布鲁菌S2株感染巨噬细胞后对小胶质细胞极化的影响

Effect of the Macrophages Infected by Brucella Suis S2 on the Polarization of Microglia

目的确定布鲁菌S2株感染巨噬细胞后对BV-2小胶质细胞极化的影响。方法用布鲁菌S2株感染RAW264.7巨噬细胞24及48h后,收集未感染巨噬细胞的上清液(COU)和感染巨噬细胞24h的上清液(COB24h)及48h的上清液(COB48h),BV-2细胞经上述上清液分别处理24h。实验分为空白对照组、COU组、COB24h组、COB48h组。倒置相差显微镜观察各组细胞形态。RT-qPCR检测iNOS、TNF-α、Arg-1mRNA的表达。ELISA检测培养细胞上清液中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、白细胞介素10(IL-10)的水平。免疫组化染色法观察iNOS蛋白的表达。结果 BV-2细胞经上清处理后,镜下观察COB24h、COB48h处理的BV-2细胞胞体变大,细胞核变大,突起较多,并较空白组伸长明显,呈长杆状。与空白对照组比较,COB24h组与COB48h组BV-2细胞iNOS及TNF-αmRNA的表达升高(P均<0.01),Arg-1 mRNA的水平降低(P<0.01),且COB48h组iNOS mRNA表达升高(P<0.01);COB24h组与COB48h组上清中TNF-α、IL-1β的含量升高(P均<0.01),COB48h处理组IL-10的含量降低(P<0.05)。免疫组化染色法观察BV-2细胞中iNOS蛋白阳性表达在胞浆中,为橙色颗粒,COB48h组胞浆中iNOS蛋白的表达高于空白对照组。结论布鲁菌S2株感染巨噬细胞后的上清液可以驱动小胶质细胞向经典激活的巨噬细胞(M1)极化。

Objective To determine the effect of macrophages infected by Brucella suis S2 on the polarization of microglia. Methods RAW 264.7 cells were infected with Brucella suis S2. After 24 h and 48 h of incubation,supernatant of uninfected macrophages（COU） and supernatants infected with macrophages for 24 h（COB 24 h）and supernatants from 48 h（COB48 h） were collected. BV-2 cells were treated with COU and COB24 h,CoB48 h for 24 h, respectively.The experiment was divided into blank control group,COU group,COB24 group and COB48 group. RT-qPCR was used to detect the expression of iNOS, TNF-a and Arg-1 mRNA. The levels of TNF-α,IL-1β and IL-10 in the cell culture supernatant were determined with ELISA. The iNOS protein expression of BV-2 cells was assayed by immunohistochemical staining. Results Compared with the blank control group,the expression of iNOS and TNF-a mRNA in BV-2 cells increased and the level of Arg-1 mRNA decreased in the 24 h and 48 h supernatant group（P〈0.01）, and the expression of iNOS was significantly increased after 48 h supernatant treatment（P〈0.01）. The levels of TNF-α and IL-1 β were increased in the24 h and 48 h supernatant group（P〈0.01） and the content of IL-10 was decreased in the 48 h supernatant group（P〈0.05）.The positive expression of iNOS protein in BV-2 cells was mainly in cytoplasm, and it was orange granules. The expression of iNOS protein in cytoplasm of 48 h supernatant treatment group was significantly higher than that of blank control group. Conclusion Macrophages can induce microglia M1 polarization after Brucella suis S2 strain infection