纳米细菌对人关节软骨细胞生物学形态的影响

Influence of nanobacteria on biological morphology of human articular chondrocytes

目的观察纳米细菌损伤人膝关节软骨细胞的方式和特点。方法正常人膝关节软骨细胞分为高浓度组、中浓度组、低浓度组和对照组,分别加入吸光度值为0.280、0.035、0.002、0的纳米细菌液进行攻击,加入纳米细菌液24、48h时光镜下观察4组膝关节软骨细胞形态学变化及存活率,免疫荧光染色法观察中浓度组加入纳米细菌液0、1、2、4、8h时纳米细菌与软骨细胞的位置关系,流式细胞仪检测加入纳米细菌液24h时4组软骨细胞凋亡率。结果高浓度组加入纳米细菌液24h、中浓度组加入纳米细菌液48h,光镜下大部分软骨细胞坏死甚至未见完整细胞形态;加入纳米细菌液24、48h时,高浓度组软骨细胞存活率吸光度值(0.413±0.046、0.345±0.125)低于中浓度组(0.496±0.039、0.708±0.043)、低浓度组(0.539±0.047、1.018±0.048)和对照组(0.577±0.045、1.133±0.127)(P<0.05),中浓度组低于低浓度组和对照组(P<0.05),低浓度组与对照组比较差异无统计学意义(P>0.05);纳米细菌可在较短时间内侵入至软骨细胞中,且随时间延长软骨细胞内纳米细菌荧光颗粒数目增多;加入纳米细菌液24h,高、中、低浓度组和对照组软骨细胞凋亡率[(1.541±0.046)%、(1.546±0.039)%、(1.539±0.047)%、(1.527±0.045)%]比较差异无统计学意义(P>0.05)。结论纳米细菌可损伤人关节软骨细胞,短时间内(24h)被攻击的软骨细胞未发现明显凋亡,高浓度纳米细菌可造成人关节软骨细胞更严重损伤。

Objective To investigate the injury mode and characteristics of nanobacteria in human articular chondrocytes.Methods The normal knee articular chondrocytes were divided into high-concentration group,medium-concentration group,low-concentration group and control group,attacked by nanobacteria solution with optical density(OD)value of0.280,0.035,0.002 and 0,respectively.The morphological changes and the survival rate of chondrocytes were observed under optical microscope in 24 and 48 hin 4 groups.The spatial relations between chondrocytes and nanobacteria were identified by immunofluorescence in 0,1,2,4 and 8 hin medium-concentration group.The apoptosis rate of chondrocytes was monitored by flow cytometry in 24 hin 4 groups.Results Light microscopy showed necrosis of most chondrocytes,and rare complete cell morphology in 24 hin high-concentration group and 48 hin medium-concentration group.The survival rates of chondrocytes in 24 and 48 hwere significantly lower in high-concentration group(0.413±0.046,0.345±0.125)than those in medium-concentration group(0.496±0.039,0.708±0.043),low-concentration group(0.539±0.047,1.018±0.048)and control group(0.577±0.045,1.133±0.127)(P<0.05),and lower in medium-concentration group than those in low-concentration group and control group(P<0.05),while there were no significant differences between low-concentration group and control group(P>0.05).Immunofluorescence results indicated nanobacteria attacked chondrocytes quickly,and the nanoparticles increased in number as the time prolonged.The chondrocytes apoptosis rates showed no significant differences in high-,medium-and low-concentration groups and control group in 24h((1.541±0.046)%,(1.546±0.039)%,(1.539±0.047)%,(1.527±0.045)%)(P>0.05).Conclusion The articular chondrocytes can be injured by nanobacteria.No apoptosis could be found in 24 hin the attacked chondrocytes.High-concentration nanobacteria would cause severe injury of chondrocytes.