| RUNX2基因过表达载体修饰BMSC来源外泌体联合碳酸钙支架系统在骨缺损中的应用 |
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投稿时间：2020-12-20
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| 作者 | Author | 单位 | Address | E-Mail |
| 赵有顺 |
ZHAO You-shun |
浙江大学附属金华医院 金华市中心医院骨三科, 金华 浙江 321000 |
Department of Bone Surgery,Affiliated Jinhua Hospital,Zhejiang University School of Medicine,The Jinhua Municipal Central Hospital,Jinhua 321000,Zhejiang,China |
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| 林平 |
LIN Ping |
浙江大学附属金华医院 金华市中心医院骨三科, 金华 浙江 321000 |
Department of Bone Surgery,Affiliated Jinhua Hospital,Zhejiang University School of Medicine,The Jinhua Municipal Central Hospital,Jinhua 321000,Zhejiang,China |
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| 涂迎春 |
TU Ying-chun |
浙江大学附属金华医院 金华市中心医院骨三科, 金华 浙江 321000 |
Department of Bone Surgery,Affiliated Jinhua Hospital,Zhejiang University School of Medicine,The Jinhua Municipal Central Hospital,Jinhua 321000,Zhejiang,China |
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| 安涛 |
AN Tao |
浙江大学附属金华医院 金华市中心医院骨三科, 金华 浙江 321000 |
Department of Bone Surgery,Affiliated Jinhua Hospital,Zhejiang University School of Medicine,The Jinhua Municipal Central Hospital,Jinhua 321000,Zhejiang,China |
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| 吴裕平 |
WU Yu-ping |
浙江大学附属金华医院 金华市中心医院骨三科, 金华 浙江 321000 |
Department of Bone Surgery,Affiliated Jinhua Hospital,Zhejiang University School of Medicine,The Jinhua Municipal Central Hospital,Jinhua 321000,Zhejiang,China |
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| 李晓飞 |
and LI Xiao-fei |
浙江大学附属金华医院 金华市中心医院骨三科, 金华 浙江 321000 |
Department of Bone Surgery,Affiliated Jinhua Hospital,Zhejiang University School of Medicine,The Jinhua Municipal Central Hospital,Jinhua 321000,Zhejiang,China |
professorlxf@163.com |
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| 期刊信息：《中国骨伤》2022年，第35卷，第4期，第379-386页 |
| DOI：10.12200/j.issn.1003-0034.2022.04.016 |
| 基金项目:浙江省自然科学基金项目(编号:Q20H060058);浙江省医药卫生科技项目(编号:2020KY343);金华市公益类项目(编号:2019-4-002) |
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中文摘要:
目的: 探究RUNX2基因过表达载体修饰骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSC)来源的外泌体联合碳酸钙支架系统在骨缺损中的效果。
方法: 以兔源BMSCs为研究对象,利用流式细胞仪对BMSCs进行鉴定。构建RUNX2基因过表达载体,利用慢病毒转染BMSCs,采用超速离心法进行收集外泌体。利用透射电子显微镜观察外泌体的形态,利用Western blot法检测外泌体标志物CD63分子的表达,利用三室并联自动温控反应系统,构建碳酸钙支架。根据是否转染RUNX2基因过表达载体,将BMSCs与碳酸钙支架的复合物分成3组,即BMSCs组,RUNX2过表达组和外泌体组。利用油红O染色和逆转录-聚合酶链反应(reverse-transcription-polymerasechain reaction,RT-PCR)检测BMSCs的成骨分化。采购成年健康雄性新西兰大白兔9只,清洁级,年龄(12.97±1.21)个月,体质量(19.3±3.6) kg,每组3只。利用手术方法构建颅骨缺损动物模型,利用影像学,HE染色和Masson染色评估骨缺损的修复。
结果: 流式细胞仪检测结果表明,BMSCs细胞表面CD29蛋白表达为99.5%,CD44蛋白表达为100%,CD11b蛋白表达为0.1%,CD45蛋白表达为0.1%。透射电镜结果显示,外泌体为直径50~150 nm的双层膜囊泡状结构。Western blot结果显示,外泌体的分子标志物CD63表达阳性。油红O染色结果显示,外泌体组BMSCs的成骨分化要明显高于RUNX2过表达组和BMSCs组。RT-PCR检测结果显示,外泌体组BMSCs的RUNX2,BMP-2和碱性磷酸酶的mRNA相对表达量要明显高于RUNX2过表达组和BMSCs组(P<0.05)。影像学结果显示,外泌体组颅骨缺损修复效果要优于RUNX2过表达组。HE染色和Masson染色结果显示,外泌体组颅骨缺损修复效果要优于RUNX2过表达组(P<0.05)。
结论: 相较于RUNX2基因过表达载体转染,直接提取外泌体,可以更高效的促进BMSCs向成骨细胞分化,与碳酸钙支架相结合后,可以更好地促进骨缺损的愈合,从而为临床上骨缺损的治疗提供新思路和方法。 |
| 【关键词】外泌体 骨缺损 骨髓间充质干细胞 慢病毒 |
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| Application of RUNX2 gene over expression vector modified exosomes from BMSC combined with calcium carbonate scaffold system in bone defect |
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ABSTRACT
Objective: To investigate the effect of RUNX2 gene overexpression vector modified exosomes derived from bone marrow mesenchymal stem cells (BMSCs) combined with calcium carbonate scaffold system in bone defect.
Methods: Rabbit BMSCs were used as the research object,and BMSCs were identified by flow cytometry. Construct RUNX2 gene overexpression vector,transfect BMSCs with lentivirus,and collect exosomes by ultracentrifugation. The morphology of exosomes was observed by transmission electron microscope,the expression of exosome marker CD63 was detected by Western blot,and the calcium carbonate scaffold was constructed by three chamber parallel automatic temperature control reaction system. According to whether the RUNX2 gene overexpression vector was transfected or not,the complex of BMSCs and calcium carbonate scaffold was divided into three groups,namely BMSCs group,RUNX2 overexpression group and exosome group. The osteogenic differentiation of BMSCs was detected by oil red O staining and RT-PCR. There were 9 clean adult healthy male New Zealand white rabbits,aged (12.97±1.21) months,with a body weight of (19.3±3.6) kg,with 3 rabbits in each group. The animal model of skull defect was constructed by surgical method,and the repair of bone defect was evaluated by imaging,he staining and Masson staining.
Results: The results of flow cytometry showed that the expression of CD29 protein,CD44 protein,CD11b protein and CD45 protein on the surface of BMSCs were 99.5%,100%,0.1% and 0.1%,respectively. Transmission electron microscopy showed that the exosomes were bilayer vesicles with a diameter of 50 to 150 nm. Western blot showed that the molecular marker CD63 of exosomes was positive. Oil red O staining showed that the osteogenic differentiation of BMSCs in exosome group was significantly higher than that in RUNX2 overexpression group and BMSCs group. The results of RT-PCR showed that the relative expressions of RUNX2,BMP-2 and ALP mRNA in BMSCs in exosome group were significantly higher than those in RUNX2 overexpression group and BMSCs group (P<0.05). The imaging results showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group. HE staining and Masson staining showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group (P<0.05). MSCs in exosome group was significantly higher than that in RUNX2 overexpression group and BMSCs group. The results of RT-PCR showed that the relative expressions of RUNX2,BMP-2 and ALP mRNA in BMSCs in exosome group were significantly higher than those in RUNX2 overexpression group and BMSCs group(P<0.05). The imaging results showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group. HE staining and Masson staining showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group(P<0.05).
Conclusion: Compared with RUNX2 gene overexpression vector transfection,extraction of exosomes directly can promote the differentiation of BMSCs into osteoblasts more efficiently,and the combination with calcium carbonate scaffold can better promote the healing of bone defects. So as to provide new ideas and methods for the clinical treatment of bone defects. |
| KEY WORDS Exosomes Bone defects Bone marrow mesenchymal stem cells Lentivirus |
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| 引用本文，请按以下格式著录参考文献： |
| 中文格式: | 赵有顺,林平,涂迎春,安涛,吴裕平,李晓飞.RUNX2基因过表达载体修饰BMSC来源外泌体联合碳酸钙支架系统在骨缺损中的应用[J].中国骨伤,2022,35(4):379~386 |
| 英文格式: | ZHAO You-shun,LIN Ping,TU Ying-chun,AN Tao,WU Yu-ping,and LI Xiao-fei.Application of RUNX2 gene over expression vector modified exosomes from BMSC combined with calcium carbonate scaffold system in bone defect[J].zhongguo gu shang / China J Orthop Trauma ,2022,35(4):379~386 |
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