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目的探讨IgG Fc受体Ⅰ(FcγRⅠ)在脂多糖(LPS)诱导的大鼠肾上腺嗜铬细胞瘤PC12细胞凋亡中的作用。方法将PC12细胞接种于培养板中,用(50、125、250、500、1000)μg/mL LPS处理24 h,噻唑蓝(MTT)比色法检测细胞存活率,选取合适的LPS剂量。待细胞处于对数生长期,采用随机数字表法分为空白对照组:不进行任何处理,继续培养24 h;LPS组:培养液中加入500μg/mL LPS,孵育24 h;LPS联合FcγRⅠ中和抗体组:培养液中加入500μg/mL LPS和0.2μg/mL FcγRⅠ中和抗体,孵育24 h。处理结束后,实时荧光定量PCR和Western blot法检测各组FcγRⅠmRNA和蛋白含量,异硫氰酸荧光素标记的膜联素Ⅴ/碘化丙啶(annexinⅤ-FITC/PI)染色结合流式细胞术检测细胞凋亡,采用免疫组织化学染色法检测各组细胞caspase-3、Bcl-2和BAX的蛋白水平。结果随着LPS剂量的增加,PC12细胞的活力逐渐下降;与对照组相比,LPS处理组FcγRⅠmRNA和蛋白水平增加,caspase-3、Bcl-2和BAX蛋白水平增加,细胞凋亡率增加;与LPS组相比,LPS联合FcγRⅠ中和抗体组FcγRⅠmRNA和蛋白水平降低,caspase-3、BAX蛋白水平显著降低,细胞凋亡率降低,Bcl-2蛋白水平增加。结论FcγRⅠ参与LPS诱导的PC12细胞凋亡。
Objective To investigate the role of IgG Fc receptor Ⅰ (FcyRⅠ) in the apoptosis of rat adrenal pheochromocytoma PC12 cells induced by lipopolysaccharide (LPS). Methods PC12 cells were seeded into culture plates and treated with LPS (50, 125, 250, 500, 1000) μg / mL for 24 h. MTT assay was used to determine cell viability. When the cells were in logarithmic growth phase, they were divided into blank control group by random number table method: without any treatment, cultured for 24 h; LPS group: 500μg / mL LPS was added into the culture medium and incubated for 24 h; LPS combined with FcγRⅠ neutralization Antibody group: 500μg / mL LPS and 0.2μg / mL FcγRⅠ neutralizing antibody were added into the culture medium and incubated for 24 hours. After the treatment, the contents of FcyRⅠmRNA and protein in each group were detected by real-time fluorescence quantitative PCR and Western blot, and fluorescein isothiocyanate labeled annexinⅤ-FITC / PI staining combined with flow cytometry The apoptosis of cells was detected by immunohistochemistry. The protein levels of caspase-3, Bcl-2 and BAX in each group were detected by immunohistochemical staining. Results The viability of PC12 cells decreased gradually with the increase of LPS dose. Compared with the control group, the mRNA and protein levels of FcyRⅠ, the levels of caspase-3, Bcl-2 and BAX increased and the apoptosis rate increased in LPS treatment group Compared with LPS group, the expression of FcγRⅠmRNA and protein decreased, while the levels of caspase-3 and BAX protein decreased, the apoptosis rate decreased and Bcl-2 protein level increased in LPS plus FcγRⅠ neutralizing antibody group. Conclusion FcγRⅠ is involved in the apoptosis of PC12 cells induced by LPS.