Emdogain对共培养成骨样细胞及内皮细胞成骨及成血管基因表达的影响  

The effect of emdogain on proliferation and differentiation of co-cultured osteoblasts and endothelial cells

作　　者：王珊珊[1,2] Xiaohui Rausch-Fan Oleh Andrukov 林毅 林李嵩[1] 施斌[1] WANG Shan-shan;Xiaohui RAUSCH-FAN;Oleh ANDRUKOV;LIN Yi;LIN Li-song;SHI Bin(Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Fujian Medical University, Fujian Maxillofacial Medical Center, Laboratory of Facial Rehabilitation and Reconstruction of Fujian Medical University. Fuzhou 350005, Fujian Province;College of Stomatology, Fujian Medical University. Fuzhou 350005, Fujian Province;Central Research Laboratory Unit, Bernhard Gottlieb University Clinic of Dentistry, Medical University of Vienna. Vienna 1090, Austria;Department of Stomatology, Fujian Provincial Hospital. Fuzhou 350001, Fujian Province, China)

机构地区：[1]福建医科大学附属第一医院口腔颌面外科福建医科大学面部整复与重建研究室福建省颌面医学中心,福建福州350005 [2]福建医科大学口腔医学院,福建福州350005 [3]奥地利维也纳医科大学牙医学院口腔医学研究中心 [4]福建省立医院口腔科,福建福州350001

出　　处：《中国口腔颌面外科杂志》2019年第1期32-39,共8页China Journal of Oral and Maxillofacial Surgery

基　　金：福建省卫计委医学创新课题(2015-CX-26);福建省科技厅国际合作课题(2017I0003)

摘　　要：目的:评价emdogain对直接与间接共培养条件下成骨样细胞和内皮细胞成骨及成血管基因表达的影响以及其间的相关性。方法:将人脐静脉内皮细胞(HUVEC)和成骨样细胞(MG63)按2∶1比例于12孔板内进行直接及间接共培养,并使用不同浓度(0、0.1、1、10、50μg/mL)的emdogain共培养细胞72 h。采用细胞计数仪结合流式细胞仪评价细胞增殖情况,并通过荧光激活流式细胞分选技术分选共培养细胞。对分选后的成骨样细胞及内皮细胞采用实时荧光定量PCR分别检测成骨相关基因碱性磷酸酶(ALP)、I型胶原蛋白(Col-1)及成血管相关基因血管内皮细胞生长因子(VEGF)、VEGF酪氨酸激酶受体1(Flt-1)、VEGF酪氨酸激酶受体2(KDR)、von Willebrand因子(vWF)、内皮细胞C蛋白受体(EPCR)、E选择素(E-Selectin)、促血管生成素2(Ang-2)的表达水平。采用SPSS24.0软件包对数据进行统计学处理。结果:在直接与间接共培养条件下,Emdogain对MG63及HUVEC细胞增殖的作用具有浓度依赖性。随着emdogain浓度增加,细胞增殖率逐渐下降。50μg/mL浓度的emdogain对MG63及HUVEC细胞增殖抑制作用最为明显。随着emdogain浓度增加,MG63表面ALP和Col-1表达水平逐渐增加;50μg/mL浓度条件下,表达水平最高且与其他组基因表达水平差异显著(P<0.01)。同一emdogain浓度条件下,直接共培养MG63表面ALP及Col-1表达水平均显著高于间接共培养(P<0.01)。除10μg/mL组外,直接共培养条件下,VEGF表达水平均显著高于间接共培养(P<0.01)。50μg/mL组直接共培养的HUVEC表面Flt-1、KDR、vWF、E-selectin基因表达水平显著高于间接共培养及直接共培养的其他各组(P<0.01)。间接共培养条件下,HUVEC表面EPCR及Ang-2基因表达水平显著高于直接共培养(P<0.05)。结论:Emdogain对共培养MG63和HUVEC增殖的作用具有浓度依赖性,50μg/mL可能是emdogain作用的关键浓度。在直接共培养条件下,50μg/mL emdogain抑制MG63及PURPOSE: To evaluate the effect of emdogain on proliferation and differentiation of osteoblasts and endothelial cells under different co-culture conditions, and to assess their correlationship. METHODS: Human umbilical vein endothelial cells(HUVECs) and osteoblast-like cells(MG63s) were directly and indirectly co-cultured in 12-well cell culture plate at a ratio of 2∶1. The cells were treated by emdogain of different concentrations(0, 0.1, 1, 10, 50 μg/mL)for 72 h. Cell proliferation was measured by cell counter in combination with flow cytometry. Direct co-cultured HUVECs and MG63 s were separated by fluorescence-activated cell sorting. The expression level of alkaline phosphatase(ALP),collagen type I(Col-1), vascular endothelial growth factor(VEGF) in MG63 s, and vascular endothelial growth factor receptors Flt-1 and KDR, Von Willebrand factor(vWF), endothelial cell protein C receptor(EPCR), E-selectin, and angopoietin-2(Ang-2) in HUVECs were measured by real-time quantitative polymerase chain reaction(qPCR). Statistical analysis was performed using SPSS 24 software package. RESULTS: Under direct and indirect co-culture condition, the effect of emdogain on MG63 s and HUVECs proliferation was in a concentration dependent manner. The proliferation rate of cells was reduced with the increase of emdogain concentration. The inhibitory effect of emdogain on MG63 s and HUVECs proliferation was the most prominent at the concentration of 50 μg/mL. The expression of ALP and Col-1 in MG63 s was enhanced with the increase of emdogain concentration. The highest level of ALP and Col-1 was observed at the concentration of 50 μg/mL, and had significant difference compared with that in other groups(P <0.01). Under conditions of same emdogain concentration, the ALP and Col-1 level in directly co-cultured MG63 s was significantly higher than that in indirectly co-cultured MG63s(P <0.01). Apart from the group of 10 μg/mL, VEGF level was significantly higher under direct co-culture condition than under indirect co-culture con

关 键 词：EMDOGAIN 成骨细胞 内皮细胞 直接共培养 间接共培养 细胞增殖 细胞分化 细胞交联 

分 类 号：Q25[生物学—细胞生物学]