基因扫描分析维持性血液透析患者外周血T细胞克隆性分布的特点

Genescan analysis of clonality of TCR Vβ repertoire T cell in patients with maintenance hemodialysis

目的：了解慢性肾功能衰竭进行维持性血液透析(MHD)患者的外周血T细胞克隆性增殖的特性和意义。方法：通过RT-PCR方法扩增11例膜性肾病(MGN)3例、膜增殖性肾炎(MPGN)3例、IgA肾病(IgAN)4例、局灶节段性肾小球硬化症(FSGS)1例。MHD患者外周血单核细胞T细胞受体(TCR)Vβ24个亚家族基因的CDR3，阳性的PCR产物进一步经荧光素标记和基因扫描的方法分析产物CDR3的长度及序列，了解T细胞的克隆性。结果：11例MHD患者中仅表达1-10个TCRVβ的亚家族，其中2例只表达Vβ3一个亚家族，分别为寡克隆及双克隆性。最常见的TCR Vβ亚家族是Vβ3，其次Vβ8、Vβ19、Vβ22，再次为Vβ10、Vβ23。11例患者的透析时间与TCR Vβ表达呈显著负相关(r=-0．646，P=0．044)；用协方差分析MGN、MPGN、IgAN三个病种的TCR Vβ表达差别：不同透析时间的TCR Vβ表达的数量不同(F=10．193，P=0．019)，而MGN、MPGN、IgAN三个病种之间的表达量没有显著的差异(F=1．772，P=0．249)。结论：MHD患者外周血存和着TCR Vβ亚家族T细胞倾斜分布的现象，并有克隆性增殖的特征，提示大部分MHD患者存在某些细胞免疫功能低下和有相同的免疫刺激原。透析时间愈长，TCR Vβ表达的数量愈少，提示长期血液透析可能是MHD患者的细胞免疫功能低下的一个原因；而MGN、MPGN、IgAN三个病种之间TCR Vβ的表达量没有显著差异。

Objective: To investigate the clonality of TCR Vb subfamily T cell in the patients with maintenance hemodialysis. Methodology: The CDR3 of TCR Vb 24 subfamily genes were amplified in samples of peripheral blood mononuclear cells before hemodialysis, which were drawn from 11 patients, There were 4 cases of IgA nephropathy (IgAN), 3 of membranous glomerulopathy (MGN) , 3 of membranopoliferative glomerulonephritis (MPGN), and 1 of focal segmental glomerulosclerosis (FSGS), undergoing maintenance hemodialysis. In order to obverse the usage of TCR Vb repertoire, the RT-PCR products were further labeled with fluorescent and analyzed by genescan technique for CDR3 size, to evaluate the clonal expansion and distribution of the detectable TCR Vb subfamily T cells. Results: Only 1-10 TCR Vb subfamily T cells could be identified in all of the patients, and there was only one TCR Vb subfamily (Vb3) T cells detected in 2 cases, respectively expressed as oligoclonality and biclonality. The most frequent expression of TCR Vb genes was Vb 3 (in 8 patients), the second included Vb8, Vb19, Vb22 (in 6 patients), and the third included Vb10, Vb23 (in 5 patients). There was significant negative relationship between the hemodialysis time and expression of TCR Vb subfamily in all patients (r=-0.646,P=0.044) by analysis of linear correlation. Similar results were found respectively in MGN, MPGN and IgAN (F=10.193,P=0.019), but no significant expression of TCR Vb in different primary diseases (F=1.772,P=0.249) by the analysis of variance. Conclusion: The significantly skew distribution and clonal proliferation of TCR Vb subfamily T cells could be found in all of the patients with maintenance hemodialysis, which may indicate that these patients had been stimulated by similar immune stimulus and low cell immune functions. The longer the time of hemodialysis, the less expression of TCR Vb indicated that hemodialysis could be one of the causes of dysfunction of cell immunity in patients with chronic renal failure.