小鼠胸腺及其功能回復(fù)性的研究
發(fā)布時(shí)間：2018-09-08 21:27
【摘要】：第一章 評價(jià)小鼠胸腺功能和狀態(tài)準(zhǔn)確方法的建立 目的:為了準(zhǔn)確和全面地評價(jià)胸腺的功能和狀態(tài),需建立:實(shí)時(shí)定量PCR對初始T細(xì)胞標(biāo)記物(sjTRECs)進(jìn)行絕對定量以評價(jià)胸腺的生成和輸出功能的研究方法;實(shí)時(shí)定量逆轉(zhuǎn)錄PCR對幾種胸腺狀念和功能相關(guān)基因的表達(dá)進(jìn)行相對定量以直接評價(jià)胸腺微環(huán)境的研究方法。 方法: 實(shí)時(shí)定量PCR檢測sjTRECs的方法:提取小鼠胸腺和脾臟淋巴細(xì)胞基因組DNA;普通PCR擴(kuò)增目的片段;純化構(gòu)建標(biāo)準(zhǔn)質(zhì)粒的RAG_2片段;構(gòu)建標(biāo)準(zhǔn)重組質(zhì)粒并鑒定;優(yōu)化PCR體系;進(jìn)行實(shí)時(shí)定量PCR反應(yīng),建立標(biāo)準(zhǔn)曲線;檢測小鼠胸腺及脾臟淋巴細(xì)胞sjTRECs含量。 實(shí)時(shí)定量RT-PCR法定量檢測GAPDH、LMO2、Foxn1、IL-7基因表達(dá)的方法:提取小鼠胸腺淋巴細(xì)胞RNA;逆轉(zhuǎn)錄成為cDNA:PCR擴(kuò)增目的片段;優(yōu)化PCR體系;進(jìn)行實(shí)時(shí)定量PCR反應(yīng),建立擴(kuò)增曲線;反應(yīng)結(jié)束后,改定閾值,軟件輸出Ct值;根據(jù)比較Ct值法公式,獲得不同處理組間相關(guān)基因表達(dá)比值。 結(jié)果: 實(shí)時(shí)定量PCR檢測sjTRECs的方法:確定最適反應(yīng)條件和體系后實(shí)時(shí)定量PCR獲得可信度高的標(biāo)準(zhǔn)曲線(斜率為-3.738,R~2=0.998)和具產(chǎn)物單一峰的熔解曲線;通過標(biāo)準(zhǔn)曲線獲得TREC和RAG的拷貝數(shù);計(jì)算出樣本的sjTRECs含量。 實(shí)時(shí)定量RT-PCR法定量檢測基因表達(dá)的方法:普通RT-PCR擴(kuò)增出目的片段;確定最適反應(yīng)條件和體系后實(shí)時(shí)定量PCR獲得S型的擴(kuò)增曲線和具產(chǎn)物單一峰的熔解曲線;輸出不同樣本Ct值,根據(jù)比較Ct值法計(jì)算基因表達(dá)的比值。 結(jié)論:成功建立利用實(shí)時(shí)定量PCR檢測小鼠T淋巴細(xì)胞中sjTRECs含量以及胸腺相關(guān)基因表達(dá)的方法。
[Abstract]:Chapter 1: establishment of an accurate method for evaluating thymus function and status in mice objective: to evaluate the function and state of thymus accurately and comprehensively, It is necessary to establish a real-time quantitative PCR method to evaluate the function of thymus production and output by absolutely quantifying the initial T cell marker (sjTRECs). Real-time quantitative reverse transcription (PCR) was used to quantify the expression of several thymic genes in order to evaluate the thymic microenvironment directly. Methods: Real-time quantitative PCR was used to detect sjTRECs. The genomic DNA; of mouse thymus and spleen lymphocytes was amplified by normal PCR. The RAG_2 fragment of the standard plasmid was purified, the standard recombinant plasmid was constructed and identified, the PCR system was optimized, the real-time quantitative PCR reaction was carried out, the standard curve was established, and the content of sjTRECs in mouse thymus and spleen lymphocytes was detected. Real-time quantitative RT-PCR assay for quantitative detection of GAPDH,LMO2,Foxn1,IL-7 gene expression: reverse transcription of RNA; from mouse thymic lymphocytes into cDNA:PCR amplification target fragment; optimization of PCR system; real-time quantitative PCR reaction; establishment of amplification curve; According to the formula of comparing Ct value, the relative gene expression ratio among different treatment groups was obtained. Results: the method of detecting sjTRECs by real-time quantitative PCR: determine the optimum reaction conditions and obtain the standard curve with high reliability after determining the optimal reaction conditions and the system. -3.738N ~ (2 +) -0.998) and the melting curve with a single product peak; The copy numbers of TREC and RAG were obtained by the standard curve, and the sjTRECs content of the sample was calculated. Methods for quantitative detection of gene expression by real-time quantitative RT-PCR: the target fragment was amplified by ordinary RT-PCR, the S-type amplification curve and melting curve with a single product peak were obtained by determining the optimal reaction conditions and real-time quantitative PCR, and the Ct values of different samples were outputted. The ratio of gene expression was calculated by comparing Ct value method. Conclusion: a real-time quantitative PCR method was established for the detection of sjTRECs content and thymus related gene expression in T lymphocytes of mice.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392
[Abstract]:Chapter 1: establishment of an accurate method for evaluating thymus function and status in mice objective: to evaluate the function and state of thymus accurately and comprehensively, It is necessary to establish a real-time quantitative PCR method to evaluate the function of thymus production and output by absolutely quantifying the initial T cell marker (sjTRECs). Real-time quantitative reverse transcription (PCR) was used to quantify the expression of several thymic genes in order to evaluate the thymic microenvironment directly. Methods: Real-time quantitative PCR was used to detect sjTRECs. The genomic DNA; of mouse thymus and spleen lymphocytes was amplified by normal PCR. The RAG_2 fragment of the standard plasmid was purified, the standard recombinant plasmid was constructed and identified, the PCR system was optimized, the real-time quantitative PCR reaction was carried out, the standard curve was established, and the content of sjTRECs in mouse thymus and spleen lymphocytes was detected. Real-time quantitative RT-PCR assay for quantitative detection of GAPDH,LMO2,Foxn1,IL-7 gene expression: reverse transcription of RNA; from mouse thymic lymphocytes into cDNA:PCR amplification target fragment; optimization of PCR system; real-time quantitative PCR reaction; establishment of amplification curve; According to the formula of comparing Ct value, the relative gene expression ratio among different treatment groups was obtained. Results: the method of detecting sjTRECs by real-time quantitative PCR: determine the optimum reaction conditions and obtain the standard curve with high reliability after determining the optimal reaction conditions and the system. -3.738N ~ (2 +) -0.998) and the melting curve with a single product peak; The copy numbers of TREC and RAG were obtained by the standard curve, and the sjTRECs content of the sample was calculated. Methods for quantitative detection of gene expression by real-time quantitative RT-PCR: the target fragment was amplified by ordinary RT-PCR, the S-type amplification curve and melting curve with a single product peak were obtained by determining the optimal reaction conditions and real-time quantitative PCR, and the Ct values of different samples were outputted. The ratio of gene expression was calculated by comparing Ct value method. Conclusion: a real-time quantitative PCR method was established for the detection of sjTRECs content and thymus related gene expression in T lymphocytes of mice.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392
【共引文獻(xiàn)】
相關(guān)期刊論文 前10條
1 ;Thymic Output: Influence Factors and Molecular Mechanism[J];Cellular & Molecular Immunology;2006年05期
2 歐雪玲;伍新堯;童大躍;時(shí)燕薇;孫宏鈺;;人外周血sjTREC水平與年齡的相關(guān)性調(diào)查[J];解剖學(xué)研究;2010年03期
3 曾慧蘭;蔣建偉;李揚(yáng)秋;;胸腺輸出及sjTRECs與移植后 T 細(xì)胞免疫重建[J];國外醫(yī)學(xué)(內(nèi)科學(xué)分冊);2005年10期
4 陳東,山岸舞,曾我浩之,伊藤恒敏;IL-6陽性細(xì)胞在小鼠胸腺內(nèi)的定位[J];解剖學(xué)雜志;2002年06期
5 郭海燕;陳明;汪莉萍;葛國洪;潘修成;李麗;李秀華;;原發(fā)性肝癌和乙型肝炎肝硬化患者胸腺近期輸出功能初步探討[J];臨床薈萃;2007年10期
6 葛國洪;陳明;李紅兵;郭海燕;汪莉萍;;慢性乙型肝炎患者抗病毒治療前后TRECs含量的變化[J];臨床肝膽病雜志;2007年03期
7 郭海燕;陳明;汪莉萍;葛國洪;張言超;;實(shí)時(shí)熒光定量PCR分析慢性丙型肝炎患者胸腺近期輸出功能[J];臨床肝膽病雜志;2007年06期
8 李揚(yáng)秋,陳少華,楊力建,周羽z，
本文編號(hào)：2231748
本文鏈接：http://sikaile.net/yixuelunwen/binglixuelunwen/2231748.html
最近更新
教材專著
