本文關(guān)鍵詞：鈣激活氯通道TMEM16A在小鼠心肌組織的表達(dá)　出處：《青島大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： TMEM16A蛋白 氯通道 小鼠 心肌組織

【摘要】：目的：鈣激活氯通道(Calcium-activated Cholirde Channels, CaCC)是心肌主要的氯離子通道之一,該通道與正常心肌細(xì)胞的電活動(dòng)和某些心臟疾病(如心肌梗死,心力衰竭)關(guān)系密切。本實(shí)驗(yàn)探討鈣激活氯通道蛋白TMEM16A在小鼠心肌組織的表達(dá)。 材料和方法：1.清潔級(jí)昆明種小鼠購(gòu)自青島市藥檢所。成年小鼠,體重25-35mg,新生小鼠出生1-3天內(nèi)使用。2.拉頸處死后迅速取出心臟,取50-100mg心肌組織以Trizol試劑提取總RNA。以提取的DNA為模板,采用RT-PCR檢測(cè)心肌TMEM16A mRNA的表達(dá)。3.成年小鼠快速處死后取出100mg心肌組織,4g/L多聚甲醛(4℃)固定6-8h,轉(zhuǎn)入300g/L蔗糖放置24h至沉底,做14um厚連續(xù)冷凍切片。免疫熒光法檢測(cè)心肌TMEM16A蛋白的表達(dá)。4.成年小鼠取出心臟后,用RIPA裂解液裂解后提取蛋白,酶標(biāo)儀測(cè)出蛋白濃度,免疫印跡法檢測(cè)成年小鼠心肌組織的TMEM16A蛋白的表達(dá)。 結(jié)果：1.新生及成年小鼠TMEM16A(?)的基因的擴(kuò)增條帶為330bp,與TMEM16AcDNA陽(yáng)性對(duì)照位于同一水平,提示二者心肌均有TMEM16A mRNA的表達(dá)。差異無(wú)顯著性(n=4,t=0.7698,P0.05)。2.成年小鼠心肌切片用兔抗鼠TMEM16A抗體(一抗)處理后再加FITC標(biāo)記的山羊抗兔IgG(二抗)染色,激光共聚焦顯微鏡下顯示綠色熒光信號(hào),WGA-TMR標(biāo)記的細(xì)胞膜呈紅色熒光,二者融合呈黃色。陰性對(duì)照(PBS代替一抗)未見(jiàn)綠色熒光,僅見(jiàn)紅色的細(xì)胞膜。提示小鼠心肌組織存在TMEM16A蛋白的表達(dá),且主要定位于細(xì)胞膜上。3.成年小鼠TMEM16A目的基因的擴(kuò)增條帶是130kb,與TMEM16A陽(yáng)性對(duì)照位于同一水平,提示成年小鼠心肌組織中有TMEM16A的表達(dá)。 結(jié)論：本研究利用分子生物學(xué)、免疫熒光染色技術(shù)和免疫印跡法,在小鼠心肌組織成功地檢測(cè)到TMEM16A mRNA和蛋白的表達(dá)。新生小鼠和成年小鼠TMEM16A mRNA水平無(wú)明顯差異,提示TMEM16A氯通道蛋白在小鼠心肌組織的生長(zhǎng)發(fā)育過(guò)程中相對(duì)恒定。
[Abstract]:Objective: calcium activated chloride channels (Calcium-activated Cholirde, Channels, CaCC) is one of the major cardiac chloride ion channel, the channel and the normal electrical activity of myocardial cells and some heart diseases (such as myocardial infarction, heart failure) are closely related. The effects of calcium activated chloride channel protein TMEM16A expression in the myocardial tissue of mice.
Materials and methods: 1. Kunming mice of clean grade were purchased from Qingdao Institute of adult mice, weight 25-35mg, newborn mouse.2. were executed within 1-3 days after quickly remove the heart, myocardium 50-100mg total RNA. was extracted with Trizol reagent with the extracted DNA as template, take the myocardial tissue 100mg with rapid death the expression of.3. in adult mice RT-PCR detection of myocardial TMEM16A mRNA after 4g/L paraformaldehyde fixed 6-8h (4 C), to 300g/L 24h to be placed in sucrose sink, 14um thick continuous frozen sections. The expression of.4. in adult mice to detect myocardial TMEM16A protein by immunofluorescence after removing the heart, the extraction of protein by RIPA lysis, measure the concentration of protein expression was detected by eliasa, immunoblotting in adult mice myocardium TMEM16A protein.
Results: 1. newborn and adult mice (TMEM16A?) gene amplified bands of 330bp, TMEM16AcDNA and positive control are at the same level, suggesting that expression of two TMEM16A were myocardial mRNA. No significant difference (n=4, t=0.7698, P0.05).2. in adult mouse myocardial sections with Rabbit anti mouse TMEM16A antibody (anti) treatment plus FITC labeled Goat anti rabbit IgG (two -) staining showed green fluorescence signal under confocal laser microscopy, cell membrane WGA-TMR labeled red fluorescence, two fusion yellow. The negative control (PBS instead of primary antibody) showed no green fluorescence, only to see the red cell membrane. The expression of TMEM16A protein that in mouse myocardium, and mainly localized in the cell membrane of.3. adult mice TMEM16A gene amplification band is 130kb, and the TMEM16A positive control are at the same level, suggesting that adult mice myocardial tissues expressed TMEM16A.
Conclusion: This study using molecular biology, immunofluorescence staining and Western blot method successfully detected in myocardial tissue of mice to the expression of TMEM16A protein and mRNA. There is no significant difference in neonatal mice and adult mice TMEM16A mRNA levels, suggesting a relatively constant TMEM16A chloride channel protein in myocardial tissue of mice in the process of growth and development.

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

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本文編號(hào)：1377076