Effects Of Primary Cilia On The Antioxidant Activity Of PC12 Cells In Hypoxia

Objective To investigate effect of hypoxia environment on primary cilia length of PC12 and the function of its primary cilium as an oxygen sensor playiny a role in the activation of cellular defense mechanism against hypoxia.Methods 1.PC12 cells were cultured under hypoxia environment,and the cultivation included physical hypoxia and simulated chemical hypoxia using Cobalt Chloride for 0h,6 h and 18 h,and then the morphology of cells was observed by optical microscope,and we will decide which way to be used in the next experiment by observing the degree of hypoxia injury induced by two different ways of hypoxia on PC12 cells.2.After PC12 cells were cultured under physical hypoxia for 0h,6 h and 18 h,primary cilia length of PC12 cells were observed and measured as well as the growth rate.3.After PC12 cells were cultured under physical hypoxia for 0h,6 h and 18 h,detected the gene expression of HIF-1? ? Nrf2 ? VEGF and SOD in intracellular by RT-PCR and detected the protein expression of HIF-1??Nrf2?VEGF and SOD in intracellular by WsternBlot.4.After inhibit the ciliogenesis by IFT 88 si RNA,firstly we observed whether the length of primary cilia was increased after hypoxia.Occurrence rate was also measured after inhibit the ciliogenesis.5.After inhibit the ciliogenesis by IFT 88 siRNA,we observed the nuclear translocation in cells of HIF-1??HIF-2? and Nrf2 in hypoxia by immunofluorescence staining.,then detected the expression of HIF-1??Nrf2?VEGF and SOD in intracellular by RT-PCR and WeaternBlot.Finally,the outcome were compared with the experimental results of the control group which without RNA interference of IFT88.Results 1.After cells were cultured under physical hypoxia environment or simulated chemical hypoxia using Cobalt Chloride for 6 h,there was no obvious hypoxia injure,but after hypoxia for 18 h,the cell morphology was changed significantly.In particular,chemical hypoxia by Cobalt Chloridecan cause a severe hypoxia injure.2.After hypoxia,the growth rate of primary cilia decreased with the increase of hypoxia time.3.The length of primary cilia was increased after physical hypoxia.The average length of cilia which without hypoxia was 0.64?m while it was1.34?m after hypoxia for 6h and 2.02?m after hypoxia for 18 h.4.After inhibit the ciliogenesis by IFT 88 siRNA,primary cilia of scrambled control group were growing normally and the length of cilia was increase after hypoxia while the length of si RNA group was no longer increase after hypoxia.5.After inhibit the ciliogenesis by IFT 88 si RNA,the nuclear translocation in cells of HIF-1??HIF-2? and Nrf2 were no longer occurred,and at the same time,the expression of HIF-1?,Nrf2,VEGF was inhibited while the expression of SOD was increased.On the contrary,while without RNA interference of IFT88,hypoxia can induce the nuclear translocation in cells of HIF-1??HIF-2? and Nrf2.In the meantime,the expression of HIF-1?,VEGF in cells correspondingly first increasing after hypoxia for 6h and then decreasing after hypoxia for 18 h,and the expression of SOD was decreased compared with the normal oxygen concentration.Conclusion 1.Hypoxia environment can induce increasing primary cilia length of PC12 cells.But after inhibit the ciliogenesis by IFT 88 siRNA,primary cilia were also inhibited,what is more,the length was no longer increase after hypoxia.2.What we've done possibly identify the function of primary cilia as an oxygen sensor and primary cilia can transfer the hypoxic and oxidative stress information into the cell body after sense the extracellular oxygen concentration,and help to activate the intracellular defense mechanism against the hypoxic injuries.In this experiment,we observed the changes of primary cilia length,the morphology of primary cilia and the internal signal protein during hypoxia after inhibite the cilia,we also observed the change of cilia intracellular hypoxia related factors during hypoxia after inhibite the cilia.The results showed us that hypoxia environment can induce increasing primary cilia length of PC12 cells.,and it also identified that the function of primary cilia as an oxygen sensor and primary cilia can transfer the hypoxic and oxidative stress information into the cell body.The results are of great significance to the research and development of new drugs for the prevention and treatment of high altitude diseases and the economic development in Plateau Areas.