骨骼肌中筋膜源性细胞分化为软骨细胞的能力

Chondrogenic progenitor cells isolated from the fascia of skeletal muscle

背景:骨骼肌中的肌源性细胞在骨形态发生蛋白4与转化生长因子β3的作用下具有成软骨分化的能力,但由于骨骼肌组成复杂,其中包括肌纤维组织、筋膜组织、血管组织、神经组织等,所以其中具备向软骨分化能力的细胞组织来源尚未确定。目的:观察骨骼肌中筋膜源性细胞分化成软骨细胞的能力,以明确骨骼肌来源的细胞中具有成软骨分化能力的种子细胞。方法:分离大鼠的臀大肌筋膜组织,剪成细小组织块后于含骨形态发生蛋白4和转化生长因子β3的软骨形成培养基中培养,在第14天收集样本,使用阿尔新蓝、苏木精-伊红染色和番红O染色来鉴定筋膜源性细胞的成软骨细胞分化能力;使用结蛋白、CD34、波形蛋白、v-WF和α平滑肌肌动蛋白相应抗体的免疫染色来研究筋膜源性细胞上各种不同细胞标志物在的表达情况;筋膜源性细胞与L6大鼠成肌细胞不同比例混合(1∶0,4∶1,1∶1,1∶4,0∶1),用免疫染色方法分析筋膜源性细胞和L6细胞分化成软骨细胞的能力。结果与结论:(1)当在软骨形成培养基中培养第14天时,在阿尔新蓝和番红O免疫染色为阳性结果,表明筋膜组织块中的细胞具有分化成软骨细胞的能力;(2)分离的筋膜源性细胞的结蛋白,CD34、v-WF抗原和α平滑肌肌动蛋白的表达是阴性的,但超过90%的细胞表达波形蛋白;(3)纯筋膜源性细胞成软骨分化能力最强,随着L6大鼠成肌细胞比例增高,其成软骨分化能力逐渐下降;(4)结果提示,从骨骼肌中筋膜源性细胞表面标记物分析、免疫染色及筋膜源性细胞与L6大鼠成肌细胞混合培养对比后,表明筋膜源性细胞具有分化成软骨细胞的能力,是骨骼肌来源的细胞中具有成软骨能力的种子细胞。

BACKGROUND:Muscle derived cells in the skeletal muscle have the ability to differentiate into chondrocytes under the induction of bone morphogenic protein 4 and transforming growth factorβ3.However,due to the complexity of the skeletal muscle(consisting of muscle fiber,fascia,blood vessel and nerves),the origin of cells has not been identified.OBJECTIVE:To observe the potential for chondrogenic differentiation of fascia-derived cells(FDCs)in order to clarify the chondrogenic ability of the cells derived from the skeletal muscle.METHODS:The fascia was isolated from the rat gluteus maximus,and was then cultured in chondrogenic medium containing bone morphogenic protein 4 and transforming growth factorβ3.Samples were harvested on day 14.The chondrogenic differentiation of FDCs was identified by alcian blue,hematoxylin-eosin and safranin O stainings.Cell markers were investigated by immunostaining using the antibodies for desmin,CD34,vimentin,vWF and a-SMA.Immunostaining was used to assay the chondrogenic potential of FDCs cultured with L6 rat myoblasts at varying ratios(1:0,4:1;1:1,1:4 and 0:1).RESULTS AND CONCLUSION:FDCs harvested from the skeletal muscle displayed chondrogenic differentiation and formed cartilaginous tissue when cultured for 14 days in chondrogenic medium after alcian blue and safranin O stainings.Cells isolated from the fascia were negative for desmin,CD34,vWF and a-SMA,but over 90%of the cells were positive for vimentin.Mixed FDC and L6 myoblast pellets showed chondrogenic potential decreased with the increasing ratio of L6 myoblasts.From the analysis of cell surface marker of FDCs in the skeletal muscle,immunostaining,and the comparison with L6 rat myoblasts,it shows that FDCs are the seed cells with chondrogenic potential in the skeletal muscle.