华西口腔医学杂志 ›› 2017, Vol. 35 ›› Issue (4): 362-367.doi: 10.7518/hxkq.2017.04.004

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不同体积分数氧气对山羊颞下颌关节盘细胞骨架改建的影响

何晓兰1(), 包广洁1,2, 孙凌璐1, 张雪1, 保善英1, 康宏1()   

  1. 1.兰州大学口腔医学研究所,兰州 730000
    2.西北民族大学甘肃省口腔疾病研究重点实验室,西北民族大学口腔医学国家民委重点实验室,兰州 730030
  • 收稿日期:2017-01-11 修回日期:2017-04-01 出版日期:2017-08-01 发布日期:2017-08-01
  • 作者简介:

    何晓兰,硕士,E-mail:hexl2014@lzu.edu.cn

  • 基金资助:
    国家自然科学基金(81160139,81660189)

Effect of different oxygen tension on the cytoskeleton remodeling of goat temporomandibular joint disc cells

Xiao-lan He1(), Guangjie Bao1,2, Linglu Sun1, Xue Zhang1, Shanying Bao1, Hong Kang1()   

  1. 1. Institute of Stomatology, Lanzhou University, Lanzhou 730000, China
    2. Key Laboratory of Oral Diseases of Gansu Provincial, Key Laboratory of Stomatology of State Ethnic Affairs Commission, Northwest Minzu University, Lanzhou 730030, China
  • Received:2017-01-11 Revised:2017-04-01 Online:2017-08-01 Published:2017-08-01
  • Supported by:
    Natural Nation Science Foundation of China (81160139, 81660189)

摘要:

目的 探讨不同体积分数的氧气对山羊颞下颌关节盘细胞3种细胞骨架蛋白改建的影响。方法 体外分离、培养山羊颞下颌关节盘细胞,传代至第2代,分别于常氧21%O2及低氧8%O2、4%O2、2%O2下培养。甲苯胺蓝、天狼星红及Ⅰ型胶原免疫细胞化学染色观察不同氧体积分数下细胞显型的变化。细胞免疫荧光染色和实时定量逆转录聚合酶链反应检测细胞骨架蛋白,包括肌动蛋白、微管蛋白和波形蛋白的表达情况。结果 不同氧体积分数下关节盘细胞仍然具有成纤维特性,3种细胞骨架蛋白有规律地排列。4%O2时,肌动蛋白和波形蛋白荧光强度最低(P<0.05);2%O2时,微管蛋白荧光强度最高(P<0.05),其他各组间差异无统计学意义(P>0.05)。检测肌动蛋白mRNA表达,21%O2组最高,而2%O2组和4%O2组最低(P<0.05);微管蛋白mRNA表达在2%O2组最高,8%O2组最低(P<0.05);波形蛋白mRNA表达,在4%O2组最低,21%O2组最高,差异有统计学意义(P<0.05)。结论 在不同氧体积分数条件下,细胞骨架蛋白有不同程度的改建,2%O2可能是适合颞下颌关节盘细胞扩增的最佳氧体积分数。

关键词: 颞下颌关节盘, 低氧, 细胞骨架, 肌动蛋白, 微管蛋白, 波形蛋白

Abstract:

Objective The effect of different oxygen tensions on the cytoskeleton remodeling of goat temporomandibular joint (TMJ) disc cells were investigated. Methods Goat TMJ disc cells were cultured under normoxia (21% O2) and hypoxia (2%, 4%, and 8% O2). Toluidine blue, picrosirius red, and type Ⅰ collagen immunocytochemical staining were performed to observe the changes in cell phenotype under different oxygen levels. Immunofluorescent staining and real-time reverse trans-cription-polymerase chain reaction analysis were then performed to identify actin, tubulin, and vimentin in the cultured disc cells. Results TMJ disc cells still displayed fibroblast characteristics under different oxygen levels and their cytoskeletons had regular arrangement. The fluorescence intensities of actin and vimentin were lowest at 4% O2 (P<0.05), whereas that of tubulin was highest at 2% O2 (P<0.05). No significant difference among the other groups was observed (P>0.05). Actin mRNA levels were considerably decreased at 2% O2 and 4% O2 in hypoxic conditions, while actin mRNA expression was highest in 21% O2. Tubulin mRNA levels considerably increased at 2% O2, while tubulin mRNA expression was lowest in 8% O2 (P<0.05). Vimentin mRNA expression was lowest at 4% O2 and highest at 21% O2, and significant differences were observed between vimentin mRNA expression levels among these oxygen levels (P<0.05). Conclusion Cytoskeletons were recon-structed in different oxygen tensions, and 2% O2 may be the optimal oxygen level required to proliferate TMJ disc cells.

Key words: temporomandibular joint disc, hypoxia, cytoskeleton, actin, tubulin, vimentin

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