系统性红斑狼疮患者骨髓间充质干细胞凋亡相关研究

Apoptosis of bone marrow-derived mesenchymal stem cells in patients with systemic lupus erythem-atosus

目的探讨系统性红斑狼疮（SLE）患者骨髓间充质干细胞（BMSCs）的凋亡变化及凋亡相关因子的表达。方法密度梯度离心和贴壁筛选法分离培养SLE患者及健康人骨髓MSCs，末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）法检测细胞凋亡；流式细胞术检测细胞表面Fas、Bcl—2表达水平及Caspase 8活性；实时荧光定量聚合酶链反应（PCR）技术检测Fas、Bcl-2、Bax、Bcl—w、Caspase 8及凋亡细胞蛋白酶激活因子（Apaf）-1mRNA表达水平；免疫组织化学染色检测胞质细胞色素C表达水平；统计学采用Mann—Whitnev秩和检验。结果SLE患者BMSCs凋亡明显高于健康对照组[（64±10）％和（14±9）％，U=0，P〈0．05]；SLE患者BMSCs抗凋亡因子Bcl-2蛋白表达显著低于健康对照组[（11±9）％和（56±18）％，U=0，P〈0．05]，其mRNA表达水平同样显著低于健康对照（0．2±0．2和2．4±0．7，U=24，P〈0．05）；SLE患者BMSCs胞质中细胞色索C表达显著增多[（56±21）％和（16＋16）％，U=1，P〈O．05]。SLE患者BMSCs细胞内Caspase 8活性增强[（49±14）％和（16±12）％，U=0，P〈0．05]，但基因表达水平与健康对照组比较差异无统计学意义（U=28，P〉O．05）；SLE患者与健康对照组BMSCs均高表达Fas，差异无统计学意义（U=19，P〉0．05）。结论SLE患者BMSCs凋亡增多，可能与Bcl-2表达减少、胞质中细胞色素C表达增多和Caspase 8活性增强，从而激活内源性及外源性凋亡途径相关。

Objective To investigate the apoptosis of bone marrow mesenchymal stem ceils （BMSCs） from systemic lupUs erythematosus （SLE） patients and the expression of apoptotie molecules. Methods BMSCs were isolated from bone marrow of SLE patients and normal controls by density centrifugation and adhesive culture in vitro. The apoptosis of BMSCs were evaluated by TUNEL assay. The expressions of Fas, Bcl-2 and the activity of Caspase 8 were detected by flow cytometry. Real-time PCR technique was used to determine the gene expressions of Fas, Bel-2, Bax, Bei-w, Caspase 8 and Apaf-1. Meanwhile, cytochrome C was detected by immunocytochemistry. Statistical analysis was conducted with t-test and Mann-Whitney rank test. Results The percentage of apoptotic BMSCs increased in SLE patients compared with healthy donors [（64±10）6/o vs （14±9）%, U-O, P〈0.05]. The expression of Bel-2 in BMSCs of SLE patients was lower than the normal controls at protein level [（ 11±9）% vs （56±18）%, U=O, P〈0.05], and mRNA level （0.2±0.2 vs 2.4±0.7, U= 24, P〈0.05）. More eytochrome C positive pellets in the cytosolic fraction could be detected in BMSCs from SLE patients compared with healthy controls [（56±21）% vs （16±16）%, U=I, P〈0.05]. The activity of Caspase 8 was enhanced [ （49±14） % vs （ 16±12）%, U=0, P〈0.05 ], although with no significant difference at mRNA level. Both groups expressed Fas but with no significant difference （U=19, P〉0.05）. Conclusion BMSCs from SLE patients undergo more apoptosis, the mechanisms may be associated with the down regulationof Bcl-2, up-regulation of Cytochrome C in cytoplasm and the activation of Caspase 8, which directs the intrinsic and extrinsic apoptosis pathways.