多次胶原酶消化法培养小鼠椎间盘髓核细胞

In vitro culture of mouse nucleus pulposus cells of intervertebral disc by multiple enzymatic digestions

背景:椎间盘为无血运组织,椎间盘髓核细胞为分化终末细胞,细胞增殖能力较差,体外培养难度较大。目的:探索小鼠椎间盘髓核细胞体外分离培养的方法。方法:取小鼠椎间盘髓核组织,使用多次胶原酶消化的方法,分离培养髓核组织细胞,接种,传代,取第2代细胞,分别采用免疫细胞化学和RT-PCR方法检测椎间盘髓核细胞特征性分泌物Ⅱ型胶原和聚合蛋白的分泌量及mRNA的表达,并与软骨细胞,成骨细胞及成纤维细胞进行比较。结果与结论:椎间盘髓核细胞贴壁后呈现软骨细胞的形态;Ⅱ型胶原和聚合蛋白染色均为阳性;Ⅱ型胶原和聚合蛋白mRNA表达与软骨细胞相同,与成纤维细胞和成骨细胞存在明显差别。说明多次胶原酶消化的方法可以获得大量纯净的椎间盘髓核细胞,性状稳定。

BACKGROUND：In vitro culture of nucleus pulposus cells of intervertebral disc is an important method to study degeneration of intervertebral disc. However, it is difficult to culture nucleus pulposus cells in vitro because the intervertebral disc is an avascular organ and nucleus pulposus cells poorly differentiate and proliferate. OBJECTIVE：To establish the method for in vitro culture of nucleus pulposus cells of intervertebral disc, and provide a reliable tool for research of phenotype changes of nucleus pulposus cells in degenerative disc and disc cell transplantation for treatment of degenerative disc diseases.METHODS：Mouse nucleus pulposus tissue was collected and repeatedly digested using collagenase. Cells were cultured and subcultured. The secretion of collagen Ⅱ and aggrecan of passage 2 cells were detected by immunohistochemistry and RT-PCR, The results were compared with other types of cells. RESULTS AND CONCLUSION：Nucleus pulposus cells of intervertebral disc exhibit a chondrocyte-like shape after adhesion. Immunohistochemistry study showed that the cells were positive for collagen Ⅱ and aggrecan staining. RT-PCR study showed that secretions of collagen Ⅱ and aggrecan in cultured cells were equal to those of chondrocyes and had significant difference from those of osteoblasts and fibroblasts. Multiple enzymatic digestions of nucleus pulposus can release a large amount of pure nucleus pulposus cells which had stable phenotype and can settle basis for research and treatment of intervertebal disc diseases.