摘要
背景:组织工程模块为股骨头坏死的治疗提供了新的思路,将血管化支架移植于坏死区域,在生长因子及支架的作用下诱导骨髓间充质干细胞向血管内皮细胞分化,促进微血管生成改善局部血运能够促进坏死区域的修复。目的:探讨体外构建诱导骨髓间充质干细胞向血管内皮细胞分化的树枝状两亲性肽自组装凝胶模块的可行性。方法:分离提取4-6周龄SD大鼠骨髓间充质干细胞,采用流式细胞术进行鉴定,体外定向成脂、成骨诱导分化检测细胞多向分化潜能。采用固相法合成肽IKVAVAK-/RGDK-KGGGGAAAA(K)-C16H31O,调配质量浓度为10 mg/mL多肽溶液,加入生理盐水后触发多肽自组装,形成半透明的水凝胶,透射电子显微镜下观察凝胶结构。用携带血管内皮生长因子165基因的腺病毒转染大鼠骨髓间充质干细胞,然后将10 mg/mL多肽溶液和细胞悬液混合,细胞分布于凝胶内部形成三维培养体系,设置为实验组;将转染空病毒的骨髓间充质干细胞与10 mg/mL多肽溶液混合形成三维培养体系,设置为对照组。体外培养7 d后采用免疫荧光及RT-qPCR检测血管内皮细胞分化情况。结果与结论:(1)流式细胞术结果显示间质干细胞表面分子CD29、CD44表达≥95%,造血干细胞表面分子CD34、CD45表达≤2%;在体外能够向成骨、成脂诱导分化;(2)树枝状多肽溶液在离子触发下形成的凝胶呈透明状,透射电镜下观察呈形态均一的小尺寸纳米胶束;(3)免疫荧光及RT-qPCR结果显示,实验组血管内皮生长因子165表达水平高于对照组,且血管内皮特异性标记物CD31、CD34表达水平高于对照组。结果表明,血管内皮生长因子165基因转染骨髓间充质干细胞种植于多肽凝胶构建组织工程模块,能够诱导骨髓间充质干细胞向血管内皮细胞分化。
BACKGROUND:The tissue engineering module provides a new idea for the treatment of femoral head necrosis.A vascularized scaffold was constructed and transplanted into the necrotic area,and the bone marrow mesenchymal stem cells were induced to differentiate into vascular endothelial cells under the action of growth factors and scaffolds,which promoted the generation of microvessels and improved local blood vascularity,thereby promoting the repair of the necrotic area.OBJECTIVE:To investigate the feasibility of constructing a dendritic amphiphilic peptide self-assembly gel module with the ability to induce the differentiation of bone marrow mesenchymal stem cells into vascular endothelial cells in vitro.METHODS:Bone marrow mesenchymal stem cells were isolated and extracted from SD rats aged 4-6 weeks and identified by flow cytometry.In vitro directed adipogenic and osteogenic induced differentiation was used to detect the multidirectional differentiation potential of cells.Peptides KGGGGAAAA(K)-C16H31O were synthesized by solid-phase method.The concentration was 10 mg/mL peptide solution.After the addition of normal salt solution,the peptide self-assembly was triggered,forming a translucent hydrogel.Transmission electron microscopy was utilized to observe the gel structure.In the experimental group,rat bone marrow mesenchymal stem cells were transfected with adenovirus carrying vascular endothelial growth factor 165 gene.The 10 mg/mL peptide solution was mixed with the cell suspension,and the cells were distributed inside the gel to form a three-dimensional culture system.In the control group,bone marrow mesenchymal stem cells transfected with empty virus were mixed with 10 mg/mL peptide solution to form a three-dimensional culture system.Immunofluorescence and RT-qPCR were used to detect the differentiation of vascular endothelial cells after 7 days of in vitro culture.RESULTS AND CONCLUSION:(1)Flow cytometry results showed that the expression of CD29 and CD44 on the surface of mesenchymal stem cells was≥95%,and the expression of CD34 and CD45 on the surface of hematopoietic stem cells was≤2%.Bone marrow mesenchymal stem cells were successfully differentiated by osteogenic induction and adipogenic induction in vitro.(2)The dendritic peptide solution was triggered by ions to form a transparent gel,and the small-sized nanomicelles with uniform morphology were observed under electron microscopy.(3)The results of immunofluorescence and RT-qPCR showed that the expression level of vascular endothelial growth factor 165 in the experimental group was higher than that in the control group,and the expression levels of CD31 and CD34 were higher than those in the control group.These results suggest that vascular endothelial growth factor 165 gene transfected bone marrow mesenchymal stem cells implanted in a tissue engineering module constructed by peptide gel can induce the differentiation of bone marrow mesenchymal stem cells into vascular endothelial cells.
作者
蒋星海
宋玉林
李德津
邵建敏
徐军志
刘华凯
吴应国
沈岳辉
冯思诚
Jiang Xinghai;Song Yulin;Li Dejin;Shao Jianmin;Xu Junzhi;Liu Huakai;Wu Yingguo;Shen Yuehui;Feng Sicheng(The Third People’s Hospital of Jingdezhen,Jingdezhen 333000,Jiangxi Province,China;The Second Affiliated Hospital of Nanchang University,Nanchang 330000,Jiangxi Province,China)
出处
《中国组织工程研究》
北大核心
2026年第8期1903-1911,共9页
Chinese Journal of Tissue Engineering Research
基金
景德镇市科技计划项目(2022SFZC012),项目负责人:蒋星海。
关键词
树枝状两亲性肽
骨髓间充质干细胞
基因转染
血管内皮生长因子165
三维培养
血管化
股骨头坏死
dendritic amphiphilic peptide
bone marrow mesenchymal stem cell
gene transfection
vascular endothelial growth factor 165
three-dimensional culture
vascularization
femoral head necrosis
