牵张应力对人骺板软骨细胞分化及凋亡的影响

Effects of mechanical stretch stress on the differentiation and apoptosis of human growth plate chondroeytes

目的观察不同强度的机械牵张应力刺激对体外培养的小儿指骨骺板软骨细胞凋亡及软骨分化的影响，并探讨其相关机制。方法体外分离培养人骺板软骨细胞，使用Ⅱ型胶原（COLⅡ）免疫组化法及甲苯胺蓝染色进行鉴定。使用四点弯曲细胞力学加载装置分别对细胞进行不同强度的周期性牵张应力刺激（刺激强度分别为0μstrain、2000μstrain、4000μstrain，刺激时间6h、刺激频率0．5Hz），AnnexinV／PI双染法流式细胞仪检测细胞凋亡情况，荧光定量PCR技术检测各组细胞细胞增殖核抗原（PCNA）、凋亡相关基因（BAX、BCL-2）、软骨特征性细胞外基质（COLⅡ、COLX）及甲状旁腺激素相关蛋白（PThrP）的mRNA表达情况。结果Ⅱ型胶原免疫组化法可见COLⅡ大量表达。2000μstrain组骺板软骨细胞PCNA、COLⅡ、COLX、PThrPmRNA相对表达量分别为1．13±0．14、9．98±0．74、9．88±1．80和5．18±0．86，在4000μstrain组分别为0．06±0．叭、2．19±0．08、0．95±0．09和0．29±0．04，在对照组分别为1．00±0．12、6．73±0．62、1．00±0．08和1．00±0．14。2000μstrain组COLⅡ、COLX、PThrPmRNA相对表达量及4000μstrain组PCNA、COLⅡ、PThrPmRNA相对表达量，与对照组比较，差异均有统计学意义（P〈0．05）。在4000μstrain的周期性牵张应力刺激下，细胞早期凋亡率、晚期凋亡和总凋亡率分别为（4．60±0．81）％、（8．39±2．08）％和（12．99±1．48）％，与对照组（1．01±0．32）％、（4．30±0．71）％和（5．61±1．04）％比较，差异均有统计学意义（P〈0．01）。2000μstrain组BCL-2、BAXmRNA比值为0．75±0．08，4000μstrain组为0．39±0．11，与对照组1．00±0．23比较，差异均有统计学意义（P〈0．05）。结论小儿指骨骺板软骨细胞的分化和凋亡受机械牵张应力所调节，适当的牵张应力能有效促进其其软骨基质形成，而过大牵张应力则促进细胞凋亡，软骨相关基因表达减少，而PThrP在这一过程中可能起重要调控作用。

Objective To observe the effects of mechanical stretch stress with different intensities on the in vitro differentiation and apoptosis of human plate ehondrocytes and explore its mechanism. Methods The human plate chondrocytes were isolated and cultured in vitro. The stains of toluidine blue and type Ⅱ collagen immunohistochemistry were used for identifying chondrocytes. Mechanical stretch stresses with different intensities were applied at 0, 2 000 and 4 000μ strain for 6 h using four-point bending system. The expression levels of COLⅡ, COLX, BAX, BCL-2 and PTHrP were detected by quantitative reverse transcription-polymerase chain reaction （ RT-PCR ）. Results Under2 000μ strain, the expression levels of PCNA, COLⅡ, COLX and PThrP mRNA were 1.13 ± 0. 14,9. 98 ± 0. 74,9. 88 ± 1.80 and 5.18 ± 0. 86; under 4 000μ strain, 0. 06 ± 0, 01,2. 19 ± 0. 08,0. 95 ± 0. 09 and 和0. 29 ± 0. 04 versus 1.00 ± 0. 12,6. 73 ± 0. 62,1.00 ± 0. 08 and 1, 00 ± 0. 14 in control group. Compared with control group, the expression levels of COLⅡ, COLX and PTHrP in 2 000 9 and 4 000 9 strain groups were statistically significant （P〈0. 05）. Under 4 000μ strain, the rates of apoptosis, apoptosis＆ necrosis and total cell apoptosis were （4.60 ± 0. 81）%, （8.39 ±2.08）% and（12.99± 1.48）% versus （1.01±0.32）%, （4.30±0.71）% and（5.61± 1.04）% in control group （P〈0. 01）. In 2 000 μ and 4 000 μ strain and control groups, the ratios of BCL-2/BAX were 0. 75 ± 0. 08, 0. 39 ± 0. 11 and 1.00 ± 0. 23 respectively. All differences were statistically significant （P〈0. 05）. Conclusions The apoptosis of growth plate chondrocytes is regulated by mechanical stretch stress. Appropriate stretch stress can effectively promote cell proliferation and differentiation. And excessive stretch stress inhibits cell proliferation and differentiation and promotes cell apoptosis. And PThrP may play important roles in this process.