钙激活氯通道TMEM16A在小鼠心肌组织的表达

发布时间：2018-01-04 04:36

本文关键词：钙激活氯通道TMEM16A在小鼠心肌组织的表达　出处：《青岛大学》2012年硕士论文　论文类型：学位论文

更多相关文章： TMEM16A蛋白 氯通道 小鼠 心肌组织

【摘要】：目的：钙激活氯通道(Calcium-activated Cholirde Channels, CaCC)是心肌主要的氯离子通道之一,该通道与正常心肌细胞的电活动和某些心脏疾病(如心肌梗死,心力衰竭)关系密切。本实验探讨钙激活氯通道蛋白TMEM16A在小鼠心肌组织的表达。材料和方法：1.清洁级昆明种小鼠购自青岛市药检所。成年小鼠,体重25-35mg,新生小鼠出生1-3天内使用。2.拉颈处死后迅速取出心脏,取50-100mg心肌组织以Trizol试剂提取总RNA。以提取的DNA为模板,采用RT-PCR检测心肌TMEM16A mRNA的表达。3.成年小鼠快速处死后取出100mg心肌组织,4g/L多聚甲醛(4℃)固定6-8h,转入300g/L蔗糖放置24h至沉底,做14um厚连续冷冻切片。免疫荧光法检测心肌TMEM16A蛋白的表达。4.成年小鼠取出心脏后,用RIPA裂解液裂解后提取蛋白,酶标仪测出蛋白浓度,免疫印迹法检测成年小鼠心肌组织的TMEM16A蛋白的表达。结果：1.新生及成年小鼠TMEM16A(?)的基因的扩增条带为330bp,与TMEM16AcDNA阳性对照位于同一水平,提示二者心肌均有TMEM16A mRNA的表达。差异无显著性(n=4,t=0.7698,P0.05)。2.成年小鼠心肌切片用兔抗鼠TMEM16A抗体(一抗)处理后再加FITC标记的山羊抗兔IgG(二抗)染色,激光共聚焦显微镜下显示绿色荧光信号,WGA-TMR标记的细胞膜呈红色荧光,二者融合呈黄色。阴性对照(PBS代替一抗)未见绿色荧光,仅见红色的细胞膜。提示小鼠心肌组织存在TMEM16A蛋白的表达,且主要定位于细胞膜上。3.成年小鼠TMEM16A目的基因的扩增条带是130kb,与TMEM16A阳性对照位于同一水平,提示成年小鼠心肌组织中有TMEM16A的表达。结论：本研究利用分子生物学、免疫荧光染色技术和免疫印迹法,在小鼠心肌组织成功地检测到TMEM16A mRNA和蛋白的表达。新生小鼠和成年小鼠TMEM16A mRNA水平无明显差异,提示TMEM16A氯通道蛋白在小鼠心肌组织的生长发育过程中相对恒定。
[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.

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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