人血管周围干细胞成骨分化及Wnt/β-catenin信号通路的调控

Osteogenic differentiation of human perivascular stem cells and its regulation based on Wnt/beta-catenin signaling pathway

背景:血管周围干细胞来源于成体脂肪组织,获取容易且干细胞比较均质,具有较强的增殖和多向分化潜能。目的:通过体外、体内实验探讨人血管周围干细胞的成骨分化能力,以及Wnt/β-catenin信号通路的调控作用。方法:抽脂来源的人脂肪组织,经荧光激活细胞分选法提取血管周围干细胞,第1代血管周围干细胞进行成骨诱导分化,分为空白对照组、成骨诱导组及Wnt信号通路抑制组,成骨诱导第5天行碱性磷酸酶染色,第10天行茜素红染色,第7天通过Western blot检测Runt相关转录因子2蛋白表达。制备血管周围干细胞复合纳米羟基磷灰石/聚乳酸-羟基乙酸共聚物支架材料,通过无胸腺小鼠胫骨单皮质缺损模型,研究血管周围干细胞在体内的骨修复效果。结果与结论:①每100 mL脂肪中含有基质血管成分细胞为(1.82±0.32)×10^(7),活细胞占(82.72±5.37)%,经荧光激活细胞分选法分选,血管外膜细胞(CD34^(+)、CD45^(-)、CD146^(-))比例为(17.66±1.05)%、血管外周细胞(CD146^(+)、CD45^(-)、CD34^(-))比例为(7.18±0.52)%,血管周围干细胞体外扩增迅速,10代之内保持较强的增殖能力;②细胞实验显示血管周围干细胞具有成骨分化能力,抑制Wnt信号通路后Runt相关转录因子2表达明显减少,血管周围干细胞成骨分化受到抑制,动物实验进一步证实血管周围干细胞复合支架材料的成骨效果;③结果表明:Wnt/β-catenin信号通路在血管周围干细胞成骨分化中发挥重要作用,血管周围干细胞为骨修复提供一种新的治疗选择。

BACKGROUND:Perivascular stem cells are derived from adult adipose tissue,which can be easily purified.Perivascular stem cells represent a comparatively homogenous population and have the ability of strong proliferation and multidirectional differentiation.OBJECTIVE:To explore the osteogenic differentiation ability of human perivascular stem cells and the regulation by Wnt/β-catenin signaling pathway by in vitro and in vivo experiments.METHODS:Perivascular stem cells were extracted from human lipoaspirate via fluorescence-activated cell sorting.Osteogenic differentiation of passage 1 perivascular stem cells was observed.Cells were randomly divided into blank control group,osteogenic differentiation group and Wnt/β-catenin signaling inhibition group.Alkaline phosphatase staining was performed on day 5 of osteogenic induction.Alizarin red staining was performed on day 10.On day 7,the protein expression of Runt related transcription factor 2 was detected by western blot assay.Perivascular stem cells+nano-hydroxyapatite/poly lactic-co-glycolic acid scaffold was prepared.Bone repair effect of perivascular stem cells in vivo was evaluated by using the model of tibia monolayer cortical bone defect in athymic mice.RESULTS AND CONCLUSION:(1)There were about(1.82±0.32)×10^(7)stromal vascular fraction cells per 100 mL of fat tissue,and of which the living cells accounted for(82.72±5.37)%.Through fluorescence-activated cell sorting,the proportion of adventitial cells(CD34^(+),CD45^(-),CD146^(-))was(17.66±1.05)%;and the proportion of pericytes(CD146^(+),CD45^(-),CD34^(-))was(7.18±0.52)%.Perivascular stem cells proliferated rapidly and still maintained strong proliferative ability within 10 generations.(2)In vitro studies had confirmed that perivascular stem cells had the ability of osteogenic differentiation.In addition,the expression of Runt-related transcription factor 2(RUNX2)was significantly reduced and osteogenic differentiation of perivascular stem cells was attenuated by inhibition of Wnt signaling pathway.Animal experiments further confirmed the osteogenic effect of perivascular stem cell composite scaffolds.(3)These results indicate that Wnt/β-catenin signaling pathway plays an important role in the osteogenic differentiation of perivascular stem cells,which provides a new therapeutic option for bone repair.