Ag85B蛋白诱导小鼠脾淋巴细胞表达IL-2、IFN-γ及促进细胞增殖的实验研究

Experimental Study on IFN-γ and IL-2 Expression in BALB/c Splenic Lymphocytes and Promotion of Cell Proliferation in Mice Induced by Mycobacterium tuberculosis Ag85B Protein

目的探讨结核分枝杆菌Ag85B蛋白在体外诱导BALB/c小鼠脾淋巴细胞表达IL-2和IFN-γ的能力及对细胞增殖的影响,以了解Ag85B基因表达在特异性细胞免疫应答中的作用。方法体外分离培养BALB/c小鼠脾淋巴细胞,除去蛋白中的内毒素,采用BCA法对Ag85B蛋白进行定量,10μg/ml浓度的蛋白与小鼠脾淋巴细胞共同孵育24、72 h,RT-PCR方法检测细胞培养液上清中IL-2和IFN-γ的表达;用不同浓度的Ag85B蛋白刺激小鼠脾淋巴细胞24、48、72 h,MTT法检测其对小鼠脾淋巴细胞增殖能力的影响。结果小鼠脾淋巴细胞在体外分离培养成功;经RT-PCR检测,Ag85B蛋白能诱导小鼠脾淋巴细胞表达IL-2和IFN-γ,对照组表达呈阴性;MTT法检测到Ag85B蛋白对小鼠脾淋巴细胞的增殖具有促进作用,在一定范围内呈时间和剂量依赖性,在同对照组的增殖比较中,除0.5μg/ml组在24、48 h差异无统计学意义外,其他浓度组在各个时间点差异均有统计学意义(P<0.05)。结论结核分枝杆菌Ag85B蛋白能诱导小鼠脾细胞产生IL-2和IFN-γ因子,能在体外促进小鼠脾淋巴细胞增殖,诱导了特异性的T细胞免疫应答。

Objective To explore the cytokines of IL-2 and IFN-γ expressed in BALB/c splenic lymphocytes which induced by Ag85B protein of Mycobacterium tuberculosis,and to know the function of Ag85B gene expression in specific cell-mediated immune response. Methods BALB/c splenic lymphocytes were isolated in vitro and the endotoxin was removed from Ag85B protein.The concentration of the protein was quantitated by BCA assay.The mouse splenic lymphocytes were incubated with 10μg/ml Ag85B protein for 24 and 72 hrs.The expression of IL-2 and IFN-γ in the cell culture supernate was detected by RT-PCR.BALB/c splenic lymphocytes were stimulated with different concentrations of Ag85B protein for 24,48 and 72 hrs.MTT assay was used to detect the cell proliferation. Results The BALB/c splenic lymphocyte was isolated and cultured in vitro successfully.The results of RT-PCR showed that Ag85B protein could stimulate the expression of IL-2 and IFN-γ in BALB/c splenic lymphocytes,but the expression in the control group was negative.The results of MTT assay showed that Ag85B protein has an auxo-action on BALB/c splenic lymphocyte multiplication in a time-and dose-dependent manner.There were statistically significant differences in the cell proliferation at different time points between different concentrations groups and the control group（P〈0.05） except the 0.5 ug/ml group at 24 and 48 hrs. Conclusions Mycobacterium tuberculosis Ag85B protein could induce BALB/c splenic lymphocyte to generate the cytokines of IL-2 and IFN-γ,motivate the cell proliferation,and generate specific T cell immune response.