细胞密度对髓核细胞产生蛋白聚糖影响的体外实验

Effect of cell density on the glycosaminoglycan production by nucleus pulposus cells in vitro

背景:退变的椎间盘组织一个重要改变是聚集蛋白聚糖明显减少,如何能够刺激髓核细胞产生更多的蛋白聚糖是学者们一直探索的问题。目的:将椎间盘髓核细胞以不同的密度植入凝胶材料中,评价细胞密度对蛋白聚糖含量的影响。设计、时间及地点:对比观察实验,于2007-08/2008-02在解放军北京军区总医院骨科实验室完成。材料:清洁级3月龄日本大耳兔3只。Live/DeadAssaykit,sGAGAssaykit试剂盒由北京纵横洋洲医药生物科技有限公司提供。方法:无菌条件下取兔椎间盘,常规分离消化其髓核细胞,传第2代时以1.0×109L-1密度接种在自制的壳聚糖支架中,细胞密度扩增至4×109L-1和25×109L-1时进行检测。主要观察指标:阿利辛蓝法测蛋白聚糖含量。氪氩激光器共聚焦显微镜下观察培养细胞存活情况。结果:培养14d,细胞密度为4×109L-1时,聚集的蛋白聚糖总量为(551.65±70.10)mg/L,当细胞密度增加到25×109L-1时,聚集蛋白聚糖总量为(2245.28±462.52)mg/L。但蛋白聚糖增加的量和细胞密度并不成比例,细胞密度增加了6倍,蛋白聚糖含量只增加了两三倍。低密度培养7d时,结果显示100%的细胞存活。高密度培养7d时,30%的细胞在材料中央死亡,边缘部位的细胞几乎全部存活。结论:体外三维培养髓核细胞产生的蛋白聚糖随种植细胞密度增加而缓慢增加,高细胞密度对蛋白聚糖聚集有限制作用。

BACKGROUND： Decrease of aggrecan markedly is an important change of degenerative intervertebral disc tissue. How to stimulate nucleus pulposus cells to produce more glycosaminoglycan has been a difficult problem for researchers. OBJECTIVE： To implant the intervertebral disc nucleus pulposus cells into gel materials, and to evaluate the effect of cell density on the content of glycosaminoglycan. DESIGN, TIME AND SETTING： A control observational experiment was performed at the Orthopaedic Laboratory in General Hospital of Beijing Military Area Command of Chinese PLA from January to July 2008. MATERIALS： Three 3-month old Japanese rabbits of clean grade were selected in this study. Uve/Dead Assay kit and sGAG Assay kit were provided by Beijing Transglobal Biomedical Technology Co., Ltd （China）. METHODS： Under aseptic condition, the nucleus pulposus cells were isolated from intervertebral discs of rabbits, and then digested The secondary generation nucleus pulposus cells were inoculated on the self-made chitosan-gel scaffold at a concentration of 1×10^9/L. The detection was performed when the cell densities were 4×10^9/L and 25×10^9/L. MAIN OUTCOME MEASURES： The contents of glycosaminoglycan were detected by alcian blue method, and the survival of nucleus pulposus cells was observed by confocal microscope of krypton-argon laser. RESULTS： At 14 days after cultured, the content of glycosaminoglycan was （551.65±70.10） mg/L at the cell density of 4×10^9/L, and （2245.28±462.52） mg/L at the cell density of 25×10^9/L. But the increase of content of glycosaminoglycan was disproportionate to the cell density. The cell density increased to 6 folds while the content only increased 2-3 folds. At 7 days after cultured in the cell density of 4×10^9/L, the survival rate of nucleus pulposus cells was 100%; while cultured in the cell density of 25×10^9/L, 30% nucleus pulposus cells were dead at the center of material, and nearly all nucleus pulposus cells at the peripheral area were survived. CONCLUSION： The content of glycosaminoglycan in this culture system in vitro increase slowly with the increase of cell density, and the high cell density is limited the aggregation of glycosaminoglycan.