肾小球白介素-10基因表达的检测及其在狼疮性肾炎分子诊断中的应用

Interleukin-10 gene expression on glomeruli in patients with lupus nephritis analyzed by laser microdissection and real-time PCR

目的 :探讨狼疮性肾炎 (LN)患者肾活检组织切片中肾小球IL 10mRNA表达与临床病理的联系 ,及其在LN分子诊断中的意义。 方法 :对 2 0例病理证实的Ⅳ型LN患者 ,运用激光微分离系统分离其肾活检组织切片中的肾小球 ,提取总RNA ,用荧光定量PCR检测IL 10mRNA的表达。 结果 :12例患者的肾小球中检测到IL 10mRNA表达。肾小球IL 10CT 比值与尿IL 10水平、血IgG水平和肾小球CD6 8平均计数呈显著正相关 ,与血C3水平呈显著负相关。 结论 :应用激光微分离技术精确获取肾小球检测基因表达 ,发现肾小球IL 10mRNA表达与Ⅳ型LN患者的免疫损伤表现及肾小球病理改变特点显著关联。肾小球原位IL 10mRNA表达的检测为判断LN患者病情 。

Objective:To investigate the relationship between glomerular interleukin 10 mRNA expression and the clinico pathological characteristics of patients with lupus nephritis, and to assess its significance in the molecular diagnosis of lupus nephritis. Methodology: Twenty lupus patients who pathologically proved diffuse proliferative lupus nephritis (LN Ⅳ) were studied. Their glomeruli were isolated by laser microdissection from each renal biopsy slides. The total RNA was extracted for reverse transcription and the cDNA was then amplified for IL 10 and GAPDH using real time PCR. The threshold cycle (C T) values were reported and the ratio of the C T values for GAPDH versus IL 10 was regarded as the relative expression of IL 10. Glomerular IL 10 mRNA expression was analyzed compared with the patients' clinical parameters and pathological features. Results: Twelve specimens were found to have IL 10 amplification. Their C T ratios were positively correlated with urinary IL 10 levels, serum IgG levels and glomerular infiltration of CD68 positive cells. They were also reversely associated with serum C3 level. Glomerular IL 10 mRNA expression was significantly correlated with immunological injuries and pathological variations in the glomeruli of patients with LN Ⅳ. Conclusion: We established a viable method to detect the mRNA expression in the renal biopsy specimens by laser microdissection and real time PCR. Determination of in situ IL 10 expression in the glomeruli may provide a new molecular marker for assessing the immunological conditions and directing the therapeutical strategies in patients with LN Ⅳ.