ARI篩選模型的建立及黃芪甲苷對VSMC生長的影響和機(jī)制研究
發(fā)布時間：2018-07-25 10:49
【摘要】：糖尿病是一種伴有諸多嚴(yán)重并發(fā)癥的疾病,在退行性糖尿病并發(fā)癥病變的發(fā)生和發(fā)展過程中,多元醇代謝通路(polyol pathway)起了十分重要的作用。醛糖還原酶(aldose reductase,AR)是多元醇代謝通路中的關(guān)鍵限速酶,是治療糖尿病并發(fā)癥的靶位酶,該酶以NADPH為輔酶,使葡萄糖還原為不易透過細(xì)胞膜的山梨醇,引起細(xì)胞的滲透性損傷,是糖尿病并發(fā)癥發(fā)生的重要原因。目前許多前沿研究已經(jīng)證實(shí),糖尿病慢性并發(fā)癥如白內(nèi)障,糖尿病視網(wǎng)膜病,糖尿病神經(jīng)病,糖尿病腎病等,都與體內(nèi)山梨醇的蓄積密切相關(guān)。大量動物實(shí)驗(yàn)和臨床研究表明,醛糖還原酶抑制劑(aldose reductase inhibitor,ARI)可以有效地改善糖尿病患者聚醇代謝異常,從而預(yù)防與延緩糖尿病并發(fā)癥的發(fā)生與發(fā)展。由于目前可用于治療糖尿病并發(fā)癥的藥物不足以滿足臨床需要,現(xiàn)有的醛糖還原酶抑制劑索比尼爾和托瑞司他等有不同程度的副作用。因而發(fā)現(xiàn)和尋找新的安全有效的醛糖還原酶抑制劑顯得非常有必要。本研究的目的就是建立一種簡便、可靠并可以用于高通量篩選的醛糖還原酶抑制劑篩選模型,并在模型的基礎(chǔ)上進(jìn)行了相關(guān)的藥物篩選以及藥物作用機(jī)制的研究。 1、建立了可用于從中藥及其他化合物中篩選醛糖還原酶抑制劑的細(xì)胞模型。通過組織貼塊法培養(yǎng)大鼠主動脈平滑肌細(xì)胞,高效液相色譜測定反應(yīng)體系中反應(yīng)后剩余的醛糖還原酶的輔酶NADPH的熒光強(qiáng)度,推算出反應(yīng)體系中醛糖還原酶的活性,逆轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)檢測醛糖還原酶mRNA的表達(dá)。用這些方法檢測了培養(yǎng)在含有不同濃度葡萄糖的DMEM中平滑肌細(xì)胞不同時間點(diǎn)的醛糖還原酶活性及醛糖還原酶mRNA表達(dá)情況,結(jié)果發(fā)現(xiàn),當(dāng)平滑肌細(xì)胞在葡萄糖濃度為37.5mmol/L,胎牛血清(fetal bovine serum,FBS)濃度為10%的DMEM中孵育48小時后醛糖還原酶活性最強(qiáng)(P0.01),醛糖還原酶mRNA的表達(dá)量最高,從而確定了促使平滑肌細(xì)胞內(nèi)AR活性增強(qiáng)的最佳葡萄糖濃度和最佳孵育時間。已知的醛糖還原酶抑制劑Epalrestat對在葡萄糖濃度為37.5mmol/L,FBS濃度為10%的DMEM中孵育48小時的平滑肌細(xì)胞醛糖還原酶抑制劑活性有顯著的抑制作用(P0.01),驗(yàn)證了模型的可行性。根據(jù)這些實(shí)驗(yàn)結(jié)果建立了基于平滑肌細(xì)胞的醛糖還原酶抑制劑篩選的細(xì)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：R-331;R285
本文編號：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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：R-331;R285
【引證文獻(xiàn)】
相關(guān)期刊論文 前5條
1 黃偉;唐燦;;黃酮類醛糖還原酶抑制劑的研究進(jìn)展[J];時珍國醫(yī)國藥;2009年06期
2 孫豪棟;龐曉斌;李繼揚(yáng);;黃芪甲苷生物活性研究進(jìn)展[J];中國藥房;2011年07期
3 汪學(xué)軍;陳代杰;楊志鈞;;微生物來源的醛糖還原酶抑制劑的研究進(jìn)展[J];中國抗生素雜志;2008年11期
4 唐章勇;唐燦;;醛糖還原酶抑制劑篩選的研究進(jìn)展[J];中國新藥雜志;2007年19期
5 王振;宋洋;彭坤;;酶抑制劑篩選模型研究進(jìn)展[J];中國當(dāng)代醫(yī)藥;2013年22期
相關(guān)碩士學(xué)位論文 前1條
1 黃偉;ARIs篩選模型的建立及相關(guān)藥物的篩選[D];西華大學(xué);2009年
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