胰酶联合Ⅱ型胶原酶分离培养髓核细胞:培养基中葡萄糖浓度的选择

Trypsin plus type Ⅱ collagenase digestion for isolation of nucleus pulposus cells: the optimal glucose concentration in complete medium

背景:椎间盘退变是脊柱退行性疾病的发病基础,研究椎间盘的退变因素,对脊柱退行性的疾病的预防和治疗有重要意义。目的:采用胰酶联合Ⅱ型胶原酶消化法体外分离培养兔髓核细胞,并观察髓核细胞在不同葡萄糖浓度培养基中的生长及增殖状况,确立最适宜髓核细胞生长的葡萄糖浓度。方法:胰酶联合Ⅱ型胶原酶消化法分离、培养兔髓核细胞,倒置显微镜观察细胞形态并计数细胞数量,绘制细胞生长曲线。苏木精-伊红和甲苯胺蓝染色观察细胞形态。细胞免疫组织化学染色结合免疫荧光染色检测Ⅱ型胶原蛋白表达情况。分别使用0,6.25,12.5,17.5,25 mmol/L葡萄糖培养基培养髓核细胞24 h后,检测各组髓核细胞增殖及凋亡情况。结果与结论:(1)髓核细胞形态:兔髓核细胞染色后镜下观察呈多角形、短梭形,有一两个核仁,随着传代次数增加,细胞伸出伪足,胞体逐渐变细长;(2)基因表达:体外分离培养髓核细胞的Ⅱ型胶原蛋白及聚集蛋白多糖基因均可稳定表达;(3)细胞增殖:各组中细胞增殖比率以葡萄糖17.5 mmol/L组最高,显著高于葡萄糖0,25 mmol/L组(P<0.05或P<0.01);(4)细胞凋亡:葡萄糖0 mmol/L组细胞凋亡率最高(P<0.05),其他各组差异无显著性意义(P>0.05)。(4)结果证实:胰酶联合Ⅱ型胶原酶消化法可收获较多的原代髓核细胞,葡萄糖浓度17.5 mmol/L比较适宜髓核细胞体外培养。

BACKGROUND： Intervertebral disc degeneration is the pathological basis of degenerative spinal diseases. Studies on the influential factors of intervertebral disc degeneration contribute to the prevention and treatment of degenerative spinal disease. OBJECTIVE： To observe the growth and proliferation of nucleus pulposus cells isolated by trypsin plus type II collagenase digestion in complete medium with different glucose concentrations, exploring the optimal glucose concentration for growth of nucleus pulposus cells. METHODS： Nucleus pulposus cells isolated and cultured by trypsin plus type II collagenase digestion method were observed under an inverted microscope, and the cell number was counted. Morphology of nucleus pulposus cells was observed after hematoxylin-eosin staining and toluidine blue staining. Collagen type II immunoreactivity was detected by immunohistochemical staining combined with immunofluorescent staining. Nucleus pulposus cells were incubated in complete medium containing various glucose concentrations（0, 6.25, 12.5, 17.5, and 25 mmol/L） for 24 hours, and then cell proliferation and apoptosis were determined. RESULTS AND CONCLUSION： The stained nucleus pulposus cells showed polygonal and short spindle, with one or two nuclei. Cellular pseudopod appeared gradually and then became slim with increased passage numbers. The isolated and cultured nucleus pulposus cells positively expressed collagen type II and aggrecan Proliferative activity of nucleus pulposus cells cultured in medium with 17.5 mmol/L glucose was significantly higher than that in medium with 0 and 25 mmol/L glucose（P〈0.05 or P〈0.01）. There was no significant difference in cell apoptosis between these groups except for 0 mmol/L glucose（P〈0.05）. These results confirm that a large number of nucleus pulposus cells can be harvested by trypsin plus type II collagenase digestion and the optimal glucose concentration is 17.5 mmol/L.