IgA1与系膜细胞共培养上清对足细胞分泌TNF-α的影响及机制探讨

目的： 探讨IgA肾病血清IgA1与系膜细胞共培养上清对足细胞分泌TNF-α的影响及机制。方法：Jacalin 亲和层析柱和Sephacryl S-200 分子筛用来纯化蛋白，单体IgA1（mIgA1)热聚合为聚合体IgA1(aIgA1), 同步化的系膜细胞与患者和健康对照来源的aIgA1共培养，收集上清，分别与同步化的足细胞作用；ELISA检测足细胞上清TNF-α水平，real time PCR 检测凋亡相关基因Ang、ACE mRNA表达情况。结果：IgAN患者来源的aIgA1与系膜细胞共培养得到上清可刺激足细胞分泌TNF-α，其水平显著高于对照组［(12.47±1.45) ng/L vs (2.33±0.65) ng/L，P<0.05］。与对照和健康上清组相比，该上清可上调足细胞Ang和ACE mRNA显著升高(P<0.05)。依那普利拉(10-5 mol/L)可使该上清作用后的足细胞分泌的TNF-α显著下降至(7.52±1.12) ng/L (P<0.05)；而缬沙坦(10-5 mol/L)则使之下降至(6.64±0.68) ng/L (P<0.05)；而依那普利拉(0.5×10-5 mol/L)和缬沙坦(0.5×10-5 mol/L)联合治疗可使足细胞TNF-α下降至(2.72±0.55) ng/L，与对照无显著差异(P>0.05)。结论：IgA肾病患者血清IgA1与系膜细胞共培养上清可通过激活肾素-血管紧张素系统而刺激足细胞TNF-α分泌，参与IgAN的进展。

Release of tumor necrosis factor - a in podocytes and its mechanism by mesangial cell cultured medium with IgAl from IgA nephropathy

AIM: To explore the effect and mechanism on tumor necrosis factor-α production in podocytes by medium from growth arrested mesangial cells incubated with aIgA1 isolated from IgAN patients and normal control. METHODS: Jacalin affinity chromatography and Sephacryl S-200 molecular sieve chromatography were used to isolate IgA1. Monomeric IgA1 (mIgA1) was transformed to aggregated IgA1 (aIgA1) by heating. IgA-mesangial cell medium was prepared by collecting medium in which growth arrested mesangial cell were incubated with different aIgA1, then the medium with RPMI-1640 containing 0.5% FBS was exposed to podocytes. Real time PCR was used to detect the mRNA expression of Ang and ACE, and TNF-α was measured by ELISA assay. RESULTS: TNF-α level of podocytes by medium from aIgA1 from IgAN cultured with mesangial cells was higher than that in control (6.47±0.45) ng/L vs (1.33±0.65) ng/L, P<0.05. Ang and ACE mRNA in podocytes exposed to the special medium were higher than those in podocytes exposed to control and medium from mesangial cells incubated with aIgA1 from healthy control (P<0.05). Enalaprilat decreased the TNF-α to (7.52±1.12) ng/L (P<0.05), and valsartan decreased TNF-α in the podocytes to (6.64±0.68) ng/L (P<0.05), while enalaprilat and valsartan restored the level of TNF-α in podocytes to normal level (2.72±0.55) ng/L, P>0.05. CONCLUSION: Our findings implicate that medium from mesangial cells cultured with IgA1 from IgA nephropathy stimulates the release of TNF-α in podocytes by the activation of renin-angiotensin system, which might accelerate the progression of IgAN.