免疫性血小板减少症患者来源的骨髓间充质细胞对巨核细胞生物学行为的影响
Effects of bone marrow mesenchymal cells immune thrombocytopenia patients on the biological behaviors of megakaryocytes

目的·研究免疫性血小板减少症（immune thrombocytopenia，ITP）患者来源的骨髓间充质细胞（bone marrow mesenchymal cells，BMCs）体外生存能力、细胞因子表达的变化以及对巨核细胞生物学行为的影响。方法·全骨髓贴壁培养法培养7名ITP患者及5名正常对照者（normal controls，NC）来源的BMCs，流式细胞术检测BMCs基础凋亡率并进行表型鉴定，CCK-8法检测细胞增殖能力。佛波酯诱导HEL细胞分化并将诱导后的HEL（induced HEL，inHEL）分为3组：inHEL单独培养组（a组）、inHEL与ITP患者来源的BMCs共培养组（b组）、inHEL与NC来源的BMCs共培养组（c组），72 h后检测3组inHEL的凋亡率及CD41a、CD42b的表达状况。实时荧光定量PCR及酶联免疫吸附试验检测BMCs中细胞因子IL6、IL11、TPO、SCF的基因及蛋白表达水平。结果·与NC相比，ITP患者来源的BMCs体外增殖减弱（第4日，P0.039；第6、10日，P0.009；第8日，P0.007），基础凋亡率增加[早期凋亡率（AV+PI-），P0.036；晚期凋亡率（AV+PI+），P0.003；总凋亡率（AV+PI-/+），P0.004]；IL6基因及蛋白表达减少、SCF基因表达减少（均P0.000），而TPO及IL11表达量无明显差别。c组细胞CD41a表达量与a组相比显著增加（P0.000）；b组细胞CD41a表达量与a组相比有所增加（P0.015），但仍显著少于c组（P0.000）。与a组相比，b组、c组细胞的早期及总凋亡率以及c组细胞的晚期凋亡率均明显降低（均P0.000），而b组细胞晚期凋亡率无明显变化；与c组相比，b组细胞的晚期及总凋亡率均明显升高（均P0.000）。结论· ITP患者来源的BMCs在体外共培养条件下对巨核细胞分化及生存的支持能力减弱，其机制与ITP来源的BMCs细胞因子IL6、SCF的表达量减少相关。
：Objective · To investigate changes of immune thrombocytopenia (ITP) patients-derived bone marrow mesenchymal cells (BMCs) in cells survival, cytokines as well as the effects of BMCs on the biological behaviors of megakaryocytes. Methods · BMCs were collected 7ITP patients and 5 normal controls (NC), and cultivatedthe whole marrow adherent method. Surface markers and basal apoptosis rate of BMCs were analyzedflow cytometry (FCM). Proliferation of BMCs was assessedCCK-8 method. Phorbol 12-myristate 13-acetate (PMA) was used to stimulate differentiation of HEL cells. The induced HEL cells (inHEL) were divided into 3 groups inHEL cultured alone (group a), inHEL co-cultured with BMCs derived ITP patients (group b), inHEL co-cultured with BMCs derived NC (group c). After 72 h incubation, the of cell surface proteins (CD41a, CD42b) and cell apoptosis rate were analyzedFCM. The mRNA and proteins levels of cytokines IL6, IL11, TPO, SCF were detectedreal-time fluorescent quantitative PCR (RT-qPCR) and enzyme linked immunosorbent assay (ELISA), respectively. Results · Compared with NC, BMCs ITP patients grew progressively slowly (Day 4, P0.039; Day 6, 10, P0.009; Day 8, P0.007), cell basal apoptosis rates were increased [AV+PI-(early apoptosis rate), P0.036; AV+PI+ (late apoptosis rate), P0.003; AV+PI-/+ (total apoptosis rate), P0.004]. Compared with group a, the of CD41a in group c was much higher (P0.000). The of CD41a in group b was higher than that in group a (P0.015), but still much less than that in group c (P0.000). Compared with group a, the early and total apoptosis rate in group b, c and the late apoptosis rate in groupc were decreased obviously (all