共培养成纤维细胞及血管内皮细胞对脂肪干细胞增殖和成骨作用的影响

Co-culture of fibroblasts and vascular endothelial cells affects proliferation and osteogenesis of adipose stem cells

背景:组织工程的发展为临床骨缺损治疗提供了新方法,但组织工程骨形成缓慢、血管化缓慢等问题一直存在。已有研究表明血管内皮细胞和脂肪干细胞联合培养体系修复骨缺损能力优于单种细胞。成纤维细胞具有优良增殖能力、分泌合成胶原能力,包含多种调控因子,可向成骨分化,具有成为优良种子细胞参与构建组织工程骨的潜力。目的:探讨成纤维细胞、血管内皮细胞和脂肪干细胞联合培养对脂肪干细胞增殖和成骨分化的影响。方法:①将细胞分为4组:脂肪干细胞组、脂肪干细胞+血管内皮细胞共培养组、脂肪干细胞+成纤维细胞共培养组、脂肪干细胞+血管内皮细胞+成纤维细胞共培养组,倒置显微镜下观察细胞的形态变化;②共培养第1,3,5,7,9天,采用CCK-8法检测各组脂肪干细胞增殖情况并绘制生长曲线;③共培养第7,14,21,28天,各组脂肪干细胞进行茜素红染色、碱性磷酸酶染色;④共培养第3周,采用Western blot检测各组脂肪干细胞中骨形态发生蛋白2的表达水平。结果与结论:①培养14 d时,脂肪干细胞+血管内皮细胞+成纤维细胞共培养组部分细胞融合成团块状,呈巢状分布,而脂肪干细胞组细胞形态单一,未见细胞团出现;②各组细胞生长曲线形态基本相同,细胞吸光度值逐渐升高,脂肪干细胞+血管内皮细胞+成纤维细胞共培养组吸光度值最高,脂肪干细胞+血管内皮细胞共培养组次之,其次是脂肪干细胞+成纤维细胞共培养组;③共培养第28天时茜素红染色,各组细胞均有红色阳性细胞,以脂肪干细胞+血管内皮细胞+成纤维细胞共培养组最多,脂肪干细胞组最少。共培养第28天时碱性磷酸酶染色,各组细胞均有红色阳性颗粒,以脂肪干细胞+血管内皮细胞+成纤维细胞共培养组和脂肪干细胞+成纤维细胞共培养组最多,脂肪干细胞组最少;④各组细胞均有骨形态发生蛋白2表达,以脂肪干细胞+成纤维细胞共培养组和脂肪干细胞+血管内皮细胞+成纤维细胞共培养组较明显;⑤结果表明,体外共培养条件下成纤维细胞促进脂肪干细胞成骨分化的能力比血管内皮细胞强,但促增殖效果不如血管内皮细胞;成纤维细胞联合血管内皮细胞与脂肪干细胞共培养体系促进脂肪干细胞大量增殖、快速高效向成骨细胞分化的能力最强。

BACKGROUND:The development of tissue engineering has provided a new method for the clinical treatment of bone defects,but the problems of slow formation and slow vascularization of tissue engineered bone have always existed.Previous studies have shown that the combined culture system of vascular endothelial cells and adipose stem cells has better ability to repair bone defects than the single type of cells.Fibroblasts have excellent proliferation characteristics,the ability to secrete and synthesize collagen,contain a variety of regulatory factors,can differentiate into osteogenesis,and have the potential to become excellent seed cells to participate in the construction of tissue engineered bone.OBJECTIVE:To investigate the effects of combined culture of fibroblasts,vascular endothelial cells,and adipose stem cells on the proliferation and osteogenic differentiation of adipose stem cells.METHODS:(1)The cells were divided into four groups:adipose stem cell group,adipose stem cell+vascular endothelial cell co-culture group,adipose stem cell+fibroblast co-culture group,adipose stem cell+vascular endothelial cell+fibroblast co-culture group.The morphological changes of the cells were observed under an inverted microscope.(2)At 1,3,5,7,and 9 days of co-culture,the proliferation of adipose stem cells in each group was detected by CCK-8 assay and the growth curve was plotted.(3)Alizarin red staining and alkaline phosphatase staining were performed on adipose stem cells in each group at 7,14,21,and 28 days of co-culture.(4)At 3 weeks of co-culture,the expression level of bone morphogenetic protein 2 in adipose stem cells in each group was detected by western blot assay.RESULTS AND CONCLUSION:(1)After 14 days of culture,in the adipose stem cell+vascular endothelial cell+fibroblast co-culture group,some cells fused into clumps and distributed in nests,while in the adipose stem cell group,the cell morphology was single and no cell clusters were found.(2)The morphology of the cell growth curve was basically the same in each group,and the absorbance value increased gradually.The absorbance value of the adipose stem cell+vascular endothelial cell+fibroblast co-culture group was highest,followed by the adipose stem cell+vascular endothelial cell co-culture group,followed by the adipose stem cell+fibroblast co-culture group.(3)At 28 days of co-culture,all the cells in each group showed red positive cells,and the most cells in the adipose stem cell+vascular endothelial cell+fibroblast co-culture group showed red focus.The adipose stem cell group was the least.At 28 days of co-culture,each cell had red positive particles.The number of particles was most in the adipose stem cell+vascular endothelial cell+fibroblast group and adipose stem cell+fibroblast co-culture group,and that in the adipose stem cell group was least.(4)Bone morphogenetic protein 2 was expressed in all cells of each group,and it was more obvious in the adipose stem cell+fibroblast co-culture group and the adipose stem cell+vascular endothelial cell+fibroblast co-culture group.(5)Results confirmed that under the condition of co-culture in vitro,fibroblasts promoted osteogenic differentiation of adipose stem cells,which is more strongly than vascular endothelial cells,but the promotion of proliferation was not as good as that of vascular endothelial cells.The co-culture system of fibroblasts combined with vascular endothelial cells and adipose stem cells has the strongest ability to promote the proliferation of adipose stem cells and the rapid and efficient differentiation into osteoblasts.