EGFL6调控血管生成-成骨偶联在骨质疏松性骨缺损修复中的作用机制

杨瑞; 刘雨; 李宁宁; 高亮; 寇世杰; 张文治; 陈祥云

EGFL6调控血管生成-成骨偶联在骨质疏松性骨缺损修复中的作用机制

The mechanism of EGFL6 regulating angiogenesis-osteogenesis coupling in the repair of osteoporotic bone defects

摘要

摘要:
目的： 探究EGFL6调控血管生成-成骨偶联对骨质疏松性骨缺损（OP-BD）修复的影响及可能作用机制。
方法： 培养骨髓间充质干细胞（BMSCs），分为对照组、EGFL6-50 ng/mL组、EGFL6-200 ng/mL组及EGFL6-500 ng/mL组。培养人脐静脉内皮细胞（HUVECs）分为对照组、EGFL6-200 ng/mL组、BMSCs培养液组及EGFL6- 200 ng/mL + BMSCs培养液组。将30只大鼠分为假手术组、OP-BD + L-EGFL6组及OP-BD + H-EGFL6组，每组10只。碱性磷酸酶（ALP）染色试剂盒检测ALP活性。ARS染色检测细胞钙盐沉积水平。免疫荧光染色检测成骨标志物OCN与血管生成标志物VEGF。RT-qPCR检测血管生成因子Ang-2及Ang-4 mRNA水平。Transwell实验检测细胞侵袭能力。血管形成检测管状结构的数量变化。H&E染色检测骨缺损病理变化。免疫组织化学染色检测OCN、VEGF及CD31表达水平。
结果： 与对照组相比，EGFL6-50 ng/mL组、EGFL6-200 ng/mL组及EGFL6-500 ng/mL组BMSCs成骨相关标志物ALP活性增加，钙盐沉积增加，OCN表达增加（P ＜ 0.05），血管生成相关标志物VEGF蛋白表达增加，Ang-2及Ang-4 mRNA表达增加（P ＜ 0.05），且EGFL6-200 ng/mL对BMSCs作用的效果更明显，用于后续研究。与对照组相比，EGFL6-200 ng/mL组HUVECs的侵袭能力与管状结构数量无显著性变化（P ＞ 0.05），BMSCs培养液组与EGFL6- 200 ng/mL + BMSCs培养液组HUVECs的侵袭能力与管状结构数量增加（P ＜ 0.05），且EGFL6-200 ng/mL + BMSCs培养液组HUVECs的侵袭能力与管状结构数量增加更明显。与对照组相比，OP-BD + L-EGFL6组大鼠胫骨骨缺损区域骨小梁断裂，数量减少，骨髓腔增大，OCN、VEGF及CD31表达增加；与OP-BD + L-EGFL6组相比，OP-BD + H-EGFL6组大鼠胫骨骨缺损区域骨小梁断裂减少，数量增加，骨髓腔减小，OCN、VEGF及CD31表达增加。
结论： EGFL6可通过调控血管生成—成骨偶联促进OP-BD修复。

Abstract:
Objective To investigate the effects of EGFL6 on the repair of osteoporotic bone defects (OP-BD) by regulating the coupling of angiogenesis and osteogenesis and its possible mechanism.
Methods The cultured bone marrow mesenchymal stem cells (BMSCs) were divided into the control group, EGFL6-50 ng/mL group, EGFL6-200 ng/mL group and EGFL6-500 ng/mL group. The cultured human umbilical vein endothelial cells (HUVECs) were divided into the control group, EGFL6-200 ng/mL group, BMSCs culture medium group and EGFL6-200 ng/mL + BMSCs culture medium group. Thirty rats were divided into the sham group, OP-BD + L-EGFL6 group and OP-BD + H-EGFL6 group (10 rats each group). Alkaline phosphatase (ALP) staining kit was used to detect the ALP activity. Calcium deposition was detected by ARS staining. Immunofluorescence staining was used to detect the osteogenic marker OCN and angiogenesis marker VEGF. RT-qPCR was used to detect the mRNA levels of Ang-2 and Ang-4. Transwell assay was used to detect the cell invasion ability. Blood vessel formation was used to examine the quantitative changes of tubular structures. H&E staining was used to detect the pathological changes of bone defects. Immunohistochemical staining was used to detect the expression of OCN, VEGF and CD31.
Results Compared with the control group, the ALP activity, calcium deposition, OCN expression and VEGF protein expression significantly increased in the EGFL6-50 ng/mL, EGFL6-200 ng/mL and EGFL6-500 ng/mL groups (P ＜ 0.05). The mRNA expression of Ang-2 and Ang-4 increased (P ＜ 0.05), and the effects of EGFL6-200 ng/mL on BMSCs was more obvious. Compared with the control group, the invasion ability and number of tubular structures of HUVECs in the EGFL6-200 ng/mL group did not change significantly (P ＞ 0.05), while the invasion ability and number of tubular structures of HUVECs in the BMSCs culture medium group and EGFL6-200 ng/mL + BMSCs culture medium group increased (P ＜ 0.05). Moreover, the invasion ability and number of tubular structures of HUVECs in the EGFL6-200 ng/mL + BMSCs culture medium group were significantly increased. Compared with the control group, the trabecular bone in the tibial bone defect area of the OP-BD + L-EGFL6 group fractured, the number of bone trabeculae reduced, the bone marrow cavity enlarged, and the expression of OCN, VEGF and CD31 increased. Compared with the OP-BD + L-EGFL6 group, the number of trabecular bone fracture in the tibial bone defect area of the OP-BD + H-EGFL6 group decreased, the number of bone marrow cavity decreased, and the expression of OCN, VEGF and CD31 increased.
Conclusions EGFL6 can promote the repair of OP-BD by regulating the coupling of angiogenesis and osteogenesis.

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