目的:构建携带人活化转录因子6(activating transcription factor 6,ATF6)的重组腺病毒Ad-ATF6和Ad-ATF6 si RNA,并探讨内质网应激(ER Stress,ERS)时其对人软骨肉瘤细胞SW1353增殖凋亡的影响。方法:将ATF6基因序列与靶向ATF6的si RNA序...目的:构建携带人活化转录因子6(activating transcription factor 6,ATF6)的重组腺病毒Ad-ATF6和Ad-ATF6 si RNA,并探讨内质网应激(ER Stress,ERS)时其对人软骨肉瘤细胞SW1353增殖凋亡的影响。方法:将ATF6基因序列与靶向ATF6的si RNA序列分别克隆到p Ad Track-cmv和p SES-HUS穿梭质粒上,然后分别与腺病毒骨架质粒p Ad Easy-1在大肠杆菌BJ5183感受态中同源重组,得到腺病毒重组质粒p Ad-ATF6和p Ad-ATF6 si RNA,通过脂质体介导在HEK293细胞中进行包装和扩增。通过RT-PCR和Western blot检测病毒效果。流式细胞仪法(flow cytometry,FCM)、MTT法及Western blot检测ERS状态下ATF6对SW1353细胞增殖凋亡的影响。结果:成功获得了重组腺病毒Ad-ATF6和Ad-ATF6 si RNA。RT-PCR和Western blot结果表明,重组腺病毒能有效的感染细胞。FCM检测结果表明,ERS条件下,ATF6感染组S期细胞比例明显升高,凋亡率明显降低(P=0.000);Ad-ATF6 si RNA感染组S期细胞比例明显降低,凋亡率明显上升(P=0.000)。MTT与Western blot实验结果与FCM结果一致。结论:ERS状态下,ATF6能促进SW1353细胞的增殖,抑制其凋亡。展开更多
Objective:To investigate the inhibition effect of dexamethasone with different concentration gradients on the expression of MMP-1,-3,and-13 in a bone joint cell model of IL-1β-induced SW1353 cells.Methods:The SW1353 ...Objective:To investigate the inhibition effect of dexamethasone with different concentration gradients on the expression of MMP-1,-3,and-13 in a bone joint cell model of IL-1β-induced SW1353 cells.Methods:The SW1353 cells were placed in a culture medium containing 10^(1)-10^(8) nmol/L dexamethasone,and after 12 hours of culture,the appropriate intervention concentration range of dexamethasone was screened by the MTS for subsequent experiments.After SW1353 cells were induced with 10 ng/mL IL-1β,10^(1)-10^(7) nmol/L dexamethasone was given for intervention,and RT-PCR was used to detect the mRNA expression of MMP-1,-3,and-13.The protein was extracted after the intervention of 104 nmol/L dexamethasone,and western blot was used to detect the protein expression of MMP-1,-3,and-13.Results:MTS proliferation experiment results showed that 10^(1)-10^(7) nmol/L dexamethasone had no significant effect on SW1353 cell viability,while 10^(8) nmol/L dexamethasone had a significant inhibitory effect on SW1353(p<0.05).There was no obvious effect on cell viability after adding 10 ng/mL IL-1β.RT-PCR results indicated that the IL-1βgroup had a significant increase in the mRNA expression of MMP-1,-3,and-13 compared with the blank control group,and the difference was statistically significant(p<0.05).After intervention with 102-10^(7) nmol/L dexamethasone,MMP-1 mRNA expression decreased(p<0.05);after intervention with 103-10^(7) nmol/L dexamethasone,MMP-3 mRNA expression decreased(p<0.05);after intervention with 10-10^(7) nmol/L dexamethasone,the mRNA expression of MMP-13 decreased(p<0.05).The Western blot results showed that compared with the blank control group,the protein levels of MMP-1,-3,and-13 in the IL-1βgroup were significantly increased;the protein levels of MMP-1,-3,and-13 were significantly reduced after the intervention of 104 nmol/L dexamethasone.Conclusion:Dexamethasone can inhibit the expression of MMP1,MMP-3,and MMP-13 in the IL-1β-induced OA cell model at the mRNA and protein levels.展开更多
文摘目的:构建携带人活化转录因子6(activating transcription factor 6,ATF6)的重组腺病毒Ad-ATF6和Ad-ATF6 si RNA,并探讨内质网应激(ER Stress,ERS)时其对人软骨肉瘤细胞SW1353增殖凋亡的影响。方法:将ATF6基因序列与靶向ATF6的si RNA序列分别克隆到p Ad Track-cmv和p SES-HUS穿梭质粒上,然后分别与腺病毒骨架质粒p Ad Easy-1在大肠杆菌BJ5183感受态中同源重组,得到腺病毒重组质粒p Ad-ATF6和p Ad-ATF6 si RNA,通过脂质体介导在HEK293细胞中进行包装和扩增。通过RT-PCR和Western blot检测病毒效果。流式细胞仪法(flow cytometry,FCM)、MTT法及Western blot检测ERS状态下ATF6对SW1353细胞增殖凋亡的影响。结果:成功获得了重组腺病毒Ad-ATF6和Ad-ATF6 si RNA。RT-PCR和Western blot结果表明,重组腺病毒能有效的感染细胞。FCM检测结果表明,ERS条件下,ATF6感染组S期细胞比例明显升高,凋亡率明显降低(P=0.000);Ad-ATF6 si RNA感染组S期细胞比例明显降低,凋亡率明显上升(P=0.000)。MTT与Western blot实验结果与FCM结果一致。结论:ERS状态下,ATF6能促进SW1353细胞的增殖,抑制其凋亡。
基金National natural science foundation of China(No.81673941,81704050,81804042)。
文摘Objective:To investigate the inhibition effect of dexamethasone with different concentration gradients on the expression of MMP-1,-3,and-13 in a bone joint cell model of IL-1β-induced SW1353 cells.Methods:The SW1353 cells were placed in a culture medium containing 10^(1)-10^(8) nmol/L dexamethasone,and after 12 hours of culture,the appropriate intervention concentration range of dexamethasone was screened by the MTS for subsequent experiments.After SW1353 cells were induced with 10 ng/mL IL-1β,10^(1)-10^(7) nmol/L dexamethasone was given for intervention,and RT-PCR was used to detect the mRNA expression of MMP-1,-3,and-13.The protein was extracted after the intervention of 104 nmol/L dexamethasone,and western blot was used to detect the protein expression of MMP-1,-3,and-13.Results:MTS proliferation experiment results showed that 10^(1)-10^(7) nmol/L dexamethasone had no significant effect on SW1353 cell viability,while 10^(8) nmol/L dexamethasone had a significant inhibitory effect on SW1353(p<0.05).There was no obvious effect on cell viability after adding 10 ng/mL IL-1β.RT-PCR results indicated that the IL-1βgroup had a significant increase in the mRNA expression of MMP-1,-3,and-13 compared with the blank control group,and the difference was statistically significant(p<0.05).After intervention with 102-10^(7) nmol/L dexamethasone,MMP-1 mRNA expression decreased(p<0.05);after intervention with 103-10^(7) nmol/L dexamethasone,MMP-3 mRNA expression decreased(p<0.05);after intervention with 10-10^(7) nmol/L dexamethasone,the mRNA expression of MMP-13 decreased(p<0.05).The Western blot results showed that compared with the blank control group,the protein levels of MMP-1,-3,and-13 in the IL-1βgroup were significantly increased;the protein levels of MMP-1,-3,and-13 were significantly reduced after the intervention of 104 nmol/L dexamethasone.Conclusion:Dexamethasone can inhibit the expression of MMP1,MMP-3,and MMP-13 in the IL-1β-induced OA cell model at the mRNA and protein levels.
