摘要
目的建立大鼠终板软骨细胞体外自然传代的退变模型，探讨自然退变过程中内源性转化生长因子（TGF）-β1与钙化基因表达变化的关系。方法取大鼠腰椎终板软骨细胞，酶消化法及自然传代法分离培养大鼠终板软骨细胞，选取P2代和P4代，在体外均培养6d，观察细胞表型及利用茜素红染色，观察P2与P4代细胞的变化。用实时PCR检测软骨标志基因Ⅱ型胶原、转录因子SOX-9及代谢相关基因蛋白多糖、基质金属蛋白酶（MMP）-13、含I型血小板结合蛋白基序的解聚蛋白样金属蛋白酶（ADAMTS）4、5的变化，验证体外退变模型的建立，在此基础上，继续检测生长因子TGF．131及钙化相关基因（ANK）、胞外核苷酸焦磷酸酶（ENPP）、组织非特异性碱性磷酸酶（TNAP）的变化。结果相对于P2代细胞，P4代细胞形态上有梭形变趋势，茜素红染色无明显变化。P4代转录因子SOX-9表达明显低于P2代（P4／P2=0．0690，P=0．0489），Ⅱ型胶原（P4／P2=0．0535，P=0．009）及蛋白多糖（P4／P2=0．2672，P=0．0343）表达也均明显低于P2代，其他代谢相关基因无明显改变。P4代细胞TGF-131（P4／P2=0．5934，P=0．0482）、TNAP（P4／P2=0．0385，P=0．0139）和ANK（P4／P2=0．2121，P=0．0009）表达均明显低于P2代，ENPP表达无明显改变（P〉0．05）。结论终板软骨随着传代次数增多，从P2代到P4代，开始发生体外自然退变，Ⅱ型胶原、SOX-9及蛋白多糖明显下调。内源性TGF-β1与钙化相关基因表达均下调，钙化相关基因ANK下调可能由内源性TGF-β1下调引起，提示调节内源性TGF-β1在终板软骨中的表达有可能会阻止椎间盘的退变。
Objective To explore the relationship between endogenous transforming growth factor （TGF） -β1 and calcification-related genes through an in vitro degeneration model by propagating rat endplate chondrocytes during a natural degeneration process. Methods Endplate chondrocytes were extracted from rat lumbar vertebrae, isolated by enzyme digestion and P2 and P4 generations selected for a 6-day in vitro culture. The specimens were photographed microscopically to observe the cellular differences by alizarin red staining. Type lI collagen marker gene, transcription factor SOX-9 gene and metabolism-related genes proteoglycan, matrix metalloproteinase （MMP）-13, a disintegrin and metalloproteinase with thrombospondin motifs （ADAMTS）-4 and ADAMTS-5 were detected by RT-PCR to verify the degeneration model. Based on this model, the changes of growth factor TGF-β1 and calcification-related genes ankyrin （ANK）, ectonucleotidepyrophosphatase （ENPP）, tissue-nonspecific alkaline phosphatase （TNAP） were continuously tested. Results Compared with P2 cells, P4 cells tended to assume a spindle-shaped morphology. And alizarin red staining showed no change between them. The level of transcription factor SOX-9 of P4 cells（ P4/P2 = 0. 0690, P = 0. 0489） was significantly lower than that of P2 ceils. Type Ⅱ collagen （ P4/P2 = 0. 0535, P = 0. 009 ） and proteoglycan（ P4/P2 = 0. 2672, P = 0. 0343 ）were also siguificantly lower than those of P2 cells. No significant changes were observed in other metabolism-related genes. TGF-β1 （ P4/P2 = 0. 5934, P = 0. 0482） was significantly lower. The expressions of TNAP （ P4/P2 = 0. 0385, P = 0. 0139 ） and ANK （P4/P2 = 0. 2121, P = 0. 0009） were significantly lower. But ENPP showed no significant change. Conclusion P4 endplate chondrocytes undergo natural degeneration in vitro with the rising passage number. Type Ⅱ collagen, SOX-9 and proteoglycan are significantly reduced. Endogenous TGF-β1 gene and calcificationrelated genes are down-regulated. The decrease of ANK gene may be caused by the down-regulation of endogenous TGF-β1. Modulating the expression of endogenous TGF-β1 gene in endplate chondrocytes may become a new therapeutic approach for the degeneration of intervertebral disc.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2011年第31期2181-2185,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）
关键词
软骨细胞
转化生长因子Β
锚蛋白类
Chondrocytes
Transforming growth factor beta
Ankyrins
