本文選題：親磷脂酸磷脂酶A1　切入點：基因表達(dá)　出處：《桂林醫(yī)學(xué)院》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：研究親磷脂酸磷脂酶A1(phosphatidic acid-preferring phospholipase Al, PA-PLA1)基因沉默對小鼠胰島p細(xì)胞株MIN6胰島素分泌的影響。方法：采用invitrogen網(wǎng)站提供的在線RNAi設(shè)計軟件設(shè)計、合成小鼠PA-PLA1靶向shRNA,以Lipofectamin2000為轉(zhuǎn)染試劑轉(zhuǎn)染小鼠胰島素分泌細(xì)胞MIN6,用熒光顯微鏡檢測干擾載體的轉(zhuǎn)染效率,實時熒光定量PCR檢測MIN6細(xì)胞中PA-PLA1mRNA的表達(dá)水平,Western Blotting檢測PA-PLA1蛋白的表達(dá)水平,同時對MIN6細(xì)胞行葡萄糖刺激胰島素分泌實驗,放免法測定細(xì)胞上清液中胰島素分泌量。結(jié)果：本實驗設(shè)計、合成了小鼠PA-PLA1靶向shRNA1、2,測序結(jié)果證實shRNA編碼序列與設(shè)計的片段完全一致。轉(zhuǎn)染MIN6細(xì)胞48h后熒光顯微鏡檢測其干擾載體的轉(zhuǎn)染效率約為50%。實時熒光定量PCR和Western Blotting檢測,干擾載體1、2PA-PLA1mRNA表達(dá)水平分別下降11%和53%,蛋白表達(dá)水平分別下降53%和59%。對經(jīng)RNAi處理過的MIN6細(xì)胞行葡萄糖刺激胰島素分泌實驗,測得細(xì)胞胰島素分泌水平下降30%。結(jié)論：本文設(shè)計、合成了小鼠PA-PLA1靶向shRNA。所構(gòu)建的小鼠PA-PLA1靶向shRNA干擾載體能有效轉(zhuǎn)染小鼠胰島分泌細(xì)胞MIN6。PA-PL A1基因沉默后的MIN6細(xì)胞胰島素的分泌水平明顯下降,表明PA-PLA1基因表達(dá)與MIN6細(xì)胞胰島素分泌存在相關(guān)性,提示該酶對胰島β細(xì)胞胰島素分泌可能發(fā)揮正調(diào)節(jié)作用。本研究結(jié)果為進一步研究胰島β細(xì)胞信號轉(zhuǎn)導(dǎo)和胰島素分泌的分子機制提供了新的線索。
[Abstract]:Objective: To study the phosphatidic acid phospholipase A1 (phosphatidic acid-preferring phospholipase Al, PA-PLA1) of MIN6 gene silencing on insulin secretion in mouse islet cell line P. Methods: the design of online RNAi design software available on the Invitrogen website, the synthesis of mouse PA-PLA1 targeting shRNA to Lipofectamin2000, and then was transfected into mouse insulin secreting cells by fluorescence MIN6. Microscope interference vector transfection efficiency and expression level of PA-PLA1mRNA was detected by real-time fluorescent quantitative PCR in MIN6 cells, the expression level of PA-PLA1 protein was detected by Western Blotting, while the MIN6 cells for glucose stimulated insulin secretion test, radioimmunoassay in insulin secretion in the supernatant. Results: the experimental design, synthesis of mouse PA-PLA1 targeting shRNA1,2 sequencing results confirmed that the design of shRNA encoding sequence and fragment identical. Transfection of MI N6 cells were detected by fluorescence microscopy after 48h interference vector transfection efficiency was about 50%. PCR and Western Blotting real-time fluorescence quantitative detection, interference vector 1,2PA-PLA1mRNA expression levels were decreased to 11% and 53%, protein expression levels were decreased by 53% and 59%. RNAi treated MIN6 cells for glucose stimulated insulin secretion test measured insulin conclusion: the decreased 30%. secretion of mouse PA-PLA1 mouse PA-PLA1 target design, target construction to synthesize shRNA. to shRNA interference vector can effectively transfected MIN6 cells insulin secretion of mouse islet secreting cells MIN6.PA-PL A1 gene silence was obviously decreased, suggesting that the PA-PLA1 gene expression correlated with MIN6 cell insulin secretion, suggesting that the enzyme on the islet beta cell insulin secretion may play a positive regulatory role. The results of this study for further study of islet beta cell signal The molecular mechanisms of signal transduction and insulin secretion provide new clues.

【學(xué)位授予單位】：桂林醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R346

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