热休克蛋白60及重组热休克蛋白60对U937细胞IL-6分泌量的影响

Effects of recombinant heat shock protein 60 and heat shock protein 60 on the secretion of IL-6

目的观察重组热休克蛋白60(hsp60)对U937细胞分泌细胞因子IL-6的影响。方法对幽门螺杆菌标准株26695的基因进行定点突变,利用重叠延伸PCR法使1398和1399位点的AG突变为GC,获得重组hsp60基因片段并构建原核表达质粒pEASY-E1-rhsp60,经IPTG诱导在大肠杆菌表达突变型rhsp60,将50、100、200μg/mL的rhsp60(rhsp60组)、hsp60(hsp60组)分别与U937细胞共培养,于12、18、24 h收集上清液,ELISA法检测上清中细胞因子IL-6水平,设PBS对照组。结果 rhsp60组、hsp60组IL-6分泌水平均明显高于对照组(P均<0.05),且同时点同浓度下rhsp60组IL-6分泌水平明显高于hsp60组(P均<0.05),IL-6的分泌随着蛋白刺激浓度的升高而增高。结论 rhsp60与hsp60皆可促进U937细胞分泌IL-6,胃癌来源的rhsp60使IL-6分泌量更多。

Objective To investigate the effects of recombinant heat shock protein 60 （hsp60） on the secretory cell factor interlukin-6 of U937 cells. Methods The nucleotide sequences of hsp60 were changed from AG to GC at sites 1398 and 1399 by using three-step PCR. The recombinant hsp60 gene segment was obtained, and then its prokaryotic expression plasmid pEASY-El-rhsp60 was constructed and induced by IPTG for expression of mutant rhsp60 in Escherichia coli. Then, the U937 cells were co-cultured with 50, 100,200 μg/mL of rhsp60 （rhsp60 group） and hsp60 （ hsp60 group）, at 12, 18 and 24 h, the supernatant was collected, and IL-6 secretion was measured by ELISA by setting the PBS as the con- trol group. Results IL-6 secretion in the rhsp60 and hsp60 groups were significantly higher than that in the control group （ all P 〈 0.05） . The rhsp60 group was higher than that in the hsp60 group at the same time and the same concentration （ all P 〈 0.05 ）. Furthermore, the secretion of IL-6 increased with the increase of protein stimulating concentration. Con- clusion The hsp60 and rhsp60 can both promote the secretion of IL-6 and rhsp60 derived from gastric cancer can secrete more IL-6 after stimulating U937 cells.