摘要:
Objective To explore the effects of cyclic tensile strain (CTS) with varied intensity on glycosaminoglycan (GAG) synthesis of chondrocytes in primary cultured rabbits' knees.Methods Articular cartilages of two knees in six New Zealand rabbits (at the age of one month) were obtained through aseptic surgery.Chondrocytes were resolved and isolated from articular cartilages by using 0.4% pronase and 0.025% 11 collagenase.Then chondrocytes from the same rabbit were divided into three parts and inoculated separately on three BioFlex culture plates.After this,stimulation of cyclic tensile strain (sinusoidal wave,0.3 Hz,6 h/d)with different intensity (0%,5% and 15%) was loaded to chondrocytes in monolayer cultured primary rabbits by means of a Flexercell 5 000 strain unit.The morphology of the cells was detected using inverted microscope at 24,36,48,and 60 hours of loaded CTS respectively.In the meanwhile,supernatants were drawn separately and GAG concentration was measured with Alcian blue precipitation.Finally,variance analysis of repeated measurement data was adopted to compare the variation of GAG concentrations in supernatants among CTS stimulation groups with different intensity.Results After cellular mechanical loading,it could be observed that chondrocytes exhibited a morphologic change from polygon to spindle shape,arranging perpendicularly along the radius of the cultured plates.Following the raising of strain intensity,GAG concentrations in supernatants were on the rise in proper order,with average concentration in each group being [(4.7 ± 1.3),(6.3 ± 1.1) and (8.0 ± 1.8) ng/ml,F =15.970,P =0.000] respectively.Consistently,at the same time point,there were significant differences of GAG concentrations in supernatants anmong the three groups,with average concentration of 24 hour [(2.3 ± 0.5) vs.(3.3±0.6) vs.(3.9±0.6) ng/ml,F=6.062,P=0.036],36 hour [(4.0±0.7) vs.(4.6± 0.8) vs.(6.8±0.2) ng/ml,F=16.720,P=0.004],48 hour [(5.5±0.7) vs.(6.8±0.7) vs.(8.5±0.8) ng/ml,F=12.570,P=0.007],and 60 hour [(6.9 ± 1.2) vs.(10.8 ±1.3) vs.(13.0 ± 1.1) ng/ml,F =19.790,P =0.002] respectively.Meanwhile,there was a significant difference between two groups by using L-S-D method.In pace with the increase of total loading time,GAG concentrations in supernatants were gradually multiplied,concentration in each group being [0% CTS group (2.3±0.5) vs.(4.0±0.7) vs.(5.5±0.7) vs.(6.9±1.2) ng/ml,F=4.640,P=0.037],[5% CTS group (3.3±0.6) vs.(4.6±0.8) vs.(6.8±0.7) vs.(10.8±1.3) ng/ml,F=23.580,P=0.000],and [15% CTSgroup (3.9±0.6) vs.(6.8±0.2) vs.(8.5±0.8) vs.(13.0±1.1) ng/ml,F =9.638,P =0.005] separately.It could be stated that there exists an interaction between total loading time and loading intensity.This suggests that the longer,the total loading time,the bigger,the values of GAG concentrations in supernatants comparing with those in control group.Conclusion CTS may promote GAG synthesis of chondrocyte in primary monolayer cultured rabbits' knees.The effects will increase along with the raising of strain intensity and duration.%目的 观察不同强度的循环动态拉伸(CTS)对原代培养的兔膝关节软骨细胞糖胺多糖(GAG)合成的影响.方法 1个月龄新西兰兔6只,无菌手术切取双膝关节软骨,采用0.4%Pronase酶和0.025%Ⅱ型胶原酶消化分离得到关节软骨细胞,来源于同一只兔的软骨细胞分为3部分,分别接种在3块BioFlex培养板上,通过Flexercell 5000系统对单层培养的兔膝软骨细胞施加CTS刺激,加载条件为0.3Hz正弦波形,时长为6 h/d,加载强度分别为0%、5%、15%的CTS刺激.于加载后24、36、48和60 h用倒置显微镜观察细胞形态,并分别抽取上清用阿尔新蓝染色沉淀法测量GAG浓度.采用重复测量资料的单因素方差分析比较不同强度CTS刺激组上清液中GAG浓度的差异.结果 细胞力学加载后可见周边部软骨细胞由多角形变为纺锤形,沿培养皿半径垂直方向排列.随拉伸强度的增加,上清中GAG浓度依次上升,各组总平均浓度分别为[(4.7±1.3)比(6.3±1.1)比(8.0±1.8) ng/ml],差异有统计学意义(F=15.970,P=0.000);同一时间点,随CTS拉伸强度的增大,上清液中GAG的含量逐渐增高,各时间点3组浓度分别为[24 h:(2.3±0.5)比(3.3±0.6)比(3.9 ±0.6)ng/ml;36 h:(4.0 ±0.7)比(4.6±0.8)比(6.8 ±0.2) ng/ml;48 h:(5.5±0.7)比(6.8±0.7)比(8.5±0.8)ng/ml;60 h:(6.9±1.2)比(10.8±1.3)比(13.0 ±1.1) ng/ml],且差异有统计学意义(F=6.062,P=0.036;F=16.720,P=0.004;F=12.570,P=0.007;F=19.790,P=0.002),同一采样时间采用组间LSD法两两比较差异均有统计学意义.随加载总时间增加,上清中GAG浓度逐渐增加,各组分别为[0% CTS组:(2.3±0.5)比(4.0±0.7)比(5.5±0.7)比(6.9±1.2) ng/ml;5% CTS组:(3.3±0.6)比(4.6±0.8)比(6.8±0.7)比(10.8±1.3)ng/ml;15% CTS组:(3.9±0.6)比(6.8±0.2)比(8.5±0.8)比(13.0±1.1)ng/ml],且差异有统计学意义(0% CTS组:F=4.640,P =0.037;5% CTS组:F=23.580,P=0.000;15% CTS组:F=9.638,P=0.005).且加载总时间与加载强度之间存在交互效应,时间越长,加载组上清中GAG浓度越大.结论 CTS可促进单层贴壁兔膝关节软骨细胞合成GAG,作用效果随拉伸强度和时间的增加而增大.