不同代次兔髓核细胞中Ⅱ型胶原和SOX9的表达

Expressions of collegen Ⅱ and SOX9 in different generations of rabbit nucleus pulposus cells

目的：通过培养不同代次兔髓核细胞，检测其Ⅱ型胶原和SOX9的含量变化，观察其细胞生长规律。方法：取2～3个月龄新西兰大白兔8只，空气栓塞致死后取出胸、腰椎段髓核进行细胞传代培养。将原代细胞作为对照组（A组．n=8），传代后的2、3、4、5代细胞分别为B、c、D、E组，显微镜下观察不同代次细胞的形态变化：MTF法绘制细胞生长曲线：RT—PCR和免疫组化法检测不同代次细胞Ⅱ型胶原和SOX9的表达。结果：原代细胞（A组）生长缓慢，20～30d达80％融合可传代；B组生长速度最快，7～10d可传代，C、D、E组生长速度依次减慢。RT—PCR和免疫组化法检测可知A组和B组的Ⅱ型胶原及SOX9的表达明显高于C、D、E三组（P〈0．05），A、B组之间差异无显著性（P〉0．05），C、D、E组之间差异无显著性（P〉0．05）。结论：不同代次兔髓核细胞的生长规律、Ⅱ型胶原及SOX9含量不同，第2代兔髓核细胞生物学特性与原代细胞相似．可作为种子细胞进行实验研究。

Objective To observe the growth law of rabbit nucleus pulposus cells （NPCs） through detecting the expressions of collegen Ⅱ and SOX9 in them. Methods 8 New Zealand white rabbits （2 - 3 months old） were sacrificed by air embolism, then the thoracic and lumbar NPCs were separated and cultured in vitro. The original generational cells were included as control group （Group A, n = 8）, and the second, third, forth and fifth generational cells were included as Group B, C, D, E, respectively. Cell morphology was observed by microscopy, and the growth curve was drawn by MTT The expressions of collagen Ⅱ and SOX9 were evaluated by RT-PCR and immunohistochemistry. Results Cells in Group A grew slowly and took 20 - 30 days to reach 80% confluence, and those in Group B grew the fastest to perform cell passage by 7 -10 days; the growth rates in Group C, D and E slowed down in turn. The expressions of collagen Ⅱ and SOX9 in Group A and B were obviously higher than those in Group C, D and E （P 〈 0.05） ; and were not significantly different between Group A and B （P 〉 0.05） and among Group C, D, E （P 〉 0.05）. Conclusion Different generational cells presented varied expressions of collagen Ⅱ and SOX9 and growth law. The second generational cells were suitable to be used as seed cells.