Novel multi-probe RNase protection assay set for detection of endotoxin associated receptors gene expression

Objective: To construct the multi-probe ribonuclease protection assay (RPA) template set to be used for detecting expression patterns of MD-2, TLR4, CD14 mRNAs in human peripheral blood mononuclear cells. Methods : The designed cDNA fragments of the three genes were generated by polymerase chain reaction (PCR)using specific primers and directionally cloned into EcoR I and Hind III sites of expression plasmid pSP72 containing the T7/ promoter, the linearized plasmids was used as template to synthesize anti-sense RNA probes. Then we extracted total RNA from peripheral blood mononuclear cells (PBMC) and detected the dynamic expression patterns of the three genes with RPA method. Results: The proper sequence and orientation of the template set were confirmed by sequencing and the template set was successfully used to assay TLR4, MD-2 and CD14 mRNAs in human PBMC. The results showed that the three detected genes decreased transiently 1-3 hours after 100 ng/ml LPS stimulation. Conclusions: These new RPA multi-probe set provided valuable tool for the simultaneous quantitative determination of expression of TLR4, CD14 and MD-2 mRNAs in both constitutive and inducible types.     

Human μ-opioid receptor overexpressed in baculovirus system and its pharmacological characterizations

目的：高效表达具有类似哺乳动物特性的人μ阿片受体．方法：人μ阿片受体表达在重组杆状病毒感染的Ｓｆ９昆虫细胞中，用受体结合分析和ｃＡＭＰ分析研究表达产物的药理学特征．结果：［３Ｈ］二丙诺啡及［３Ｈ］羟甲芬太尼（Ｏｈｍ）的最大结合能力分别是９．１±０．７，６５２±０．２３ｎｍｏｌ／ｇ蛋白．μ选择性激动剂对［３Ｈ］二丙诺啡或［３Ｈ］Ｏｈｍ与表达受体的结合均有很强的抑制作用，而δ及κ激动剂则均无抑制作用．μ选择性激动剂有效抑制ｆｏｒｓｋｏｌｉｎ刺激的ｃＡＭＰ聚集，这种作用能被拮抗剂纳洛酮阻断．结论：在Ｓｆ９昆虫细胞中高效表达的人μ阿片受体保持着野生型人μ阿片受体的特征     

核糖核酸酶保护法MD-2探针的制备

目的 构建用于核糖核酸酶保护法的MD-2转录质粒并制备反义RNA探针。方法 PCR扩增目的片段，并定向亚克隆至pSP72质粒T7启动子下游的多克隆位点，以T7 RNA聚合酶转录合成反义RNA探针。结果 亚克隆插入的片段经测序、酶切鉴定，序列及方向完全正确，成功制备了用于的反义RNA探针。结论 成功地制备了用于核糖核酸酶保护法的MD-2转录模板和探针，为深入研究内毒素信号受体相关机制以及MD-2的基因表达创造了必要的条件。