软骨终板干细胞及骨髓间充质干细胞对软骨终板细胞增殖分化的影响  

Effects of cartilage endplate stem cells and bone marrow mesenchymal stem cells on proliferation and differentiation of cartilage endplate cells

作　　者：高健明 汪靖[1,2] 刘晨 肖良 彭江[2] 汪爱媛[2] 徐宏光[1] GAO Jianming;WANG Jing;LIU Chen;XIAO Liang;PENG Jiang;WANG Aiyuan;XU Hongguang(Spine Research Center of Wannan Medical College,Department of Spine Surgery,Yijishan Hospital,Wuhu 241001,Anhui Province,China;Key Laboratory of Musculoskeletal Trauma&War Injuries PLA,Beijing Key Lab of Regenerative Medicine in Orthopedics,Chinese PLA General Hospital,Beijing 100853,China)

机构地区：[1]皖南医学院脊柱研究中心,皖南医学院第一附属医院弋矶山医院脊柱外科,安徽芜湖241001 [2]解放军总医院全军骨科战创伤重点实验室,骨科再生医学北京市重点实验室,北京100853

出　　处：《解放军医学院学报》2021年第5期548-554,共7页Academic Journal of Chinese PLA Medical School

基　　金：国家自然科学基金面上项目(81972108)。

摘　　要：背景组织工程作为一项年轻的技术获得了越来越多脊柱医学研究者的关注。但组织工程种子细胞的选择仍无明确标准。目的获取并鉴定软骨终板干细胞(cartilage endplate stem cell,CESC)和骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC),通过对比探明CESC是否有作为组织工程种子细胞的潜在可能。方法原代培养SD大鼠软骨终板细胞(cartilage endplate cell,CEP),甲苯胺蓝染色鉴定软骨细胞表型,通过单克隆法筛选培养CESC和BMSC;三系诱导分化验证CESC和MSC干细胞特性;取P3代CEP、CESC和BMSC进行非接触式共培养,在1 d、3 d、5 d、7 d,CCK-8检测三组Transwell小室下层CEP增殖,并绘制增殖曲线;流式细胞仪检测共培养7 d后三组下层小室CEP凋亡变化,RT-qPCR检测CEP表型基因ACAN、COLⅡ、Sox9表达变化。结果甲苯胺蓝染色显示原代培养的CEP能够分泌软骨相关的多糖物质,如糖胺聚糖等,且具有特征性多角形形态,呈铺路石样分布;通过单克隆法分离出的CESC和BMSC经三系诱导分化后,茜素红染色能够观察到被染成红色的钙结节,油红O染色观察到细胞周围出现大量类圆形红色脂滴形成,番红染色观察到软骨细胞特异性分泌的聚集蛋白聚糖出现。CCK-8检测表明CESC组和BMSC组增殖能力均强于CEP组,且CESC组增殖能力强于BMSC组,三组CEP凋亡水平无统计学差异;RT-qPCR结果表明三组CEP共培养第1-7天ACAN、COL2A1和Sox9表达均呈波峰状改变,第3天表达水平最高。第1天、第7天三组软骨表型基因表达ACAN、COL2A1和Sox9差异无统计学意义;第3天、第5天CESC组表达高于BMSC组和CEP组,差异有统计学意义(P<0.05)。结论 CESC和BMSC通过非接触式共培养均能够增强CEP增殖和软骨表型的表达,且前者作用明显更强。Background Tissue engineering,as a new technique,has attracted more and more attention from spinal medicine researchers.However,there is still no clear standard in the selection of seed cells in tissue engineering.Objective To obtain and identify cartilage end plate stem cells(CESC)and bone marrow mesenchymal stem cells(BMSC),and find out whether CESC has the potential as seed cells for tissue engineering by comparing with BMSC.Methods The phenotype of chondrocytes in primary cultured SD rats was identified by toluidine blue staining,and the characteristics of CESC and BMSC stem cells were identified by single clone screening.Then,the three lines cells were induced to verify the characteristics of CESC and BMSC stem cells;and the P3 generation of CEP,CESC and BMSC were taken for non-contact co-cultivation.At 1 d,3 d,5 d,and 7 d,CCK-8 was used to detect the proliferation of CEP in the lower layer of Transwell chamber in the three groups and the proliferation curve was drawn.Flow cytometry was used to detect the changes of CEP apoptosis in the middle and lower compartments of the three groups after 7 days of co-cultivation.Finally,RT-qPCR was used to detect the changes in the expression of CEP phenotype gene proteoglycan(ACAN),typeⅡcollagen(COLⅡ),and sex determining region Y box protein 9(Sox9).Results Toluidine blue staining showed that the primary cultured CEP secreted cartilage-related polysaccharides such as glycosaminoglycan(GAG),and had characteristics of polygonal shape and paving stone-like distribution.After the three-line induced differentiation of CESC and BMSC isolated by monoclonal method,red calcium nodules were observed by alizarin red staining,a large number of round red lipid droplets around the cells were observed by oil red O staining,and aggregate proteoglycans secreted specifically by chondrocytes were observed by fuchsin staining.CCK-8 assay showed that the proliferation ability of CESC group and BMSC group was stronger than that of CEP group,and the proliferation ability of CESC group was

关 键 词：软骨终板细胞 软骨终板干细胞 骨髓间充质干细胞 Transwell共培养 细胞增殖 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]