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

doi:10.7518/hxkq.2017.04.004

摘要/Abstract

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

中图分类号:

何晓兰, 包广洁, 孙凌璐, 张雪, 保善英, 康宏. 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[J]. 华西口腔医学杂志, 2017, 35(4): 362-367.

Xiao-lan He, Guangjie Bao, Linglu Sun, Xue Zhang, Shanying Bao, Hong Kang. Effect of different oxygen tension on the cytoskeleton remodeling of goat temporomandibular joint disc cells[J]. West China Journal of Stomatology, 2017, 35(4): 362-367.

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目的基因	5’—3’	熔解温度/℃	产物长度/bp
GAPDH	F：CAAGTTCCACGGCACAGTCA	59.9	248
	R：GGTTCACGCCCATCACAAA	59.6
肌动蛋白	F：ATCAAGATTATCGCTCCTCCC	57.7	164
	R：TGCTCGCAGTCCGTTTAGA	57.5
微管蛋白	F：CCCAGGGCAGTGTTTGTAGA	58.3	134
	R：TGACCTCGGGCATAGTTGTT	57.9
波形蛋白	F：ATGACCGCTTCGCCAACTA	59.0	136
	R：TCTCGCATCTCCTCCTCGTA	58.1

目标基因	氧体积分数/%				F值	P值
目标基因	21	8	4	2	F值	P值
肌动蛋白	1	0.510±0.095^ac	0.427±0.402^ab	0.418±0.191^a	7.925	0.009
微管蛋白	1	0.746±0.069^ad	0.980±0.164^d	1.247±0.070^abc	39.600	0.000
波形蛋白	1	0.635±0.099^ac	0.567±0.157^abd	0.625±0.025^ac	19.362	0.010