实时相对定量反转录PCR检测小鼠阴道黏膜炎症因子的表达

Determination of the expressions of inflammatory cytokines in mouse vaginal mucosa with real-time relative quantitative RT PCR

目的建立实时相对定量反转录聚合酶链反应(real-time RQ RT-PCR)方法,能快速准确地检测各种炎症因子在阴道黏膜的转录表达情况,从而评价包括杀微生物剂在内的生殖道或直肠黏膜用候选药物的刺激毒性。方法以Eppendorf Mastercycler ep realplex实时荧光定量PCR仪为检测平台,选择小鼠β-actin基因为内参,建立基于SYBR GreenⅠ荧光染料的real-time PCR检测方法,对白介素2(Interleukin 2,IL-2)、IL-4、IL-6、IL-10、IL-17A、肿瘤坏死因子α(Tumor necrosis factorα,TNF-α)、γ干扰素(Interferonγ,IFN-γ)等炎症因子在生殖道黏膜的mRNA转录水平,同时进行检测,并用2-⊿⊿Ct方法计算实验组小鼠目的基因相对于空白组小鼠的表达差异情况。用微量样本多指标流式蛋白定量技术(Cytometric Bead Array,CBA)对结果进行验证。结果使用real-time RQRT-PCR能对小鼠阴道黏膜炎症因子的表达情况进行快速检测,结果与CBA检测结果相吻合。结论该方法快速、灵敏、特异性强、价格低廉、高通量。可用于杀微生物剂等生殖道或直肠黏膜用候选药物的临床前安全性评价工作。

Objective To determine expressions of inflammatory cytokines in mouse vaginal mucosa which are important endpoints in the preclinical safety evaluation of mucosa applied agents including microbicide.Methods Relative gene expressions of seven cytokines in vaginal mucosa,including interleukin（IL）-2,IL-4,IL-6,IL-10,IL-17A,tumor necrosis factor（TNF）-α and interferon（IFN）-γ,were analyzed using real-time relative quantification reverse transtription PCR（real-time RQ RT PCR） and the 2-⊿⊿Ct method.The results were verified by Cytometric Bead Array（CBA）.Results The expressions of inflammatory cytokines in mouse vaginal mucosa can be quantified by real-time RQ RT PCR in a rapid and accurate way,the result of which is corroborate with that of CBA.Conclusions The developed real-time RQ RT PCR can be used to determine change of inflammatory factors in mouse vaginal mucosa.