本文選題：糖尿病視網膜病變　切入點：氧化應激　出處：《新疆醫(yī)科大學》2011年碩士論文

【摘要】：目的:從整體,細胞及分子水平,研究糖康對糖尿病大鼠視網膜組織和高糖環(huán)境培養(yǎng)的人臍靜脈內皮細胞(ECV-304)的作用及其機制研究。方法:1)動物實驗:60只雄性Wistar大鼠隨機選取10只作為正常對照組,50只大鼠用鏈脲佐菌素(STZ 50mg/kg)一次性腹腔注射的方法建立糖尿病動物模型。造模成功后又隨機分為糖康高、中。低劑量組和達納康組。采用消化鋪片方法觀察大鼠視網膜微血管組織形態(tài)變化;化學比色法檢測大鼠視網膜組織及血清中丙二醛(Malondialdehyde,MDA)含量以及超氧化物歧化酶(SOD)活性。采用western blot、RT-PCR技術分別檢測視網膜組織中NF-κB p65蛋白表達及NF-κB、IL-1β、BAX和VEGF基因的表達;2)細胞試驗:體外培養(yǎng)人臍靜脈內皮細胞(ECV-304),用不同濃度的葡萄糖培養(yǎng)液干預48h至96h后用四甲基偶氮唑藍法(MTT)檢測不同濃度的葡萄糖對細胞增殖能力的影響;采用化學比色法檢測細胞中的MDA和還原型谷胱甘肽(GSH)含量。運用血清藥理學方法制備不同濃度的糖康含藥血清,采用MTT法檢測各組糖康含藥血清對細胞增殖的影響,取各組細胞進一步檢測MDA和GSH含量。結果:1)動物實驗:通過STZ誘導糖尿病動物模型,造模一周左右即出現糖尿病典型的“三多,一少”癥狀。與模型組比較糖康高劑量與低劑量組癥狀明顯減輕,而達納康組和糖康低劑量組癥狀無改善。與正常對照組比較模型組和糖康各治療組大鼠體重明顯降低而血糖顯著升高(P0.01)。與正常組比較,模型組大鼠視網膜呈現較多的無細胞毛細血管條索數(約為正常組的5.33倍)。糖康高劑量組和中劑量組與模型組比較無細胞毛細血管數顯著減少(P0.01),糖康各治療組大鼠視網膜組織和血清中SOD活力逐步上升,MDA含量降低。與模型組比較各治療組大鼠視網膜組織中NF-κB P65蛋白和NF-κB、IL-1β、BAX和VEGF基因的表達明顯下調,以糖康高劑量組效果顯著(P0.01);2)細胞試驗:隨葡萄糖濃度的升高和培養(yǎng)時間的延長各組細胞OD值明顯降低(P0.01),胞內MDA含量增加和GSH含量降低(P0.01),具有明顯的時間和濃度依賴性。與模型組比較,糖康含藥血清各治療組明顯抑制細胞增殖,不同程度的降低細胞內MDA含量和提高GSH含量,以糖康高劑量組效果明顯(P0.01)。結論:1)糖康能夠改善DM大鼠的一般癥狀,從病理形態(tài)學觀察視網膜微血管可減輕糖尿病大鼠早期視網膜病變微血管的損傷;2)糖康能顯著增加糖尿病大鼠血清及視網膜組織中SOD活性,降低MDA含量;3)糖康能夠下調DM大鼠視網膜組織中NF-κB P65蛋白表達及NF-κB、IL-1β、BAX和VEGF基因的表達,說明該藥對氧化損傷具有保護作用;4)糖康含藥血清顯著降低高糖環(huán)境培養(yǎng)的細胞內MDA含量及提高胞內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.
【學位授予單位】：新疆醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R285.5;R774.1

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