摘要
目的建立人颈椎椎体终板软骨细胞退变模型，观察人正常颈椎椎体和退变颈椎椎体终板软骨细胞的形态及表征。方法选择2010年7月至2011年7月49例颈椎骨折、脱位（19例）及颈椎病（30例）患者术中取出的颈椎终板软骨，用酶消化法分别分离培养人正常颈椎椎体终板软骨细胞（对照组）和退变颈椎椎体终板软骨细胞（颈椎病组）；用倒置显微镜和HE染色法观察细胞形态学变化；四甲基偶氮唑蓝（MTT）法绘制细胞生长曲线；甲苯蓝染色及反转录-PCR（RT—PCR）法对终板软骨细胞进行鉴定；RT—PCR法检测终板软骨细胞特征性基因蛋白多糖、Ⅱ型胶原及Ⅰ型胶原的表达。结果人颈椎椎体终板软骨细胞表达特征性蛋白多糖、Ⅱ型胶原及Ⅰ型胶原，其生长情况及细胞表型类似于关节软骨细胞。对照组原代终板软骨细胞以多角形为主，增殖速度较快；而颈椎病组原代终板软骨细胞以梭形为主，细胞增殖速度较慢。颈椎病组原代终板软骨细胞表达的蛋白多糖基因（0．695±0．052）和Ⅱ型胶原基因（0．726±0．035）均低于对照组（0．950±0．032、0．907±0．078，t=7．263、3．681，P=0．002、0．021），Ⅰ型胶原基因则高于对照组（0．795±0．028比0．552±0．070，t=-5．560，P=0．005）。结论成功建立了人颈椎椎体终板软骨细胞退变模型，为椎间盘退变机制研究提供了较好的细胞学基础，解决了以前一直以动物细胞模型为研究对象的局限性。
Objective To establish an in vitro model of degeneration of human cervical endplate chondrocytes and observe the morphology and phenotypes of endplate cbondrocytes in normal and degenerative cervical vertebral endplates. Methods Cartilage endplates of 49 patients were divided into control group （n = 19） with cervical vertebral fracture or dislocation and experiment group （n = 30） with cervical spondylotic myelopathy. Endplate chondrocytes were isolated by enzyme digestion and cultured in vitro. The morphological appearances, growth curve and biological characteristics of endplate chondrocytes from normal and degenerative cartilage endplate were observed by inverted phase contrast microscope, HE staining, MTT, teluidine blue staining and reverse transcription-polymerase chain reaction （RT-PCR） respectively. RT-PCR was used to detect the mRNA expression of aggrecan, type Ⅱ collagen and type Ⅰ collagen. Results The endplate chondrocytes expressed aggrecan, type Ⅱ collagen and type Ⅰ collagen. The phenotypes and biological characteristics were similar to those of articular chondrocytes. The morphological appearance of primary endplate chondrocytes in the control group were mostly polygons, nucelus with round or ellipse, sometimes nuclei, vacuolus in intracytoplasm, expressing a high proliferating rate. The cells of the experiment group were fusiform and their proliferating rates decreased. Compared with the control group, the mRNA expression of aggrecan （0. 695 ± 0. 052 vs 0. 950 ±0. 032, t = 7.263, P = 0. 002） and type Ⅱ collagen （0. 726 ± 0. 035, 0. 907 ±0. 078, t = 3. 681, P = 0. 021 ） markedly decreased. And the mRNA expression of type Ⅰ collagen （0. 795 ±0. 028 vs 0. 552 ±0. 070, t = -5. 560, P =0. 005） increased in the experiment group. Conclusion A degenerative cell model of human cervical endplate chondrocytes has been established successfully in vitro. It may offer the cytological rationales for exploring the mechanism of intervertebral disc degeneration. And the previous restrictions of studying only the model of animal cells shall be resolved.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2011年第41期2912-2916,共5页
National Medical Journal of China
基金
国家自然科学基金（30973025）
安徽省教育厅自然科学基金（KJ2010A320）志谢弋矶山医院人才基金（YR2009013）
