摘要
目的研究大鼠髓核间充质干细胞(NPMSCs)体外培养的最佳血清体积浓度并探讨椎间盘退变的机制。方法用胶原酶、胰酶序贯消化法从SD大鼠腰部和尾部的椎间盘组织中提取NPMSCs,并进行体外培养,将其传代。①利用流式细胞仪对第3代传代NPMSCs细胞表型CD29、CD34、CD24、CD90、CD105进行鉴定;②利用普通PCR鉴定体外培养的NPMSCs细胞基因Sox2、Nanog的表达;③在37℃、21%O2、5%CO2的细胞培养箱中用成骨、成软骨、成脂培养液诱导培养第3代传代NPMSCs,2周以后,用染色剂对其染色,观察其成脂、成骨、成软骨能力。④在37℃、21%O2、5%CO2的细胞培养箱中用不同血清体积浓度(0%、5%、10%、20%、30%)的DMEM-F12血清培养液培养第3代传代NPMSCs,72h后利用CCK-8法检测细胞的增殖,并采用RT-PCR检测细胞蛋白多糖、Ⅱ型胶原、SOX9的mRNA表达,借助流式细胞仪对细胞进行细胞凋亡的检测。结果①大鼠NPMSCs的成骨、成软骨能力强,成脂能力较差,且其细胞表面高度表达CD29、CD90、CD105,低度表达CD24、CD34,细胞基因Sox2、Nanog高度表达。②随着培养液血清体积浓度增加,OD值增大,NPMSCs的凋亡率降低,细胞蛋白多糖、Ⅱ型胶原、SOX9基因的mRNA表达增加。结论①大鼠NPMSCs具有间充质干细胞特性;②细胞外营养影响大鼠NPMSCs的增殖、分泌、凋亡。
Objective To investigate the optimum volume concentration of serum for the culture of the rat nucleus mesenchymal stem cells（NPMSCs） in vitro and the mechanism of intervertebral disc degeneration. Methods NPMSCs was isolated and cultured from the lumbar and caudal of the SD rats by Collagenase,trypsin sequential digestion method,and then they were cultured subcultured in vitro. ①Flow cytometry was used to identify the cell phenotype CD29,CD34,CD24,CD90,CD105 of the third generation NPMSCs. ②The expression of Sox2,Nanog were assessed by PCR. ③The third generation NPMSCs were induced and cultured by osteogenesis,chondroblastomas and lipoblast culture in incubator with 37℃,21%O2,5%CO2condition. After 2 weeks,dyed them and observed the ability of osteoporosis,chondroblastomas and lipoblast. ④Cultured the third generation NPMSCs with different volume concentrations of the DMEM-F12 serum（0%,5%,10%,20%,30%） under condition of 37℃,21%O2,5%CO2for 72 hours. The cell proliferation was detected by CCK-8 method in different groups,and the proteoglycan expression of the mRNA of SOX9,collagenⅡ, were assessed by RT-PCR,and flow cytometry was used to detect apoptosis. Results ①The osteogenic and cartilage ability of the rat NPMSCs were strong,and adipogenic capacity was poor. CD29,CD90, CD105 were highly expressed, CD24 and CD34 were poorly expressed,and Sox2,Nanog were highly expressed. ②With the volume of serum concentration increasing,OD value increased,and the apoptosis rate of NPMSCs decreased,expression of the mRNA of cell proteoglycans,collagen type Ⅱ,SOX9 gene increased. Conclusion ①The rat NPMSCs has the biological characteristics like mesenchymal stem cells;②Extracellular nutrition affects the proliferation,secretion,apoptosis of the rat NPMSCs.
出处
《中国现代医药杂志》
2014年第4期17-22,共6页
Modern Medicine Journal of China
关键词
椎间盘退变
髓核间充质干细胞
营养
增殖
凋亡
Intervertebral disc degeneration Nucleus mesenchymal stem cells Nutrition Proliferation Apoptosis
