Effect Of Down-regulation Of HSP60 On Apoptosis Of MDCC-MSB1 Cells

Heat shock protens(HSPs),know as stress protein,are ubiquitous in all organism and highly conservative.HSPs are classified based on their molecular weights and include HSP110,HSP90,HSP70,HSP60,and small HSPs.In normal physiological conditions,HSPs is expressed in the cell and facilitateprotein folding,transport and maintenance of naturalstructures.The expression level of HSPs will be significent changed when the cells exposed to homeostatic challenges.The gene transcription and expression level of HSP60is significantly increased during establishment and development of tumor induced by MDV in chicken.And the intracellular localization of HSP60 is notable correlation with the localization of tumor cell in the tissues.The biological effects and molecular mechanism of HSP60 to apoptosis of MD tumor cells is not well understood.MDCC-MSB1 cell line is one of Marek’s disease lymphoblastoid cell line.Small interfering RNAs to HSP60 mRNA were synthetised and used to construct lentivirus to reduce the transcription and expression of HSP60.To study the relationship between expression level of HSP60 and the apoptosis in MDCC-MSB1 cell,the methods of fluorescence quantification RT-PCR,Western Blot and flow sytometry were used to detect transcription and expression level of HSP60 and apoptosis changes.Six pairs of primers were designed and synthesized according to the chicken HSP60and GAPDH gene RNA sequences published by NCBI,using Primer premier 5.0biological software and references.The amplification conditions of primers were optimized by PCR and fluorescence quantitative PCR.The gene fragments amplified by PCR were recovered,connected to pGEM-T vectors,and transfected to DH5αcells.Positive plasmid were identified and diluted as positive standard materials to make standard curves of fluorescence quantitative RT-PCR.The amplification efficiency,sensitivity and specificity of fluorescence quantitative RT-PCR were detected.The results showed that the established fluorescence quantitative RT-PCR method could reflect the initial number of templates in the reaction tube.The slope of standard curves of HSP60 and GAPDH was close,and the amplification efficiency was good.It could be used to detect HSP60 and GAPDH gene in MDCC-MSB1 cells.According to the HSP60 sequence published by NCBI,three small interfering RNA sequences were designed to construct lentivirus.optimize The conditions of lentivirus transfected to MDCC-MSB1 cells were optimized.The results showed that when the concentration of lentivirus was 1×10~8TU/mL,andwithout any assistant reagent,the transfection efficiency was the best.The interference effect of 5147 sequence lentivirus was the best.At 36 hours after transfection,the level of transcription and expression decreased by 88.6%and 70.6%respectively compared with the control sequence lentivirus group.At 48 hours after transfection,the level of transcription and protein expression decreased by 87.7%and 75.9%respectively compared with the control sequence lentivirus group.The MDCC-MSB1 cells were collected at 48 hours after transfectionwith 5147sequence lentivirus.The transcription and expression level of HSP60 and apoptosis level of MDCC-MSB1 cells were detected by fluorescence quantitative PCR,Western Blot and flow cytometry.The results showed that the transcription and expression of HSP60decreased 81.1%and 85.2%respectively,and the apoptotic rate increased 46.9%when the5147 sequence lentivirus interfered for 48 hours.It was proved that the apoptosis of MDCC-MSB1 cells could be increased by decreasing the expression level of HSP60.