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目的:建立一种可用于体外研究椎间盘退变的椎间盘器官培养模型,探讨渗透压负荷对模型椎间盘细胞活力和代谢的影响。方法:4～6月龄新西兰大白兔10只,均分为两组,处死后立即手术切取胸腰段椎间盘,每只9个,分别在等渗(300mOsm/kg,等渗组)或高渗(410mOsm/kg,高渗组)培养基中进行整体器官培养,在培养前和培养后第7、14、21、28天,利用MitotrackerGreen荧光探针、组织化学和生物化学方法评估两组椎间盘髓核细胞的活力、结构的完整性以及蛋白多糖含量的变化。结果:取材后培养前椎间盘髓核细胞的荧光强度为11503±402,在体外培养过程中,高渗组第14天荧光强度为9202±907,与培养前比较差异有显著性意义(P<0.05),第7、21和28天分别为10504±710、10860±711、10713±953,与培养前相比较无显著性差异(P>0.05);等渗组第7、14、21、28天分别为11350±351、11207±385、10914±300、10862±229,与培养前比较无显著性差异(P>0.05);两组在第7、21和28天时无显著性意义(P>0.05)。在培养过程中,两组椎间盘髓核和纤维环的组织结构能够基本保持完整,髓核蛋白多糖含量在培养第7天高渗组和等渗组分别为3.33±0.28mg/100mg和2.83±0.25mg/100mg,均较培养前(5.03±0.37mg/100mg)明显降低(P<0.01),第14、21、28天时与第7天相比下降不明显(P>0.05),两组相同时间点的差异无显著性意义(P>0.05)。结论:椎间盘器官培养模型可以在等渗或高渗环境中有效维持兔椎间盘结构的完整性和髓核细胞的活力至少4周。
OBJECTIVE: To establish a culture model of disc organ that can be used to study disc degeneration in vitro and to investigate the effect of osmotic pressure on cell viability and metabolism of intervertebral discs. Methods: Ten New Zealand white rabbits (4-6 months old) were divided into two groups. Immediately after the operation, thoracic and lumbar intervertebral discs were obtained by surgical operation. Nine of them were treated with isotonic (300mOsm / kg) or hypertonic (410mOsm / kg, hypertonic group) medium, organ culture was performed before and at 7th, 14th, 21st and 28th day after culture. Mitotracker Green fluorescence probe, histochemical and biochemical methods were used to evaluate the bone marrow Nuclear cell viability, structural integrity, and changes in proteoglycan content. Results: The fluorescence intensity of the nucleus pulposus cells before culture was 11503 ± 402. The fluorescent intensity of the hypertonic group on day 14 was 9202 ± 907, which was significantly different from that before culture (P <0.05) ) On day 7, 21 and 28 were 10504 ± 710, 10860 ± 711 and 10713 ± 953, respectively. There was no significant difference between before and after culture (P> 0.05). On the 7th, 14th, 21st and 28th day Respectively, 11350 ± 351,11207 ± 385,10914 ± 300,10862 ± 229, no significant difference compared with that before culture (P> 0.05). There was no significant difference between the two groups on the 7th, 21st and 28th day (P> 0.05 ). In the process of culture, the structure of nucleus pulposus and annulus fibrosus in both groups remained basically intact. The content of proteoglycan in nucleus pulposus was 3.33 ± 0.28mg / 100mg and 2.83 ± 0.25 (P <0.01). Compared with the control group (P <0.01), the decrease was not significant at the 14th, 21st and 28th day (P> 0.05) There was no significant difference between the two groups (P> 0.05). CONCLUSION: The intervertebral disc organ culture model can effectively maintain the integrity of the intervertebral disc structure and the activity of nucleus pulposus cells in isotonic or hypertonic environment for at least 4 weeks.