糖康对糖尿病大鼠和血管内皮细胞的作用及其机制研究

发布时间：2018-04-08 15:47

本文选题：糖尿病视网膜病变　切入点：氧化应激　出处：《新疆医科大学》2011年硕士论文

【摘要】：目的:从整体,细胞及分子水平,研究糖康对糖尿病大鼠视网膜组织和高糖环境培养的人脐静脉内皮细胞(ECV-304)的作用及其机制研究。方法:1)动物实验:60只雄性Wistar大鼠随机选取10只作为正常对照组,50只大鼠用链脲佐菌素(STZ 50mg/kg)一次性腹腔注射的方法建立糖尿病动物模型。造模成功后又随机分为糖康高、中。低剂量组和达纳康组。采用消化铺片方法观察大鼠视网膜微血管组织形态变化;化学比色法检测大鼠视网膜组织及血清中丙二醛(Malondialdehyde,MDA)含量以及超氧化物歧化酶(SOD)活性。采用western blot、RT-PCR技术分别检测视网膜组织中NF-κB p65蛋白表达及NF-κB、IL-1β、BAX和VEGF基因的表达;2)细胞试验:体外培养人脐静脉内皮细胞(ECV-304),用不同浓度的葡萄糖培养液干预48h至96h后用四甲基偶氮唑蓝法(MTT)检测不同浓度的葡萄糖对细胞增殖能力的影响;采用化学比色法检测细胞中的MDA和还原型谷胱甘肽(GSH)含量。运用血清药理学方法制备不同浓度的糖康含药血清,采用MTT法检测各组糖康含药血清对细胞增殖的影响,取各组细胞进一步检测MDA和GSH含量。结果:1)动物实验:通过STZ诱导糖尿病动物模型,造模一周左右即出现糖尿病典型的“三多,一少”症状。与模型组比较糖康高剂量与低剂量组症状明显减轻,而达纳康组和糖康低剂量组症状无改善。与正常对照组比较模型组和糖康各治疗组大鼠体重明显降低而血糖显著升高(P0.01)。与正常组比较,模型组大鼠视网膜呈现较多的无细胞毛细血管条索数(约为正常组的5.33倍)。糖康高剂量组和中剂量组与模型组比较无细胞毛细血管数显著减少(P0.01),糖康各治疗组大鼠视网膜组织和血清中SOD活力逐步上升,MDA含量降低。与模型组比较各治疗组大鼠视网膜组织中NF-κB P65蛋白和NF-κB、IL-1β、BAX和VEGF基因的表达明显下调,以糖康高剂量组效果显著(P0.01);2)细胞试验:随葡萄糖浓度的升高和培养时间的延长各组细胞OD值明显降低(P0.01),胞内MDA含量增加和GSH含量降低(P0.01),具有明显的时间和浓度依赖性。与模型组比较,糖康含药血清各治疗组明显抑制细胞增殖,不同程度的降低细胞内MDA含量和提高GSH含量,以糖康高剂量组效果明显(P0.01)。结论:1)糖康能够改善DM大鼠的一般症状,从病理形态学观察视网膜微血管可减轻糖尿病大鼠早期视网膜病变微血管的损伤;2)糖康能显著增加糖尿病大鼠血清及视网膜组织中SOD活性,降低MDA含量;3)糖康能够下调DM大鼠视网膜组织中NF-κB P65蛋白表达及NF-κB、IL-1β、BAX和VEGF基因的表达,说明该药对氧化损伤具有保护作用;4)糖康含药血清显著降低高糖环境培养的细胞内MDA含量及提高胞内GSH含量。
[Abstract]:Aim: to study the effect and mechanism of Tangkang on retinal tissue of diabetic rats and human umbilical vein endothelial cells (ECV-304) cultured in high glucose environment.Methods Twenty six male Wistar rats were randomly selected as normal control group. 50 rats were injected with STZ 50 mg / kg to establish diabetic animal model by intraperitoneal injection of streptozotocin (STZ) 50 mg / kg.After the success of the model, it was randomly divided into Tangkang Gao and Zhong.Low dose group and Danakam group.The morphological changes of retinal microvessels in rats were observed by using digestion-spreading method, and the content of malondialdehyde malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in the retina and serum of rats were detected by chemical colorimetry.The expression of NF- 魏 B p65 protein in retinal tissue and the expression of IL-1 尾 -Bax and VEGF genes in retinal tissue were detected by western blottir RT-PCR, respectively. In vitro, cultured human umbilical vein endothelial cells (HUVEC) were cultured with ECV-304, then treated with different concentrations of glucose for 48h to 96h.The effect of glucose concentration on cell proliferation was detected by MTT.The content of MDA and reduced glutathione (Glutathione) in the cells were detected by chemical colorimetry.The different concentrations of Tangkang serum were prepared by serum pharmacology method. The effect of Tangkang serum on cell proliferation was detected by MTT method. The contents of MDA and GSH in each group were further detected.Results: the animal model of diabetes induced by STZ appeared the typical symptoms of "more than three, one less" in one week.Compared with the model group, the symptoms of the high dose and low dose groups of Tangkang were obviously alleviated, while the symptoms of the Danakang group and the low dose group of Tangkang were not improved.Compared with the normal control group, the model group and Tangkang treatment group decreased the body weight and increased the blood glucose significantly (P 0.01).Compared with the normal group, the retina of the model group showed more acellular capillary bands (5.33 times as much as the normal group).Compared with the model group, the number of acellular capillaries in the Tangkang high dose group and the middle dose group decreased significantly (P 0.01), and the SOD activity in the retina and serum decreased gradually in the Tangkang treatment group.Compared with the model group, the expression of NF- 魏 B p65, NF- 魏 B, IL-1 尾, Bax and VEGF genes were down-regulated in the retina of rats in each treatment group.The cell test showed that with the increase of glucose concentration and the prolongation of culture time, the OD value of the cells decreased significantly, the intracellular MDA content increased and the GSH content decreased in a time-and concentration-dependent manner.Compared with the model group, each treatment group of Tangkang serum obviously inhibited cell proliferation, decreased the content of MDA and increased the content of GSH in different degree, especially in the high dose group of Tangkang.Conclusion: 1) Tangkang can improve the general symptoms of DM rats.Observation of retinal microvessels by pathomorphology can reduce the damage of microvessels in early diabetic rats. Tangkang can significantly increase the activity of SOD in serum and retina of diabetic rats.The expression of NF- 魏 B p65 protein and the expression of NF- 魏 B 尾 IL-1 尾 -Bax and VEGF genes were down-regulated by Tangkang in retina of DM rats.The results showed that the drug had protective effect on oxidative damage. The serum containing Tangkang significantly decreased the content of MDA and increased the content of GSH in the cells cultured in high glucose environment.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R285.5;R774.1

【参考文献】