摘要
目的为韧带细胞的病理、生理及组织工程学等研究提供方法及体外模型。方法采用胶原酶分离新西兰白兔的膝关节前十字韧带(anteriorcruciateligament,ACL)、内侧副韧带(medialcol-lateralligament,MCL)及人膝关节ACL和后十字韧带(posteriorcruciateligament,PCL)细胞,选择合适的条件进行体外培养,分析细胞的生物学特性。结果在含体积分数为10%胎牛血清的DMEM培养液中细胞生长良好、增殖快,但RPMI1640及F12培养液不适宜这些细胞的生长。在体外培养条件下兔ACL和MCL细胞形态相似,但MCL较ACL生长快,两种细胞合成胶原的总量相同。相对MCL细胞,ACL合成的Ⅰ型胶原较多而Ⅲ型胶原较少。人ACL和PCL细胞形态及生长特性基本相似,免疫细胞学方法检测表明两种细胞均有Ⅰ、Ⅲ型胶原及纤维连接蛋白表达。结论体外培养的人ACL和PCL细胞生物学特性相似。兔ACL和MCL细胞形态相似,但生物学特性存在差异。本实验为韧带成纤维细胞的培养及相关研究提供了简单、有效的方法。
Objective Anterior cruciate ligament(ACL) , posterior cruciate ligament(PCL) and medial collateral ligament(MCL) are the important anatomical structures for the stability of the knee joint, the injury of the these ligaments can result in instability, functional loss and secondary osteoarthritis of the knee joint. The injured ACL and PCL could not get a satisfactory process of self healing, or a high healing rate after end to end suturing. Although, allografting or autografting is a common method to repair the injured ACL and PCL, the clinical result is not satisfactory. The purpose of the present is aimed to establish fibroblast cell lines from the knee ligaments and study the biological characters of these cell lines, in order to contribute to physiologic, pathological and tissue engineering research for ligament cell, search for an ideal material of reparation for the injured ligament. Methods ACL and MCL tissues of knee joint were harvested from New Zealand white rabbit in aseptic condition, and ACL and PCL tissues were obtained from the human knee joint during the operation of total knee replacement because of knee injury as well. The ligament tissues were cleaned off and cut into a pieces of 1 mm3, and were cultured in DMEM medium in vitro. Culture conditions were optimized, three kinds of mediums, 10% fetal bovine serum, RPMI 1640 and Hams F12 medium, were used, in order to observe the effects of different medium on the growth of fibroblast cell lines. The cellular growth was observed in T-25 culture bottle, and the morphology of fibroblast cell lines were examined under contrast phase microscope. The synthesis of type Ⅰ collagen, type Ⅲ collagen and total collagen were measured according to Sykes method. Furthermore, the expression of type Ⅰ collagen, type Ⅲ collagen and fibronectin in fibroblast cell lines were observed with immunocytochemstry. Results The cells were released from the ligament matrix with 1 to 2 hours process of collagenase at 37 ℃, the survival rate of the cells was 95% demonstrated by the staining with trypan blue. The cell appearance was similar to the classical fibroblast. The Maximal growth for all these cells were obtained with Dulbeccos modified Eagle s medium supplemented with 10% fetal bovine serum, but RPMI 1640 and Hams F12 medium were not favorable for maintaining these cells. No significant difference in morphology and cellular growth was noticed between ACL cells and PCL cells from human in the culture condition. Expressions of type Ⅰ collagen, type Ⅲ collagen and fibronectin were shown on both cell types of ACL and PCL from human proven by immunocytochemstry. Morphology of both ACL and MCL cells from New Zealand white rabbit was also alike in vitro, but MCL cells grew faster than ACL cells. Both cell types produced similar amount of collagen in culture, but the ratio of collage typeⅠ to type Ⅲ produced by ACL cells was higher than MCL cells. Conclusion ACL and PCL cells from human shows no significant difference in morphology and growth in vitro, both cell types produces fibronectin, collagen type Ⅰ and type Ⅲ. Despite the ACL cells and the MCL cells from New Zealand white rabbit show similar appearance in morphology in culture, the cellular growth and the biochemical synthesis of collagen were significantly different. This cell culture system provides a simple method for maintain fibroblasts of ligaments in vitro.
出处
《中华骨科杂志》
CAS
CSCD
北大核心
2002年第1期40-44,共5页
Chinese Journal of Orthopaedics
基金
广东省科委资助项目
关键词
膝关节
韧带
成纤维细胞
生物学特性
细胞培养
Cell culture
Anterior cruciate ligament
Medial collateral ligament,knee
Posterior cruciate ligament
Fibroblasts
