【摘要】：研究背景:小檗堿(berberine,BBR),別名黃連素,異喹啉生物堿類,提取于中國傳統(tǒng)中草藥黃連、黃柏等。中外大量研究證實小檗堿具有調(diào)節(jié)腸道菌群、降低血漿葡萄糖、調(diào)節(jié)脂代謝紊亂、減輕體重、促進(jìn)胰島素分泌、改善胰島素抵抗、抑制細(xì)胞凋亡等能力,已然成為2型糖尿病、肥胖、代謝綜合征等疾病新藥防治的新寵。但小檗堿對脂毒性損傷的胰島β細(xì)胞功能的影響及其潛在的分子機制尚不清楚。第十號染色體同源丟失性磷酸酶-張力蛋白基因(Phosphatase and tensin homolog deleted in chromosome10,PTEN)是一種抑癌基因,具有脂質(zhì)及蛋白質(zhì)雙特異性磷酸酶活性,在多種腫瘤的發(fā)生發(fā)展中發(fā)揮關(guān)鍵作用。研究表明,PTEN不但參與細(xì)胞凋亡、增殖、遷徙過程,還調(diào)節(jié)PI3K-AKT信號通路。本課題組前期研究表明PTEN增加會抑制AKT信號通路激活,參與游離脂肪酸(FFAs)造成的胰島β細(xì)胞損傷;而阻斷PTEN表達(dá)可改善FFAs引起的β細(xì)胞損害;提示PTEN可能可以作為2型糖尿病治療的新靶點。本實驗觀察小檗堿在β細(xì)胞脂性凋亡中的相關(guān)作用,進(jìn)一步明確小檗堿拮抗脂毒性損傷的胰島β細(xì)胞的可能分子機制,并觀察PTEN是否參與該過程。目的:觀察小檗堿和PTEN在FFAs造成β細(xì)胞損傷中的作用,分析小檗堿抗糖尿病、保護(hù)胰島β細(xì)胞功能的可能分子機制。方法:實驗一:小檗堿對高濃度棕櫚酸(pa)條件下胰島βtc3細(xì)胞增殖、凋亡的影響。采用mtt法篩查小檗堿對βtc3細(xì)胞增殖起保護(hù)作用的濃度,流式細(xì)胞技術(shù)對比各組細(xì)胞凋亡情況;進(jìn)一步找到小檗堿對游離脂肪酸介導(dǎo)的βtc3細(xì)胞功能改變起保護(hù)作用的合適濃度與作用時間。實驗二:放射免疫法測定小檗堿對脂毒性損傷的胰島βtc3細(xì)胞胰島素分泌的影響。實驗三:分子機制研究:westernblot法檢測小檗堿干預(yù)與否對脂毒性損傷βtc3細(xì)胞氧化應(yīng)激相關(guān)蛋白pten、akt、p-akt以及凋亡相關(guān)蛋白bax、bcl-2、active-caspase3表達(dá)水平變化的影響。結(jié)果:1.mtt結(jié)果表明:(1)0.001-1μmol/l小檗堿作用βtc3細(xì)胞24、48、72h,對細(xì)胞增殖有不同程度促進(jìn)作用(與對照組比較p0.05);從24h增加到72h后,0.001-1μmol/l小檗堿對βtc3細(xì)胞增殖的促進(jìn)作用增加,而1μmol/l濃度細(xì)胞增殖下降,10μmol/l及以上濃度則抑制細(xì)胞增殖(與對照組相比p0.01),并呈濃度依賴性。(2)pa(0.2-1.0mmol/l)對βtc3細(xì)胞增殖的抑制作用具有濃度以及時間依賴性(與對照組比較p0.05),劑量越高、時間越久,抑制作用越大。2.流式細(xì)胞術(shù)結(jié)果顯示:pa孵育βtc3細(xì)胞后,pa組細(xì)胞凋亡率較對照組明顯升高,添加小檗堿處理后細(xì)胞凋亡率顯著降低(p0.01)。3.胰島素分泌結(jié)果顯示:pa組在5.6、16.7mmol/l葡萄糖刺激下胰島素分泌較正常對照組顯著減少(p0.001);添加小檗堿后胰島素分泌回升(p0.001),但較對照組減少。4.westernblot結(jié)果:pa處理βtc3細(xì)胞24、48、72h后,pa組的pten、bax、激活型caspase3表達(dá)水平顯著升高,p-akt、bcl-2水平下降;bbr治療組pten、bax表達(dá)水平較pa組下降,p-akt、bcl-2水平升高(p均0.01)。結(jié)論:bbr可顯著減少ffas誘導(dǎo)的β細(xì)胞凋亡,促進(jìn)細(xì)胞增殖,改善ffas引起的胰島素分泌減少;可能的機制為小檗堿抑制ffas引起的pten過表達(dá),降低促凋亡基因bax、active-caspase3表達(dá),促進(jìn)akt活化并增加抑凋亡基因bcl-2表達(dá);提示PTEN參與了小檗堿減輕游離脂肪酸誘導(dǎo)的β細(xì)胞損害過程。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R587.1
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本文編號：2148525