非接触共培养条件下软骨细胞诱导骨膜细胞向软骨细胞分化

Indirect co-culture with chondrocytes induces periosteum-derived cells to differentiate into chondrocytes

背景:在非接触共培养条件下,软骨细胞可诱导骨髓间充质干细胞分化为软骨细胞。骨膜细胞作为软骨修复的种子细胞,其与软骨细胞共培养的作用及影响鲜有研究。目的:通过软骨细胞与骨膜细胞非接触共培养,利用软骨细胞诱导骨膜细胞向软骨细胞分化。方法:采用胰酶、Ⅱ型胶原酶消化法分离兔关节软骨细胞;组织块培养法分离兔骨膜细胞。分别取第2代软骨细胞和骨膜细胞,按1∶1比例通过transwell培养板进行非接触共培养作为实验组,单纯骨膜细胞培养作为对照组。培养2周后,倒置显微镜观察2组细胞形态;Ⅱ型胶原免疫组化染色和甲苯胺蓝染色检测2组细胞的Ⅱ型胶原和蛋白聚糖的合成;RT-PCR检测2组细胞的蛋白聚糖、Ⅱ型胶原α1和性别决定区Y框蛋白9 m RNA表达水平。结果与结论:(1)培养2周后,实验组共培养骨膜细胞逐渐变为类圆形软骨细胞形态,对照组骨膜细胞仍为长梭形;(2)实验组共培养骨膜细胞的Ⅱ型胶原免疫组化、甲苯胺蓝染色均为阳性,对照组为阴性;(3)RT-PCR结果显示,实验组共培养骨膜细胞的蛋白聚糖、Ⅱ型胶原α1、性别决定区Y框蛋白9 mR NA相对表达量明显高于对照组;(4)结果提示,非接触共培养条件下,软骨细胞可以诱导骨膜细胞向软骨细胞分化。

BACKGROUND:Under indirect co-culture conditions,chondrocytes can induce bone marrow mesenchymal stem cells to differentiate into chondrocytes.Osteocytes are the seed cells of chondrocytes,but co-culture of osteocytes with chondrocytes is rarely reported.OBJECTIVE:To induce the chondrogenic differentiation of periosteum-derived cells(PDCs)by indirect co-culture with chondrocytes.METHODS:Chondrocytes were isolated from the rabbits by trypsin and collagenase II digestion.Rabbit PDCs were obtained by the explants culture method.Passage 2 chondrocytes and PDCs underwent indirect co-culture at 1:1 in Transwell system as experimental group.PDCs cultured alone were as control group.After 2 weeks of culture,the cellular morphological changes were observed by inverted contrast phase microscope.The expression levels of collagen type II and proteoglycan were detected by collagen type II immunohistochemistry and toluidine blue staining.The mRNA expression levels of proteoglycan,collagen type II and SRY-related protein-9 were detected by RT-PCR.RESULTS AND CONCLUSION:After 2 weeks of culture,PDCs in the experimental group were gradually transformed to chondrocyte-like cells,while PDCs in the control group still remained long spindle-shaped.Collagen type II immunohistochemistry staining and toluidine blue staining were positive in the experimental group,while the results were negative in the control group.Additionally,RT-PCR results indicated that the relative mRNA expression levels of proteoglycan,collagen type II and SRY-related protein-9 in the experimental group were significantly higher than those in the control group.Therefore,chondrocytes can induce PDCs to differentiate into chondrocytes under indirect co-culture conditions.