低氧培养对骨膜细胞成软骨分化的影响  

The effect of hypoxia on the differentiation ability of periosteal cells into cartilage

作　　者：张峻玮[1] 陆海涛[1] 杨宇明[1] 袁峰[2] 郭开今[2] 邓斌[2] 

机构地区：[1]江苏省徐州医学院研究生学院,徐州221000 [2]江苏省徐州医学院附属医院脊柱外科,徐州221006

出　　处：《中华实验外科杂志》2016年第5期1281-1283,共3页Chinese Journal of Experimental Surgery

基　　金：江苏省“科教兴卫”医学重点建设人才项目（H201129）

摘　　要：目的 体外分离培养骨膜细胞,探讨低氧对其成软骨分化的影响.方法 采用兔胫骨骨膜为材料分离骨膜细胞,完全培养基培养,倒置显微镜观察细胞形态;成软骨诱导培养基诱导分化,根据培养环境氧浓度分为实验组（低氧组,3％O2）及对照组（常氧组,20％O2）;诱导2周后甲苯胺蓝及Ⅱ型胶原免疫组织化学染色观察细胞Ⅱ型胶原、蛋白聚糖的合成;反转录-聚合酶链反应（RT-PCR）检测成软骨相关基因Ⅱ型胶原α1（Col2α1）、性别决定区Y框蛋白9（Sox9）、蛋白聚糖（Aggrecan）以及低氧诱导因子-1α（HIF-1α）的表达变化.结果 原代培养骨膜细胞生长良好,呈梭形旋涡状或平形状生长;诱导后实验组甲苯胺蓝染色、Ⅱ型胶原免疫组织化学染色均强于对照组;成软骨指标Col2α1（1.00±0.17）、Sox9（0.77±0.13）、Aggrecan（1.12 ±0.10）及HIF-1α（1.45±0.28） mRNA表达水平高于对照组（0.64 ±0.12、0.38±0.01、0.68±0.03、0.89±0.20）,呈一致的上调趋势（P＜0.05）.结论 骨膜细胞体外生长良好,低氧具有明显促进骨膜细胞分化为软骨细胞的作用.Objective Separation and culture of rabbit tibial periosteal cells in vitro and to further explore the effect of hypoxia on the differentiation ability into cartilage.Methods Rabbit tibial periosteum was used to separate the periosteal cells,after which complete medium was used for cell culture and inverted microscope was used for cell observation.Defined medium was applied to make periosteal cells differentiate into cartilage in hypoxia group （3% O2 included） and normoxia group （20% O2 included）.After inducing for 2 weeks,cells were harvest for toluidine blue staining and immunohistochemical staining to detect proteoglycan and collagen type Ⅱ （Col2）.reverse transcriptase-polymerase chain reaction （RT-PCR） was used to determine the mRNA expression of type 2 collagen alpha 1 （Col2α1）,sex determining region Y 9 （Sox9）,aggrecan and hypoxia inducible factor-1α （HIF-1 α）.Results Primary cultured periosteal cells were in good condition with spindle spiral or parallel growth form.Toluidine blue stain and immunohistochemical stain of defined medium induced periosteal cells were stronger than control.The mRNA level of Col2α1 （1.00 ± 0.17）,Sox9 （0.77 ± 0.13）,aggrecan （1.12 ± 0.10） and HIF-1 α （1.45 ± 0.28） that indicated tbe cells ability of differentiating into cartilage were upregulated in induced periosteal cells than control （0.64 ± 0.12,0.38 ± 0.01,0.68 ± 0.03,0.89 ± 0.20,P 〈 0.05）.Conclusion The in vitro reproductive activity of rabbit periosteal cells were in good condition.Hypoxia could significantly promote periosteal cells to differentiate into cartilage cells.

关 键 词：低氧 骨膜细胞 成软骨分化 低氧诱导因子-1Α 

分 类 号：R684[医药卫生—骨科学]