ARI筛选模型的建立及黄芪甲苷对VSMC生长的影响和机制研究
发布时间：2018-07-25 10:49
【摘要】：糖尿病是一种伴有诸多严重并发症的疾病,在退行性糖尿病并发症病变的发生和发展过程中,多元醇代谢通路(polyol pathway)起了十分重要的作用。醛糖还原酶(aldose reductase,AR)是多元醇代谢通路中的关键限速酶,是治疗糖尿病并发症的靶位酶,该酶以NADPH为辅酶,使葡萄糖还原为不易透过细胞膜的山梨醇,引起细胞的渗透性损伤,是糖尿病并发症发生的重要原因。目前许多前沿研究已经证实,糖尿病慢性并发症如白内障,糖尿病视网膜病,糖尿病神经病,糖尿病肾病等,都与体内山梨醇的蓄积密切相关。大量动物实验和临床研究表明,醛糖还原酶抑制剂(aldose reductase inhibitor,ARI)可以有效地改善糖尿病患者聚醇代谢异常,从而预防与延缓糖尿病并发症的发生与发展。由于目前可用于治疗糖尿病并发症的药物不足以满足临床需要,现有的醛糖还原酶抑制剂索比尼尔和托瑞司他等有不同程度的副作用。因而发现和寻找新的安全有效的醛糖还原酶抑制剂显得非常有必要。本研究的目的就是建立一种简便、可靠并可以用于高通量筛选的醛糖还原酶抑制剂筛选模型,并在模型的基础上进行了相关的药物筛选以及药物作用机制的研究。 1、建立了可用于从中药及其他化合物中筛选醛糖还原酶抑制剂的细胞模型。通过组织贴块法培养大鼠主动脉平滑肌细胞,高效液相色谱测定反应体系中反应后剩余的醛糖还原酶的辅酶NADPH的荧光强度,推算出反应体系中醛糖还原酶的活性,逆转录聚合酶链式反应(RT-PCR)检测醛糖还原酶mRNA的表达。用这些方法检测了培养在含有不同浓度葡萄糖的DMEM中平滑肌细胞不同时间点的醛糖还原酶活性及醛糖还原酶mRNA表达情况,结果发现,当平滑肌细胞在葡萄糖浓度为37.5mmol/L,胎牛血清(fetal bovine serum,FBS)浓度为10%的DMEM中孵育48小时后醛糖还原酶活性最强(P0.01),醛糖还原酶mRNA的表达量最高,从而确定了促使平滑肌细胞内AR活性增强的最佳葡萄糖浓度和最佳孵育时间。已知的醛糖还原酶抑制剂Epalrestat对在葡萄糖浓度为37.5mmol/L,FBS浓度为10%的DMEM中孵育48小时的平滑肌细胞醛糖还原酶抑制剂活性有显著的抑制作用(P0.01),验证了模型的可行性。根据这些实验结果建立了基于平滑肌细胞的醛糖还原酶抑制剂筛选的细胞模型。
[Abstract]:Diabetes is a disease with many serious complications. The polyol metabolic pathway (polyol pathway) plays a very important role in the development and development of the complications of degenerative diabetes. Aldose reductase (AR) is the key rate limiting enzyme in the multi polyol Xie Tong Road, which is the target for the treatment of diabetic complications. The enzyme, which uses NADPH as a coenzyme, makes glucose reduced to sorbitol that is not easy to permeate the cell membrane, causing cell permeability damage and is an important cause of diabetes complications. Many recent frontier studies have confirmed that chronic diabetic complications such as cataracts, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy and so on are all confirmed. It is closely related to the accumulation of sorbitol in the body. A large number of animal experiments and clinical studies have shown that aldose reductase inhibitor (aldose reductase inhibitor, ARI) can effectively improve the abnormality of polyol metabolism in diabetic patients, thus preventing and retarding the occurrence and development of diabetic complications. Drugs are not sufficient to meet clinical needs. The existing aldose reductase inhibitors, Sobhi Neil and Torre, have different levels of side effects. Therefore, it is necessary to find and find new safe and effective aldose reductase inhibitors. The purpose of this study is to establish a simple, reliable and high throughput screening aldehyde. Screening models of sugar reductase inhibitors, and based on the model, the related drug screening and drug action mechanism were studied.
1, a cell model which can be used to screen aldose reductase inhibitors from traditional Chinese medicine and other compounds was established. The rat aortic smooth muscle cells were cultured by tissue patch method. The fluorescence intensity of the coenzyme NADPH of the residual aldose reductase in the reaction system was determined by HPLC, and the aldose reductase in the reaction system was calculated. Activity, reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the expression of aldose reductase mRNA. These methods were used to detect aldose reductase activity and aldose reductase mRNA expression in different time points of smooth muscle cells in DMEM containing different concentrations of glucose. The results showed that the smooth muscle cells were in the glucose concentration of 37.5mmol. /L, the activity of aldose reductase (aldose reductase) is the strongest (P0.01) and the highest expression of aldose reductase mRNA in fetal bovine serum (FBS) concentration of 10%, and the highest concentration of aldose reductase mRNA, which determines the optimal concentration and optimum incubation time for enhancing the AR activity in smooth muscle cells. The known aldose reductase inhibitor Epalrestat is the same The activity of aldose reductase inhibitor (aldose reductase inhibitor), which was incubated for 48 hours in DMEM with glucose concentration of 37.5mmol/L and 10% FBS, had significant inhibitory effect (P0.01), which verified the feasibility of the model. Based on these results, a cell model was established for the screening of aldose reductase inhibitor based on smooth muscle cells.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R-331;R285
本文编号：2143596
[Abstract]:Diabetes is a disease with many serious complications. The polyol metabolic pathway (polyol pathway) plays a very important role in the development and development of the complications of degenerative diabetes. Aldose reductase (AR) is the key rate limiting enzyme in the multi polyol Xie Tong Road, which is the target for the treatment of diabetic complications. The enzyme, which uses NADPH as a coenzyme, makes glucose reduced to sorbitol that is not easy to permeate the cell membrane, causing cell permeability damage and is an important cause of diabetes complications. Many recent frontier studies have confirmed that chronic diabetic complications such as cataracts, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy and so on are all confirmed. It is closely related to the accumulation of sorbitol in the body. A large number of animal experiments and clinical studies have shown that aldose reductase inhibitor (aldose reductase inhibitor, ARI) can effectively improve the abnormality of polyol metabolism in diabetic patients, thus preventing and retarding the occurrence and development of diabetic complications. Drugs are not sufficient to meet clinical needs. The existing aldose reductase inhibitors, Sobhi Neil and Torre, have different levels of side effects. Therefore, it is necessary to find and find new safe and effective aldose reductase inhibitors. The purpose of this study is to establish a simple, reliable and high throughput screening aldehyde. Screening models of sugar reductase inhibitors, and based on the model, the related drug screening and drug action mechanism were studied.
1, a cell model which can be used to screen aldose reductase inhibitors from traditional Chinese medicine and other compounds was established. The rat aortic smooth muscle cells were cultured by tissue patch method. The fluorescence intensity of the coenzyme NADPH of the residual aldose reductase in the reaction system was determined by HPLC, and the aldose reductase in the reaction system was calculated. Activity, reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the expression of aldose reductase mRNA. These methods were used to detect aldose reductase activity and aldose reductase mRNA expression in different time points of smooth muscle cells in DMEM containing different concentrations of glucose. The results showed that the smooth muscle cells were in the glucose concentration of 37.5mmol. /L, the activity of aldose reductase (aldose reductase) is the strongest (P0.01) and the highest expression of aldose reductase mRNA in fetal bovine serum (FBS) concentration of 10%, and the highest concentration of aldose reductase mRNA, which determines the optimal concentration and optimum incubation time for enhancing the AR activity in smooth muscle cells. The known aldose reductase inhibitor Epalrestat is the same The activity of aldose reductase inhibitor (aldose reductase inhibitor), which was incubated for 48 hours in DMEM with glucose concentration of 37.5mmol/L and 10% FBS, had significant inhibitory effect (P0.01), which verified the feasibility of the model. Based on these results, a cell model was established for the screening of aldose reductase inhibitor based on smooth muscle cells.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R-331;R285
【引证文献】
相关期刊论文 前5条
1 黄伟;唐灿;;黄酮类醛糖还原酶抑制剂的研究进展[J];时珍国医国药;2009年06期
2 孙豪栋;庞晓斌;李继扬;;黄芪甲苷生物活性研究进展[J];中国药房;2011年07期
3 汪学军;陈代杰;杨志钧;;微生物来源的醛糖还原酶抑制剂的研究进展[J];中国抗生素杂志;2008年11期
4 唐章勇;唐灿;;醛糖还原酶抑制剂筛选的研究进展[J];中国新药杂志;2007年19期
5 王振;宋洋;彭坤;;酶抑制剂筛选模型研究进展[J];中国当代医药;2013年22期
相关硕士学位论文 前1条
1 黄伟;ARIs筛选模型的建立及相关药物的筛选[D];西华大学;2009年
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