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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。
Objective To study the tissue engineering seed cells of disc tissue and observe the effects of spinal cord cell culture medium on the proliferation and differentiation of BMSCs. Methods Chordate cells were isolated from the thoracolumbar discs of 4-week-old Japanese white rabbits. BMSCs were isolated from bilateral femurs and cultured in DMEM / F12 medium containing 15% FBS. After 5 days, spinal cord cells . The experiment was divided into two groups. BMSCs in experiment group were cultured in spinal cord cell culture medium, BMSCs in control group were cultured in DMEM / F12 medium containing 15% FBS. Cell viability was detected by cell viability cytotoxicity assay. The expression of proteoglycan and type Ⅱ collagen in BMSCs were detected by immunofluorescence and real-time fluorescence quantitative PCR. Results The spinal cord cells and BMSCs were successfully isolated. Cell proliferation assay showed that the number of cells in the experimental group was significantly more than that of the control group at 5, 7, 9 and 14 days (P <0.05). Immunofluorescence assay showed that there was no or less type II collagen and proteoglycan expression in the control group at 7 and 14 days, both of which were more expressed in the experimental group and significantly more than 7 days after 14 days of culture. Real-time quantitative PCR showed that the expression of proteoglycan and type Ⅱ collagen mRNA in experimental group was significantly higher than that in control group on the 7th and 14th day (P <0.05), and the expression of proteoglycan and type Ⅱ collagen mRNA in the experimental group at 14th day was significantly higher than that of the control group d (P <0.05). Conclusion The culture media of spinal cord cells can promote the proliferation of BMSCs and induce the differentiation of BMSCs into chondrocytes. It provides the basis for the treatment of disc degeneration of spinal cord cells and BMSCs as seed cells.