褪黑素对小鼠软骨细胞系ATDC5活性的影响

Melatonin effect on the activity of mouse ATDC5 cells

背景:目前在青少年特发性脊柱侧凸的研究中,主要使用从青少年髂软骨中提取的人原代软骨细胞,然而由于家长顾虑及伦理限制等原因,极大限制了青少年特发性脊柱侧凸的研究。因此,寻找一种可以代替人原代软骨细胞进行青少年特发性脊柱侧凸研究的细胞具有重大科研和社会意义。目的:观察小鼠软骨细胞系ATDC5在褪黑素刺激下的活性、受体及信号通路蛋白的改变,探究是否可以使用ATDC5细胞代替人软骨细胞。方法:将ATDC5细胞接种于96孔板中,给予不同浓度(10^(-5),10^(-6),10^(-7),10^(-8),10^(-9),10^(-10),10^(-11),10^(-12)mol/L)的褪黑素刺激,以不给予褪黑素刺激的细胞为对照,培养72 h后,采用MTT法检测ATDC5细胞增殖率。将ATDC5细胞接种于96孔板中,对照组不给予褪黑素刺激,实验组给予10^(-5)mol/L的褪黑素刺激,培养24 h后,采用免疫印迹法检测褪黑素受体及AMPK通路的蛋白表达。结果与结论:①随着褪黑素刺激浓度的增加,ATDC5细胞增殖率升高;②实验组Ⅱ型胶原、p-AMPK、褪黑素受体基因1B的蛋白表达高于对照组(P<0.05),两组AMPK、褪黑素受体基因1A的蛋白表达比较差异无显著性意义(P>0.05);③结果表明,褪黑素可促进小鼠软骨细胞系ATDC5增殖,其增殖率与文献报道的相同浓度褪黑素刺激人软骨细胞类似,提示存在使用小鼠软骨细胞系ATDC5代替人软骨细胞进行研究的可能性。

BACKGROUND:Human primary chondrocytes extra cted from adolescent iliac cartilage are currently used in the study of adolescent idiopathic scoliosis.Howeve r,research on adolescent idiopathic scoliosis is largely limited due to parental concerns and ethical restrictions.Therefo re,it is of great scientific and social significance to find a kind of cells that can replace human primary chondrocytes in the study of adolescent idiopathic scoliosis.OBJECTIVE:To study the activity,receptor and signaling pathway changes of ATDC5 cells stimulated by melatonin,and to explore whether ATDC5 cells can be used to replace human chondrocytes in comparison with the response of human chondrocytes under melatonin stimulation.METHODS:(1)ATDC5 cells were seeded into 96-well plates and stimulated by melatonin at different concentrations(10^(-5),10^(-6),10^(-7),10^(-8),10^(-9),10^(-10),10^(-11),10^(-12)mol/L).Cells without melatonin stimulation were used as the control.After 72 hours of culture,the prolife ration rate of ATDC5 cells was detected by MTT method.(2)ATDC5 cells were seeded into 96-well plates.The control group was not stimulated with melatonin,and the experimental group was stimulated with 10^(5)mol/L melatonin.After culture for 24 hours,the protein expression of melatonin receptor and AMPK pathway was detected by western blot assay.RESULTS AND CONCLUSION:After melatonin stimulation,the prolife rative rate of ATDC5 cells was increased with melatonin concentration increasing.After melatonin stimulation,the protein expressions of typeⅡcollagen,p-AMPK and melatonin receptor 1 B in the experimental group were higher than those in the control group(P<0.05),while there was no significant difference in the protein expressions of AMPK and melatonin receptor 1 A between the two groups(P>0.05).Therefore,melatonin can promote the prolife ration of mouse ATDC5 cells,and the proliferation rate is similar to that of human chondrocytes stimulated by melatonin at the same concentration,suggesting there is a possibility that mouse chondrocyte ATDC5 can be a substitute of human chondrocytes.