氧环境对成骨细胞活性的影响

Viability of osteoblasts under cell hypoxia condition

目的对比观察缺氧及缺氧再复氧环境下新生大鼠成骨细胞的细胞活性、凋亡率及基因表达的变化。方法取48 h内新生SD大鼠的颅骨，组织块法培养和纯化成骨细胞，采用茜素红染色、碱性磷酸酶染色行细胞形态学鉴定。以正常培养成骨细胞36 h为对照组（A组）；取第3代成骨细胞，分别于缺氧24 h（B组）、缺氧36 h（C组）、缺氧24 h再复氧12 h（D组）培养后，采用MTT法检测细胞活性、流式细胞技术检测细胞凋亡率；采用实时定量聚合酶链反应和免疫印迹技术检测下游基因胶原（COL）Ⅰ、骨形态发生蛋白（BMP）-2、核心结合因子（RUNX）-2、转化生长因子（TGF）β1 mRNA表达和相关蛋白的变化。结果缺氧及缺氧复氧环境下，成骨细胞活性降低，A组为（99.1%±8.3%），B组为（90.9%±9.4%），C组（79.9%±8.7%），D组为（73.0%±8.2%），D组〈C组〈B组〈A组（F=37.886，P=0.000）；细胞凋亡率增加，A组为（1.9%±1.3%），B组为（16.3%±2.5%），C组（28.2%±4.2%），D组为（33.5%±3.6%），D组〉C组〉B组〉A组（F=26.198，P=0.000）；BMP-2、RUNX-2、Collagen type Ⅰ、TGF-β1 mRNA表达量降低，且D组〈C组〈B组〈A组（F=13.082, P=0.006; F=7.088, P=0.017; F=6.857, P=0.038; F=51.368, P=0.000）；BMP-2、RUNX-2、Collagen type Ⅰ、TGF-β1蛋白表达量降低，且D组〈C组〈B组〈A组（F=8.114, P=0.013; F=28.935, P=0.000; F=9.857, P=0.007; F=46.541, P=0.000）。结论缺氧及缺氧再复氧环境会降低成骨细胞活性，增加细胞凋亡率，下调相关基因的表达。缺氧再复氧环境会加重缺氧状态下成骨细胞损伤。

ObjectiveTo investigate the effects of hypoxia condition and hypoxia-reoxygenation condition on the cell viability, apoptosis rate and gene expression of osteoblasts cultured in vitro.MethodsThe cranium osteoblasts from newborn Sprague Dawley rats within 48 hours were cultured and purified through tissue block method.The morphological changes of cells were evaluated by the Alizarin Red S staining and Alkaline phosphatase staining.The third-generation osteoblasts were cultured in normal condition for 36 hours （group A）, in hypoxic condition for 24hours （group B）, in hypoxic condition for 24hours thereafter reoxygenated for 12 hours （group D）, in hypoxic condition for 36 hours （group C）. The cell viability of osteoblasts was tested via MTT assay.The apoptosis rate of osteoblasts was tested by FCM （flow cytometry）. Quantitative PCR and Western blot methods were used to determine Collagen type Ⅰ, Bone morphogenetic protein 2 （BMP-2）, Runt-related transcription factor 2 （RUNX-2）, Transforming growth factor-β1（TGF-β1） expression levels.ResultsThe cell viability of osteoblasts decreased, group A（99.1%±8.3%）, group B（90.9%±9.4%）, group C（79.9%±8.7%）, group D（73.0%±8.2%）, group D 〈group C 〈group B 〈group A （F=37.886, P=0.000）; the apoptosis rate of osteoblasts increased, group A（1.9%±1.3%）, group B（16.3%±2.5%）, group C（28.2%±4.2%）, group D（33.5%±3.6%）, group D 〉group C 〉group B 〉group A（F=26.198, P=0.000）; the mRNA expressions of Col Ⅰ, BMP-2, RUNX-2, TGF-β1 decreased under hypoxia and hypoxia-reoxygenation condition, and group D〈 group C〈 group B〈 group A （F=13.082, P=0.006; F=7.088, P=0.017; F=6.857, P=0.038; F=51.368, P=0.000）; the protein expressions of Col Ⅰ, BMP-2, RUNX-2, TGF-β1 decreased under hypoxia and hypoxia-reoxygenation condition, and group D〈 group C〈 group B〈 group A （F=8.114, P=0.013; F=28.935, P=0.000; F=9.857, P=0.007; F=46.541, P=0.000）.ConclusionHypoxia condition and hypoxia-reoxygenation condition decreased the cell viability, increase the apoptosis rate and suppress the expressions of associated genes.Hypoxia-reoxygenation condition aggravate the injure of osteoblasts preconditioning under hypoxia condition.