本文選題：心肌細(xì)胞分離 + Langendorff灌流系統(tǒng)�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2011年碩士論文

【摘要】：背景：心肌細(xì)胞轉(zhuǎn)錄水平是心臟病領(lǐng)域的重要研究方法,幫助認(rèn)識疾病的發(fā)病機(jī)制。傳統(tǒng)研究所用的對象多是心肌組織,但是在正常的心室壁組織中,除心肌細(xì)胞外,還有成纖維細(xì)胞、平滑肌細(xì)胞以及其它類型細(xì)胞,這些細(xì)胞存在會對分析心肌細(xì)胞基因表達(dá)水平造成很大的干擾。所以我們更需要分離心肌細(xì)胞后再進(jìn)行基因表達(dá)分析。既往許多研究者設(shè)計(jì)過不同的分離心肌細(xì)胞的方法,但均有各種的不足,如對于操作技術(shù)、設(shè)備要求較高、分離時間過長、細(xì)胞得出量較少等。酶解法是一種常用的分離心肌方法,但技術(shù)操作細(xì)節(jié)的差異使其分離的結(jié)果不盡一致,且尚無對于分離后提取RNA進(jìn)行基因表達(dá)分析的效果進(jìn)行詳細(xì)的研究。本文將對該方法進(jìn)行深入研究,并進(jìn)行轉(zhuǎn)錄水平分析,為進(jìn)一步使用純化的心肌細(xì)胞進(jìn)行轉(zhuǎn)錄水平研究提供基礎(chǔ)和依據(jù)。 方法：對8周雄性SD大鼠的心臟應(yīng)用Langendorff灌流酶解法進(jìn)行心肌細(xì)胞分離純化,分離得到的細(xì)胞進(jìn)行普通光學(xué)顯微鏡觀察并細(xì)胞計(jì)數(shù)；吖啶橙染色后用熒光顯微鏡觀察細(xì)胞形態(tài)；提取總RNA后應(yīng)用分光光度計(jì)、瓊脂糖凝膠電泳和測RNA完整指數(shù)(RNA integrity number, RIN)的方法檢測RNA完整性；用Real-time RT-PCR的方法檢測心肌組織中三種細(xì)胞特異性標(biāo)志的相對表達(dá)量：肌鈣蛋白T (TnT,心肌細(xì)胞)、波形蛋白(Vim,成纖維細(xì)胞)和平滑肌伐肌動蛋白(a-SMA平滑肌細(xì)胞),作為內(nèi)參的看家基因分別為肽脯氨酰異構(gòu)酶A(PPIA)和核糖體蛋白(RPLPO)。此外選用2周齡、3周齡和8周齡的雄性Wistar大鼠進(jìn)行心肌細(xì)胞分離,分離后提取RNA,用Real-time RT-PCR的方法檢測心肌組織中腺嘌呤核苷酸易位酶(ANT)的兩種亞型ANT-1和ANT-2的表達(dá)量,內(nèi)參基因選擇PPIA。 結(jié)果：整個心肌分離過程大約需要30分鐘左右,可以得到比較好的心肌產(chǎn)出率,普通光學(xué)顯微鏡下觀察絕大部分細(xì)胞形態(tài)完整呈桿狀,有明顯的橫紋,吖啶橙染色后在熒光顯微鏡下觀察亦可見細(xì)胞形態(tài)完整,細(xì)胞核被染成明亮的綠色,而細(xì)胞外的背景幾乎沒有熒光產(chǎn)生。將形態(tài)典型的心肌細(xì)胞記為心肌細(xì)胞,其它均作為不典型細(xì)胞,3只大鼠的細(xì)胞計(jì)數(shù)結(jié)果n心肌細(xì)胞/n所有細(xì)胞分別為：87.3±3.2%,91.8±5.1%,88.1±3.3%。提取的總RNA的A260/280在1.8-2.0之間,符合Real-time RT-PCR需要的RNA純度；電泳結(jié)果提示RNA沒有明顯的降解,與心肌組織提取的RNA無明顯差異(n=3)；RIN值在7-8之間,亦適于進(jìn)行Real-time RT-PCR,與心肌組織提取的RNA無明顯差異(n=6,P0.05)。通過Real-time RT-PCR檢測TnT、Vim和α-SMA相對于PPIA和RPLPO的表達(dá)量,均為經(jīng)過分離Vim和α-SMA的表達(dá)量顯著下降,而TnT的表達(dá)量顯著上升(n=6,P0.05),三者的變化倍數(shù)分別為0.38、0.16、2.56(PPIA作為內(nèi)參)和0.35、0.17、2.33(RPLPO作為內(nèi)參)。ANT-1在心肌細(xì)胞中的表達(dá)量要遠(yuǎn)高于ANT-2(10±1.3倍,n=6),高的倍數(shù)比之前文獻(xiàn)報(bào)道(以心肌組織作為樣本)的結(jié)果要更顯著,ANT-1和ANT-2在心肌發(fā)育過程中(2周、3周和12周)表達(dá)量的變化(n=6)也與之前文獻(xiàn)報(bào)道(以心肌組織作為樣本)有所差異。 結(jié)論： 1.通過對既往文獻(xiàn)的總結(jié),完善了Langendorff灌流酶解分離大鼠心肌細(xì)胞的方法,提純度可達(dá)90%,細(xì)胞產(chǎn)量較高,適于提取RNA進(jìn)行進(jìn)一步研究； 2.該方法在分離過程中RNA沒有明顯的降解,適用于心肌基因表達(dá)分析； 3.基因表達(dá)分析的結(jié)果優(yōu)于以心肌組織作為樣本獲取RNA,單純以心肌組織為樣本進(jìn)行心肌細(xì)胞缺血再灌注損傷機(jī)制和心肌保護(hù)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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329.2

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