脱细胞软骨基质对于大鼠腰椎终板软骨干细胞分化的影响  

Effect of decellular endplate cartilage matrix on differentiation of lumbar endplate derived cartilage stem cells in rat

作　　者：王效 徐宏光[1] 刘晨[1] 肖良 金中行 沈阳 WANG Xiao;XU Hong-guang;LIU Chen;XIAO Liang;JIN Zhong-xing;SHEN Yang(Department of Spinal Surgeo,Yijis-han Hospital,Wannan Medical College,Wuhu 241001,China)

机构地区：[1]皖南医学院弋矶山医院脊柱外科,安徽芜湖241001

出　　处：《中国矫形外科杂志》2018年第13期1215-1221,共7页Orthopedic Journal of China

基　　金：国家自然科学基金面上项目(编号:81572185);国家自然科学青年基金项目(编号:81702158);安徽省自然科学基金面上项目(编号:1708085MH185);安徽省科技厅对外科技合作项目(编号:1704e1002229)

摘　　要：[目的]制备家猪脱细胞软骨基质,探索其对大鼠腰椎终板软骨干细胞分化的影响。[方法]通过胰酶、核酶及Triton X-100制备脱细胞软骨基质,将软骨基质溶于双蒸水制得脱细胞软骨基质膜。将大鼠腰椎终板软骨干细胞种植于基质膜上,CCK-8检测1、3、5 d的增殖情况,并与空白板对照。取P3代终板软骨干细胞种植于铺有软骨基质的孔板(DEPM组)和两组空白板(正常诱导组和无诱导组),然后进行成骨、成脂肪及成软骨分化诱导,2周后,茜素红、油红O及番红染色,分别鉴定其成骨、成脂肪及成软骨能力;每组取4孔按500 ul/孔加入DMSO,置于酶标仪上,在450 nm波长下检测各组的吸光度。同时Real-Time PCR分别检测成骨、成脂肪及成软骨特征性基因Runx2、Col-2a1和PPAR?在各组中的相对表达量,分析干细胞在软骨基质上的分化倾向性。[结果]大鼠腰椎终板软骨干细胞能够在脱细胞软骨基质上生长,并与其有很好的组织相容性。将终板软骨干细胞进行诱导后染色发现,DEPM组的干细胞具有更强的成骨分化能力,较低的成脂肪及成软骨分化能力。Real-Time PCR检测成骨基因Runx2在DEPM组较其他两组呈较高表达,而Col-2a1和PPAR?基因表达较低。在450 nm波长下,DEPM组中的成骨吸光度最大,大于其他两组,成脂肪及成软骨的吸光度小于正常诱导组。[结论]脱细胞软骨基质能够较好的保持干细胞成脂肪及成软骨的分化特性,但是促进干细胞向成骨方向分化。[Objective] To prepare of decellularized endplate cartilage matrix（DEPM） and explore its effects on the differentiation of lumbar endplate cartilage stem cells in rat. [Methods] The DEPM prepared by trypsin, ribozyme and Triton X-100 was dissolved in double distilled water to make decellularized endplate cartilage matrix membrane（DEPMM）, the rat lumbar endplate derived cartilage stem cells（EPCSCs） were seeded on the DEPMM and incubated. Subsequently, the proliferation of the cells was evaluated by CCK-8 assay at 1, 3 and 5 days, and compared with blank panel. The third generation of EPCSCs was seeded in the cartilage matrix-plated wells（the DEPM group） and other two groups of blank plates, including the induction group and the non-induction group, where the osteogenic, adipogenic and chondrogenic differentiation were induced. Two weeks later, Alizarin red, Oil red and Safranin O staining were done to identify osteogenic, adipogenic and cartilage capacity of the cells. The expression of Runx2, Col-2 a1 and PPARY in osteogenic, adipogenic and chondrogenic tissues was detected by Real-Time PCR to analyze differentiation tendency of stem cells on the DEPMM. [Results] The EPCSCs grew well on DEPMM with good histocompatibility. After induction of EPCSCs, the stem cells of the DEPM group had stronger osteogenic differentiation capacity, lower adipogenic and chondrogenic differentiation ability compared with the blank groups. RealTime PCR revealed that osteoblast Runx2 in DEPM group was higher expression, while Col-2 a1 and PPARY gene were lower expression than the other two groups. Under the wavelength of 450 nm, the absorbance of osteoblast in the DEPM group was the largest, which was significantly greater than that in the other two groups, whereas the absorbance of adipocytes and cartilage in the DEPM group was lower than that of normal induction group. [Conclusion] The DEPM maintains stem cells adipogenic and cartilage differentiation characteristics,promote the differentiation of stem cells to the os

关 键 词：脱细胞软骨基质 终板软骨干细胞 多向分化 

分 类 号：R318[医药卫生—生物医学工程]