非接触性共培养BMSCs和髓核细胞时间差异性的实验研究

TIME DIFFERENCES OF BONE MARROW MESENCHYMAL STEM CELLS AND NUCLEUS PULPOSUS-LIKE CELLS IN A NON CONTACT CO-CULTURE SYSTEM

目的探讨在非接触性共培养环境下,BMSCs定向分化为类髓核细胞在共培养时间上的差异性,寻找适合体内移植的最佳时间。方法取6只8周龄健康新西兰大白兔(体重1.5～2.0 kg)骨髓及椎间盘髓核,分离、培养BMSCs和髓核细胞并进行免疫细胞化学鉴定。取原代髓核细胞和第2代生长良好的BMSCs体外建立非接触性共培养模型。观察共培养后第1、3、5代BMSCs的形态学变化并绘制生长曲线;RT-PCR检测共培养5、10、15 d BMSCsⅡ型胶原和蛋白聚糖mRNA表达;Western blot检测共培养5、10、15、20、25、30 d BMSCsⅡ型胶原和蛋白聚糖蛋白的表达。结果 BMSCs相对特异性标记物CD44、CD90表达阳性,造血细胞表面标记物CD34、CD45表达阴性。髓核细胞Ⅱ型胶原、蛋白聚糖表达阳性。共培养后2周BMSCs形态发生明显变化,呈多角形、不规则形;共培养后3代内,BMSCs生长速度无明显差异,随着传代次数增加,细胞增殖明显减慢。RT-PCR检测示共培养后10、15 d BMSCs蛋白聚糖和Ⅱ型胶原mRNA表达明显高于5 d时(P<0.05),而10 d与15 d时差异无统计学意义(P>0.05)。Western blot检测示共培养后随时间延长细胞表达Ⅱ型胶原和蛋白聚糖蛋白逐渐增加,5、10、15 d间差异有统计学意义(P<0.05),15 d后各时间点间比较差异无统计学意义(P>0.05)。结论在非接触性共培养环境下,BMSCs在髓核细胞诱导下可向类髓核细胞分化,表达Ⅱ型胶原和蛋白聚糖,在共培养15 d时达到相对稳定,此时较适合进行体内移植。

Objective To investigate time differences in directional differentiation of bone marrow mesenchymal stem cells（BMSCs） into nucleus pulposus-like cells（NPCs） in a non contact co-culture system so as to search for the best time for transplantation in vivo.Methods Six New Zealand white rabbits（aged 6 weeks,weighing 1.5-2.0 kg） were selected.BMSCs were collected and cultured for immunocytochemistry identification of CD34,CD44,CD45,and CD90;NPCs were isolated and identified immunocytochemically by RT-PCR.The 2nd passage BMSCs and the primary NPCs were co-cultured in a non contact co-culture system.The cell morphological changes were observed and the cell growth curves were made at 1,3,and 5 passages after co-culture.The expressions of the aggrecan and collagen type Ⅱ genes were detected by RT-PCR in BMSCs at 5,10,and 15 days after co-culture;the expressions of the aggrecan and collagen type Ⅱ proteins were detected by Western blot at 5,10,15,20,25,and 30 days after co-culture.Results The expressions of CD44 and CD90 were positive,CD34 and CD45 were negative in BMSCs.The expressions of the collagen type Ⅱ and aggrecan were positive in NPCs.At 2 weeks after co-culture,the morphology of BMSCs changed obviously,the cells were polygonal and irregular shape.The cell growth rate showed no difference within 3 passages,but decreased obviously after 3 passages.RT-PCR showed that the expressions of collagen type Ⅱ and aggrecan genes at 10 and 15 days were significantly higher than those at 5 days（P 0.05）,no significant difference was found between at 10 days and at 15 days（P 0.05）.Western blot showed that the expressions of collagen type Ⅱ and aggrecan proteins gradually increased with time,and there was significant difference within 5,10,and 15 days（P 0.05）,but no significant difference was found after 15 days of co-culture（P0.05）.Conclusion In a non contact co-culture system,BMSCs can differentiate into the NPCs.The expression of collagen type Ⅱ and aggrecan can reach a stable level at 15 days after co-culture,and it is the suitable time for transplantation in vivo.