小檗碱对游离脂肪酸损伤的胰岛βTC3细胞的保护作用
发布时间：2018-07-27 16:58
【摘要】：研究背景:小檗碱(berberine,BBR),别名黄连素,异喹啉生物碱类,提取于中国传统中草药黄连、黄柏等。中外大量研究证实小檗碱具有调节肠道菌群、降低血浆葡萄糖、调节脂代谢紊乱、减轻体重、促进胰岛素分泌、改善胰岛素抵抗、抑制细胞凋亡等能力,已然成为2型糖尿病、肥胖、代谢综合征等疾病新药防治的新宠。但小檗碱对脂毒性损伤的胰岛β细胞功能的影响及其潜在的分子机制尚不清楚。第十号染色体同源丢失性磷酸酶-张力蛋白基因(Phosphatase and tensin homolog deleted in chromosome10,PTEN)是一种抑癌基因,具有脂质及蛋白质双特异性磷酸酶活性,在多种肿瘤的发生发展中发挥关键作用。研究表明,PTEN不但参与细胞凋亡、增殖、迁徙过程,还调节PI3K-AKT信号通路。本课题组前期研究表明PTEN增加会抑制AKT信号通路激活,参与游离脂肪酸(FFAs)造成的胰岛β细胞损伤;而阻断PTEN表达可改善FFAs引起的β细胞损害;提示PTEN可能可以作为2型糖尿病治疗的新靶点。本实验观察小檗碱在β细胞脂性凋亡中的相关作用,进一步明确小檗碱拮抗脂毒性损伤的胰岛β细胞的可能分子机制,并观察PTEN是否参与该过程。目的:观察小檗碱和PTEN在FFAs造成β细胞损伤中的作用,分析小檗碱抗糖尿病、保护胰岛β细胞功能的可能分子机制。方法:实验一:小檗碱对高浓度棕榈酸(pa)条件下胰岛βtc3细胞增殖、凋亡的影响。采用mtt法筛查小檗碱对βtc3细胞增殖起保护作用的浓度,流式细胞技术对比各组细胞凋亡情况;进一步找到小檗碱对游离脂肪酸介导的βtc3细胞功能改变起保护作用的合适浓度与作用时间。实验二:放射免疫法测定小檗碱对脂毒性损伤的胰岛βtc3细胞胰岛素分泌的影响。实验三:分子机制研究:westernblot法检测小檗碱干预与否对脂毒性损伤βtc3细胞氧化应激相关蛋白pten、akt、p-akt以及凋亡相关蛋白bax、bcl-2、active-caspase3表达水平变化的影响。结果:1.mtt结果表明:(1)0.001-1μmol/l小檗碱作用βtc3细胞24、48、72h,对细胞增殖有不同程度促进作用(与对照组比较p0.05);从24h增加到72h后,0.001-1μmol/l小檗碱对βtc3细胞增殖的促进作用增加,而1μmol/l浓度细胞增殖下降,10μmol/l及以上浓度则抑制细胞增殖(与对照组相比p0.01),并呈浓度依赖性。(2)pa(0.2-1.0mmol/l)对βtc3细胞增殖的抑制作用具有浓度以及时间依赖性(与对照组比较p0.05),剂量越高、时间越久,抑制作用越大。2.流式细胞术结果显示:pa孵育βtc3细胞后,pa组细胞凋亡率较对照组明显升高,添加小檗碱处理后细胞凋亡率显著降低(p0.01)。3.胰岛素分泌结果显示:pa组在5.6、16.7mmol/l葡萄糖刺激下胰岛素分泌较正常对照组显著减少(p0.001);添加小檗碱后胰岛素分泌回升(p0.001),但较对照组减少。4.westernblot结果:pa处理βtc3细胞24、48、72h后,pa组的pten、bax、激活型caspase3表达水平显著升高,p-akt、bcl-2水平下降;bbr治疗组pten、bax表达水平较pa组下降,p-akt、bcl-2水平升高(p均0.01)。结论:bbr可显著减少ffas诱导的β细胞凋亡,促进细胞增殖,改善ffas引起的胰岛素分泌减少;可能的机制为小檗碱抑制ffas引起的pten过表达,降低促凋亡基因bax、active-caspase3表达,促进akt活化并增加抑凋亡基因bcl-2表达;提示PTEN参与了小檗碱减轻游离脂肪酸诱导的β细胞损害过程。
[Abstract]:Research background: berberine (berberine, BBR), berberine and isoquinoline alkaloids are extracted from Chinese traditional Chinese herbal medicine Coptis chinensis and cortex Phellodendron. A lot of studies have proved that berberine can regulate intestinal flora, reduce plasma glucose, regulate lipid metabolism disorder, reduce weight, promote insulin secretion, improve insulin resistance and inhibit cell withering. The ability to death has become a new favorite in the prevention and treatment of new drugs such as type 2 diabetes, obesity and metabolic syndrome. However, the effect of berberine on the islet beta cell function of lipotoxic damage and its potential molecular mechanism are still not clear. The Phosphatase and tensin homolog deleted in C is a homologous loss of chromosome tenth. Hromosome10, PTEN, a tumor suppressor gene, has the activity of lipid and protein bispecific phosphatase, which plays a key role in the development of various tumors. The study shows that PTEN not only participates in cell apoptosis, proliferation and migration, but also regulates the PI3K-AKT signaling pathway. The previous study in our group showed that the increase of PTEN would inhibit the AKT signaling pathway. Activate, participate in the pancreatic islet beta cell damage caused by free fatty acid (FFAs), and blocking the expression of PTEN can improve the damage of beta cell caused by FFAs, suggesting that PTEN may be a new target for the treatment of type 2 diabetes. The correlation of berberine in the lipid apoptosis of beta cells was observed and the pancreatic berberine was further identified by the antagonistic toxicity of berberine. The possible molecular mechanism of islet beta cells and observe whether PTEN participates in the process. Objective: To observe the role of berberine and PTEN in the damage of beta cells caused by FFAs and to analyze the possible molecular mechanism of berberine against diabetes and protect the function of islet beta cells. Effect of apoptosis. MTT method was used to screen the protective effect of berberine on the proliferation of beta TC3 cells. Flow cytometry was used to compare the apoptosis of each group, and the appropriate concentration and time of berberine to protect the function of beta TC3 cells mediated by free fatty acid were further found. Experiment two: the determination of Berberine by radioimmunoassay Effect on insulin secretion of islet beta TC3 cells induced by lipid toxicity. Experiment three: molecular mechanism study: the effect of berberine intervention on the changes of oxidative stress related protein PTEN, Akt, p-Akt and apoptosis related protein Bax, Bcl-2, active-caspase3 expression level in beta TC3 cells of lipotoxic damage. Results: the result: 1.mtt result table (1) (1) the effect of berberine (berberine) on beta TC3 cell 24,48,72h (24,48,72h) promoted the proliferation of cells in different degrees (compared with that of the control group (P0.05). After the increase of 24h to the control group, the promoting effect of 0.001-1 u mol/l berberine on the proliferation of beta TC3 cells increased, while the proliferation of cell proliferation decreased by 1 mu mol/l concentration, and 10 mu mol/l and above concentration inhibited cell proliferation. The control group compared with P0.01) and showed a concentration dependence. (2) the inhibitory effect of PA (0.2-1.0mmol/l) on the proliferation of beta TC3 cells was concentration and time dependent (compared with that of the control group (P0.05). The higher the dose, the longer the time, the greater the inhibitory action, the more.2. flow cytometry showed that the apoptosis rate of the PA group was significantly higher than the control group after PA incubated the beta TC3 cells. " The apoptosis rate of the cells decreased significantly after the addition of berberine (P0.01).3. insulin secretion results showed that the insulin secretion of PA group was significantly decreased under the 5.6,16.7mmol/l glucose stimulation (p0.001), and the insulin secretion recovered after the addition of berberine (p0.001), but the result of.4.westernblot in the control group was reduced by the control group: PA treated beta TC3 cells 24, After 48,72h, the expression level of PTEN, Bax, activator Caspase3 in group PA was significantly higher, p-Akt, bcl-2 level decreased, BBR treatment group PTEN, Bax expression level was lower than PA group, p-Akt, increased (all 0.01). Conclusion: it can significantly reduce the apoptosis induced by beta cells, promote cell proliferation, improve the decrease of insulin secretion, and possible machine Berberine inhibits the overexpression of PTEN induced by FFAs, reduces the expression of Bax, active-caspase3, activates Akt activation and increases the expression of Bcl-2 in the anti apoptotic gene, suggesting that PTEN participates in berberine to reduce the process of beta cell damage induced by free fatty acids.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R587.1
，
本文编号：2148525
[Abstract]:Research background: berberine (berberine, BBR), berberine and isoquinoline alkaloids are extracted from Chinese traditional Chinese herbal medicine Coptis chinensis and cortex Phellodendron. A lot of studies have proved that berberine can regulate intestinal flora, reduce plasma glucose, regulate lipid metabolism disorder, reduce weight, promote insulin secretion, improve insulin resistance and inhibit cell withering. The ability to death has become a new favorite in the prevention and treatment of new drugs such as type 2 diabetes, obesity and metabolic syndrome. However, the effect of berberine on the islet beta cell function of lipotoxic damage and its potential molecular mechanism are still not clear. The Phosphatase and tensin homolog deleted in C is a homologous loss of chromosome tenth. Hromosome10, PTEN, a tumor suppressor gene, has the activity of lipid and protein bispecific phosphatase, which plays a key role in the development of various tumors. The study shows that PTEN not only participates in cell apoptosis, proliferation and migration, but also regulates the PI3K-AKT signaling pathway. The previous study in our group showed that the increase of PTEN would inhibit the AKT signaling pathway. Activate, participate in the pancreatic islet beta cell damage caused by free fatty acid (FFAs), and blocking the expression of PTEN can improve the damage of beta cell caused by FFAs, suggesting that PTEN may be a new target for the treatment of type 2 diabetes. The correlation of berberine in the lipid apoptosis of beta cells was observed and the pancreatic berberine was further identified by the antagonistic toxicity of berberine. The possible molecular mechanism of islet beta cells and observe whether PTEN participates in the process. Objective: To observe the role of berberine and PTEN in the damage of beta cells caused by FFAs and to analyze the possible molecular mechanism of berberine against diabetes and protect the function of islet beta cells. Effect of apoptosis. MTT method was used to screen the protective effect of berberine on the proliferation of beta TC3 cells. Flow cytometry was used to compare the apoptosis of each group, and the appropriate concentration and time of berberine to protect the function of beta TC3 cells mediated by free fatty acid were further found. Experiment two: the determination of Berberine by radioimmunoassay Effect on insulin secretion of islet beta TC3 cells induced by lipid toxicity. Experiment three: molecular mechanism study: the effect of berberine intervention on the changes of oxidative stress related protein PTEN, Akt, p-Akt and apoptosis related protein Bax, Bcl-2, active-caspase3 expression level in beta TC3 cells of lipotoxic damage. Results: the result: 1.mtt result table (1) (1) the effect of berberine (berberine) on beta TC3 cell 24,48,72h (24,48,72h) promoted the proliferation of cells in different degrees (compared with that of the control group (P0.05). After the increase of 24h to the control group, the promoting effect of 0.001-1 u mol/l berberine on the proliferation of beta TC3 cells increased, while the proliferation of cell proliferation decreased by 1 mu mol/l concentration, and 10 mu mol/l and above concentration inhibited cell proliferation. The control group compared with P0.01) and showed a concentration dependence. (2) the inhibitory effect of PA (0.2-1.0mmol/l) on the proliferation of beta TC3 cells was concentration and time dependent (compared with that of the control group (P0.05). The higher the dose, the longer the time, the greater the inhibitory action, the more.2. flow cytometry showed that the apoptosis rate of the PA group was significantly higher than the control group after PA incubated the beta TC3 cells. " The apoptosis rate of the cells decreased significantly after the addition of berberine (P0.01).3. insulin secretion results showed that the insulin secretion of PA group was significantly decreased under the 5.6,16.7mmol/l glucose stimulation (p0.001), and the insulin secretion recovered after the addition of berberine (p0.001), but the result of.4.westernblot in the control group was reduced by the control group: PA treated beta TC3 cells 24, After 48,72h, the expression level of PTEN, Bax, activator Caspase3 in group PA was significantly higher, p-Akt, bcl-2 level decreased, BBR treatment group PTEN, Bax expression level was lower than PA group, p-Akt, increased (all 0.01). Conclusion: it can significantly reduce the apoptosis induced by beta cells, promote cell proliferation, improve the decrease of insulin secretion, and possible machine Berberine inhibits the overexpression of PTEN induced by FFAs, reduces the expression of Bax, active-caspase3, activates Akt activation and increases the expression of Bcl-2 in the anti apoptotic gene, suggesting that PTEN participates in berberine to reduce the process of beta cell damage induced by free fatty acids.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R587.1
，
本文编号：2148525
本文链接：https://www.wllwen.com/yixuelunwen/nfm/2148525.html
最近更新
教材专著
