摘要
[目的]观察检测低氧和常氧条件下原代和传代后软骨细胞各特异性基因及蛋白表达的改变。[方法]采用3～5日龄C57BL/6小鼠,取四肢关节软骨,经机械分离和酶消化法获得关节软骨细胞,将原代细胞P0和传代后细胞P1、P2分别在普通和低氧培养箱中培养2d,用RT-PCR检测II型胶原、可聚蛋白聚糖和sox9特异性基因及Ihh、PTHrP、bmp4、wnt5a分化相关基因的表达差异,原代软骨细胞用免疫组化和阿利新蓝染色检测II型胶原、可聚蛋白聚糖的蛋白表达。[结果]低氧条件下,免疫组化显示II型胶原和可聚蛋白聚糖的表达较普通培养增强,II型胶原、wnt5a的基因表达增强,Ihh的表达降低;传代后软骨细胞的蛋白、特异性基因和分化相关基因表达未见明显差异。[结论]低氧条件下,原代软骨细胞的II型胶原表达增强,且可能主要是受Ihh、wnt5a等分化相关基因的调控。短期单层培养方式不能明显改善、恢复特异性基因表达降低的传代后软骨细胞表型。
[Objective] To examine the specific gene and protein expression of primary and passaged chondrocytes in normal oxygen tension and hypoxia.[Methods]Articular chondrocytes were isolated from limb joint cartilage of C57BL/6 mice（35 days）.Primary chondrocytes（P0）and passaled chondrocytes（P1,P2）were cultured in normal oxygen tension and hypoxia respectively for two days.The mRNA levels of collagen II,aggrecan,sox9,Indian hedgehog（ihh）,parathyroid hormone-related protein（PTHrP）,bone morphogenetic protein（BMP）-4 and wnt5a were examined with RT-PCR,and protein levels of collagen II,aggrecan were examined with immunohistochemistry and toluidine blue staining.[Results]During the culture of primary chondrocyte,the expression of collagen II and aggrecan increased under hypoxia,mRNA levels of collagen II,wnt5a increased and ihh decreased at the same time.The mRNA levels of special genes and differentiation related genes of passaged chondrocytes were not altered under hypoxia.[Conclusion]Protein and mRNA level of Collagen II of primary chondrocyte under hypoxia increased.It may be regulated by chondrocyte differentiation gene ihh,and wnt5a.The chondrocyte phenotype could not be resumed under hypoxia through short-term monolayer culture in vitro.
出处
《中国矫形外科杂志》
CAS
CSCD
北大核心
2009年第23期1811-1814,共4页
Orthopedic Journal of China
基金
上海交通大学医学院科技基金项目(编号:05XJ21035)
作者简介
梁静（1979-），女，山东泰安人，硕士，（电话）021—64370045×663338，（电子信箱）jingliang—lj@yahoo．com．cn
