两种棘球绦虫原头节促进骨髓间充质干细胞钙化的差异性分析

Mesenchymal stem cell calcification induced by protoscolex of two species of Echinococcus:a differential analysis

背景:细粒棘球蚴与泡状棘球蚴的生长方式不同,肝细粒棘球蚴病可以形成完整的纤维钙化囊壁,肝泡状棘球蚴病呈浸润性生长,不能形成完整的纤维钙化囊壁。骨髓间充质干细胞参与棘球蚴病纤维钙化囊壁的形成,但细粒棘球蚴和泡状棘球蚴钙化特征不同与骨髓间充质干细胞的作用尚不清楚。目的:比较2种棘球绦虫原头节对骨髓间充质干细胞钙化的作用,初步探讨2种棘球蚴钙化灶差异的形成机制。方法:提取、培养、鉴定C57BL/6小鼠骨髓间充质干细胞。将骨髓间充质干细胞分别与泡状棘球绦虫原头节、细粒棘球绦虫原头节共培养,以骨髓间充质干细胞单独培养为对照组。共培养1,4,7 d取骨髓间充质干细胞通过微量酶标法检测成骨标记物碱性磷酸酶活性,RT-qPCR检测BMP2和RUNX2 mRNA的表达,Western blot检测BMP2、RUNX2和P-Smad1/5/8的蛋白表达。结果与结论:①细粒棘球绦虫原头节共培养组、泡状棘球绦虫原头节共培养组1,4 d的碱性磷酸酶活性均高于对照组(P <0.05),且细粒棘球绦虫原头节共培养组1,4,7 d的碱性磷酸酶活性高于泡状棘球绦虫原头节共培养组(P <0.05)。②Western blot结果显示:细粒棘球绦虫原头节共培养组和泡状棘球绦虫原头节共培养组1,4 d的BMP2、RUNX2、P-Smad1/5/8蛋白表达高于对照组(P <0.05),细粒棘球绦虫原头节共培养组高于泡状棘球绦虫原头节共培养组(P <0.05)。③RT-qPCR结果显示:细粒棘球绦虫原头节共培养组1,4,7 d的BMP2、RUNX2mRNA表达水平显著高于对照组(P <0.05),细粒棘球绦虫原头节共培养组4,7 d的BMP2、RUNX2 mRNA表达水平显著高于泡状棘球绦虫原头节共培养组(P <0.05)。泡状棘球绦虫原头节共培养组1,4,7 d的RUNX2 mRNA表达水平显著高于对照组(P <0.05)。④结果表明,棘球绦虫原头节与骨髓间充质干细胞共培养通过上调BMP-Smad1/5/8通路促进骨髓间充质干细胞钙化因子碱性磷酸酶和RUNX2的表达,共培养后期泡状棘球蚴促钙化作用明显减弱,细粒棘球蚴促钙化作用保持不变,提示该机制可能与2种棘球蚴生长方式不同相关。

BACKGROUND:The growth pattern of Echinococcus granulosus is different from that of Echinococcus alveolaris.Hepatic echinococcosis can form a complete fibrous calcified cyst wall,while hepatic alveolar echinococcosis can grow infiltratively and cannot form a complete fibrous calcified cyst wall.Bone marrow mesenchymal stem cells(BMSCs)are involved in the formation of calcified wall of hydatidosis,but the calcification characteristics of Echinococcus granulosus and Echinococcus alveolaris are different and the role of BMSCs is still unclear.OBJECTIVE:To compare the effects of Echinococcus granulosus and Echinococcus alveolaris on the calcification of BMSCs and to preliminarily investigate the formation mechanism of echinococcosis calcifications.METHODS:BMSCs of C57BL/6 mice were extracted,cultured and identified,followed by co-culture with the protoscolex of Echinococcus granulosus(BMSC+CE group)and Echinococcus alveolaris(BMSC+AE group),respectively.BMSCs cultured alone were used as control group.After 1,4,and 7 days of co-culture,alkaline phosphatase activity was detected by a microplate reader,the expression of BMP2 and RUNX2 mRNA was detected by RT-q PCR,and the expression of BMP2,RUNX2 and phosphorylated Smad1/5/8(P-Smad1/5/8)proteins was detected by western blot assay.RESULTS AND CONCLUSION:(1)The alkaline phosphatase activity of the BMSC+CE group and BMSC+AE group was significantly higher than that of the control group at 1 and 4 days after culture(P<0.05),and the alkaline phosphatase activity of the BMSC+CE group was significantly higher than that of BMSC+AE group(P<0.05).(2)Western blot results showed that the expression of BMP2,RUNX2,and P-Smad1/5/8 protein in the BMSC+CE group and BMSC+AE group was significantly higher than that in the control group at 1 and 4 days after culture(P<0.05),while the expression of BMP2,RUNX2,and P-Smad1/5/8 protein in the BMSC+CE group was significantly higher than that in the BMSC+AE group(P<0.05).(3)RT-qPCR results showed that the expression of BMP2 and RUNX2 mRNA in the BMSC+CE group was significantly higher than that in the control group at 1,4 and 7 days after culture(P<0.05),and was significantly higher than that in the BMSC+AE group at 4 and 7 days after culture(P<0.05).The expression of RUNX2 mRNA in the BMSC+AE group was significantly higher than that in the control group at 1,4,and 7 days after culture(P<0.05).(4)To conclude,co-culture of the protoscolex of Echinococcus alveolaris and BMSCs promotes the expression of alkaline phosphatase and RUNX2 in BMSCs by up-regulating BMP-Smad1/5/8 pathway.At the later stage of co-culture,the effect of Echinococcus alveolaris on BMSCs calcification is significantly weakened,while the effect of Echinococcus granulosus on BMSCs calcification remains unchanged,suggesting that this mechanism may be related to the different growth patterns of two kinds of hydatids.