原发性痛风患者外周血单个核细胞PYCARD基因及其转录剪接体的表达变化

Changes in Expression of PYCARD Gene and Its Transcript Variant mRNA in Peripheral Blood Mononuclear Cells of Patients with Primary Gout

目的了解凋亡相关斑点样蛋白(PYCARD)基因及其转录剪接体-1、-2(PYCARD-1、-2)在原发性痛风(PG)患者外周血单个核细胞(PBMCs)中的表达水平及作用。方法采用半定量聚合酶链反应(RT-PCR)法检测95例PG患者〔原发性痛风急性期(APPG)44例、原发性痛风非急性期(NAPPG)51例〕和87例健康对照PBMCs中PYCARD基因及其PYCARD-1、-2mRNA表达水平;Western blot法检测PG患者和健康对照PBMCs的PYCARD、核因子-κB(p105/p50)〔NF-κB(p105/p50)〕蛋白表达水平;检测APPG、NAPPG患者与健康对照血尿酸及部分血常规指标,并分析PYCARD基因及其PYCARD-1、-2 mRNA表达与各指标的相关性。结果 APPG与NAPPG患者PYCARD基因及其PYCARD-1、-2mRNA相对表达量均高于健康对照组(P<0.01),NAPPG患者PBMCs中PYCARD、2xmRNA和2y mRNA的相对表达量高于健康对照和APPG患者组(P<0.05);PG患者PYCARD、NF-κB(p105/p50)蛋白表达均高于健康对照组〔分别为(4.900±1.324)vs.(3.975±0.210)和(0.263±0.106)vs.(0.127±0.008),P均<0.05〕;相关性分析显示NAPPG组PYCARD-2mRNA表达与嗜中性粒细胞绝对值呈正相关(r=0.378 5,P<0.01)。结论 PG患者PBMCs中PYCARD基因及其转录剪接体mRNA表达以及PYCARD蛋白表达均异常,提示在PG患者的炎症反应中PYCARD基因及其转录剪接体可能发挥重要的调控作用。

Objective To determine the expression level and role of PYCARD CPYRIN-PAAD-DAPIN domain （PYD） and a C-terminal caspase recruitment domain （CARD）, PYCARD3 gene and its transcript variant mRNA in peripheral blood mononuclear cells （PBMCs） of patients with primary gout （PG）. Methods PYCARD gene and its transcript variant mRNA were measured using reverse transcription-polymerase chain reaction （RT- PCR） in PBMCs. The expression of PYCARD gene and PYCARD-1,-2 mRNA in PBMCs was compared between the patients with acute phase PG （APPG） （n= 44）, non-acute phase PG （NAPPG） （n = 51） and healthy controls （HC） （n=87）. PYCARD and NF-κB （p105/p50） protein expressions were measured using Western blot in the PBMCs of participants in the PG and HC groups. Routine blood tests and blood uric acid test were undertaken in all participants. Differences in the indicators were examined among the three groups. Correlations between the expression of PYCARD gene and PYCARD-1,-2 mRNA and other indicators were analyzed. Results The expression level of PYCARD gene, PYCARD-1 ,-2 mRNA was significantly higher in the APPG and NAPPG group than in the HC group（P〈0.01）. The NAPPG group had significantly higher levels of PYCARD gene transcript variant 2x mRNA and 2y mRNA in the HC and APPG groups （P〈0.05）. The expression of PYCARD and NF-κB （p105/pS0） protein was significantly higher in the PG group compared with the HC group [（4. 900 ±1. 324） vs. （3.975±0. 210） and （0. 263±0. 106） vs. （0. 127±0. 008）, respectively P〈0. 05], The expression level of PYCARD-2 mRNA and granulocyte were positively correlated in the NAPPG group. Conclusion Abnormalexpression of PYCARD gene and its transcript variant and PYCARD protein in PG patients suggests that PYCARD gene and its transcript variant may play an important role in regulating the inflammatory response of PG patients.