發(fā)布時(shí)間：2018-05-13 04:11

  本文選題：水通道蛋白 + RNA干擾　； 參考：《南京醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的：探討RNA干擾下調(diào)人AQP3基因表達(dá)對(duì)腸粘膜上皮屏障的影響，并探討其可能的作用機(jī)制。 方法：在Caco-2細(xì)胞系中檢測(cè)AQP3基因的表達(dá)，構(gòu)建沉默AQP3mRNA表達(dá)的shRNA慢病毒載體，，轉(zhuǎn)染Caco-2細(xì)胞系，建立穩(wěn)定轉(zhuǎn)染細(xì)胞系，熒光定量PCR以及Western blot驗(yàn)證其在mRNA和蛋白水平的干擾效率。體外建立腸粘膜上皮屏障，分為實(shí)驗(yàn)分為三組：空白對(duì)照組（CON）、陰性對(duì)照組(NC)、AQP3干擾組(RNAi)，分別在第7，14，21，28天檢測(cè)細(xì)胞跨膜電阻（TEER），熒光黃(Luciferyellow，LY)的通透性和易位的大腸桿菌（E.coli C25）的數(shù)量。Western blot驗(yàn)證緊密連接（TJ）相關(guān)蛋白的表達(dá)情況。并且采用免疫細(xì)胞化學(xué)法觀察緊密連接相關(guān)蛋白，如Occludin蛋白，Claudin-1蛋白的分布和結(jié)構(gòu)變化。 結(jié)果：熒光定量PCR及Western blot結(jié)果顯示shRNA慢病毒載體可有效沉默AQP3在Caco-2細(xì)胞系中的表達(dá)，與空載質(zhì)粒組相比，RNA干擾組在第7，14，21天TEER明顯降低，熒光黃的通透性增大并且易位的大腸桿菌（E.coli C25）的數(shù)量明顯增多，各檢測(cè)時(shí)間點(diǎn)RNAi組和空載質(zhì)粒組比較均存在統(tǒng)計(jì)學(xué)差異（P㩳0.05)。Western blot結(jié)果顯示AQP3干擾組緊密連接相關(guān)蛋白Occludin以及Claudin-1的表達(dá)明顯降低。免疫細(xì)胞化學(xué)結(jié)果顯示Caco-2細(xì)胞間Occludin以及Claudin-1主要表達(dá)在細(xì)胞膜和/或胞漿中，相鄰Caco-2細(xì)胞間TJ結(jié)構(gòu)遭到破壞。 結(jié)論：靶向AQP3的shRNA技術(shù)通過(guò)對(duì)AQP3的有效沉默可明顯降低腸粘膜屏障的完整性，為進(jìn)一步探討腸粘膜屏障損傷治療提供新的思路。
[Abstract]:Aim: to investigate the effect of RNA interference on intestinal mucosal epithelial barrier and its possible mechanism. Methods: to detect the expression of AQP3 gene in Caco-2 cell line, construct the shRNA lentivirus vector which silenced the expression of AQP3mRNA, transfect Caco-2 cell line, establish stable transfection cell line, test its interference efficiency at mRNA and protein level by fluorescence quantitative PCR and Western blot. To establish intestinal mucosal epithelial barrier in vitro, The experiment was divided into three groups: blank control group (Conn), negative control group (control group) and negative control group (control group). The transmembrane resistance (TJ) and the translocation of E. coli C25 (E. coli C25) were detected on day 714, 21 and 28, respectively. Western blot was used to verify the close connection of TJ). Expression of related proteins. Immunocytochemistry was used to observe the distribution and structural changes of tightly linked proteins, such as Occludin protein Claudin-1. Results: the results of fluorescence quantitative PCR and Western blot showed that shRNA lentivirus vector could effectively silence the expression of AQP3 in Caco-2 cell line. Compared with the control group, the TEER of shRNA lentivirus interference group was significantly lower than that of the blank plasmid group on day 7, 14 and 21. The permeability of fluorescent yellow increased and the number of E. coli C25 translocated increased significantly. There was statistical difference between RNAi group and no-load plasmid group at each time point. The results of Western blot showed that the expression of Occludin and Claudin-1 in AQP3 interference group was significantly lower than that in control group. The results of immunocytochemistry showed that Occludin and Claudin-1 were mainly expressed in the cell membrane and / or cytoplasm of Caco-2, and the structure of TJ between adjacent Caco-2 cells was destroyed. Conclusion: shRNA targeting AQP3 can significantly reduce the integrity of intestinal mucosal barrier through the effective silencing of AQP3, which provides a new idea for further study on the treatment of intestinal mucosal barrier injury.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 隋海心,任罡;水分子通道蛋白的結(jié)構(gòu)與功能[J];化學(xué)進(jìn)展;2004年02期

2 關(guān)兵;張慶豐;董震;楊占泉;;水通道蛋白1及4在大鼠喉組織中的表達(dá)[J];臨床耳鼻咽喉科雜志;2006年03期

3 王石生;顏春松;;肺水通道蛋白與肺疾病[J];實(shí)用臨床醫(yī)學(xué);2006年05期

4 路煒,漆洪波;水通道蛋白調(diào)節(jié)的研究進(jìn)展[J];國(guó)外醫(yī)學(xué).婦幼保健分冊(cè);2005年01期

5 關(guān)兵,董震;水通道蛋白與腫瘤[J];中國(guó)腫瘤臨床;2005年13期

6 李燕;漆洪波;;水通道蛋白質(zhì)的生物學(xué)作用[J];中華婦幼臨床醫(yī)學(xué)雜志(電子版);2006年02期

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