| Objectives:1.Chapter 1 To optimize the structure of cerebral infarction model and the construction of(MCAO)model of middle cerebral artery occlusion in rats.2.Chapter 2 To study the role of HSP70-hom+2437 SNPs in SD rat MCAO model and its potential mechanism.Methods:1.Chapter 1.(1)Preparation of rat MCAO model:50 male SD rats weighing 260g-280g(SPF grade)were divided into two groups:sham operation group(n=25)and model group(n=25).All the rats in the model group were occluded by thread occlusion of the right middle cerebral artery to block the blood flow of the middle cerebral artery to form a permanent MCAO model.The sham operation group did not place the thread or ligate the blood vessels,and the other steps were the same.(2)Neurological deficit score and TTC staining were used to measure the volume of cerebral infarction:24 hours after the establishment of MCAO model,the neurological function was evaluated by Bederson method.Immediately after scoring,the rats were killed to take the brain tissue,and the brain tissue was cut into thin slices of 2mm thickness for TTC staining,and the percentage of cerebral infarction volume in each group was analyzed.(3)Observation of histomorphological changes by HE staining:the third brain section of each group was fixed for 3 days and then embedded in paraffin.The embedded paraffin sections were dewaxed,hematoxylin staining,eosin staining,dehydration and sealing,and the morphological changes were observed under Nikon inverted fluorescence microscope and the images were collected and analyzed.2.Chapter 2(1)Construction of lentivirus:construction of negative control virus,over-expression of C allele lentivirus and over-expression of T allele lentivirus.The titer of the lentivirus was determined by limited dilution method.When the fluorescence infection efficiency was 60%,the optimal titer of overexpressed lentivirus in the two groups was(1E+5)x 60%/(IE-1)=6E+5,in TU/μL,6E+8TU/mL.(2)Intracerebroventricular injection:150 male SD rats weighing 210g-230g were randomly divided into five groups:MCAO model group,MCAO model+normal saline group,MCAO model+negative virus control group,MCAO model+overexpression C allele genome and MCAO model+ overexpression T allele group,with 30 rats in each group.MCAO model group did not receive intracerebroventricular injection,MCAO model+saline group,MCAO model+negative virus control group,MCAO model+ overexpression of C allele genome group,MCAO model+overexpression of T allele genome group were injected with saline,negative control virus,overexpression of C allele lentivirus and overexpression of T allele lentivirus,respectively.(3)After intracerebroventricular injection,the rats were fed to the body weight of 260-280g to establish the MCAO model.24 hours later,the neurological damage of each group was scored,the cerebral infarction area of each group was measured by TTC staining,and the histomorphological changes of each group were observed by HE staining.(4)The relative expressions of HSPA1L and apoptosis-related factors were analyzed after total RNA and total protein were extracted from ischemic penumbra.The mRNA and protein expression of HSPA1L,pro-apoptotic factors Bax and cleaved-caspase3,and anti-apoptotic factor Bcl-2 in the periinfarct area of rats in each group were measured by qPCR and Western blot methodsResults:1.Chapter 1.(1)Verify the success of the rat MCAO model:compared with the sham operation group,there were obvious symptoms of neurological impairment in the model group,and the neurological deficit score in the model group was significantly higher,the difference was statistically significant(P<0.001).-(2)The location and volume of infarction were verified by TTC staining.The location of infarction was often located in the cortex and subcortex in TTC staining,and there was obvious infarct area in the model group,which was significantly different from that in the normal group(P<0.05).(3)HE staining was used to verify the pathological changes at the cellular level.The experimental results showed that most of the nerve cells in the infarct core area of the model group were necrotic,while most of the neurons in the ischemic penumbra were apoptotic,which further verified the success of the cerebral infarction model.2.Chapter 2.(1)HSP70-hom+2437 C and T allele lentiviruses were successfully constructed and transfected.In the experiment,negative control viruses were constructed,C allele lentiviruses and T allele lentiviruses were overexpressed and transfected into rat brain tissue.The expression of C allele and T allele in brain tissue was detected by qPCR and Western blot:there was no significant difference in HSP70-hom mRNA and HSPA1L protein levels among model group,normal saline group and negative control virus group.Compared with the model group,saline group and negative control group.HSP70-Hom mRNA and HSPA1L protein levels of the C and T allelic genomes were significantly higher than those of the model group(p<0.05),but there was no significant difference between C and T allele genome mRNA and protein levels.(P>0.05).(2)Neurological deficit score:after 24 hours of MCAO model,there was no significant difference in neurological deficit score among model group,normal saline group and negative control virus group(P>0.05).Compared with model group,normal saline group and negative control virus group,overexpression of C and T alleles could significantly reduce neurological deficit score(P<0.01).There was no significant difference in neurological deficit score between overexpressed C allele group and overexpressed T allele group(P>0.05).(3)Infarct size measured by TTC staining:the percentage of cerebral infarction volume in each group after 24 hours of MCAO model was 43.28%,43.62%,42.52%,35.60%and 30.81%,respectively.There was no significant difference in cerebral infarction volume among the first three groups(P>0.05).Compared with the first three groups,the infarction volume was reduced after overexpression of C and T alleles,(P<0.05).The infarct volume of overexpressed T allele was significantly smaller than that of overexpressed C allele(P<0.05).(4)The pathological changes of histomorphology were observed by HE staining:the morphological changes such as membrane shrinkage staining,nuclear pyknosis and fragmentation of neurons in the ischemic penumbra of over-expressed C and T alleles were lighter than those of the former three groups,and the neuron density was significantly higher than that of the former three groups.(5)qPCR and Western blot were used to detect the expression of HSPA1L and apoptosis-related factors in brain tissue:in qPCR experiment,compared with model group,normal saline group and negative control group,the mRNA expression of pro-apoptotic factor Bax decreased after overexpression of C and T alleles(P<0.05),but there was no significant difference between the two groups(P>0.05).In the protein detection experiment,compared with the model group,saline group and negative control group,the expression of pro-apoptotic protein Bax decreased after overexpression of C and T alleles(P<0.05),and the decrease of overexpression of T allele genomic Bax protein was more significant than that of overexpression of C allele genomes(P<0.05).However,there was no significant difference in the expression of Bcl-2,cleaved-caspase3 mRNA and protein among the five groups(P>0.05).Conclusions:1.Chapter 1(1)Successfully construct the rat MCAO model and verify the successful preparation of the model.(2)The construction of rat MCAO model was successfully improved from the aspects of preoperative preparation,intraoperative operation and postoperative nursing,.Improve the key steps and methods to raising the Survival rate and Molding rate in the Construction of MCAO Model2.Chapter 2.(1)The overexpression of HSP70-hom+2437 C and T alleles decreased the neurological damage score,alleviated the pathological changes of ischemic penumbra and reduced the infarct volume in MCAO rats,and the effect of overexpression of T allele was more obvious than that of overexpression of C allele.(2)Overexpression of HSP70-hom+2437 C and T alleles decreased the expression of pro-apoptotic factor Bax in rat brain,especially in overexpressed HSP70-hom+2437 T alleles,suggesting that overexpression of HSP70-hom+2437 C and T alleles may play a protective role in rat MCAO model by down-regulating Bax expression. |
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