摘要
目的观察体外经传代培养去分化的成人关节软骨细胞,在生物反应器培养后生物学性状的变化,探索去分化软骨细胞反分化的手段,为软骨细胞移植修复关节软骨缺损建立合适的体外培养方法。方法无菌条件下取成人关节软骨组织,型胶原酶消化法(0.2%,37℃,3h)分离软骨细胞,分成两组:一组常规单层传代培养,另一组添加重组人的生长因子(1ng/ml转化生长因子β1+5ng/ml成纤维细胞生长因子2)体外培养传代大量扩增后,无微载体生物反应器内培养3周。血小板计数器行细胞计数,计算各代细胞倍增时间;细胞爬片和石蜡、冰冻切片进行HE、蕃红O、阿利新蓝染色,、型胶原和aggrecan免疫组织化学检测,观察细胞表型变化。结果成人关节软骨细胞体外培养3代后迅速去分化,增殖缓慢。添加生长因子培养细胞去分化速度减缓;传10代,细胞扩增2000倍以上,部分去分化,但细胞扩增增殖能力仍很强;传20代软骨细胞表型基本丢失,但仍有增殖能力;置于生物反应器继续培养3周,细胞番红O染色强阳性、aggrecan和型胶原阳性,型胶原阴性,表型恢复良好。结论软骨细胞在体外大量扩增后,在生物反应器培养,可恢复其表型,可望用于在体外培养时去分化软骨细胞的再分化。
Objective To examine the biological characteristic changes in the dedifferenciated human articular chondrocytes by the bioreactor culturing in vitvo. Methods The cartilage tissue was obtained from the joints of the adult human. The chondrocytes were isolated from the cartilage tissue with the type Ⅱ collagenase digestion（0. 2%, 37℃, 3 h）and were cultured in DMEM-F12 supplemented with 20% fetal bovine serum （FBS） with 1 ng/ml of TGF-β1 and 5 ng/ml of FGF-2. After about 20 passages by the monolayer culture, the cells were then transferred to the bioreactor culturing of the rotational cell culture system （RCCS） for a 3-week sequence culture. The cell counting was performed with the platelet counter, and the doubling time for each passage of the cells was determined. The frozen section was stained with HE. The differentiated phenotype was evaluated by histochemistry or immunohistochemistry. Results When the monolayer culture was performed without any growth factors, the chondrocytes were rapidly proliferated within 3 passages （average doubling time, 59 h）, but at the same time, dedifferentiation was also progressing rapidly. After the 4th passage, most of the cells were dedifferenciated and the proliferation was decreased. With the growth factors （TGF-β1/FGF 2）, the speed of the expansion was accelerated （average doubling time, 47 h）, but the speed of the dedifferentiation was slowed down. After 20 passages were performed with the monolayer culture, the dedifferentiated chondrocytes could be redifferentiated when they were cultured for 3 weeks with RCCS. Then, the Safranine-O staining was strongly positive for the cells, positive for aggrecan and collagen Ⅱ , but negative for collagen Ⅰ , with a well-regained phenotype. Conclusion The bioreactor culturing of the dedifferenciated human articular condrocytes can regain the differentiated phenotype and it is a useful method of obtaining the human articular chondrocytes in large amounts and in a differentiated phenotype in vitro.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2006年第8期840-844,共5页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家高技术研究发展计划(863)资助项目(2002AA205021)~~
