| 张涛贠喆蔡承魁马云雷姬振伟裘秀春范清宇钱济先.大鼠髓核细胞原代培养及表型鉴定[J].现代生物医学进展英文版,2012,12(3):430-434. |
| 大鼠髓核细胞原代培养及表型鉴定 |
| The Primary Culture of Rats Nucleus Pulposus Cells andthe Phenotype Identifying |
| |
| DOI： |
| 中文关键词: 髓核细胞 原代培养 HIF-1α MMP2 Ⅱ型胶原 |
| 英文关键词: Nucleus pulposus cells Primary culture Hypoxia inducible factor-1α Matrix Metalloproteinases 2 TypeⅡCollagen |
| 基金项目: |
|
| Hits: 1191 |
| Download times: 0 |
| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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 type Ⅱ 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. |
|
View Full Text
View/Add Comment Download reader |
| Close |
|
|
|
