实时定量RT-PCR评价小鼠胸腺功能和状态方法的建立

Establishing of Real-time Quantitative RT-PCR Method Evaluating Murine Thymus Function and Status

目的建立实时定量RT-PCR的分析方法,对胸腺微环境内的相关基因的表达进行准确的定性和定量,以分析胸腺微环境及其中细胞的状态,直接有效地评价胸腺功能和状态。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RT-PCR反应,建立扩增曲线;反应结束后,设定阈值,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RT-PCR的扩增曲线为S型的动力学曲线,熔解曲线为单一峰,证实反应的准确性和特异性;36周小鼠相对于1周小鼠的胸腺相关基因表达变化比值为:LMO2 2.54±0.08、Foxn1 0.68±0.02I、L-7 0.15±0.03。结论36周小鼠发生了胸腺衰老,胸腺的功能和状态均出现退化;本研究成功建立利用实时定量RT-PCR评价小鼠胸腺功能和状态方法。

Objective establishing real-time RT-PCR method to qualitatively and quantitatively analyze thymus-associated genes expression for valuing directly thymus microenvironment and evaluating accurately the thymus function and status. Methods The murine thymocytes RNA was extracted and the first-strand of cDNA was synthesized by RT. PCR amplification was performed and the curve output and Ct data were obtained and calculated by comparative C（T） method. Results The real-time PCR obtained S-shaped amplification curve and single peak dissociation curve to demonstrate PCR accuracy and speciality; Different samples＇ Ct data could be exported; According comparative C（T） method, the ratio of gene expression could be calculated and compared with 1 week-old mouse. The gene expression ratios of 36 weeks-old mouse： LMO2 2.54±0.08, Foxnl 0. 68±0. 02,IL-7 0.15±0. 03. Conclusion Thymus atrophy occurred in 36 weeks mouse and the thymus function and status have age-associated decline; The methods to evaluate thymus function and status by real-time RT-PCR are developed successfully.