| Nowadays, as the aging of the population acceleration all over the world,Alzheimer's disease (AD) has become the major global healthy problem. Itinfluence patients' life quality, and make the family and society's burdenmuch heavier not only in spirit but also medical cost that greatly improved. Soit take hugeous challenge to medical researches.Prevention and intervention ofAD is a pressing task,so early diagnosis,early treatment and looking for neweffective therapy is important. As the majority of elderly dementia, AD is aneurodegenerative disease characterized by aggravated overall cognitivedysfunction and behavior damaged.It becomes a serious threat to humanhealth with increasing incidence. The earlier treat, the better effect.The latest research confirmed that cell cycle disorders exist in AD thathappened in neurodegenerative early phase. It provides a new approach todiagnosis and treatment. PI3K/Akt-p21/p27-RB access is involved in cellcycle regulation, especially in the G1/S, of which the cell cycle inhibitingprotein p21/p27played a crucial role. The cell cycle disorder is systematic,besides brain, the related peripheral cells such as lymphocytes and fibroblastsalso have changes. In some aspects, AD patient's lymphocyte have samedefects with nerve cells, is a promising in vitro model to study brain'smetabolism and biochemistry. The clinical studies of AD often focus on theoxidative stress, cell apoptosis, etc, however, the cell cycle and relatedresearch is few. Because it's difficult to get brain from patients, the experimentchoose the peripheral blood lymphocytes(PBLs) as model, using flowcytometry to research cell cycle changes in patient's lymphocytes, and Westernblotting p21,p27protein level for further study of cell cycle disorders,especially G1/S checkpoint dysfunction in AD. It may provide experimental data for basic study of AD and new clues to early diagnosis and treatment.Objective: This experiment study AD patient's peripheral bloodlymphocyte as model to evaluation cell cycle changes, and measure theexpression of the cell cycle G1/S phase related factors such as p21p27, andfurther study between severity and mild groups. In addition to set normalcontrol group, we also analyze and compare the other neurodegenerativedisorder-Parkinson's disease(PD) in the level of p21, p27change, to explorewhether it has the specificity in AD. Thus a new method and interventiontarget especially early diagnosis and treatment could be provided.Methods: Collect20sporadic AD,20PD and20healthy controls, usingsimple mental state scale (MMSE) and clinical dementia scale (CDR) tocomprehensive evaluate for cognitive impairment, then collecting allinformation, clinical characteristicsh, history and neuroimaging data, etc.Lymphocytes isolated immediately from peripheral blood, then prepare asingle-celled levitation liquid after it was washed by buffer solution and thenimpurities was remove, fixed, For nucleus emitted fluorescence afterpropidium iodide (PI) dyeing, cell cycle distribution of lymphocytes wasanalyzeby flow cytometry test, and relative contents of DNA and proliferationability of cells could be detected with analysis in patients with AD and healthycontrols. Further withdraw fresh total protein from lymphocyte, using westernblotting to detect the level of p21p27protein expression.Results:1The detection of cell cycle distribution showed obvious differencesbetween AD and healthy control 's PBLs. G1and S phase difference wasstatistically significant, G1phase ratio drops, S phase ratio increases, andlymphocyte proliferation index and relative contents of DNA increases, thedifference was statistically significant (P <0.05), G2period was notstatistically significant (P>0.05).2In the PBLs from AD patiens, p21p27level is lower than healthycontrol and PD group, with a statistical significance,P <0.05. PD groupcompared with normal control group, p21p27level is no statistical difference (P>0.05).(p21/gapdh HC0.942±0.177, AD0.564±0.273, PD0.805±0.205,which AD vs HC, P <0.05; AD vs PD, P <0.05; PD vs HC, P>0.05)(p27/gapdh HC0.974±0.334, AD0.458±0.204, PD0.781±0.287, which ADvs HC, P <0.05; AD vs PD, P <0.05; PD vs HC, P>0.05)3In the peripheral blood lymphocyte, p21, p27level were not correlationwith age, MMSE score and CDR score (P>0.05).4Comparison between severity and mild groups of AD haven't shownstatistical difference in p21, p27level (P>0.05).Conclusions：In peripheral lymphocytes of AD, there may have similarcell cycle changes with central neurons. The cell cycle re-entry causedifferentiation, then resulting lack of neurons. We use flow cytometrydetection cells cycle changes, found that AD not yet block in Gl period,but Sphase has a significantly higher proportion. By software analysis, AD showsenhanced lymphocyte proliferation capacity, may provides new ideas todiagnosis and treatment of AD. In addition, the cell cycle distribution andlymphocyte proliferation ability not statistically significant differ AD in severedegree, to hint the cell cycle disorders may be already exists in the early AD,and have nothing to do with progression.In PI3K/Akt-p21/p27-RB pathways, the cell cycle inhibiting protein p21p27, are important molecules in the process of cell cycle participating grow,differentiation, aging and death process, make the cell cycle stagnation in Glperiod by forming cyclin/CDK complexes. The experiment find a differentdegree of decline of cellular p21,p27protein in AD compare to PD andhealthy controls,and they are echo to the cell cycle test results. This may beperipheral blood markers of AD, and differ from other neurodegenerativedisease.but the expression level of two kinds protein have no correlation withMMSE, CDR rates, and there is no significant differences between differentsevere degree patients, So we can't think it as a progress monitoring index. |
