脊索细胞培养基促进BMSCs增殖分化的研究

目的为研究椎间盘组织工程种子细胞,观察脊索细胞培养基对BMSCs增殖分化的影响。方法取4周龄日本大耳白兔胸腰段椎间盘分离培养脊索细胞,取双侧股骨分离培养BMSCs,用含15%FBS的DMEM/F12培养基培养脊索细胞,5 d后制备脊索细胞培养基。实验分为两组,实验组BMSCs中加入脊索细胞培养基培养,对照组BMSCs中加入含15%FBS的DMEM/F12培养基培养。使用细胞活力细胞毒性检测检测细胞增殖情况,采用免疫荧光及实时荧光定量PCR检测BMSCs蛋白多糖及Ⅱ型胶原表达情况。结果成功分离脊索细胞及BMSCs。细胞增殖检测示,培养5、7、9、14 d,实验组细胞数量明显多于对照组(P<0.05)。免疫荧光检测示对照组培养7、14 d细胞内均无或者有较少Ⅱ型胶原及蛋白多糖表达,实验组二者均有较多表达,且培养14 d时表达明显多于7 d。实时荧光定量PCR检测示,培养7、14 d实验组蛋白多糖和Ⅱ型胶原mRNA表达均显著高于对照组(P<0.05);实验组14 d蛋白多糖和Ⅱ型胶原mRNA表达显著高于7 d(P<0.05)。结论脊索细胞培养基可促进BMSCs增殖,并诱导BMSCs向类软骨细胞分化,为脊索细胞和BMSCs作为种子细胞治疗椎间盘退变提供了依据。

Promotion effect of notochordal cells conditioned medium on proliferation and differentiation of bone marrow mesenchymal stem cells

Objective To investigate the effect of notochordal cells(NCs) conditioned medium(NCCM) on the proliferation and differentiation of bone marrow mesenchymal stem cells(BMSCs). Methods NCs and BMSCs were isolated from the thoracolumbar intervertebral disc and the femurs of 4-week-old Japanese white rabbits,respectively.NCs were cultured with DMEM/F12 medium containing 15% FBS for 5 days to prepare NCCM.The experiment consisted of 2 groups: BMSCs were cultured with NCCM in experimental group and with DMEM/F12 medium containing 15% FBS in control group.The proliferation of BMSCs was assessed by cell counting kit-8 at 1,3,5,7,9,and 14 days after culture;the expression of proteoglycan and collagen type II were determined by immunofluorescence and real-time fluorescent quantitative PCR at 7 and 14 days after culture. Results NCs and BMSCs were successfully isloated.At 5,7,9,and 14 days,the number of BMSCs in the experimental group was significantly more than those in the control group(P < 0.05).At 7 and 14 days,there was no expression or less expression of proteoglycan and collagen type II in the control group;however,there was a lot of expression of proteoglycan and collagen type II in the experimental group,and the expressions were higher at 14 days than at 7 days.At 7 and 14 days after culture,the mRNA expressions of proteoglycan and collagen type II were significantly higher in the experimental group than in the control group(P < 0.05),and at 14 days than at 7 days in the experimental group(P < 0.05).Conclusion NCCM can promote the proliferation and the differentiation of BMSCs into chondroyte-like cells,which provides the basis for NCs and BMSCs as seed cells in the treatment of degenerative disc disease.