CAJ | 학술논문

目的:观察高迁移率蛋白B1-A盒蛋白(HMGB1-Abox)对小鼠单核细胞系在脂多糖(LPS)刺激下表达HMGB1的影响和动态变化以及对炎性因子分泌的抑制作用.方法:利用克隆载体构建PET28a-HMGB1-Abox原核质粒,转化大肠杆菌BL21(DE3),以异丙基-β-D-硫代半乳糖苷(ITPG)诱导后经Ni~(2+)-NTA树脂亲和纯化得到目的蛋白;以不同浓度的重组HMGB1Abox蛋白作用于LPS刺激的小鼠单核细胞系RAW264.7,CCK8试剂盒检测其对小鼠单核细胞活力的影响.以PBS和重组HMGB1-Abox蛋白为对照组和实验组,于2、6、12、24、48小时检测其细胞上清中的TNF-α、IL-1β的含量,Western blot、免疫荧光法观察细胞中HMGB1的表达、RT-PCR检测细胞中HMGB1-mRNA变化趋势.结果:成功构建PET28a-HMGB1-Abox原核质粒并纯化出大约为14 kD的目的蛋白.小鼠单核细胞活力与重组HMGB1-Abox蛋白浓度及作用时间呈正相关;实验组细胞上清TNFα、IL-1β的含量较对照组显著下降;Western blot及免疫荧光检测提示实验组细胞中HMGB1表达不断减少;细胞中HMGB1-mRNA含量及峰值均较对照组显著减少且延后.结论:重组HMGB1-Abox蛋白可以显著减少小鼠单核细胞在受到LPS刺激时HMGB1的表达和分泌,从而有效的阻断HMGB1对其他早期炎性因子的促进作用,减少炎性因子的分泌,有较强的抗炎作用.
목적:관찰고천이솔단백B1-A합단백(HMGB1-Abox)대소서단핵세포계재지다당(LPS)자격하표체HMGB1적영향화동태변화이급대염성인자분비적억제작용.방법:이용극륭재체구건PET28a-HMGB1-Abox원핵질립,전화대장간균BL21(DE3),이이병기-β-D-류대반유당감(ITPG)유도후경Ni~(2+)-NTA수지친화순화득도목적단백;이불동농도적중조HMGB1Abox단백작용우LPS자격적소서단핵세포계RAW264.7,CCK8시제합검측기대소서단핵세포활력적영향.이PBS화중조HMGB1-Abox단백위대조조화실험조,우2、6、12、24、48소시검측기세포상청중적TNF-α、IL-1β적함량,Western blot、면역형광법관찰세포중HMGB1적표체、RT-PCR검측세포중HMGB1-mRNA변화추세.결과:성공구건PET28a-HMGB1-Abox원핵질립병순화출대약위14 kD적목적단백.소서단핵세포활력여중조HMGB1-Abox단백농도급작용시간정정상관;실험조세포상청TNFα、IL-1β적함량교대조조현저하강;Western blot급면역형광검측제시실험조세포중HMGB1표체불단감소;세포중HMGB1-mRNA함량급봉치균교대조조현저감소차연후.결론:중조HMGB1-Abox단백가이현저감소소서단핵세포재수도LPS자격시HMGB1적표체화분비,종이유효적조단HMGB1대기타조기염성인자적촉진작용,감소염성인자적분비,유교강적항염작용.
Objective: To study the influence of HMGB1-Abox on the expression of HMGB-1 in RAW264.7 cells stimulated by lipopolysaccharide(LPS)and its inhibition on the secretion of inflammatory factor.Methods: Use cloning vector to make up prokaryotic plasmid PET28a-HMGB1-Abox, which included the gene fragment of the A box of HMGB1, and recombinant vector was further transformed into Escherichia coil strain BL21(DE3),the recombinant plasmid was induced by isopropylthiogalactoside(IPTG)to express target protein.The protein was purified by Ni-column.We tested the effect of the different concentration of rHMGB1-Abox on the viabihty of RAW264.7 cells stimulated by LPS using CCK-8.Experimental group(EG)was treated with rHMGB41-Abox and control group(CG)was deh with PBS.TNF-α and IL-1β levels were detected by the enzyme linked immunosorbent assay(ELISA).Western blot and immunofluorescence staining method were used to compare the expression of HMGB1 in RAW264.7 cells with the experiment group and control group.Reverse transcription polymerase chain reaction(RT-PCR)was used to detect the variation tendency of HMGB1-mRNA.All procedures were manipulated at 2 \ 6 \ 12 \ 24 \ 48 h after treatment.Results: The recombinant prokaryotic expression vector PET28a-HMGB1-Abox was successfully constructed and mouse HMGB1-Abox protein about 14 kD was successfully expressed.There was a positive correlation between the viability of RAW264.7 cells and the concentration of protein and acting time.Compared to CG, TNF-α and IL-1β levels in EG declined significantly.In EG, Western blot and Immunofluorescence staining showed that the expression of HMGB1 protein was decreased and the expression of HMGB1-mRNA was reduced markedly and delayed.Conclusion: The rHMGB1-Abox could reduce expression and secretion of HMGB1 in RAW264.7 cells stimulated by LPS significantly,thereby to prevented the promotion of HMGB1 on pro-inflammatory mediator and inhibit the expression and secretion of inflammatory factors,which has some anti-inflammatory action.