强直性脊柱炎患者蛋白酶体基因表达的研究

The expression of proteasome gene in the patients with ankylosing spondylitis

目的 分析比较强直性脊柱炎 (AS)患者与正常对照者之间蛋白酶体 (proteasome)的基因表达及生物学功能的差异。方法 用快速蛋白质液相色谱法 (FPLC法 )分别从 10个HLA B2 7阳性家族中的AS患者及正常对照者 (同一级亲属 )的B淋巴细胞系中制备和纯化蛋白酶体。用反转录 聚合酶链式反应 (RT PCR)测定蛋白酶体基因的表达。用三氯乙酸 (TCA)沉淀法测定不同的蛋白酶体对12 5I标记的HLA B2 7的降解程度。结果 B2 7阳性家族中 ,AS患者与非AS对照者的B细胞内蛋白酶体亚单位LMP2mRNA的表达差异无显著性 ,而LPM 7和MECL1mRNA的表达AS患者明显高于非AS对照者 (P均 <0 0 0 0 1)。经干扰素 (IFN) γ刺激培养后 ,非AS对照者的LMP2、LMP7和MECL1mRNA的表达均显著增加 (P值分别为 0 0 0 7,<0 0 0 0 1和 <0 0 0 0 1) ,而AS组这三种亚单位基因的表达增加均不明显。因此 ,在IFN γ刺激下AS与非AS组蛋白酶体基因的表达差异无显著性。免疫印迹分析表明AS患者LMP7及MECL1蛋白水平的表达也明显高于非AS对照者 ,LMP2蛋白的表达两组无明显差别。AS患者蛋白酶体的糜蛋白酶样活性明显增强 ,对B2 7的降解作用明显强于非AS的蛋白酶体 ,孵育 4h时的平均降解率为 81 8% ,而非AS对照者的蛋白酶体的平均降解率为 4 1 9% (P <0 0

Objective To investigate the differences of proteasome gene expression and biological function between ankylosing spondylitis (AS) patients and normal controls.Methods Proteasomes were purified from the 10 HLA B27 positive family individuals B lymphoblasts (10 AS and 10 normal controls),respectively.The gene expressions of the proteasome subsets were detected by reverse transcript polymerase chain reaction (RT PCR).The in vitro reaction systems of the proteasomal mediated B27 proteolysis were reconstituted.The degradation of 125 I labeling sB27 by proteasome was determined by measuring the amount of soluble radioactivity after addition of trichloroacetic acid.Results The RT PCR values for the low molecular weight protein (LMP)7 and multicatalytic endopeptidase complex like 1 (MECL1) genes were increased in AS patients compared with non AS individuals (both P <0 000 1),but there was no statistical difference of the LMP2 gene expression between two groups.Upon IFN γ treatment,up regulation of LMP2,LMP7 and MECL1 gene expressions were observed in non AS individuals,and there was no significant difference of LMP2,LMP7 and MECL1 mRNA expressions between AS and non AS groups.Western blot analysis showed that LMP7 and MECL1 protein expressions were also significantly increased in AS patients.The proteasome purified from the AS patients showed increased chymotrypsin like activity as compared with that from non AS individuals.B27 degraded by proteasome was observed in both groups,and it was of linear change with the incubation time during the first four hours.However the degradation rate by the proteasome from AS patients was significantly higher than that from non AS individuals [(82±5)% vs (42±8)%, P <0 000 1].Conclusion Our findings indicate that up regulation of proteasome gene expres expressionandfunctionmayplayanimportantroleforB2 7positiveimdividualsinthedevelopmentofAS .