口腔疾病防治2025，Vol.33Issue(9)：744-756,13.DOI:10.12016/j.issn.2096-1456.202550104
HIF-1α对BMSCs膜片成骨-成血管耦联的效应研究
Effect of HIF-1α on osteogenic-angiogenic coupling response in BMSCs sheets
摘要
Abstract
Objective To explore the effect of HIF-1α on osteogenic-angiogenic coupling response in bone mes-enchymal stem cells(BMSCs)and provide new concepts for engineered bone tissue in vitro.Methods With the ap-proval 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-1α transfection 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 abil-ity.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 min-eralization 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 lenti-viral 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 differ-entiation rate as high as 91.81%.Transwell assay verified that HIF-1α could recruit iECs in vitro.Alkaline phosphatase staining and alizarin red staining confirmed that OCTs formed by LV-HIF-1α-BMSCs had a statistically significant os-teogenic 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 signifi-cantly 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-1α have good osteogenic-angiogenic effect after in-duction and differentiation,which provides experimental foundation for optimizing the construction of three-dimensional prevascularized bone tissue.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Key words
hypoxia-inducible factor-1α/bone marrow mesenchymal stem cells/cell sheet/prevasculariza-tion/osteogenic-angiogenic coupling response/bone tissue engineering/alkaline phosphatase staining/alizarin red staining/osteopontin/osteocalcin分类
医药卫生引用本文复制引用
张丹,黄银莉,滕雍辉,韩畅..HIF-1α对BMSCs膜片成骨-成血管耦联的效应研究[J].口腔疾病防治,2025,33(9):744-756,13.基金项目
宁夏回族自治区自然科学基金(2023AAC03880) This study was supported by the grants from Natural Science Foundation of Ningxia Hui Autonomous Region(No.2023AAC03880). （2023AAC03880）
