摘要
目的分离纯化兔髓核中的细胞成分,观察脊索细胞形态及对软骨样细胞增殖及细胞表型的影响。方法6只4周龄新西兰兔胸腰段脊柱,获取髓核,0.2%Ⅱ型胶原酶消化法及不连续密度梯度法分离纯化脊索细胞及软骨样细胞。实验分为单纯软骨样细胞培养组(A组)及脊索细胞、软骨样细胞(1∶1)共培养组(B组)。倒置相差显微镜观察两组细胞生长情况,测定两组原代及传代细胞成活率,MTT法测定两组第2代细胞增殖曲线,并以甲苯胺蓝染色和免疫细胞化学染色鉴定两组原代及传代细胞蛋白多糖及Ⅱ型胶原的表达。结果成功分离、纯化脊索细胞和软骨样细胞。细胞培养观察,原代脊索细胞直径10～15μm,胞浆内富含大小不等的囊泡;原代软骨样细胞直径4～6μm,胞浆内无囊泡。各代细胞成活率A组为89.0%～95.3%,B组为91.3%～96.3%,各代细胞成活率两组间比较差异无统计学意义(P>0.05)。A组细胞培养3～4d进入对数生长期,B组细胞培养2d后进入对数生长期;培养4d后,B组细胞增殖能力高于A组,差异有统计学意义(P<0.05)。A组3代以内细胞表达蛋白多糖及Ⅱ型胶原,B组细胞表型维持至5代。结论脊索细胞能促进髓核软骨样细胞的增殖及表型维持;脊索细胞可能在防止椎间盘退变中具有重要意义。
Objective To isolate and culture the chondroid cells and notochord cells from New Zealand rabbit immature nucleus pulposus （NP） in monolayer, and to valuate the responsiveness of rabbit disc-derived chondroid cells to notochord cells with respect to cell proliferation and phenotype. Methods The NP cells were released from the minced immature NP of 6 New Zealand rabbits （4-week-old） by 0.2% collagenase II digestion. The chondroid cells and notochord cells were purified by discontinuous gradient density centrifugation. The chondroid cells were cultured alone （group A） and co-cultured with notochord cells （group B） （1 ： 1）, and cell proliferation and phenotype including proteoglycan and collagen II were evaluated. The cells in both groups were observed by the inverted microscope, and the survival rates of the primary and passage cells were detected by toluidine blue staining. The growth curves of the second passage cells in both groups were determined by MTT. Besides, the expressions of proteoglycan and collagen II of the primary and passage cells were examined by toluidine blue and immunocytochemistry staining. Results The notochord cells and chondroid cells were isolated and purified. With the diameter of 10-15 μm, the notochord cell had abundant intracytoplasmic vesicles, while the chondroid cell, with the diameter of 4-6 μm, had no intracytoplasmic vesicle. The cell survival rate was 89.0%-95.3% in group A and 91.3%-96.3% in group B. There was no significant difference between the same passages in both groups （P 〉 0.05）. The co-cultured cells （group B） increased in cell proliferation compared with the chondroid cells alone （group A） in repeated experiments. The cells in group A reached their logarithmic growth phase after 3-4 days of culture, while the cells in group B did after 2 days of culture. The cell proliferation in group B was more than that in group A after 4-day culture （P 〈 0.05）. The cocultured cells retained their phenotype for 5 passages, while parallel-c
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2008年第8期939-943,共5页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家自然科学基金资助项目(30572163/C030320)~~
关键词
椎间盘
髓核
脊索细胞
软骨样细胞
细胞培养
Intervertebral disc Nucleus pulposus Notochord cell Chondroid cell Cell culture
