大鼠髓核细胞原代培养及表型鉴定

目的:探讨Ⅱ型胶原酶联合透明质酸酶消化分离培养髓核细胞及免疫细胞化学表型鉴定的可行性。方法:无菌条件下分离SD大鼠凝胶状髓核,采用Ⅱ型胶原酶联合透明质酸酶消化分离髓核细胞并连续培养,倒置相差显微镜下观察,随后进行免疫细胞化学染色检测不同代次髓核细胞HIF-1、Ⅰ、Ⅱ型胶原、MMP2及蛋白聚糖的表达情况,并给予MTT法测定髓核细胞生长曲线。结果:Ⅱ型胶原酶联合透明质酸酶分离培养原代髓核细胞需要12 d左右贴壁,达95%融合需要34 d,而传代髓核细胞贴壁速率明显增快至10 h,且其倍增时间约为2.5 d;免疫细胞化学显示髓核细胞均表达HIF-1、Ⅰ、Ⅱ型胶原、MMP2和蛋白聚糖,且随着髓核细胞传代其HIF-1α、HIF-1β、Ⅰ型胶原及MMP2表达均增加,但Ⅱ型胶原表达降低,而蛋白聚糖表达无明显差异;MTT法显示随着髓核细胞传代其增殖有所减缓。结论:Ⅱ型胶原酶联合透明质酸酶可成功分离髓核细胞,提高培养效率,且HIF-1α、HIF-1β、Ⅰ、Ⅱ型胶原及MMP2可作为髓核细胞表型分子用于髓核细胞的鉴定。

The Primary Culture of Rats Nucleus Pulposus Cells and the Phenotype Identifying

Objective: To investigate the feasibility of isolating and culturing nucleus pulposus(NP) cells from rats intervertebral discs by type Ⅱ collagenase and hyaluronidase and identifying the phenotype of NP by immunocytochemistry.Methods: The gel NP was isolated under sterile circumstance and digested by type Ⅱ collagenase and hyaluronidase,which was continuous cultured and viewed under inverted phase contrast microscope.The expression of hypoxia inducible factor-1(HIF-1,typeⅠcollagen,typeⅡcollagen,matrix metalloproteinases 2(MMP2) and proteoglycans in different generations of NP cells were detected,and the growth curve of NP cells was assayed by MTT.Results: The NP cells approximately needed 12 days to adhere,and needed 34 days to reach 95% confluence in primary culture.After transfer of culture,the cells needed 10 hours to adhere while the doubling generation time was 2.5 days.With serial subcultivation of NP cells,the expression of HIF-1α,HIF-1β,typeⅠ collagen and MMP2 all increased,typeⅡ collagen decreased,proteoglycans had no marked variance,and the growth of NP cells gradually slowed down.Conclusion: The way of isolating and culturing NP cells by typeⅡ collagenase and hyaluronidase is successful,and it improves culture efficiency.Moreover,HIF-1α,HIF-1β,typeⅠcollagen,typeⅡcollagen and MMP2 were the characteristic phenotype of NP cells and can be used to identify NP cells.