大鼠心肌细胞分离和基因表达的实验研究

发布时间：2018-06-29 18:44

本文选题：心肌细胞分离 + Langendorff灌流系统　；参考：《北京协和医学院》2011年硕士论文

【摘要】：背景：心肌细胞转录水平是心脏病领域的重要研究方法,帮助认识疾病的发病机制。传统研究所用的对象多是心肌组织,但是在正常的心室壁组织中,除心肌细胞外,还有成纤维细胞、平滑肌细胞以及其它类型细胞,这些细胞存在会对分析心肌细胞基因表达水平造成很大的干扰。所以我们更需要分离心肌细胞后再进行基因表达分析。既往许多研究者设计过不同的分离心肌细胞的方法,但均有各种的不足,如对于操作技术、设备要求较高、分离时间过长、细胞得出量较少等。酶解法是一种常用的分离心肌方法,但技术操作细节的差异使其分离的结果不尽一致,且尚无对于分离后提取RNA进行基因表达分析的效果进行详细的研究。本文将对该方法进行深入研究,并进行转录水平分析,为进一步使用纯化的心肌细胞进行转录水平研究提供基础和依据。方法：对8周雄性SD大鼠的心脏应用Langendorff灌流酶解法进行心肌细胞分离纯化,分离得到的细胞进行普通光学显微镜观察并细胞计数；吖啶橙染色后用荧光显微镜观察细胞形态；提取总RNA后应用分光光度计、琼脂糖凝胶电泳和测RNA完整指数(RNA integrity number, RIN)的方法检测RNA完整性；用Real-time RT-PCR的方法检测心肌组织中三种细胞特异性标志的相对表达量：肌钙蛋白T (TnT,心肌细胞)、波形蛋白(Vim,成纤维细胞)和平滑肌伐肌动蛋白(a-SMA平滑肌细胞),作为内参的看家基因分别为肽脯氨酰异构酶A(PPIA)和核糖体蛋白(RPLPO)。此外选用2周龄、3周龄和8周龄的雄性Wistar大鼠进行心肌细胞分离,分离后提取RNA,用Real-time RT-PCR的方法检测心肌组织中腺嘌呤核苷酸易位酶(ANT)的两种亚型ANT-1和ANT-2的表达量,内参基因选择PPIA。结果：整个心肌分离过程大约需要30分钟左右,可以得到比较好的心肌产出率,普通光学显微镜下观察绝大部分细胞形态完整呈杆状,有明显的横纹,吖啶橙染色后在荧光显微镜下观察亦可见细胞形态完整,细胞核被染成明亮的绿色,而细胞外的背景几乎没有荧光产生。将形态典型的心肌细胞记为心肌细胞,其它均作为不典型细胞,3只大鼠的细胞计数结果n心肌细胞/n所有细胞分别为：87.3±3.2%,91.8±5.1%,88.1±3.3%。提取的总RNA的A260/280在1.8-2.0之间,符合Real-time RT-PCR需要的RNA纯度；电泳结果提示RNA没有明显的降解,与心肌组织提取的RNA无明显差异(n=3)；RIN值在7-8之间,亦适于进行Real-time RT-PCR,与心肌组织提取的RNA无明显差异(n=6,P0.05)。通过Real-time RT-PCR检测TnT、Vim和α-SMA相对于PPIA和RPLPO的表达量,均为经过分离Vim和α-SMA的表达量显著下降,而TnT的表达量显著上升(n=6,P0.05),三者的变化倍数分别为0.38、0.16、2.56(PPIA作为内参)和0.35、0.17、2.33(RPLPO作为内参)。ANT-1在心肌细胞中的表达量要远高于ANT-2(10±1.3倍,n=6),高的倍数比之前文献报道(以心肌组织作为样本)的结果要更显著,ANT-1和ANT-2在心肌发育过程中(2周、3周和12周)表达量的变化(n=6)也与之前文献报道(以心肌组织作为样本)有所差异。结论： 1.通过对既往文献的总结,完善了Langendorff灌流酶解分离大鼠心肌细胞的方法,提纯度可达90%,细胞产量较高,适于提取RNA进行进一步研究； 2.该方法在分离过程中RNA没有明显的降解,适用于心肌基因表达分析； 3.基因表达分析的结果优于以心肌组织作为样本获取RNA,单纯以心肌组织为样本进行心肌细胞缺血再灌注损伤机制和心肌保护策略的研究是需要警惕的。
[Abstract]:Background: the transcriptional level of cardiac myocytes is an important research method in the field of heart disease. It helps to understand the pathogenesis of the disease. Most of the objects used in traditional research are myocardial tissue, but in normal ventricular wall tissue, besides myocardial cells, there are fibroblasts, smooth muscle cells, and other types of cells. The level of gene expression in cardiac myocytes is very disturbing. So we need to separate cardiomyocytes and then analyze the gene expression. Many researchers have designed different methods of separating cardiomyocytes, but there are various deficiencies, such as high equipment, long separation time and less cell quantity for operation technology. Enzyme hydrolysis is a common method of isolation of myocardium, but the differences in technical details make the separation results unanimously, and there is no detailed study on the effect of gene expression analysis after isolation of RNA. This method will be studied in depth, and the transcriptional level analysis will be carried out for further use of purification. Myocardial cells provide basis and basis for transcriptional studies.
Methods: the hearts of the male SD rats were isolated and purified by Langendorff perfusion method for 8 weeks. The isolated cells were observed and counted by ordinary optical microscope. The morphology of the cells was observed with fluorescence microscope after the acridine orange staining. The total RNA was extracted with the spectrophotometer, agarose gel electrophoresis and RNA measurement. The integrity index (RNA integrity number, RIN) was used to detect RNA integrity; the relative expression of three cell specific markers in myocardial tissue was detected by Real-time RT-PCR: troponin T (TnT, cardiac myocytes), vimentin (Vim, fibroblasts) and smooth muscle actin (a-SMA smooth muscle cells), as the internal reference The family genes were peptide prolyl isomerase A (PPIA) and ribosomal protein (RPLPO) respectively. In addition, cardiomyocytes were isolated from male Wistar rats of 2 weeks old, 3 weeks old and 8 weeks old, and RNA was extracted after separation. Real-time RT-PCR was used to detect the expression of two subtypes of adenosine ribosomal translocation enzyme (ANT) in myocardium. Selection of PPIA. for internal reference gene
Results: the whole process of isolation of the whole myocardium takes about 30 minutes, and the rate of cardiac output is better. Under the ordinary optical microscope, most of the cells are rod-shaped and have a clear pattern. The morphology of the cells under the fluorescence microscope is complete and the nucleus is dyed bright green after the fluorescence microscope. The extracellular background was almost without fluorescence. The typical cardiomyocytes were recorded as cardiac myocytes, and the other were used as atypical cells. The cell count results of 3 rats in the 3 rats showed that all /n cells of N cardiomyocytes were 87.3 + 3.2%, 91.8 + 5.1%, and 88.1 + 3.3%. of total RNA extracted from 1.8-2.0, which conformed to the Real-time RT-PCR needs. The RNA purity of RNA showed no obvious degradation and no significant difference from RNA extracted from myocardium (n=3), RIN value was between 7-8 and Real-time RT-PCR, and there was no significant difference between RNA and cardiac tissue (n=6, P0.05). The expression of Vim and alpha -SMA decreased significantly, while the expression of TnT increased significantly (n=6, P0.05). The number of changes of the three were 0.38,0.16,2.56 (PPIA as internal reference) and 0.35,0.17,2.33 (RPLPO as internal reference).ANT-1 in cardiac myocytes was far higher than ANT-2 (10 + 1.3 times, n=6). The results of the muscle tissue as samples were more significant. The changes in the expression of ANT-1 and ANT-2 during the development of the myocardium (2, 3 and 12 weeks) (n=6) were also different from the previous literature (with myocardial tissue as a sample).
Conclusion:
1. through the summary of previous literature, the method of Langendorff perfusion enzyme hydrolysis to isolate rat cardiac myocytes was perfected. The purification degree was up to 90%, and the cell production was high. It was suitable for the extraction of RNA for further study.
2. the RNA did not degrade significantly during the separation process, and was suitable for myocardial gene expression analysis.
The result of 3. gene expression analysis is better than that of myocardial tissue as a sample to obtain RNA. It is necessary to be vigilant to study the mechanism of myocardial ischemia reperfusion injury and the strategy of myocardial protection only with myocardial tissue as sample.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329.2

【参考文献】