文章摘要
马骢,李刚,马亮亮,等.人脐带间充质干细胞来源的外泌体调控血管内皮生长因子对小鼠胫骨骨折的修复作用研究[J].安徽医药,2024,28(6):1092-1097.
人脐带间充质干细胞来源的外泌体调控血管内皮生长因子对小鼠胫骨骨折的修复作用研究
Study on the reparative effect of VEGF regulated by human umbilical cord mesenchymal stem cell-derived exosomes on tibial fracture mice
  
DOI:10.3969/j.issn.1009-6469.2024.06.007
中文关键词: 胫骨骨折  外泌体  人脐带间充质干细胞  血管内皮生长因子  修复
英文关键词: Tibial fractures  Exosomes  Human umbilical cord mesenchymal stem cells  Vascular endothelial growth factor  Reparation
基金项目:2019年河南省医学科技攻关计划联合共建项目( LHGJ20190171)
作者单位
马骢 河南省洛阳正骨医院河南省骨科医院康复医学科河南洛阳 450003 
李刚 河南省洛阳正骨医院河南省骨科医院小儿外科河南洛阳 450003 
马亮亮 河南省组织细胞库临床科研部河南郑州450000 
李想 河南省洛阳正骨医院河南省骨科医院康复医学科河南洛阳 450003 
张永泼 河南省洛阳正骨医院河南省骨科医院康复医学科河南洛阳 450003 
冷子宽 郑州大学第一附属医院骨科河南郑州 450052 
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中文摘要:
      目的探讨人脐带间充质干细胞( huc-MSC)来源外泌体( Exo)对小鼠胫骨骨折的修复作用及可能机制。方法于 2018年 12月至 2022年 1月,体外分离、培养 hucMSCs,超速离心法收集 Exo并鉴定。 24只 8周龄雄性 C57BL/6J小鼠行胫骨骨折,并采用随机数字表法分为模型对照组、磷酸缓冲盐溶液( PBS)对照组及 huc-MSC-Exo组,每组各 8只,其中 huc-MSC-Exo组小鼠骨髓腔内注射 huc-MSC-Exo(100 μg溶解于 100 μL PBS),PBS对照组小鼠注射等量 PBS,模型对照组小鼠不进行处理。 21 d后, X线观察骨折愈合情况;双能 X线检测骨矿物质密度( BMD);苏木精 -伊红( HE)染色观察胫骨组织学变化;免疫组织化学染色检测骨折区骨组织中血管内皮生长因子 A(VEGFA)阳性表达情况;蛋白质印迹法检测骨折区骨组织中 VEGFA、Runt相关转录因子 2(RUNX2)、骨形成蛋白 2(BMP-2)和骨桥蛋白( OPN)蛋白表达水平。结果经鉴定,成功提取 huc-MSC-Exo;模型对照组和 PBS对照组小鼠胫骨骨折断端明显、骨膜增厚,骨折带的两侧可见大量未分化的间充质细胞及部分软骨细胞,骨痂的软骨内骨区域软骨细胞形态肥大,而 huc-MSC-Exo组小鼠胫骨骨折区愈合明显,外侧骨皮质连续性良好,部分软骨细胞已被吸收,膜内成骨区域出现骨痂;另与模型对照组(VEGFA 0.23±0.02)比较, PBS对照组(VEGFA 0.24±0.02)无明显变化(P>0.05)huc-MSC-Exo组小鼠胫骨骨折断端恢复良好,骨折线基本消失,同时骨折愈合评分、 BMD、VEGFA IRS评分、 VEGFA(0.48±0.04,)、 RUNX2、 BMP-2和 OPN蛋白相对表达水平均升高( P<0.05)。结论 huc-MSC-Exo可加快小鼠胫骨骨折修复,其作用机制可能与提高 VEGFA表达水平,促进血管生成有关。
英文摘要:
      Objective To investigate the reparative effects and possible mechanisms of human umbilical cord mesenchymal stem cell (huc-MSC)-derived exosomes (Exos) on tibial fracture mice.Methods From December 2018 to January 2022, huc-MSCs were isolated and cultured in vitro, and Exos were collected and identified by ultracentrifugation. A total of 24 8-week-old male C57BL/6J mice thatunderwent tibial fracture were randomly divided into a model control group, a phosphate buffer solution (PBS) control group and a huc-MSC-Exo group using a random number table method, with 8 mice in each group. Mice in the huc-MSC-Exo group were injected with huc-MSC-Exo (100 μg dissolved in 100 μL of PBS) in the bone marrow cavity, and mice in the PBS control group were injected with anequal amount of PBS, while the mice in the model control group were left untreated. After 21 d, fracture healing was observed by X-ray; bone mineral density (BMD) was measured by dual-energy X-ray; histological changes in the tibia were observed by hematoxylin-eosin (H&E) staining; positive expression of vascular endothelial growth factor A (VEGFA) was detected by immunohistochemical staining ofbone tissues in the fracture area; and the protein expression levels of VEGFA, runt-related transcription factor 2 (RUNX2), bone-form ing protein 2 (BMP-2) and osteoblastic polypeptide (OPN) were detected in the fracture area of bone tissue by Western blotting.Results After identification, huc-MSC-Exos were successfully extracted. In model-control and PBS-treated mice, the fracture ends of thetibia were obvious, the periosteum was thickened, and a large number of undifferentiated mesenchymal cells and some chondrocyteswere visible on both sides of the fracture zone. The chondrocyte morphology in the endochondral bone region of the bone scab was hypertrophic, while the tibia fracture area of mice in the huc-MSC-Exo group healed significantly, with good continuity of the outer bonecortex, some chondrocytes were resorbed, and bone scabs appeared in the endochondral bone region of the periosteum. Compared withthe model control group (VEGFA 0.23±0.02), the PBS control group (VEGFA 0.24±0.02) showed no significant difference (P>0.05). The mice in the huc-MSC-Exo group had good recovery of tibial fracture breaks, and the fracture line basically disappeared, while therelative expression levels of fracture healing score, BMD, VEGFA IRS score, VEGFA (0.48±0.04), RUNX2, BMP-2, and OPN proteins were all elevated (P<0.05).Conclusion Huc-MSC-Exos accelerate the repair of tibial fractures in mice, possibly through increasingthe expression level of VEGFA and promoting angiogenesis.
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