电刺激对软骨细胞增殖及细胞内Ca^(2+)浓度的影响

Effects of electrical stimulation on chondrocyte proliferation and intracellular Ca^(2+) concentration

目的寻求促进体外培养的软骨细胞增殖的适宜电刺激参数,探讨软骨细胞增殖与细胞内Ca^(2+)浓度之间的关系。方法使用3-5日龄家兔肋骨软骨细胞(Pri Cells),经过2次传代培养,将细胞按照4×10~4/孔的密度接种于6孔培养板内,次日利用函数信号发生器刺激细胞。采用占空比20%的方波波形,频率1 Hz,按照电压大小将细胞分为5组:电压1 V组、2V组、3 V组、5 V组和0 V组(无刺激组)。各组细胞每日刺激20 min,连续刺激3 d。CCK-8法检测细胞增殖,用钙离子荧光探针Fluo-3 AM和多功能酶标仪检测细胞内的Ca^(2+)浓度。结果占空比20%的方波波形,频率1 Hz,刺激电压1 V时,细胞吸光度值和细胞内Ca^(2+)浓度与无刺激组相比差异无统计学意义(P>0.05);电压2 V和3 V时细胞吸光度值和细胞内Ca^(2+)浓度与无刺激组相比均有提高(P<0.05),电压3 V时有显著提高(P<0.01);但当电压增大到5 V时抑制细胞增殖,细胞吸光度值与无刺激组相比降低(P<0.01)。结论适宜的电刺激能够提高软骨细胞胞内Ca^(2+)浓度进而促进软骨细胞增殖,其适宜条件为方波波形,占空比20%,频率1 Hz,电压3 V。

Objective To screen suitable electrical stimulation parameters for chondrocytes cultured in vitro,and explore the relationship between cell proliferation and intracellular Ca^2＋concentration.Methods The costal chondrocytes were obtained from the Pri Cells of 3-5-day-old rabbits.The cells at passage 2 were seeded into the 6 wells plate at initial seeding density of 4 × 104 cells/well.Next day the costal chondrocytes were stimulated by signal generator with square wave at duty cycle of 20%,frequency of 1 Hz.According to the voltage,the cells were divided into five groups：1 V,2 V,3 V,5 V and 0 V（non-stimulation） groups.The cells in each group were stimulated for 20 min per day for 3 d.Cell proliferation was detected by CCK-8.The concentration of intracellular Ca^2＋was detected by Fluo-3 AM and Multiscan Spectrum.Results Compared with non-stimulation group,the absorbance value and intracellular Ca^2＋concentration in 1 V group showed no significant difference（P〉0.05）,the absorbance value and intracellular Ca^2＋concentration were improved in 2 V group（P〈0.05） and 3 V group（P〈0.01）.Compared with non-stimulation group,the cell proliferation was inhibited in 5 V group,and the cell absorbance was decreased（P〈0.01）.Conclusion The suitable electrical stimulation could promote the intracellular Ca^2＋concentration and improve the chondrocyte proliferation.The appropriate electrical stimulation condition of electrical stimulation on chondrocyte is square waveform,20% duty cycle,frequency of 1 Hz and voltage of 3 V.