Molecular Biological Function And Its Mechanism Of RBM3 In Resisting Lower Temperature Stress

Objective: RNA binding motif protein 3(RNA binding motif protein 3,RBM3)is an important cold stress protein in the presence of organism.It is up-regulated by low temperature,playing an important role of protecting cells from harm stimulate in lower temperature,but the regulation mechanism for the protective effect remains to be further clarification.In this study,we successfully constructed lentiviral vector of overexpression of rat RBM3 gene,by using the vector infected in hippocampal neurons and intraperitoneal injected in animals.The protective effect and the mechanism of RBM3 were comprehensively researched,the results could provide theoretical basis in clinical treatment of mild hypothermia,and also provide theoretical basis for resisting cold stress in development of animal husbandry and aquaculture.Methods:(1)Constructed RBM3 overexpression lentiviral vector: cDNA sequences were obtained from SD rat testis,digested and sequenced accurately and cloned into the lentiviral vector of LV5 plasmid;Transfected LV5-RBM3 recombinant vector and p Gag/Pol,p Rev,p VSV-G three plasmids into 293 T cells by Using liposome respectively,packaged,produced lentivirus and detected titer.(2)In vitro test: hippocampal neurons of SD rats were infected with the successful construction of lentiviral vector of RBM3 overexpression.Neurons were treated by different temperature,and the level of neuronal cell apoptosis were detected for the mechanism of RBM3 on cell protection under mild hypothermia.(3)In vivo test: by intraperitoneal injection of the lentiviral vector and treated by low temperature stimulation for different temperature and time,redox indicators,apoptosis related index and cytokine expression levels were detected to explore the mechanism of RBM3 in body under cold stimulation.Results:(1)The lentiviral vector of overexpressing RBM3 were constructed Successfully;(2)Hippocampal neurons were infected with lentiviral vector of overexpression successfully,and the value of best MOI was 140:1;(3)In vitro test:(1)The RBM3 expression in the overexpression group and the mild hypothermia group were significantly higher than the control group(P<0.01,P<0.05).(2)The RBM3 overexpression group and the mild hypothermia group were significantly lower than that of control group for neuronal apoptosis rate(P<0.01).(3)The level of caspase-3 and the ratio of bax/bcl-2 of RBM3 overexpression group and mild hypothermia group in neurons were significantly lower than that of control group respectively(P<0.05,P<0.01).(4)In vivo test:(1)Compared with the group of cold treated only,the level of T-AOC,SOD and IL-2,IL-6 were significantly lower(P<0.05 or P<0.01)than the group of cold treatment in the RBM3 overexpression,and the level of MDA,IL-6 were significantly higher(P<0.05)than the group of cold treatment in the RBM3 overexpression.(2)There was no significant change of caspase-3 between all groups(P>0.05).Conclusion: In vitro and In vivo comprehensive tests showed that RBM3 could effectively avoid the stress injury caused by cold stress,and play a protective role in the body cells.