本文選題：黃連素　切入點(diǎn)：cAMP　出處：《天津醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的黃連素(BBR),主要活性成分是一種生物堿,它可以有效的治療2型糖尿病(T2DM)患者。大多研究表明:它改善胰島素抵抗,而它對(duì)胰島β細(xì)胞胰島素分泌作用的影響還未明確。本研究在確定黃連素促進(jìn)胰島素分泌的同時(shí),初步探討c AMP-PKA-calcium channel通路在其中所起的調(diào)節(jié)作用。方法(1)培養(yǎng)大鼠胰島素瘤細(xì)胞株INS-1 832/13;(2)黃連素不同濃度(1μM、5μM、10μM、20μM)干預(yù)細(xì)胞72小時(shí);(3)采用3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazoliμM bromide(MTT)法檢測(cè)細(xì)胞活性;(4)利用BD FACSAriaTM Cell Sorter檢測(cè)細(xì)胞凋亡情況;(5)試劑盒提取細(xì)胞總RNA,應(yīng)用實(shí)時(shí)定量聚合酶鏈反應(yīng)(q PCR)檢測(cè)細(xì)胞胰島素合成、凋亡以及鈣離子通道相關(guān)基因的表達(dá),包括pdx1,ins1,ins2,bcl-2、cav1.2;(6)萃取及分離細(xì)胞膜蛋白和細(xì)胞漿蛋白,靶蛋白特性抗體檢測(cè)蛋白表達(dá)的變化,包括L型鈣離子通道(L-type calcium channel protein,Cav1.2)、AMPK、P-AMPK、CREB和P-CREB;(7)體內(nèi)膠原酶注射,分離消化SD大鼠胰腺,體視鏡下手工摘取大鼠胰島,過(guò)夜培養(yǎng)后,黃連素不同濃度(1μM、5μM、10μM、20μM)干預(yù),然后進(jìn)行高糖刺激胰島素分泌試驗(yàn)(glucose stimulated insulin secretion,GSIS),放免法檢測(cè)胰島的胰島素分泌量;(8)試劑盒細(xì)胞c AMP含量測(cè)定。結(jié)果(1)MTT測(cè)定細(xì)胞活性的結(jié)果表明:BBR對(duì)INS-1 832/13細(xì)胞干預(yù)72 h后,細(xì)胞的活性未受到明顯變化(與對(duì)照組相比p0.05);(2)BD FACSAriaTM Cell Sorter分析儀探測(cè)細(xì)胞凋亡的結(jié)果表現(xiàn)為:BBR干預(yù)(濃度分別為1μM、5μM、10μM、20μM)INS-1 832/13細(xì)胞72 h后,早期和晚期凋亡細(xì)胞的百分比總和分別為12.9%、13.2%、14.3%和15.6%,與對(duì)照組凋亡細(xì)胞的百分比總和13.9%相比,差異不明顯,沒(méi)有統(tǒng)計(jì)學(xué)意義(p0.05);(3)q PCR實(shí)驗(yàn)探測(cè)不同基因的m RNA表達(dá)水平表明:BBR干預(yù)72小時(shí)后(濃度同上),20μM BBR導(dǎo)致ins1和ins2 m RNA表達(dá)顯著下降到47%和48%,差異顯著,具有統(tǒng)計(jì)學(xué)意義(與對(duì)照組相比p0.01);其他的BBR干預(yù)濃度,1μM,5μM,10μM均沒(méi)有對(duì)ins1、ins2 m RNA表達(dá)產(chǎn)生顯著影響(與對(duì)照組相比p0.05);本實(shí)驗(yàn)所用BBR濃度沒(méi)有對(duì)pdx1的轉(zhuǎn)錄水平產(chǎn)生明顯的影響,(與對(duì)照組相比p0.05);(4)GSIS實(shí)驗(yàn)結(jié)果表明:2.5m M濃度的葡萄糖孵育大鼠胰島后,各BBR濃度干預(yù)(同上述)均未對(duì)基礎(chǔ)胰島素分泌產(chǎn)生顯著影響(與對(duì)照組比較p0.05);當(dāng)葡萄糖孵育濃度提高到16.7m M時(shí),BBR干預(yù)濃度為1μM、5μM及10μM時(shí)胰島的胰島素分泌量明顯增加,差異顯著,具有統(tǒng)計(jì)學(xué)意義(p0.05),但是當(dāng)BBR濃度增至20μM時(shí),胰島的胰島素分泌與對(duì)照組相比,沒(méi)有明顯的變化(p0.05);(5)q PCR實(shí)驗(yàn)結(jié)果顯示:BBR(1μM、5μM、10μM)顯著增加cav1.2的m RNA的表達(dá)(p0.01);(6)免疫印記方法表明:分別探測(cè)L型鈣離子通道1.2(Cav1.2)和1.3(Cav1.3)揭示出INS-1 832/13細(xì)胞Cav1.2的含量高于Cav1.3;BBR干預(yù)(1μM、5μM、10μM)顯著增加Cav1.2蛋白表達(dá),而當(dāng)BBR濃度為20μM時(shí),未能增加Cav1.2蛋白表達(dá)量(p0.05),與q PCR實(shí)驗(yàn)結(jié)果取得一致;(7)細(xì)胞內(nèi)c AMP含量測(cè)定結(jié)果顯示:5μM和10μM的BBR干預(yù)72 h后,顯著增加INS-1 832/13細(xì)胞中c AMP含量;(8)細(xì)胞信號(hào)傳導(dǎo)通道相關(guān)實(shí)驗(yàn)表明:5μM和10μM濃度的BBR能明顯增加P-AMPK的蛋白表達(dá),干預(yù)組P-AMPK/AMPK比值高于空白對(duì)照組(p0.01);空白對(duì)照組、BBR干預(yù)組(5μM)、PKA激動(dòng)劑Forskolin干預(yù)組,BBR+PKA激動(dòng)劑Forskolin干預(yù)組、PKA抑制劑H89干預(yù)組以及BBR+H89干預(yù)組的對(duì)照實(shí)驗(yàn)提示出BBR、BBR+Forskolin刺激增加P-CREB和Cav1.2蛋白的表達(dá),相反的,H89、BBR+H89處理細(xì)胞后,P-CREB和Cav1.2蛋白表達(dá)明顯降低,差異有統(tǒng)計(jì)學(xué)意義(與BBR干預(yù)組相比較p0.01)。結(jié)論BBR可能不是通過(guò)對(duì)細(xì)胞凋亡的抑制作用和增加胰島素生物合成發(fā)揮促胰島素分泌的作用,而可能是由于增加鈣離子通道蛋白的表達(dá)�？赡艿姆肿油緩桨╟ AMP-PKA-CREB通路。
[Abstract]:The purpose of berberine (BBR), the main active ingredient is an alkaloid, it can be effective in treating type 2 diabetes mellitus (T2DM) patients. Most of the research shows that it improves insulin resistance, and its effect on insulin secretion of islet beta cells is not clear. In this study, determine the Yellow even element promoting insulin secretion at the same time, preliminary study on the role of C in regulation of AMP-PKA-calcium channel pathway in the method. (1) in cultured rat insulinoma cell line INS-1 832/13; (2) different concentrations of berberine (1 M, 5 M, 10 M, 20 M) intervention cells 72 hours; (3) using 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2-H-tetrazoli M bromide (MTT) assay cell activity; (4) using BD FACSAriaTM Cell Sorter to detect cell apoptosis; (5) cell total RNA extraction kit, using real-time quantitative polymerase chain reaction (Q PCR) to detect cell apoptosis and insulin synthesis. The expression of calcium channel genes including PDX1, ins1, Ins2, Bcl-2, Cav1.2; (6) the extraction and separation of membrane proteins and cytoplasmic proteins, changes in protein expression characteristics of target protein antibody detection, including L type calcium channel (L-type calcium channel protein, Cav1.2), AMPK, P-AMPK, and CREB P-CREB; (7) by injection of collagenase digested pancreatic body, SD rats under stereoscope manual removal of rat islets after overnight culture, different concentrations of berberine (1 M, 5 M, 10 M, 20 M) intervention, then glucose stimulated insulin secretion test (glucose stimulated insulin secretion, GSIS), radioimmunoassay of islet insulin secretion; (8) the content of AMP cell C assay kit. Results (1) MTT cell activity was determined by the results showed that the BBR of INS-1 in 832/13 cells after treatment for 72 h, the cell activity was not significantly affected by changes (compared with the control group (2 P0.05); BD FACSAriaTM) Cell Sorter鍒嗘瀽浠帰嫻嬬粏鑳?yōu)鍑嬩骸鐨劸l撴灉琛ㄧ幇涓，

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