The Correlation Between Chondrocyte Apoptosis And Mitochondrial Function In Joint Disease

Objective: To test the proportion of apoptosis for articular cartilage cells under hypoxia induced dynamic and;To measuring the change of active caspase 3C28 / I2 in articular cartilage cells in hypoxia environment and normal oxygen;Using MitoSOX kits to test hypoxia induced superoxide generation of C28 / I2 articular cartilage cells,living cell permeability,the change of mitochondrial superoxide indicator content;In order to further study on hypoxia induced molecular signaling pathways of expressing RhoA articular cartilage cells and chondrocytes apoptosis related mechanism after CREB,To detect hypoxic interference C28RhoA/I2 cells and expression of the change rule,degree of cell apoptosis and CREB phosphorylation levels,to apply the foundation for the mechanism of articular cartilage degeneration for joint degeneration and immunity and provide the basis for the prevention and treatment of inflammatory injury.Methods:Articular cartilage cells C28 / I2 was routine cultured human,theexperiment were divided into two groups,hypoxia group with 5% oxygen group and 3% CO2 in cultivation group,normal oxygen group was used conventional environment,the change of the cell vitality was researched using MTT experiment continuous observation for 12 hours,24 hours a day and 48 hours,the two groups were used flow cytometry technology to continuous observat the ratio of two groups of apoptosis,the content of Caspase 3 using Caspase SenSolyte kit using fluorescence immunoassay instrument were detected the wavelength of 460 nm,two groups of cytochrome c(Cyt c),caspase 3(CASP)3,after CREB,phosphorylation after CREB protein expression were detected in western blot method for 24 hours and 48 hours.MitoSOXTM Red mitochondrial was used to detect mitochondria superoxide superoxide,ROS-sensitive fluorescence phosphorylation 5-(and to 6)-chloromethyl-2,7-dichlorodi-hydrofluorescein kit was used to detect the levels of superoxide inside the cell,by fluorescence spectrophotometer in excitation light is 488 nm,under the condition of light is 530 nm,the results with the control group ratio was expressed as percentage.Cell mitochondrial membrane potential detection mainly adoptes TMRE-membrane membrane potential kit for testing,mitochondrial respiratory chain complexes ? content by Rodent enzyme-linked active micro plate kits was choosen for testing,by fluorescence spectrophotometer in excitation light is 575 nm,distributeddetection under the condition of light is 549 nm.Using plasmid transfection technology detected C28 / I2 cells expressing RhoA,coding sequence of RhoA gene cloning to pcDNA3.1(+)on the plasmid vector,then RhoA-pcDNA3.1(+)and contrast pcDNA3.1(+)plasmid transfected C28 / I2 cells,two groups of transfected 24 hours a day and 48 hours using western blot method to detect RhoA,CREB phosphorylation and CREB levels of change were detect;Then RhoA-pcDNA3.1(+)group of articular cartilage cells was in hypoxia environment 24 hours a day and 48 hours later,MitoSOXTM Red mitochondrial mitochondria superoxide superoxide tracer detection,the levels of superoxide cells by fluorescence spectrophotometer were detected the ROS-sensitive fluorescence phosphorylation 5-(and to 6)-chloromethyl-2,7-dichlorodi-hydrofluorescein kit in the excitation light is 488 nm,send out test under the condition of light is 530 nm,the results with the control group ratio was expressed as percentage.Cell mitochondrial membrane potential detection mainly adopts TMRE-membrane membrane potential kit for testing,mitochondrial respiratory chain complexes ? content by Rodent enzyme-linked active micro plate kits for testing,by fluorescence spectrophotometer in excitation light is 575 nm,distributed detection under the condition of light is 549 nm.Results:1.Hypoxia environment culture C28 / I2 cell 24 and 48 hours later,cell activity significantly decreased with MTT experiments(p < 0.05 or p <0.01).2.Under anoxic environment,C28 / I2 apoptosis were significantly higher than that of normal oxygen Using flow cytometry instrument to detect(p < 0.01 or p < 0.001).3.We used immunofluorescence ti detect apoptosis protein caspase 3,undedr anoxic environment develop 12,24 and 48 hours later,caspase 3 of C28 caspase 3 / I2 cells were significantly higher than that of normal oxygen(p < 0.01 or p < 0.001).4.Western blot reaction,cytochrome C and Clv-CASP 3 of C28 /I2 cell were significantly higher under hypoxia environment culture 12,24 and 48 hours later to compare with the normal oxygen environment,the difference was statistically significant;Compared with the normal oxygen concentration,phosphorylation after CREB of C28 / I2 cell were significant declined undre hypoxia environment culture,the phosphorylation CREB in hypoxia environment was no significant change.5.Articular cartilage cells was under hypoxia environment for 24 hours a day and 48 hours later,superoxide significantly increased,the comparative differences was statistically significant(p < 0.01 or p <0.001).articular cartilage cells was under hypoxia environment for 24 hours a day and 48 hours later,ROS product significantly increased(p < 0.05,p <0.01 or p < 0.001);articular cartilage cells was under hypoxia environmentfor 24 hours a day and 48 hours later,MMP for 24 H.P.T.decreased significantly(p < 0.05 or p < 0.01 for 48 H.P.T.),in the mitochondrial respiratory chain complexes ? dcreased,the comparison of difference was statistically significant.6.RhoA-pcDNA3.1(+)plasmid transfection articular cartilage cells24 hours a day and 48 hours later,the RhoA was significantly higher(all p < 0.001),the difference was statistically significant,and phosphorylation after CREB levels significantly was higher(all p < 0.01,the interference between 24 hours and 48 hours),RhoA-pcDNA3.1(+)plasmid transfected articular cartilage cell,could significantly alleviate hypoxia induced cell vitality reduced(p < 0.05,24 or 48 hours),compared with control group,RhoA were overexpression of articular cartilage cells in the group,the vigor of caspase 3 induced by hypoxia was inhibited.Compared with transfection pcDNA3.1(+)plasmid controls,transfection RhoA-pcDNA3.1(+)group of articular cartilage cells in hypoxia environment,the mitochondria superoxide products was significantly lower(p < 0.05,transfection for 24 hours,p < 0.01)after 48 hours transfection,and transfected pcDNA3.1(+)plasmid control group comparison,transfected RhoA-pcDNA3.1(+)group of articular cartilage cells in hypoxia environment after training,ROS was significantly lower(p < 0.05,transfection for 24 hours,p < 0.01)after 48 hours transfection,and we found that the transfection RhoA-pcDNA3.1(+)group of articularcartilage cells in hypoxia environment,RhoA overexpression of significancely promote the secretion of MMPS and mitochondrial respiratory chain compound ?.Conclusion:1.The hypoxia induced C28 / I2 cell activity significantly to decrease,a marked increase in the level of apoptosis,caspase 3 levels rise CASP 3were significant risen,phosphorylation after CREB significantly decreased,and There was no influence on the CREB.2.Under hypoxia environment,superoxide of articular cartilage cells significantly elevated,ROS product of articular cartilage cells significantly increased,MMP of articular cartilage cells declined significantly,the mitochondrial respiratory chain complexes ?,could inhibit the role of mitochondrial function.3.RhoA-pcDNA3.1(+)plasmid transfeced articular cartilage cell,RhoA significant rose,phosphorylation after CREB levels significantly raised,could significantly relieve the cell vigor reduce cell apoptosis induced by hypoxia.4.Transfection RhoA-pcDNA3.1(+)group of articular cartilage cells in hypoxia environment after training,mitochondria significantly lower superoxide products,ROS products significantly lower,promote the production of MMPS and mitochondrial respiratory chain compound ?.