摘要
目的探讨低氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)膜片成骨-成血管耦联的效应,为体外构建预血管化的组织工程骨提供依据。方法获得医院实验动物伦理委员会批准,体外培养Wistar大鼠的BMSCs,将携带HIF-1α的慢病毒(lentivirus,LV)和空载慢病毒稳定转染至第三代大鼠BMSCs中,形成Lv-HIF-1α-BMSCs组和Lv-BMSCs组,同时以未转染慢病毒的BMSCs为空白对照(BMSCs组),荧光定量PCR和Western Blot验证转染HIF-1α的效果。将LV-HIF-1α-BMSCs进行诱导分化形成内皮样细胞(endothelial-like cells,iECs),光学显微镜观察形态,细胞流式CD31检测分化情况,Transwell实验检测HIF-1α对iECs的迁移能力。将LV-HIF-1α-BMSCs(实验组)和LVBMSCs(对照组)连续成骨诱导培养形成成骨细胞膜片(osteogenic cell sheets,OCTs),分别在第14、21天进行碱性磷酸酶和茜素红染色、拍照并计数其矿化能力。最终将iECs植入OCTs中构建预血管化的成骨膜片(prevascularized osteogenic cell sheets,P-OCTs),在第1、3、7、14天进行免疫荧光CD31检测并计算内皮血管网形成的数量;第1、7、14天进行骨桥蛋白(osteopontin,OPN)和骨钙素(osteocalcin,OCN)的Western Blot检测,验证其成骨分化的能力。结果慢病毒转染BMSCs的最佳感染复数(multiplicity of infection,MOI)为30,转染率>80%。荧光定量PCR和Western Blot结果显示,与LV-BMSCs组和BMSCs组相比,LV-HIF-1α-BMSCs组中的HIF-1α具有稳定且高的表达(P<0.05)。LV-HIF-1α-BMSCs向iECs分化效率高达91.81%。Transwell实验结果显示HIF-1α具有体外招募iECs的作用;碱性磷酸酶染色和茜素红染色证实LV-HIF-1α-BMSCs形成的OCTs具有明显的成骨分化能力,与对照组LV-BMSCs相比差异具有统计学意义(P<0.05)。当iECs植入LV-HIF-1α-BMSCs组OCTs中形成P-OCTs时,iECs大量增殖、迁移并快速融合,免疫荧光CD31染色显示进行性管腔和血管网的形成。OPN、OCN的表达与对照组Lv-BMSCs相比显著增强,OPN在第1天表达量最高,OCN在第7天表达量最高(P<0.05)。结论HIF-1α增强表达的BMSCs经诱导分化后具有良好的成骨-成血管效应,为优化构建三维预血管化的骨组织提供实验基础。
Objective To explore the effect of HIF-1αon osteogenic-angiogenic coupling response in bone mesenchymal stem cells(BMSCs)and provide new concepts for engineered bone tissue in vitro.Methods With the approval of the hospital’s experimental animal ethics committee,BMSCs were harvested from Wistar rats.The lentivirus carrying hypoxia-inducible factor-1α(HIF-1α)and empty lentivirus were stably transfected into the third generations of BMSCs to form LV-HIF-1α-BMSCs and LV-BMSCs.Meanwhile,BMSCs without transfection of lentivirus were used as a blank control.Then,the effect of HIF-1αtransfection was verified by qPCR and Western Blot.LV-HIF-1α-BMSCs were induced to differentiate into endothelium-like cells(iECs).The morphology was observed by optical microscopy,the differentiation rate was detected by cellular flow CD31,and the Transwell test was used to detect the migration ability.At the same time,LV-HIF-1α-BMSCs and LV-BMSCs were continuously cultured to form osteogenic cell sheets(OCTs),which were stained by alkaline phosphatase on day 14 and alizarin red staining on day 21,and counted for mineralization capacity.Finally,iECs were implanted into OCTs to form prevascularized osteogenic cell sheets(P-OCTs),immunofluorescence CD31 was performed to detect the formation of vascular networks,and the results were recorded on days 1,3,7,and 14.Meanwhile,osteopontin(OPN)and osteocalcin(OCN)were detected by western blot to verify their ability for osteogenic differentiation on days 1,7,and 14.Results The optimal multiplicity of infection(MOI)for lentiviral transfection was 30,and the transfection efficiency was>80%.The results of qPCR and western blot showed that compared with the LV-BMSCs group and BMSCs group,the LV-HIF-1α-BMSCs group had stable and high expressions of HIF-1α(P<0.05).LV-HIF-1α-BMSCs showed an enhanced ability to differentiate into endothelial cells,with a differentiation rate as high as 91.81%.Transwell assay verified that HIF-1αcould recruit iECs in vitro.Alkaline phosphatase staining and alizarin red staining confirmed that OCTs formed by LV-HIF-1α-BMSCs had a statistically significant osteogenic differentiation ability compared with LV-BMSCs control group(P<0.05).When iECs were implanted into the LV-HIF-1α-BMSCs group OCTs to form P-OCTs,iECs substantially proliferated and rapidly fused,and formation of the progressive lumen was revealed by immunofluorescent CD31 staining.The expressions of OPN and OCN were significantly enhanced compared with those of the LV-BMSCs control group;OCN was the highest on day 7,and OPN was the highest on day 1(P<0.05).Conclusion BMSCs transfected by HIF-1αhave good osteogenic-angiogenic effect after induction and differentiation,which provides experimental foundation for optimizing the construction of three-dimensional prevascularized bone tissue.
作者
张丹
黄银莉
滕雍辉
韩畅
ZHANG Dan;HUANG Yinli;TENG Yonghui;HAN Chang(Department of Orthodontics,Yinchuan Stomatology Hospital,Yinchuan 750000,China)
出处
《口腔疾病防治》
2025年第9期744-756,共13页
Journal of Prevention and Treatment for Stomatological Diseases
基金
宁夏回族自治区自然科学基金(2023AAC03880)。
关键词
低氧诱导因子-1Α
骨髓间充质干细胞
细胞膜片
预血管化
成骨-成血管耦联效应
骨组织工程
碱性磷酸酶
茜素红
骨桥蛋白
骨钙素
hypoxia-inducible factor-1α
bone marrow mesenchymal stem cells
cell sheet
prevascularization
osteogenic-angiogenic coupling response
bone tissue engineering
alkaline phosphatase staining
alizarin red staining
osteopontin
osteocalcin
作者简介
通信作者:张丹,主治医师,硕士,Email:1254887917@qq.com。
