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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 the difference in the time of co-culture of BMSCs differentiated into nucleus pulposus cells in a non-contact co-culture environment and to find out the optimal time for transplantation in vivo. Methods Bone marrow and nucleus pulposus of 6 8-week-old healthy New Zealand white rabbits weighing 1.5-2.0 kg were isolated, cultured and identified. Immunocytochemistry was used to identify BMSCs and nucleus pulposus cells. Take the primary nucleus pulposus cells and the second generation of well-grown BMSCs establish a non-contact co-culture model in vitro. The morphological changes of BMSCs were observed at the 1st, 3rd, 5th passage after co-culture and the growth curves were drawn. The mRNA expression of typeⅡcollagen and proteoglycan of BMSCs were detected by RT-PCR at 5, Expression of collagen type Ⅱ and proteoglycan in BMSCs at 10, 15, 20, 25 and 30 d. Results The relative specific markers CD44 and CD90 of BMSCs were positive, while the expression of CD34 and CD45 on the surface of hematopoietic cells was negative. Nucleus pulposus cells type Ⅱ collagen, proteoglycan expression was positive. After 2 weeks of co-culture, the morphology of BMSCs changed significantly, showing polygonal and irregular shape. There was no significant difference in the growth of BMSCs within 3 passages after co-culture, with the passage number increasing, the proliferation of BMSCs significantly slowed down. RT-PCR results showed that the expression of BMSCs was significantly higher than that of BMSCs on the 10th and 15th day after co-culture (P <0.05), while there was no significant difference between 10 and 15 days (P> 0.05 ). Western blot analysis showed that the expression of type Ⅱ collagen and proteoglycan increased gradually with time after co-culture, with a significant difference between 5 and 10 days (P <0.05). After 15 days, the difference was significant No statistical significance (P> 0.05). Conclusions Under non-contact co-culture conditions, BMSCs can differentiate into nucleus pulposus cells under the induction of nucleus pulposus cells and express type II collagen and proteoglycan, which are relatively stable at 15 days of co-culture, which is more suitable for in vivo transplant.