中文摘要：

目的：研究肺泡巨噬细胞（NR8383）不同蛋白酶体激活程度对内质网应激的影响。方法：构建UbG76V-GFP融合蛋白,将含有UbG76V-GFP的质粒导入NR8383细胞,筛选出可稳定表达UbG76V-GFP的细胞系,通过蛋白酶体抑制剂（MG132）、蛋白酶体激活剂（阿霉素）干预蛋白酶体活性。荧光显微镜观察不同蛋白酶体活性下大鼠肺泡巨噬细胞在缺氧复氧2 h、4 h、6 h时蛋白酶体活性,Western blot及PCR技术检测不同蛋白酶体活性下大鼠肺泡巨噬细胞在缺氧复氧2 h、4 h、6 h时泛素化蛋白及内质网应激相关基因的表达。结果：在缺氧复氧2 h、4 h、6 h这3个时间点,加入MG132组大鼠肺泡巨噬细胞绿色荧光及泛素化蛋白（Ubiquitin）表达明显降低（P〈0.05）,而PCR及Western blot示内质网应激基因BIP（免疫球蛋白结合蛋白）、XBP-1（X-盒结合蛋白）和CHOP（C/EBP同源蛋白）平均扩增量及蛋白表达量明显增加（P〈0.05）;加入阿霉素组大鼠肺泡巨噬细胞在缺氧复氧2 h、4h、6 h表现出相反的实验结果,绿色荧光及Ubiquitin蛋白相对表达均明显增加（P〈0.05）,而PCR及Western blot示内质网应激基因BIP、XBP-1和CHOP平均扩增量及蛋白表达量明显增加（P〈0.05）。结论：本实验结果表明活细胞泛素-蛋白酶体活性程度与内质网应激存在紧密联系,外源性增强泛素蛋白酶体活性会抑制内质网应激,外源性减弱泛素蛋白酶体活性会增强内质网应激。

英文摘要：

Objective： To study the impact of different protease activated degree to endoplasmic reticulum stress in alveolar macrophage（NR8383）. Methods： After conducting ubiquitin-conjugated variant of the green fluorescent protein（UbG76V-GFP）, the plasmid contained UbG76V-GFP was imported into NR8383 cells. The stable expression cell lines were screened to further study. The protease activity was regulated by proteasome inhibitors（MG132）, protease activator（adriamycin）. Fluorescence microscope was used to analyze the expression of UbG76V-GFP. By Western blot and PCR technology, observing the different protease activated degree and endoplasmic reticulum stress in the time of hypoxia-reoxygenation（H/R） 2 hours, 4 hours, 6 hours after giving the process of proteasome inhibitors（MG132）, protease activator（adriamycin）. Results： MG132 pretreatment led to lower green fluorescent protein expression（UbG76V-GFP） and decreased ubiquitin, but increased protein expression or gene transcription of BIP（binding immunoglobulin protein）,XBP-1（X-Box Binding Protein 1） and CHOP（C/EBP homologus protein） by Western blot or PCR method following H/R treatment at 2h, 4 h and 6 h（P〈0.05）. On the contrary, green fluorescent protein expression（UbG76V-GFP） and ubiquitin protein were enhanced, and protein expression or gene transcription of BIP, XBP-1 and CHOP were decreased by treated with adriamycin following H/R treatment at2 h, 4 h and 6 h（P〈0.05）. Conclusion： A close relationship between the degree of ubiquitin proteasome activity and endoplasmic reticulum stress was showed that exogenous enhanced ubiquitin proteasome activity inhibited the endoplasmic reticulum stress,exogenous abate ubiquitin proteasome activity enhanced the endoplasmic reticulum stress.