pEGFP-C3-PENK载体的构建及在体内外的表达

Construction of eukaryotic expression vector of pEGFP-C3-PENK and its in vivo and vitro expression

目的：观察载体介导的外源性脑啡肽在细胞以及大鼠体内的表达与生物学作用.方法：将从大鼠脑组织中扩增的前脑啡肽原基因与真核绿色荧光载体连接构建新载体pEGFP-C3-PENK,体外转染NIH3T3细胞,利用荧光显微镜及免疫细胞化学观察新建载体在NIH3T3细胞内表达绿色荧光及脑啡肽;制备神经性疼痛大鼠模型并在蛛网膜下腔注射新建载体,通过大鼠疼痛阈值变化检测新合成脑啡肽的生物学作用.结果：在NIH3T3细胞与大鼠蛛网膜下腔均有大量的脑啡肽表达,大鼠的热敏阈明显提高.结论：新建载体介导的脑啡肽在生物体内能持续稳定表达并发挥生物学功能.

Objective： To observe the expression in vivo and biologic effect of vector mediated exogenous enkephalin in somatic cells of rats. Methods： The preproenkephalin gene was amplified and inserted into eukaryotic expression vector pEGFP-C3. And then, the recombinant vector pEGFP-C3-PENK was transfeeted into NIH3T3 cells to detect the expression of enkephalin using immunocytochemieal staining. Biological function of exogenous enkephalin was determined based on the change of pain thresholds in neuropathic pain rats after injected recombinant vector pEGFP-C3-PENK into subarachnoid space. Results： Enkephalin was expressed strongly in NIN3T3 cells and the subarachnoid space of rats. After injection with recombinant vector pEGFP-C3-PENK, the temperature sensitive threshold of neuropathic pain rats was improved obviously. Conclusion： New vector mediated enkephalin can be expressed continually and have biological function.