| Objectives: In this study,we used the lentiviral vector and RNA interference technology to construct LV-RFK and sh-RFK.By infecting neurons to make RFK overexpressed or downregulated.To observe the relationship between the down-regulation of RFK expression and the susceptibility to stroke,and to explore its mechanism.Chapter?: Construction of RFK lentiviral vectorMethods:(1)PCR amplification was carried out using raw cDNA of murine RFK as a template.LV-RFK was constructed using the p Lenti6.3-IRES2-EGFP/V5 DEST series plasmid vector,and the Vehicle was constructed using the Lenti-EGFP plasmid vector.(2)Firstly,the pc DNA vector was constructed and three groups of si RNA interference sequences were designed,then transfored and identified.The prepared three sets of si RNA interference sequence vectors were transfected into C6 cells,and then total RNA and protein were extracted.The expression of RFK in C6 cells after si RNA interference was analyzed by q PCR and Western Blotting,in order to find out the optimal si RNA interference sequence targeting RFK gene.Then,the plasmid vector with the best interference sequence was used as a template to construct into the p Lenti6.3-MCS/V5 DEST series plasmid vector,in order to construct sh-RFK.The LV-RFK,vehicle and sh-RFK were infected into 293 T cells,and the virus titer was detected.PCR and Western Blotting were used to detect the expression of RFK after transfection of LV-RFK and sh-RFK.Results:(1)The LV-RFK was transfected into neurons,and no fluorescence was observed in the control group,but a large amount of fluorescence was observed both in the vehicle group and the LV-RFK group.Compared with the vehicle group,the m RNA expression of RFK in the LV-RFK group was significantly increased(about 5.7 times.Western Blotting showed that the expression of RFK protein in the LV-RFK group was 1.6 times that of the Vehicle group(P<0.01).Results indicated that the RFK overexpression lentiviral vector was successfully constructed.(2)The results of q PCR and Western Blotting showed that the 2# interference target had the best effect on the RFK gene interference in rat.In the sh-RFK group,the state of 293 T cells was good,and most of the cells emitted green fluorescence indicating that the expression of GFP was positive.Western Blotting showed that the expression of RFK protein in the sh-RFK group was 0.7 times that of the negative group(P<0.01).Results indicated that the construction of the lentiviral vector that interferes with RFK expression was successful.Chapter ? The Mechanism of RFK against ischemic brain damageThe rat cortex was transfected into lentiviral vector,and the rat cortex was transfected for 21 days to prepare the model of middle cerebral artery infarction(MCAO);the neurons were infected for 12 hours and cultured for 2-3 days for oxygen and glucose deprivation(OGD)model.The experiment was divided into 5 groups: the control group,model group,Vehicle(empty virus vector)group,LV-RFK(RFK overexpression lentiviral vector)group and sh-RFK(interfering with RFK lentiviral vector)group.Methods: 1.RFK inhibits ischemic brain damage(1)Rat brain edema was detected by weighing.(2)Rat infarct size was detected by TTC staining technique.(3)The cortex of the cerebral infarction and the neurons after OGD model were observed to determine the morphology of cell.(4)MTT was used to determine the cell activity.(5)We determined the necrosis rate.The relationship between RFK and ischemic brain injury was analyzed by detecting the above indicators.2.The mechanism of action of RFK against ischemic brain damageMethods:(1)Western blotting was used to detect the expression of Ero1,CHOP and Caspase family apoptosis-related proteins.(2)Addited Ero1 inhibitor Erodoxin and detected cell viability to study the effect of RFK on ERS-induced apoptosis.(3)We used 3 months old SHR-SP rats,rats in the riboflavin group drank water containing riboflavin and continued to death.The natural death time of each group was observed.(4)Removed riboflavin and then detected cell necrosis rate and cell viability.(5)Supplement of riboflavin,FMN and FAD,detect cell activity and cell necrosis rate when RFK expression was down-regulated.Results: 1.RFK inhibits ischemic brain damage(1)Compared with the control group,the brain tissue weight of the model group,the Vehicle group and the LV-RFK group increased significantly(P<0.01);the brain tissue weight of the LV-RFK group was lighter than that of the Vehicle group(P<0.01).It is suggested that MCAO surgery leads to edema in rat brain tissue,and up-regulation of RFK has a certain inhibitory effect on MCAO-induced cerebral edema.(2)The cerebral infarct size of the LV-RFK group was significantly lower than that of the Vehicle group(P<0.01).It is suggested that the expression of RFK is up-regulated to inhibit necrosis of brain tissue around ischemia after cerebral ischemia,thereby inhibiting ischemic brain damage.(3)In the Vehicle group,the synaptic vesicles in the cerebral cortex cells expanded,and the mitochondria had vacuoles;in the sh-RFK group,the synaptic vesicles in the cerebral cortex cells were extremely dilated,and the mitochondria were extremely swollen;the cerebral cortex cells in the LV-RFK group were basically normal.In the Vehicle group,mitochondrial swelling,mitochondrial membrane rupture,synaptic vesicle content disappeared;sh-RFK group neuronal cell matrix was light,synaptic vesicles were extremely dilated,membrane separation before and after synaptic vesicles,endoplasmic reticulum expansion;LV-RFK The neuronal cell structure of the group was basically normal.The results showed that the up-regulation of RFK expression has protective effects on ischemic neurons,while the down-regulation of RFK expression aggravates ischemic injury of neurons.(4)Compared with the Vehicle group,the cell viability of the LV-RFK group was significantly increased(P<0.01),and the cell viability of the sh-RFK group was significantly decreased(P<0.05).The above conclusions are demonstrated in terms of cell activity.(5)Compared with the Vehicle group,the necrosis rate of LV-RFK group was significantly increased(P<0.01),and the necrosis rate of sh-RFK cells was significantly decreased(P<0.01).The above conclusions were confirmed from the aspect of apoptosis.2.The mechanism of action of RFK against ischemic brain damage(1)Compared with the Vehicle group,there was no change in the expression levels of Ero1,Caspase12 and Caspase3 in the LV-RFK group and the sh-RFK group compared with the Vehicle group.However,the CHOP expression level in the LV-RFK group was significantly decreased(P< 0.01).Compared with the Vehicle group,the expression of CHOP(P<0.01)and Caspase3(P<0.01)in the LV-RFK group was significantly lower than that in the Vehicle group.Caspase12(P<0.01)and Caspase3 in the sh-RFK group.(P<0.01)The expression level was significantly increased;while the expression levels of Ero1 in the LV-RFK group and the sh-RFK group were unchanged.The results showed that RFK affects the expression of CHOP,Caspase12 and Caspase3 in endoplasmic reticulum stress-related proteins,but does not affect the expression of Ero1 protein.(2)Compared with the Vehicle group,the activity of the LV-RFK group without Erodoxin was significantly increased(P<0.01),and the activity of the LV-RFK group to which Erodoxin was added was significantly decreased(P<0.01).This indicates that the role of RFK against ischemic neuronal damage is dependent on the flavoprotein Ero1.(3)Compared with the control group,the lifespan of the riboflavin group was significantly prolonged(P<0.05).It indicates that the supplementation of riboflavin during the whole life can significantly prolong the life span of SHR-SP rats with low RFK gene function.(4)When riboflavin was absent,the cell activity of LV-RFK group was significantly increased(P<0.01);when riboflavin was used,the protective effect of RFK on neurons was enhanced;the activity of RFK down-regulated was stronger than that without riboflavin.the trend of.When riboflavin was absent,the necrosis rate of LV-RFK neurons was still lower than that of the Vehicle group(P<0.05),but the necrosis rate was higher than that of the LV-RFK group with riboflavin(P<0.01).The results indicated that the protective effect of RFK was not dependent on riboflavin,and the supplementation of riboflavin had a certain compensation effect on the low RFK function.(5)Compared with the Vehicle group,the cell viability of the sh-RFK group was significantly lower(P<0.01);compared with the sh-RFK group,riboflavin supplementation(P<0.01),FMN(P<0.01),FAD(P< 0.01)The cell activity of the group was significantly increased.Compared with the vehicle group,the necrosis rate of sh-RFK group was significantly increased(P<0.01);compared with sh-RFK group,riboflavin supplementation group(P<0.01),FMN group(P<0.05),FAD group(P <0.01)The rate of cell necrosis was significantly reduced.It indicated that riboflavin,FMN and FAD could protect cells to some extent,but could not completely reverse the neuronal damage down-regulated by RFK expression.Conclusion:1.LV-RFK and sh-RFK were successfully constructed.Transfected into 293 T cells or neurons,and effectively make RFK overexpression or expression down-regulation.2.The low function of RFK gene will increase the susceptibility to stroke,and RFK is an endogenous protein against ischemic brain damage.3.RFK is resistant to ischemic brain damage through ERS-induced apoptosis,including up-regulation of CHOP and Caspase12 signaling pathways,and the protective effect of RFK is dependent on the flavinprotein Ero1.4.The protective effect of RFK against ischemic injury is not completely dependent on riboflavin,but supplementation with riboflavin can partially compensate for the low function of RFK gene. |
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