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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。
Objective: To investigate the effect of different proteasome activation of alveolar macrophages (NR8383) on endoplasmic reticulum stress. Methods: The UbG76V-GFP fusion protein was constructed and the plasmid containing UbG76V-GFP was transfected into NR8383 cells. The cell line stably expressing UbG76V-GFP was screened by proteasome inhibitor (MG132), proteasome activator (doxorubicin) Interfering proteasome activity. Fluorescence microscopy was used to observe the proteasome activity of alveolar macrophages under different proteasome activities during hypoxia and reoxygenation for 2 h, 4 h and 6 h. Western blot and PCR were used to detect the changes of alveolar macrophages in alveolar macrophages Under hypoxia and reoxygenation for 2 h, 4 h and 6 h, the expressions of ubiquitinated protein and endoplasmic reticulum stress related genes were observed. Results: The expressions of green fluorescence and Ubiquitin in alveolar macrophages of MG132 group were significantly decreased (P <0.05) at 3 h after hypoxia and reoxygenation at 2 h, 4 h and 6 h PCR and Western blot showed that the average amount of amplification and protein expression of endoplasmic reticulum stress gene BIP (immunoglobulin binding protein), XBP-1 (X-box binding protein) and CHOP (C / EBP homologous protein) (P <0.05). The alveolar macrophages in adriamycin group showed opposite results at 2 h, 4 h and 6 h after hypoxia and reoxygenation, and the relative expression of green fluorescence and Ubiquitin protein were significantly increased (P <0.05 ). PCR and Western blot showed that the average amount of BIP, XBP-1 and CHOP gene amplification and protein expression in endoplasmic reticulum increased significantly (P <0.05). CONCLUSIONS: The results of this experiment indicate that the degree of ubiquitin-proteasome activity in living cells is closely related to the endoplasmic reticulum stress. Exogenously enhancing ubiquitin proteasome activity may inhibit endoplasmic reticulum stress and exogenously decrease ubiquitin proteasome Activity increases endoplasmic reticulum stress.