短周期动态压应力通过Ca^2＋通道调控生长板软骨细胞Sox9及Ⅱ型胶原表达  

Regulated expressions of Sox9 and type Ⅱ collagen by cyclic compressive stress in growth plate chondrocytes via calcium channel

作　　者：张津铭[1] 陈安民[1] 吕正涛[1] 李兴艳[1] 郭风劲[1] 祁军[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院骨科,武汉430030

出　　处：《中华小儿外科杂志》2016年第4期296-301,共6页Chinese Journal of Pediatric Surgery

基　　金：国家自然科学基金（31200707,81371915）

摘　　要：目的研究短周期动态压应力对体外培养的大鼠生长板软骨细胞Sox9、Ⅱ型胶原mRNA及蛋白表达水平的影响，探讨L-型钙离子通道是否参与该力学信号转导过程。方法分离并体外培养两周龄SD大鼠生长板软骨细胞，传代后的生长板软骨细胞分为4组：对照组、单纯压应力组、硝苯地平组及压应力联合硝苯地平组。其中对照组不施加力学及药物干预，单纯压应力组通过四点弯曲力学加载仪予以周期性压应力（2 000 με，1.0 Hz）干预，硝苯地平组添加L-型钙离子拮抗剂硝苯地平（10 μmol/L）但不施加力学干预，压应力联合硝苯地平组添加硝苯地平（10 μmol/L）并施加周期性压应力干预（2000 με，1.0 Hz）。实验处理6 h，运用荧光定量PCR及Western blot方法检测各组细胞的Sox9、Ⅱ型胶原mRNA和蛋白表达情况。各组细胞行Fluo-3/AM染色，激光共聚焦显微镜观察并分析细胞内钙离子的荧光强度。使用SPSS17.0软件对数据进行统计学分析。结果单纯压应力组生长板软骨细胞的Sox9的mRNA与蛋白表达水平较对照组分别增加（82.97±24.95）%和（79.67±26.03）%，差异有统计学意义（P〈0.05）；Ⅱ型胶原的mRNA与蛋白表达水平较对照组分别增加（118.93±34.40）%和（95.93±29.87）%，差异有统计学意义（P〈0.05）。压应力联合硝苯地平组生长板软骨细胞的Sox9、Ⅱ型胶原的mRNA与蛋白表达水平低于单纯压应力组（P〈0.05），但高于硝苯地平组（P〈0.05）。激光共聚焦显微镜能清晰观察到细胞内钙离子染色后产生的荧光，但各组平均荧光强度差异无统计学意义（P〉0.05）。结论适当的短时间周期性压应力能上调生长板软骨细胞Sox9及Ⅱ型胶原mRNA与蛋白的表达水平，钙离子通道可能参与该力学信号转导过程。Objective To explore the effect of short-term cyclic compressive stress on the mRNA and protein expressions of Sox9 and type II collagen in growth plate chondrocytes in vitro and examine whether L-type calcium channel is involved in mechanical signal transduction. Methods The growth plate chondrocytes were harvested from 2-week-old Sprague-Dawley rats and cultured in vitro. The ehondrocytes were divided into four groups of control, cyclic compressive stress （CCS）, cyclic compressive stress plus nifedipine （CCS ± Nif） and nifedipine （Nif）. The control group received neither drug nor mechanical intervention. And the CCS group received cyclic compressive stress of 2 000με at a frequency of 1.0 Hz through a four-point bending system without drug dosing. The Nil group received 10 μmol/L nifedipine without mechanical intervention. And the CGS ± Nil group received both 10 μmol/L nifedipine and cyclic compressive stress of 2 000με at a frequency of 1.0 Hz. The duration of intervention was 6 hours. The mRNA levels and protein expressions of Sox9 and type Ⅱ collagen were measured by reabtime polymerase chain reaction （qPCR） and Western blot respectively. The chondrocytes were stained by Fluo-3/AM and the fluorescent intensity of intracellular calcium was o＇oserved by laser scanning confocal microscope. And SPSS17. 0 was employed for statistical analysis. Results The expressions of mRNA and protein of Sox9 were significantly up-regulated for （82. 97 ± 24. 95）% and （79. 67 ± 26. 03）% respectively compared withcontrol group （P〈0. 05）. The levels of mRNA and protein of type l] collagen significantly increased versus control group （118. 93 ± 34. 40） 〈 and （95.93 ±29. 87） % respectively compared with control group （P〈0. 05）. The mRNA and protein expression of Sox9 and type U collagen in growth plate chondrocytes of CCS± Nil group were lower than those of CCS group （P〈0. 05） but higher than those of Nil group （P〈0. 05）. No differences in fluorescent intens

关 键 词：骨骺生长板 软骨细胞 钙通道 

分 类 号：R726.8[医药卫生—儿科]