钙网蛋白调控抗凋亡分子的表达促进类风湿关节炎滑膜细胞存活

Calreticulin-mediated upregulation of anti-apoptotic BcI-XL and Mcl-1 promotes survival of fibroblast- like synoviocytes in rheumatoid arthritis

目的探讨钙网蛋白对RA成纤维样滑膜细胞（FLS）存活的影响，为深入揭示钙网蛋白在RA发病机制中的作用及寻找新的治疗靶点奠定实验基础。方法采用胶原酶消化法分离RA和OA患者滑膜组织中的FLS并进行体外培养。应用实时荧光定量．聚合酶链反应（q-PCR）、蛋白质印迹法以及细胞免疫荧光等技术，检测RA和OAFLS中抗凋亡分子Bcl-XL和Mcl-1的表达；进一步应用q-PCR、蛋白质印迹法检测在不同浓度钙网蛋白刺激下，RA和OAFLS中Bcl-XL和Mcl-1的表达；采用四甲基偶氮唑盐（MTT）法检测钙网蛋白对RAFLS增殖的影响；采用t检验及单因素方差分析进行统计学分析。结果①RAFLS中抗凋亡分子Bcl-XL和Mcl-1的mRNA表达水平（14．51±2．20；12．82±1．80）与OA对照组（1．00±0．39；1．00±0．46）相比均明显升高（拉10．47，11．02；P均〈0．01）；RAFLS中Bcl-XL和Mcl-1的蛋白表达水平较OA对照组也明显升高；RAFLS中Bcl-XL和Mcl-1荧光染色与OA对照组相比显著增强。②钙网蛋白可使RAFLS中Bcl-XL和Mcl-1的表达上调：钙网蛋白刺激浓度为10ng／ml和50ng／ml时，RAFLS中Bel-XL的mRNA表达水平（1．70±0．28；1．87±0．35）较对照组（1．00±0．20）明显升高（q=4．58，5．69；P〈0．05）；Mcl-1的mRNA表达水平（1．85±0．36；1．72±0．26）较对照组（1．00±0．20）也明显升高（q=5．63，4．77；P〈0．05）；不同浓度的钙网蛋白作用后，OAFLS中Bel-XL和Mel-1的mRNA表达水平没有明显的改变（F=1．49，1．60；P〉0．05）；钙网蛋白作用后，RAFLS中Bcl-XL和Mcl-1的蛋白表达水平呈浓度依赖性升高，而OAFLS中二者的蛋白表达水平没有明显的变化。③MTT结果显示钙网蛋白对RAFLS增殖没有显著作用（F=2．88，P〉0．05）。结论钙网蛋白可能通过上调抗凋亡分子Bcl-XL和Mcl-1的表达进而抑制凋亡，促进RAFLS的存活。

Objective Fibroblast-like synoviocytes （FLS） play important roles in the pathogenesis of rheumatoid arthritis （RA）. The present study was undertaken to investigate the mechanism of calreticulin （CRT） to promote FLS survival in RA. Methods FLS were isolated by enzymatic digestion of synovial tissue specimens obtained from RA and osteoarthritis （OA） patients and cultured in vitro. The expression of Bcl-XL and Mcl-1 in FLS at mRNA and protein level was detected by quantitative-polymerase chain reaction （q-PCR）, Western blotting and immunofluorescence respectively. RA and OA FIS were cultured with different concentrations of recombinant human CRT for 48-72 h, the expression of Bcl-XL and Mcl-1 was detected by q-PCR and Western blotting. The proliferation of RA FLS following CRT stimulation was determined by MTT assay. Results ①Compared with FLS from OA patients （1.00±0.39; 1.00±0.46）, the anti-apoptotic Bcl-XL and Mcl-1 mRNA expression （14.51 ±2.20; 12.82±1.80） was significantly higher in the FLS from RA patients （t=10.47, 11.02; P〈0.01）; Western blotting analysis also showed increased protein levels of Bcl-XL and Mcl-1 in RA FLS; Immunofluorescence results showed higher expression of Bcl-XL and Mcl-1 in RA at the single FLS level; ② CRT up-regulated the expression of Bcl-XL and Mcl-1 in RA FLS： compared with the control group （0 ng/ml）, CRT stimulation at 10 ng/ml and 50 ng/ml increased the levels of Bcl-XL mRNA （1.70±0.28 vs 1.00±0.20, q=4.58, P〈0.05; 1.87±0.35 vs 1.00±0.20, q=5.69, P〈0.05） and Mcl-1 mRNA （1.85±0.36 vs 1.00±0.20, q=5.63, P〈0.05; 1.72±0.26 vs 1.00±0.20, q=4.77, P〈0.05） in RA FLS, while no significant effects of CRT on Bcl-XL and Mcl-1 mRNA expression were observed in OA FLS （F=1.49, 1.60; P〉0.05）; Western blotting results showed elevated protein levels of both Bcl-XL and Mcl-1 in RA FLS after CRT treatment at a concentration dependent manner. However, neither Bcl-XL nor Mcl-1 expression was significantly changed in OA FLS. ③MTT assay showed that CRT had no significant effect on the proliferation of RA FLS （F=2.88, P〉 0.05）. Conclusion Our results indicate that CRT-mediated up-regulation of anti-apoptotic Bcl-XL and Mcl-1 may inhibit apoptosis and promote the survival of FLS from RA patients.