B细胞活化因子信号对小鼠肾小管上皮细胞生长特性的影响

Effects of B cells activating factor on growth characteristics of mouse renal tubular epithelial cells

目的观察B细胞活化因子（BAFF）及其受体在原代小鼠肾小管上皮细胞（RTECs）的表达及对其生长特性的影响。方法酶消化法分离培养原代小鼠RTECs；以500 U/ml干扰素（IFN）-γ刺激原代小鼠RTECs，48 h后流式细胞术（FCM）检测RTECs细胞膜上BAFF及BAFF受体（BAFF-R）的表达；以重组BAFF蛋白和/或阻断性B细胞活化因子受体-Fc融合蛋白（BAFF-R-Fc）刺激RTECs，48 h后以荧光素钠转运实验检测RTECs离子转运功能、细胞免疫荧光法检测角蛋白-18（CK-18）的表达、四唑氮化合物（MTS）法检测RTECs增殖、FCM法检测细胞凋亡。结果小鼠RTECs经IFN-γ作用后，BAFF-R表达水平较未受IFN-γ刺激组显著上调（4 166.250±913.914比3 602.750±875.584，t＝4.124，P＝0.026）；荧光素钠转运实验结果显示，BAFF刺激组的平均吸光度（A）值为0.011±0.003，显著低于BAFF-R-Fc＋BAFF组（0.020±0.007）和对照组（0.016±0.006），差异均有统计学意义（t＝－3.437，P＝0.041；t＝－3.572，P＝0.038）；免疫荧光结果显示，BAFF刺激组平均A值为0.032±0.005，显著低于BAFF-R-Fc＋BAFF组（0.043±0.007）和对照组（0.041±0.008），差异均有统计学意义（t＝－8.006，P＝0.015；t＝－4.603，P＝0.044）；5、20 ng/ml BAFF刺激组细胞增殖A值分别为（1.532±0.058）、（1.509±0.056）显著高于对照组（1.473±0.045），差异均有统计学意义（t＝5.636，P＝0.030；t＝5.765，P＝0.029）且5 ng/ml BAFF刺激组显著高于BAFF-R-Fc＋BAFF组（1.477±0.050），差异有统计学意义（t＝5.085，P＝0.037）；BAFF刺激组细胞凋亡率为（39.850±8.544）%，显著低于BAFF-R-Fc＋BAFF组[（42.950±8.330）%]和对照组[（44.467±7.642）%]，差异均有统计学意义（t＝－3.399，P＝0.019；t＝－3.020，P＝0.029）。结论BAFF/BAFF-R信号增强能促进RTECs增殖，但使其上皮细胞特性和离子转运功能显著下降。

ObjectiveTo investigate the expression of B cell activating factor （BAFF） and BAFF receptor （BAFF-R） on primary mouse renal tubular epithelial cells （RTECs）, and the effect on growth characteristics of RTECs.MethodsPrimary mouse RTECs were isolated by enzyme digestion method, and cultured in the conditioned medium. After stimulated by 500 U/ml Interferon-γ （IFN-γ） for 48 h, the expression of BAFF and BAFF-R on RTECs were detected by Flow Cytometry （FCM）. After treated with recombinant mouse BAFF and/or blockade B cell activating factor receptor-Fc fusion protein （BAFF-R-Fc） for 48 h, the transport ability of fluorescein sodium and cytokeratin-18 （CK-18） expression of RTECs were detected, respectively; The proliferation ability and the apoptosis rates of RTECs were tested by 3- （4, 5-dimenthylthiazol-2-yl）-5- （3-carboxymethoxyphenyl）-2- （4-sulfophenyl）-2H-tetrazolium （MTS） and FCM method, respectively; Data were analyzed by SPSS 17.0, and P〈0.05 was considered to be statistically significant.ResultsBAFF-R expression on RTECs in the IFN-γ treated group significantly up-regulated, compared with control group （4 166.250±913.914 vs. 3 602.750±875.584, t=4.124, P=0.026）. Fluorescein sodium transport experiment showed the average absorbance of BAFF stimulation group was significantly lower than BAFF-R-Fc＋ BAFF group （0.011±0.003 vs. 0.020±0.007, t=-3.437, P=0.041）, and control group （0.011±0.003 vs. 0.016±0.006, t=-3.572, P=0.038）; Immunofluorescence results showed the average absorbance of BAFF stimulation group was significantly lower than that of BAFF-R-Fc＋ BAFF group （0.032±0.005 vs. 0.043±0.007, t=-8.006, P=0.015）, and control group （0.032±0.005 vs. 0.041±0.008, t=-4.603, P=0.044）; Cell proliferation assay results showed that, A value of 5 ng/ml and 20 ng/ml BAFF group were significantly higher than that of control group （1.532±0.058 vs. 1.473±0.045; 1.509±0.056 vs. 1.473±0.045, t=5.636, P=0.030; t=5.765, P=0.029, respectively）, and A value of 5 ng/ml BAFF group were significantly higher than that of BAFF-R-Fc＋ BAFF group （1.532±0.058 vs. 1.477±0.050, t=5.085, P=0.037）; Apoptosis test results indicated that the apoptosis rate of BAFF-treated group was significantly lower than that of BAFF-R-Fc＋ BAFF group [（39.850±8.544）% vs. （42.950±8.330）%, t=-3.399, P=0.019], and control group [（39.850±8.544）% vs. （44.467±7.642）%, t=-3.020, P=0.029].ConclusionBAFF signaling could promote the proliferation ability of RTECs, but down-regulate the epithelial cell characteristics and Ion transport ability of RTECs.