| Objective: To investigate the mechanical and biological characteristics of chondrocytes after Ihh gene knockout.We using immature mouse costal chondrocytes as a source of chondrocytes,combined with Col2a1-Cre ERT2;Ihhfl / fl genetically engineered mice.The pressure used was a periodic compressive stress(Flexcell 5000 applied sine wave,0.5 Hz,5 k Pa,1 h / d compressive stress).Methods:(1)Cultivate Col2a1-Cre ERT2;Ihhfl / fl and Ihhfl / fl genetically engineered mice and identify their genotypes.(2)newborn Ihhfl/fl pure merge with Col2a1-Cre ERt2 genetically engineered mice 40,were randomly divided into control group and experimental group,experimental group(Tamoxifen),intraperitoneal injection of TM,intraperitoneal injection of the control group in the same amount of vegetable oil injection for five days in a row,acute digestive rib cartilage cells.(3)real-time PCR detection Ihh gene expression,analysis Ihh gene knockout efficiency.(4)The chondrocytes in the experimental group and the control group were divided into two groups,.With 2 × 105 / ml density and 2% sodium alginate gel mixture,made of cylindrical cell plate.One group was subjected to a sine wave,0.5 Hz,5 k Pa,1 h / d compressive stress on the cell plate using a Flexcell-5000 substrate loading system;another group of static cultured cell plates.(5)The growth status of chondrocytes at different time points between the ballast group and the static culture group was observed by inverted microscope.(6)The expression of MMP-13、 Col-10 、AGG and Col-2m RNA in experimental group and control group at the same time point was analyzed by RT-PCR.(7)The changes of mechanical properties of costal chondrocytes after Ihh gene knockout under physiological pressure were measured by microtubule sucking technique combined with semi-infinite somatic cell mechanics model.Results:(1)Through the experimental group of mice by intraperitoneal injection of TM(Tamoxifen)found that Ihh gene knockout efficiency of about 64%.(2)At the same time point,compression stimulation could promote the proliferation of chondrocytes in sodium alginate gel.Inverted microscope,the cell density of compression group was significantly higher than that of static culture group.In the static culture,the chondrocytes after Ihh knockout had no significant difference in cell density compared with chondrocytes without Ihh knockout.(3)The experimental group compared with the control group in the three-dimensional culture of the cell plate,a significant up-regulation was observed in the gene expression of AGG and Col-2 on 7th day or MMP-13 and Col-10 on 14 th day respectively;Compression cell plates in the experimental group were compared to compressed cell plates in the control group,a significant up-regulation was observed in the gene expression of AGG and Col-2 on 7th day or MMP-13 and Col-10 on 14 th day respectively;Comparison of different time points in each group: The expression level of AGG and Col II was higher than that of 14 days and 21 days,while the relative expression of Col X and MMP-13 in the compression group was higher than that in the control group.(4)The E0、E∞ andμof the cells in each group decreased with the increase of culture time,the E0、E∞ andμof the dynamic compression group were significantly lower than those of the 7 and 14 days group;Compared with the control group,the E0、E∞ and μ of the chondrocytes cultured at the same time point were higher than those in the control group,the results of the chondrocytes in the dynamic compression group were also the same,the E0、E∞ and u of the 21-day group were significantly higher than those of the control group(P <0.05);In the experimental group,the E0 of the Dynamic compression chondrocytes of in the 7-day group was significantly greater than that in the static culture group(P <0.05).Conclusion:Sodium alginate in three-dimensional culture conditionally knocked out Ihh gene in mice with costal chondrocytes during physiological dynamic compression stimulation for 7 days,which can effectively improve the Col II and Agg expression,inhibition of Col X and Mmp13 expressed,improve the mechanical properties of chondrocytes to a certain extent.But with the increase in the number of days ofchondrocyte matrix secretion gradually reduced,mechanical properties also weakened. |
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