摘要
背景:椎间盘退变是个慢性、复杂的过程,然而椎间盘退变其发生机制尚未完全阐明,很难自行修复。近年来研究细胞移植治疗椎间盘退行性变尚处在实验室阶段。研究髓核细胞的生物学性状可为研究椎间盘退变机制、组织工程构建椎间盘、基因治疗等提供理论依据。目的:研究兔不同代次髓核细胞的生物学特性,旨在找出合适的种子细胞去治疗椎间盘退变性疾病。方法:从新西兰大耳白兔椎间盘髓核组织中,分离并培养髓核细胞同时进行培养传代,对原代及第3,4代髓核细胞进行苏木精-伊红染色观察细胞形态学变化;甲苯胺蓝染色和免疫细胞化学法检测髓核细胞内聚集蛋白聚糖和Ⅱ型胶原的表达;反转录PCR法测定Ⅱ型胶原和聚合蛋白聚糖mRNA的表达水平,观察各代髓核细胞生物学特性的变化。结果与结论:兔椎间盘髓核细胞可以在体外培养并进行传代,原代髓核细胞一般需7d左右贴壁,形状呈类圆形或多角形,原代和第3代髓核细胞都呈圆形或多角形,活力较强,苏木精-伊红染色后细胞核被染成均一蓝黑色,胞浆呈现淡粉色;髓核细胞经过甲苯胺蓝染色后,胞浆内呈现天蓝色,通过Ⅱ型胶原免疫组织化学染色后,胞浆内表现为黄褐色沉淀。到第4代细胞出现退变,Ⅱ型胶原和聚合蛋白聚糖mRNA的表达水平较前几代细胞显著下降。前3代的髓核细胞代谢旺盛,表型一致,聚集蛋白聚糖和Ⅱ型胶原表达正常,传第4代后髓核细胞开始出现衰老、退变。
BACKGROUND： Intervertebral disc degeneration is a chronic and complicate procedure, but its mechanism is still unclear and difficult for self-repairs. Recent studies addressing stem cell transplantationin the treatment of disc degeneration disease are still limited to the laboratory study. Research on biological properties of nucleus pulposus cells can provide theoretical basis for studying the mechanism underlying intervertebral disc degeneration, construction of the intervertebral disc by tissue engineering, and gene therapy. OBJECTIVE： To investigate the characteristics of different generations of rabbit nucleus pulposus cells, searching for the best suitable seed cells to treat degenerative disc diseases. METHODS： Nucleus pulposus cells from New Zealand white rabbits were separated, cultured and then passaged. The morphological changes of primary, passages 3 and 4 nucleus pulposus cells were observed by hematoxylin-eosin staining under an inverted microscope. The biological properties of rabbit nucleus pulposus cells were observed. Aggrecan and type [I collagen expressions were detected by toluidine blue and immunocytochemistry staining, respectively. Type II collagen and glycosaminoglycan mRNA expressions in the nucleus pulposus cells were detected by reverse transcription-PCR. RESULTS AND CONCLUSION： Rabbit nucleus pulposus cells were successfully cultured and passaged in vitro Primary nucleus pulposus cells were round or polygonal, and the average adherence time was 7 days. The first and third generations of nucleus pulposus cells were round or polygonal, and have strong vitality. Hematoxylin-eosin staining showed that nuclei were in a uniform blue-black, and cytoplasm showed light pink. The cytoplasm of nucleus pulposus cells was sky blue stained for toluidine blue staining, and type II collagen immunohistochemical staining showed the cytoplasm of nucleus pulposus cells displayed yellowish-brown. Passage 4 nucleus pulposus cells appeared with degeneration, and typeⅡcollagen and glycosaminoglycan
出处
《中国组织工程研究》
CAS
CSCD
2013年第11期1901-1908,共8页
Chinese Journal of Tissue Engineering Research
基金
内蒙古自治区自然科学基金项目资助(2009MS1117)~~
关键词
组织构建
软骨组织构建
兔
髓核细胞
体外培养
聚集蛋白聚糖
Ⅱ型胶原
椎间盘退变
细胞培养
省级基金
组织构建图片文章
tissue construction
cartilage tissue construction
rabbits
nucleus pulposus cells
in vitro cultureaggrecan
type Ⅱ collagen
intervertebral disc degeneration
cell culture
provincial grants-supported paper
tissue construction photographs-containing paper
