小鼠胸腺及其功能回复性的研究
发布时间：2018-09-08 21:27
【摘要】：第一章 评价小鼠胸腺功能和状态准确方法的建立 目的:为了准确和全面地评价胸腺的功能和状态,需建立:实时定量PCR对初始T细胞标记物(sjTRECs)进行绝对定量以评价胸腺的生成和输出功能的研究方法;实时定量逆转录PCR对几种胸腺状念和功能相关基因的表达进行相对定量以直接评价胸腺微环境的研究方法。 方法: 实时定量PCR检测sjTRECs的方法:提取小鼠胸腺和脾脏淋巴细胞基因组DNA;普通PCR扩增目的片段;纯化构建标准质粒的RAG_2片段;构建标准重组质粒并鉴定;优化PCR体系;进行实时定量PCR反应,建立标准曲线;检测小鼠胸腺及脾脏淋巴细胞sjTRECs含量。 实时定量RT-PCR法定量检测GAPDH、LMO2、Foxn1、IL-7基因表达的方法:提取小鼠胸腺淋巴细胞RNA;逆转录成为cDNA:PCR扩增目的片段;优化PCR体系;进行实时定量PCR反应,建立扩增曲线;反应结束后,改定阈值,软件输出Ct值;根据比较Ct值法公式,获得不同处理组间相关基因表达比值。 结果: 实时定量PCR检测sjTRECs的方法:确定最适反应条件和体系后实时定量PCR获得可信度高的标准曲线(斜率为-3.738,R~2=0.998)和具产物单一峰的熔解曲线;通过标准曲线获得TREC和RAG的拷贝数;计算出样本的sjTRECs含量。 实时定量RT-PCR法定量检测基因表达的方法:普通RT-PCR扩增出目的片段;确定最适反应条件和体系后实时定量PCR获得S型的扩增曲线和具产物单一峰的熔解曲线;输出不同样本Ct值,根据比较Ct值法计算基因表达的比值。 结论:成功建立利用实时定量PCR检测小鼠T淋巴细胞中sjTRECs含量以及胸腺相关基因表达的方法。
[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.
【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：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.
【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392
【共引文献】
相关期刊论文 前10条
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