IgA肾病患者血清IgA_1活化细胞外信号调节激酶并诱发肾小球系膜细胞增殖的作用

Serum IgA_1 from patients with IgA nephropathy induces phosphorylation of extracellular signal-regulated kinase and proliferation of human mesangial cells

目的 探讨IgA肾病 (IgAN)患者血清IgA1对体外培养的正常人肾小球系膜细胞 (HMC)细胞外信号调节激酶 (ERK)磷酸化及细胞增殖的刺激作用 ,并比较其与正常人血清IgA1作用力的异同。方法 亲和层析提取正常人及患者血清IgA1,加热聚合 (aIgA1) ,原代培养正常人HMC ,Western杂交测定ERK蛋白磷酸化 ,DNA荧光标记技术结合流式细胞仪观察HMC细胞周期变化 ,直接计数检测HMC增殖。结果 (1)患者及正常人aIgA1均呈时间依赖性诱发HMCERK信号蛋白磷酸化、DNA合成和细胞增殖 ,二者作用趋势相似 ,作用高峰时间分别为孵育 15min、2 4～ 36h和 4 8～ 72h ;(2 )患者aIgA1刺激强度显著高于正常人aIgA1,在作用高峰时间 ,患者组和正常人组磷酸化ERK/总ERK的比值分别为 4 9 5 %± 10 1%和 30 7%± 4 4 % ,S G2 M期细胞数所占的比例分别为 (33 0 %± 0 3%和30 7%± 0 7% ,HMC计数分别为 (5 7 78± 2 4 5 )× 10 4/ml和 (5 0± 1 5 1)× 10 4/ml,差异均有显著意义(P <0 0 5 ) ;(3)阻断ERK通路只能部分抑制患者aIgA1对HMC增殖的诱导作用。结论 IgAN患者血清IgA1可以诱导正常人HMCERK信号蛋白磷酸化并刺激细胞增殖 ,其作用强于正常人血清IgA1。

Objective To study the effects of serum IgA 1 from patients with IgA nephropathy (IgAN) on the phosphorylation of extracellular signal regulated kinase (ERK) and proliferation of human mesangial cells(HMC) Methods Serum was taken from 10 IgAN patients and 10 healthy persons IgA 1 was isolated with jacalin column, and then heated to become aggregated form (aIgA 1) Primary HMCs were cultured and the cells of third or fourth generation were used in the test HMCs were incubated with aIgA 1 from IgAN patients or from healthy persons To do blocking test 50μmol/L PD98059 was added into the culture of HMC of the 3 groups while the HMCs were in the relatively stationary phase The phosphorylation of ERK was evaluated by Western blot; cell cycle was examined by DNA fluorescence labeling and flow cytometry HMC number was counted Results 1) Both the aIgA 1 from patients with IgAN and that from healthy persons induced the phosphorylation of ERK, DNA synthesis and proliferation of HMC in a time dependent manner and with a similar trend, however, the effects of aIgA 1 from patients with IgAN were much stronger and longer The incubation times for the maximum effect on phosphorylation of ERK were 15 minutes, 24～36 hours, and 48～72 hours in the IgAN patient group, healthy person group, and the control group with a ratio of phosphorylated ERK/ total ERK of 49 5%±10 1%, 30 7%±4 4% and 10 5%～12% respectively ( P <0 05) 2) After 18 hour incubation, the percentages of cells at the stage S were 3 48%±0 54%, 6 64%±0 96%, and 7 85%±0 71% in the control group, healthy person group, and IgAN patient group 24 hours after incubation, the percentages of cells at stage S G 2 M were 9 4%±1 86% in the control group , significantly lower than that in the healthy person group (14 5%±0 7%, P <0 05) and that of the patient group (17 2%±0 3%, significantly higher than the other 2 groups, P <0 01 and P <0 05) At the peak time, the cells at stage S G 2 M accounted for 33 0%±0 3% and 30 7%±0 7% in the patient group and healthy person group respectively ( P <0 05) with the HMC count of (57 8±2 5)×10 4/ml and (50±1 5)×10 4/ml ( P <0 05) respectively After 48, 72, and 96 hour incubation with aIgA 1 the HMC counts were (51 7±1 6)×10 4/ml, and (56 4±2 6)×10 4/ml, and (58 8±1 8)×10 4/ml, and(65 8±2 9)×10 4/ml, (60 0±1 7)×10 4/ml and(66 8±2 0)×10 4/ml in the patient group with and without pre incubation of PD98059 respectively ( P <0 05) However, the HMC count was not significantly changed in the healthy person group Blocking of ERK with PD98059 could inhibit the effect of aIgA 1 from patients with IgAN on the proliferation of HMC but could not inhibit the effects of aIgA 1 from healthy controls Conclusion IgA 1 induces the phosphorylation of ERK, DNA synthesis and proliferation of HMC, and the effects of IgA 1 from patients with IgAN are stronger than that from healthy persons