Translated Abstract
Preface: Alzheimer’s disease (AD) is a type of progressive neurodegenerative disease in center nervous system, generally occurs in old population, and is characterized by a series of clinical symptom of impairments of the short-term memory and dysfunction of cognition. With the population aging all over the world, the incidence of AD is increasing rapidly, and AD has become threatening for human health that just ranges after cardio-cerebrovascular disease and cancers. The etiology of AD hasn’t been elucidated completely, and there is no effective therapy method especially yet. Now, the study on pathogenesis, preventions and therapy of AD has been crucial issue to be explored. AD has been considered that it is a multifactors disease, and may be caused by factors of genetics, age, environment etc. At present, AD associated genes definitely located universally are following: gene App, ps1, ps2 and apoE. However, these disease associated genes together interpreted mainly abnormal increase and accumulation of Aβ, rather than molecular genetic mechanism for the formation of neurofibrillary tangles and neuronal loss. Thereby, further study on genetic risk factors should be performed. P35 is neuron specific regulative unit of CDK5. p35 gene exist several SNPs, and at some SNPs sites, the change of a single base causes the correspond change of amino acid of P35. Cleavage of P35 into P25 increases greatly kinase activity of CDK5, which in turn phosphorylates protein tau abnormally, and then contributed to the formation of neurofibrillary tangles. Several lines of evidence show that the proportion of P25/P35 in AD brain increased significantly. Study about transgenic mouse overexpressing P25 show that aberrant activity of CDK5 induced by P25 led to progressive neuronal loss in the cerebral cortex and hippocampus, accompanied by severe forebrain atrophy and reactive astrogliosis. And in p25 transgenic mouse, endogenous tau accumulated as insoluble aggregates and was hyperphosphorylated at several epitopes. Thereby, abnormally cleavage of P35 into P25 has close relation with AD pathology. What we are interested in is that whether the polymorphism of P35 gene was involved in the mechanism of AD. There are few research reports about the relationship of P35 gene polymorphism with AD.Objective: To study the association of the p35 rs17852832 polymorphism with Alzheimer’s disease. Molecular biologic techniques were adopted, such as polymerase chain reaction (PCR), the analytical method of restriction fragment length polymorphism ( RFLP) etc, and analyzed the frequency of genotypes and alleles of the p35 rs17852832 polymorphism in AD and normal senile pepole.Method: To collect 2ml venous blood of the cases of AD and controls respectively, and extract genomic DNA with rapidly salting-out method. Then obtain product of 681-940 fragment of p35 gene including rs17852832 SNP through PCR. After being identified and purified, p35 fragment was cut with restrictive enzyme MvaⅠ, then analyze RFLP of the product through PAGE, and analyze the association of the RFLP with AD. Result: 1. The genomic DNA extracted with rapidly salting-out method were electrophoresed through 1% agarose gel, and found that genomic DNA bands were clear and regular. DNA value of OD ≥40ng/µl ,260/280≥1.5 , the max. of which got to 1003.9ng/µl and 1.88 respectively.2. The PCR products were examined through 2% agarose gel electrophoresis, and found that closely after the 250bp band existed a regular and clear band, which was the intended band of 260bp fragments of p35 gene including rs17852832 SNP. Then, we purified the intended band through gel purified kit to recollect the intended p35 gene fragment. 3. The purified fragments of 681-940bp of p35 gene including rs17852832 SNP were analyzed with method of RFLP, and found that the p35 gene fragments of all cases of AD and controls were cut by MvaⅠ into three shorter fragments, which length were 114bp, 90bp and 53bp. The allele and the genotype of SNP rs17852832 were C and C/C respectively, allele A and genotype C/A or A/A hadn’t been found yet. 4. The purified and identified fragments of 681-940bp of p35 gene were cloned into pMD18-T vector for sequencing. The sequencing result showed that they were identical to the p35 gene sequence from GENBANK, which show that the sequence cloned into T vector was our intended fragments, p35 gene 681-940bp including rs17852832 SNP. And that, the sequencing result proved further that the allele and genotype of rs17852832 SNP were C and C/C respectively, allele A and genotype C/A or A/A hadn’t been found yet. Conclusion:1. Human genomic DNA was extracted from venous blood through rapidly salting-out method successfully, and the quality of products was approving.2. The fragments of p35 gene encoding region 681-940bp including rs17852832 SNP were obtained through PCR technique successfully, and were identical to the sequence from GENBANK. 3. The RFLP of p35 gene 681-940bp fragments were analyzed through restrictive enzymes and PAGE technique, and made sure that the allele of SNP at 798 site of p35 gene in all cases of AD and controls were C, which was identical to sequence provided by GENBANK Genotype of rs17852832 SNP were C/C, allele A and genotype C/A or A/A hadn’t been found yet.
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