīng)用Canstatin-N蛋白治療糖尿病視網(wǎng)膜血管增生。 應(yīng)用PCR的方法克隆Canstatin-N基因,將Canstatin-N基因重組于畢赤酵母表達(dá)載體pGAP9K的多克隆位點(diǎn),構(gòu)建畢赤酵母組成型分泌型表達(dá)載體pGAP9K-canstatin-N。通過(guò)電轉(zhuǎn)法將pGAP9K-canstatin-N轉(zhuǎn)化于畢赤酵母GS115菌種,用基因特異引物PCR驗(yàn)證重組子,用G418抗性篩選出高拷貝的重組子作為工程菌。在發(fā)酵罐中進(jìn)行高密度發(fā)酵GS115(pGAP9K-canstatin-N)工程菌分泌表達(dá)重組Canstatin-N。用SP-Sepharose Fast Flow陽(yáng)離子交換純化重組蛋白。經(jīng)過(guò)純化的重組Canstatin-N蛋白具有抑制雞胚絨毛尿囊膜血管生成的生物學(xué)活性。應(yīng)用DeadEndTM Fluorometric TUNEL System (Promega)分析證明濃度為0.12μg/mL的Canstatin-N蛋白誘發(fā)81%的血管內(nèi)皮細(xì)胞凋亡(P0.01)。 成功構(gòu)建用鏈脲佐菌素誘發(fā)SD大鼠糖尿病視網(wǎng)膜血管增生模型。分別應(yīng)用滴眼法和皮下注射法治療糖尿病SD鼠模型的視網(wǎng)膜血管增生。用藥兩月后,注射生理鹽水對(duì)照組10個(gè)視野(40×)167條血管,注射Canstatin-N蛋白組10個(gè)視野(40×)110條血管(P0.05)；滴生理鹽水對(duì)照組10個(gè)視野(40×)177條血管,滴Canstatin-N蛋白眼藥水組10個(gè)視野(40×)41條血管(P0.01)；對(duì)注射法與滴眼法治療后視網(wǎng)膜血管數(shù)量統(tǒng)計(jì)分析表明兩組之間呈顯著性差異(P0.01)。 以上結(jié)果表明Canstatin-N的注射給藥法和滴眼給藥都能有效地治療SD大鼠糖尿病視網(wǎng)膜血管增生,但是滴眼法的療效顯著高于注射給藥法。制備眼藥水法簡(jiǎn)易易行,并且療效顯著,具有深入研究和開(kāi)發(fā)Canstatin-N蛋白眼藥水作為治療人眼睛血管增生藥物的前景。
[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.
【學(xué)位授予單位】：海南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R774.1

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