中国组织工程研究

中国组织工程研究 ›› 2015, Vol. 19 ›› Issue (40): 6408-6412.doi: 10.3969/j.issn.2095-4344.2015.40.003

• 器官移植动物模型 organ transplantation and animal model • 上一篇    下一篇

构建瘤细胞模型：NS-398对低氧状态下MG-63细胞侵袭能力的影响

王一凡1，许永涛2     

  1. 1长江大学医学院，湖北省荆州市  434020； 2湖北省荆州市中心医院骨三科，湖北省荆州市  434020
  • 出版日期:2015-09-30 发布日期:2015-09-30
  • 通讯作者: 许永涛。湖北省荆州市中心医院骨三科，湖北省荆州市 434020
  • 作者简介:王一凡，长江大学医学院，湖北省荆州市 434020

Establishment of osteosarcoma cell models: effects of NS-398 on invasive ability of MG-63 cells under hypoxic condition 

Wang Yi-fan1, Xu Yong-tao2   

  1. 1Medical School of Yangtze University, Jingzhou 434020, Hubei Province, China; 2Third Department of Orthopedics, Jingzhou Central Hospital, Jingzhou 434020, Hubei Province, China
  • Online:2015-09-30 Published:2015-09-30
  • Contact: Xu Yong-tao, Third Department of Orthopedics, Jingzhou Central Hospital, Jingzhou 434020, Hubei Province, China
  • About author:Wang Yi-fan, Medical School of Yangtze University, Jingzhou 434020, Hubei Province, China

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摘要：

背景：研究表明缺氧环境中，肿瘤细胞的侵袭能力显著增加，这种侵袭能力的加强可能与包括尿激酶或基质金属蛋白酶的变化有关。低氧与肿瘤治疗尤其是对化疗影响的研究有了很大的进展，发现低氧诱导因子1是介导细胞低氧反应最关键的核转录因子，也是低氧时调控细胞凋亡的重要因素。

目的：观察低氧骨肉瘤细胞模型中环氧化酶2抑制剂NS-398对低氧状态下MG-63细胞侵袭能力的影响。

方法：实验应用化学性缺氧诱导剂氯化钴建立浓度梯度为0，100，200，400 μmol/L化学性缺氧环境，再在200 μmol/L氯化钴的浓度下建立NS-398 30，60，90 μmol/L的浓度梯度，将骨肉瘤细胞培养48 h时开始测定，使用定量PCR法检测基质金属蛋白酶9 mRNA的表达和低氧诱导因子1α mRNA的水平，Western blot测定低氧诱导因子1α的表达，流式细胞术检测各组细胞凋亡。

结果与结论：PCR和Western blot检测结果显示，随着氯化钴浓度的增加，基质金属蛋白酶9 mRNA，低氧诱导因子1α mRNA及蛋白表达升高(P < 0.05)，呈浓度依赖性；而在氯化钴浓度为200 μmol/L缺氧状态下，随着NS-398浓度的增加，基质金属蛋白酶9 mRNA，低氧诱导因子1α mRNA及蛋白表达均逐渐减小，呈浓度依赖性(P < 0.05)；流式细胞仪检测结果显示，在400 μmol/L氯化钴浓度下，细胞凋亡水平明显减少；在氯化钴浓度为200 μmol/L缺氧状态下，随着NS-398的浓度上升细胞凋亡率增加(P < 0.05)。结果证实，缺氧环境是骨肉瘤侵袭能力增加的一个重要因素，在缺氧环境下NS-398可有效抑制骨肉瘤的侵袭能力并促进细胞凋亡。

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

关键词: 实验动物, 骨及关节损伤动物模型, 缺氧, 骨肉瘤, 基质金属蛋白酶9, 低氧诱导因子1α, 细胞凋亡, 氯化钴, 侵袭

Abstract:

BACKGROUND: In anoxic environment, invasive ability of tumor cells significantly increased. The increase in above invasive ability is possibly associated with the changes in urokinase or matrix metalloproteinases. Studies on hypoxia and tumor therapy especially the effect on chemotherapy have made great progress, and found that hypoxia-inducible factor 1 is a key nuclear factor mediating cellular hypoxia response, and also an important factor for regulating cell apoptosis during hypoxia.

OBJECTIVE: To observe the effects of cyclooxygenase-2 inhibitor NS-398 on invasive ability of MG-63 cells under hypoxic condition in cell models of hypoxia osteosarcoma.

METHODS: Chemical hypoxia inducer cobalt chloride was used to establish chemical hypoxia environment of concentration gradient of 0, 100, 200, 400 μmol/L. Under 200 μmol/L cobalt chloride, concentration gradient of 30, 60, 90 μmol/L of NS-398 were set. Osteosarcoma cells were determined after 48 hours of culture. Quantitative PCR was used to detect the expression of matrix metalloproteinases 9 mRNA and hypoxia-inducible factor 1α mRNA. Western blot assay was applied to measure hypoxia-inducible factor 1α expression. Flow cytometry was utilized to examine cell apoptosis in each group.

RESULTS AND CONCLUSION: PCR and western blot assay results demonstrated that with increased concentration of cobalt chloride, matrix metalloproteinases 9 mRNA and hypoxia-inducible factor 1α mRNA and protein expression increased (P < 0.05), showing a concentration-dependent manner. Under cobalt chloride concentration of 200 μmol/L and hypoxia, with increased concentration of NS-398, matrix metalloproteinases 9 mRNA and hypoxia-inducible factor 1α mRNA and protein expression gradually reduced, showing a concentration-dependent manner (P < 0.05). Flow cytometry results showed that under 400 μmol/L cobalt chloride concentration, cell apoptosis noticeably reduced. Under cobalt chloride concentration of 200 μmol/L and hypoxia, with increased concentration of NS-398, cell apoptosis increased (P < 0.05). Results confirm that anaerobic environment is an important factor for increased invasive ability of osteosarcoma. Under the anaerobic environment, NS-398 could effectively suppress the invasive ability of osteosarcoma and promote cell apoptosis.

Key words: Tissue Engineering, Anoxia, Osteosarcoma, Apoptosis

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