静压力下缺氧诱导因子-1α信号通路对髁突软骨细胞增殖与凋亡的调控作用

doi:10.3877/cma.j.issn.2095-8757.2021.03.006

目的

探讨不同时间静压力下缺氧诱导因子-1α（hypoxia-inducible factor-1α, HIF-1α）信号通路对髁突软骨细胞增殖与凋亡的调控作用。

方法

体外分离、培养髁突软骨细胞，取第三代进行实验。采用200 kPa静压力对髁突软骨细胞进行0、1、2、4、8、12 h的加压，在不同加压时点获取细胞样本后用CCK-8法检测细胞增殖情况，流式细胞技术检测细胞凋亡情况，Western Blot及qRT-PCR检测髁突软骨细胞HIF-1α、血管内皮生长因子（vascular endothelial growth factor, VEGF）及Ⅱ型胶原蛋白（collagen type Ⅱ, COLⅡ）蛋白和mRNA的表达情况。不同时点计量资料的比较采用单因素方差分析。

结果

随着加压时间的延长，髁突软骨细胞的增殖和凋亡总体均呈上升趋势，其中各时点增殖的差异有统计学意义（F=5.336，P<0.01），加压8、12 h后髁突软骨细胞的增殖均明显强于加压前（P<0.05或0.01），各时点凋亡的差异无统计学意义（F=12.175，P>0.05）。随着加压时间的延长，髁突软骨细胞HIF-1α、VEGF、COLⅡ蛋白和mRNA的表达均呈先上升后下降趋势，不同时点表达水平的差异均有统计学意义（F=21.330、41.710、12.960，15.880、85.390、35.210；P<0.01）；加压后各时点HIF-1α、VEGF、COLⅡ蛋白的表达较之加压前均发生明显变化（P<0.05或0.01），大部分时点HIF-1α、VEGF、COLⅡ mRNA的表达较之加压前均发生明显变化（P<0.05或0.01），其中加压4 h后HIF-1α、VEGF、COLⅡ蛋白和mRNA的表达均达到最高点。

结论

适宜大小的力学刺激能促进髁突软骨细胞的增殖及细胞外基质合成，HIF-1α信号通路可能参与了髁突软骨细胞力学信号的传递。

关键词: 颞下颌关节, 髁突软骨细胞, 骨关节病, 缺氧诱导因子1α, 血管内皮生长因子, Ⅱ型胶原蛋白

Objective

To investigate the regulation of HIF-1α signaling pathway on proliferation and apoptosis of condylar chondrocytes under different time static pressure.

Methods

Condylar chondrocytes were isolated and cultured in vitro, and the third generation was used for experiment. Condylar chondrocytes were pressurized at 0, 1, 2, 4, 8 and 12 h under static pressure of 200 kPa. Cell samples were obtained at different pressurization time points and cell proliferation was detected by CCK-8 method and cell apoptosis was detected by flow cytometry. The expressions of HIF-1α, VEGF and COLⅡ protein and mRNA in condylar chondrocytes were detected by Western Blot and QRT-PCR. The measurement data at different time points were compared by one-way ANOVA.

Results

With the extension of pressurization time, the proliferation and apoptosis of condylar chondrocytes generally increased, and the difference of proliferation at each time point was statistically significant (F=5.336, P < 0.01). The proliferation of condylar chondrocytes was significantly enhanced after 8 and 12 h of compression (P < 0.05 or 0.01). The expressions of HIF-1α, VEGF and COLⅡ protein and mRNA in condylar chondrocytes increased firstly and then decreased with increasing pressure time, and the differences of expression levels at different time points were statistically significant (F=21.330, 41.710, 12.960, 15.880, 85.390, 35.210; P < 0.01). The expressions of HIF-1α, VEGF and COLⅡ protein were significantly changed at each time point after compression (P < 0.05 or 0.01), and the expressions of HIF-1α, VEGF and COLⅡ mRNA were significantly changed at most of the time points after compression (P < 0.05 or 0.01). The protein and mRNA expressions of HIF-1α, VEGF and COLⅡ reached the highest after 4 h of compression.

Conclusion

Appropriate mechanical stimulation can promote the proliferation and extracellular matrix synthesis of condylar chondrocytes, and HIF-1α signaling pathway may be involved in the transmission of mechanical signals of condylar chondrocytes.

Key words: Temporomandibular joint, Condylar chondrocytes, Osteoarthritis, Hypoxia inducible factor 1α, Vascular endothelial growth factor, Collagen typeⅡ

表1 PCR引物序列及预扩增长度

基因	引物序列（5'to 3'）	扩增长度（bp）
HIF-1α	上游引物：CGTGCCCCTACTATGTCGCTTT，下游引物：GTCTTCTGCTCCATTCCATCCTGT	80
VEGF	上游引物：GTCACCACCACACCACCATCGT，下游引物：CTCCTCTCCCTTCATGTCAGGCT	76
ColⅡ	上游引物：GAGTGGAAGAGCGGAGACTACTG，下游引物：GTCTCCATGTTGCAGAAGACTTTCA	83
β-actin	上游引物：GAATTCCCAGTAAGTGCGGGTCATA，下游引物：CGAGGGCCTCACTAAACCATC	105

表1 PCR引物序列及预扩增长度

基因	引物序列（5'to 3'）	扩增长度（bp）
HIF-1α	上游引物：CGTGCCCCTACTATGTCGCTTT，下游引物：GTCTTCTGCTCCATTCCATCCTGT	80
VEGF	上游引物：GTCACCACCACACCACCATCGT，下游引物：CTCCTCTCCCTTCATGTCAGGCT	76
ColⅡ	上游引物：GAGTGGAAGAGCGGAGACTACTG，下游引物：GTCTCCATGTTGCAGAAGACTTTCA	83
β-actin	上游引物：GAATTCCCAGTAAGTGCGGGTCATA，下游引物：CGAGGGCCTCACTAAACCATC	105

图1 髁突软骨细胞甲苯胺蓝染色镜下所见（×100）

图2 髁突软骨细胞COLⅡ免疫组织化学染色镜下所见（×200）

图3 200 kPa静压力加载前后髁突软骨细胞的增殖情况

图4 200 kPa静压力加载前后髁突软骨细胞的凋亡情况

表2 不同时间点静压力加载后髁突软骨细胞HIF-1α、VEGF和COLⅡ的表达情况（±s）

指标	加压前	加压1 h	加压2 h	加压4 h	加压8 h	加压12 h	F值	P值
HIF-1α	4.55±0.03	7.54±0.03^#	8.93±0.02^#	10.94±0.02^#	8.74±0.03^#	7.15±0.03^#	21.330	<0.01
VEGF	2.24±0.03	2.85±0.02^#	4.33±0.02^#	9.55±0.03^#	2.83±0.02^#	1.84±0.03^*	41.710	<0.01
COLⅡ	0.91±0.07	1.51±0.07^#	3.13±0.12	5.32±0.08^#	3.49±0.05^#	2.88±0.04^#	12.960	<0.01

表2 不同时间点静压力加载后髁突软骨细胞HIF-1α、VEGF和COLⅡ的表达情况（±s）

指标	加压前	加压1 h	加压2 h	加压4 h	加压8 h	加压12 h	F值	P值
HIF-1α	4.55±0.03	7.54±0.03^#	8.93±0.02^#	10.94±0.02^#	8.74±0.03^#	7.15±0.03^#	21.330	<0.01
VEGF	2.24±0.03	2.85±0.02^#	4.33±0.02^#	9.55±0.03^#	2.83±0.02^#	1.84±0.03^*	41.710	<0.01
COLⅡ	0.91±0.07	1.51±0.07^#	3.13±0.12	5.32±0.08^#	3.49±0.05^#	2.88±0.04^#	12.960	<0.01

表3 不同时间点静压力加载后髁突软骨细胞HIF-1α、VEGF和COLⅡ的表达情况（±s）

指标	加压前	加压1 h	加压2 h	加压4 h	加压8 h	加压12 h	F值	P值
HIF-1α	0.84±0.14	1.21±1.18^*	1.18±0.07^*	1.59±0.36^#	0.78±0.06	0.41±0.02^*	15.880	<0.01
VEGF	0.89±0.09	2.49±0.25^#	2.54±0.08^#	3.22±0.17^#	1.82±0.15^#	2.13±0.07^#	85.390	<0.01
COLⅡ	0.97±0.06	1.44±0.08^#	1.47±0.15^#	2.07±0.31^#	1.02±0.04	0.62±0.01^*	35.210	<0.01

表3 不同时间点静压力加载后髁突软骨细胞HIF-1α、VEGF和COLⅡ的表达情况（±s）

指标	加压前	加压1 h	加压2 h	加压4 h	加压8 h	加压12 h	F值	P值
HIF-1α	0.84±0.14	1.21±1.18^*	1.18±0.07^*	1.59±0.36^#	0.78±0.06	0.41±0.02^*	15.880	<0.01
VEGF	0.89±0.09	2.49±0.25^#	2.54±0.08^#	3.22±0.17^#	1.82±0.15^#	2.13±0.07^#	85.390	<0.01
COLⅡ	0.97±0.06	1.44±0.08^#	1.47±0.15^#	2.07±0.31^#	1.02±0.04	0.62±0.01^*	35.210	<0.01

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Regulation of HIF-1α signaling pathway on proliferation and apoptosis of condylar chondrocytes under static pressure

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Regulation of HIF-1α signaling pathway on proliferation and apoptosis of condylar chondrocytes under static pressure