Study On The Anti-apoptotic Mechanism Of MiR-7 On Mouse Neurons Under Low Temperature And Hypoxia

Chapter 1.The establishment of mimic deep hypothermic and hypoxia modelObjective To establish a deep hypothermic and hypoxia model of the original generation of neurons in mice.Methods Choose healthy clean level C57BL/6 mice rats(within 48 h after birth),get the cerebral cortex neurons,the cells will be islated and cultured.And mimic deep hypothermic and hypoxia model was established.Results We compared the morphology of neurons in each group.Normal neurons refracted three-dimensional shape,cell body,axon growth was strong,and with the surrounding interconnected network shape.In deep hypothermic and hypoxia groups,neuronal densky decreased significantly for dead floating and appeared axon disintegration or Waller degeneration.Conclusion The establishment of mimic deep hypothermic and hypoxia model is very important.It provides a new way for the research of cerebral I/R injury after deep hypothermic and hypoxia.Chapter 2.The protective effect and mechanism of miR-7 on mimic deep hypothermic and hypoxia model.Objective To investigate the cerebral protective effect of miRNA-7 on mimic deep hypothermic and hypoxia model and the related functional molecular mechanism..Methods The mimic deep hypothermic and hypoxia model were divided into 3 groups:miR-7 agamir group,miR-7 antagomir group and control group by adding miR-7 agomir,miR-7 antagomir and artificial cerebrospinal fluid.Observe each group and detect the expression of miR-7 by qRT-PCR.To detect the LDH leakage situation by ELISA and the apoptosis by FCM.The expression of p-Akt,Akt,Bcl-2 and Bax was detected by Western blot analysis.Results qRT-PCR revealed that miR-7 expression was upregulated in the miR-7 agomir group and was downregulated in the miR-7 antagomir group(p<0.05).The agomir group kept a better cell form compared to the control group after deep hypothermic and hypoxia with more cells alive,while the antagomir group showed a different situation.The agomir group kept a better cell form compared to the control group after deep hypothermic and hypoxia with more cells alive,while the antagomir group showed a different situation.The result of ELISA showed that the LDH leakage of agomir group was lower than which in control group,the antagomir group with a higher leakage level.The FCM results showed that the agomir group had less number of apoptosis and necrosis than the control group,while more apoptosis and necrosis appeared in the antagomir group.Western blot revealed that miR-7 agomir upregulated the expression of p-Akt,Bcl-2 and downregulated Bax expression(p<0.05).In contrast,miR-7 antagomir inhibited p-Akt and Bcl-2 expression,but increased Bax exression(p<0.05).Conclusion miR-7 has neuroprotective effects in the mimic deep hypothermic and hypoxia model and the mechanism may be that miR-7 could activate the PI3K/Akt signaling pathway to regulate the downstream key protein Bcl-2 and Bax.