定量测定IL-4和IFN-γ mRNA的RT-竞争PCR法及其应用

A competitive RT-PCR method for quantitative determination of IFN-γ mRNA and IL-4 mRNA and its application

目的 建立测定IFN γmRNA和IL 4mRNA和RT 竞争PCR方法 ,探讨体外诱生条件并作临床初步应用。方法 用自行设计和制备的IFN γcDNA和IL 4cDNA竞争模板 ,作RT 竞争PCR ,对健康人经PMA和calciumionophore(钙离子载体 )诱导的PBMC、11例健康人和 13例哮喘患者PBMC作IFN γmRNA和IL 4mRNA定量测定。结果 在建立了准确的定量测定mRNA方法的基础上 ,确认在calciumionophore 2 .0 μmol/L浓度时 ,用PMA 10 0ng/ml诱导 6h或PMA 80ng/ml诱导 7h ,IFN γmRNA或IL 4mRNA表达分别达最高水平 ,两种细胞因子mRNA在健康人PBMC中不表达 ,而在 10 /13例和 11/ 13例哮喘患者中分别呈不同程度的表达 ,其中IL 4mRNA表达水平高于IFN γmRNA。结论用自行设计和制备的竞争模板经RT 竞争PCR法可对细胞内IFN γmRNA和IL 4mRNA作较准确的定量测定 ,方法简便且敏感 ;健康人PBMC需经一定条件刺激方可表达两种细胞因子mRNA ;多数哮喘患者PBMC不经刺激即可表达两种细胞因子mRNA ,IL 4mRNA表达水平高于IFN γmRNA ,提示本法可用于研究细胞因子基因表达和哮喘等患者细胞因子变化动态。

Objective To establish a competitive RT PCR method for quantitative determination of IFN γ mRNA and IL 4 mRNA and explore the optimal inductive condition in vitro. Methods IFN γ mRNA and IL 4 mRNA in PBMC induced by PMA and calcium ionophore of a healthy volunteer (HV) and in unstimulated PBMC of 11 HVs and 13 asthma patients were determined quantitatively with the established competitive RT PCR method using self prepared internal standard as competitive templets. Results A simple, sensitive and accurate method for quantitatively determining cytokine mRNA has been established in the HV′s PBMC stimulated with calcium ionophore 2.0μmol/L, and PMA 100ng/ml harvested at 6?h or PMA 80ng/ml harvested at 7?h, expression of IFN γ mRNA or IL 4 mRNA reached the highest level respectively. In the HV′s unstimulated PBMC, the expression of mRNA of the two cytokines could not be detected. In unstimulated PBMC from ten and eleven of the thirteen asthma patients, IFN γ mRNA and IL 4 mRNA were expressed in various level, the former was lower. Conclusion The method was simple, sensitive and accurate, HV′s PBMC, only stimulated, expressed mRNA of the two cytokines, but unstimulated PBMC from most asthma patients expressed IFN γ mRNA and IL 4 mRNA, the former was lower. The method could be applied in exploring cytokine gene expression and monitoring the change of cytokine mRNA in PBMC of patients.