血小板衍生生长因子BB在川崎病血小板增多中的作用及机制研究

Role and mechanism of platelet-derived growth factor BB in thrombocytosis in Kawasaki disease

摘要:

目的 通过体内外实验探究血小板衍生生长因子BB（platelet-derived growth factor BB，PDGF-BB）对川崎病（Kawasaki disease，KD）小鼠和人巨核细胞株Dami细胞生成血小板的作用及机制。方法 ELISA检测KD及健康儿童各40例血清PDGF表达水平。C57BL/6小鼠构建KD模型，随机分为正常组、KD组、伊马替尼组，每组30只。检测各组血常规及PDGF-BB、巨核细胞集落形成单位（megakaryocyte colony forming unit，CFU-MK）、巨核细胞标记物CD41表达。采用CCK-8、流式细胞术、实时定量PCR、Western blot检测PDGF-BB对Dami细胞生成血小板的作用和机制。结果 PDGF-BB在KD患儿血清中高表达（P<0.001）。KD组小鼠血清PDGF-BB高表达（P<0.05）、CFU-MK及巨核细胞标记物CD41表达增加（P<0.001）；伊马替尼组CFU-MK及CD41表达减少（P<0.001）。体外实验结果显示，PDGF-BB促进Dami细胞增殖、血小板生成、PDGFR-β mRNA及p-Akt蛋白表达（P<0.05）；联合组（PDGF-BB 25 ng/mL+伊马替尼20 μmol/L）血小板生成、PDGFR-β mRNA和p-Akt蛋白表达少于PDGF-BB组（P<0.05）。结论 PDGF-BB可能通过与PDGFR-β结合，激活PI3K/Akt通路，促进巨核细胞增殖、分化及血小板生成；PDGFR-β抑制剂伊马替尼可减少血小板生成，为KD血小板增多的诊疗提供了新的策略。

Abstract:

Objective To study the role and mechanism of platelet-derived growth factor BB (PDGF-BB) on platelet production in Kawasaki disease (KD) mice and human megakaryocytic Dami cells through in vitro and in vivo experiments.Methods ELISA was used to measure the expression of PDGF in the serum of 40 children with KD and 40 healthy children. C57BL/6 mice were used to establish a model of KD and were then randomly divided into a normal group, a KD group, and an imatinib group (30 mice in each group). Routine blood test was performed for each group, and the expression of PDGF-BB, megakaryocyte colony forming unit (CFU-MK), and the megakaryocyte marker CD41 were measured. CCK-8, flow cytometry, quantitative real-time PCR, and Western blot were used to analyze the role and mechanism of PDGF-BB in platelet production in Dami cells.Results PDGF-BB was highly expressed in the serum of KD children (P<0.001). The KD group had a higher expression level of PDGF-BB in serum (P<0.05) and significant increases in the expression of CFU-MK and CD41 (P<0.001), and the imatinib group had significant reductions in the expression of CFU-MK and CD41 (P<0.001). In vitro experiments showed that PDGF-BB promoted Dami cell proliferation, platelet production, mRNA expression of PDGFR-β, and protein expression of p-Akt (P<0.05). Compared with the PDGF-BB group, the combination group (PDGF-BB 25 ng/mL + imatinib 20 μmol/L) had significantly lower levels of platelet production, mRNA expression of PDGFR-β, and protein expression of p-Akt (P<0.05).Conclusions PDGF-BB may promote megakaryocyte proliferation, differentiation, and platelet production by binding to PDGFR-β and activating the PI3K/Akt pathway, and the PDGFR-β inhibitor imatinib can reduce platelet production, which provides a new strategy for the treatment of thrombocytosis in KD.