| Objective:Amyloid cascade hypothesis theory of amyloid precursor protein after a specific enzyme shear final formation of the amyloid beta. It is the important factor to promote the formation and development of Alzheimer's Disease,(Alzheimer's diseases, AD). Outside of the Alzheimer's patients' cerebral cortex and hippocampus, The main ingredient in neurons of senile plaques, and fiber entanglement is amyloid beta which consists of the insoluble amyloid fibers, still the important markers of clinical diagnosis of alzheimer's disease. In addition in AD patients with parenchymal microvascular walls, there are a lot of amyloidosis beta protein form fibrous plaques, lead to the pathological damage to the blood vessels, local hemodynamic changes, associated with the atherosclerosis, hypertension, hyperlipidemia, and so on. Eventually reduce cerebral blood flow and the degeneration in degenerative hippocampus neuron. Synaptic death, brain neuronal pathway, eventually lead to dementia. What we have known at present in peripheral circulation is that platelets are the main carriers of the amyloid beta which accounts for about more than 95% of the total beta amyloid protein in the peripheral circulation. After the activation of the platelets. They will clung to the damaged blood vessel wall. The A beta amyloid peptide which formed in the alpha particles of platelet will be released when the platelets were activated, this article through compare and contrast the crowd of alzheimer's patients to the people of healthy control and beta peptide expression in platelets activation status. Exploring the function of platelets in the formation of the amyloid fibrous plaque in central nervous system in patients with AD and the role of plateletsin in AD's pathogenesis. To provide the scientific basis for the molecular mechanism of the formation of AD amyloid protains Methods:1. According to the strict clinical diagnostic criteria and the screening criteria for the Alzheimer's disease. Choose 40 cases of the Alzheimer's patients in the neurology department of the tianjin medical university general hospital as the experimental group(AD group) and 40 counterparts in Tianjin Jiuhua medical center according to matching criteria in addition to age, sex, in patients with AD matching criteria,compare to the AD group as healthy controls(HC) during the period between September 2013 and October 2015. Extracting whole veinal blood with EDTA- 2 Na anticoagulation about 4 ml for research; In addition to obtain the myocardial tissue with the clinical pathological changes of the Alzheimer's disease.2. We manufacture the myocardial tissue into paraffin sections, staining the tissues with Congo red, the thioflavin T dyeing, Sudan black and the immunohistochemical staining by the TTR specific antibody to exam the amyloidosis lesions of myocardial tissue in Alzheimer's patients. We compare the specificity between the Congo red and thioflavin T dyeing in Alzheimer's patients seek for the amyloidosis specific detection probe.3. Extracting the pathological changes of amyloidosis fiber aggregates in myocardial tissue, using the QCM to determinate the affinity and specificity test between the amyloidosis fibers aggregates and thioflavin T, the result confirmed that the amyloidosis fiber aggregates in the myocardial tissue have strong specificity with the thioflavin T. in addition, by using the monoclonal antibody of TTR combining the Westen Bloting, confirmed that the thioflavin T have the of specific binding of the amyloidosis fiber aggregates in the myocardial tissue.Confirmed that the thioflavin T can detect the amyloidosis fiber aggregates, and have a high specificity in this detection.4. Extracting the AD group and HC group participants' veinal whole blood for 4ml, separating 100 ul, used for the whole blood's thioflavin T fluorescent probe painting(ThT). The rest of the blood platelets wrere extracted by the density gradient centrifugation. Then washing the platelets for 3 times to dye the platelets' membrane protein and the thioflavin T dyeing for the platelets.5. Respectively dyeing the whole blood and the platelets after washing. Discussing whether the presence of plasma on the ThT recognition to platelets' experiment of using the thioflavin T to test the specificity between the amyloidosis fiber aggregates and platelets aggregates. To conform whether the presence of plasma on the ThT recognition on platelets will effect the specificity of the aggregates.6. Through the Thioflavin T,(ThT) fluorescence intensity quantitative experiments, comparing the differences Amyloidosis on platelets between the HC group with AD group, including observation of amyloid levels AD group and the relationship between the HC group. As well as in the normal control group, platelets' ThT staining positive rate between different age differences.7. Using potein immunoblot(Western blotting or WB) confirmed that there were amyloidosis fiber aggregates on platelets' membrane in plasma and using the immunofluorescence test technology of the tssue section demonstrated amyloid fiber aggregates were existed and expressed at the beginning of the formation of the platelets.8. Obtain the ThT positive percent of total platelets in detecting platelets' specificity experiments between AD group and HC group. ThT positive percent of total platelet platelet individual number, and individuals' ThT positive count percentage accounted for the overall number of platelets.9. From the physical index of platelets and comparative analysis to explore the state of blood between AD and HC group and the changes of platelets' activity. Thus for clinical diagnosis of Alzheimer's disease to provide important basis. Results:1. Congo red(CR) test results show that the Congo red staining combined with polarized light that can read the myocardial tissue's specificity of amyloid fiber structure and the tissue section after thioflavin T dyeing found amyloidosis positive part and the amyloid deposition has been stained with Congo red parts is overlapped. The non-amyloid deposition area's thioflavin T fluorescent dye are negative, Thioflavin T fluorescent dye's sensitivity to amyloid fiber aggregates in tissue are even higher than that of Congo red stain, the above results show that the thioflavin T of and tissue amyloid fiber aggregates with high affinity, can be used for specific showed amyloid fiber aggregates structure.2. Using the myloidosis dyeing combining immune antibodies against A beta cells to explore specificity of ThT dyeing conditions in amyloid fiber aggregates in platelets. Dyeing results are not affected by composition of plasma's interference, dyeing method is simple and feasible.3. ThT probe and platelets' amyloid fiber aggregates on membrane have affinity reaction, can be clearly observed on platelets' amyloidosis; Thioflavin T fluorescent probes that can read the platelets' membrane specific beta amyloid fiber/aggregates and amyloid fiber tips on platelets.4. Platelets in the AD group and HC group, Thioflavin T fluorescent probes showed that all the dyeing test results are positive, specific to the individuals, there wrere no difference in the different age stages of platelets. They all also have positive results.5. Protein immunoblot(Western bloting or WB) technology confirmed that the beta amyloid fiber/aggregates were existed in plasma and platelets. In the AD group and HC group, the beta amyloid fiber/aggregates have the different expressing forms, the expression form of AD in the group are 15 kD, 70 kD and beta amyloidosis in the HC group, the expression form of fibers/aggregates are 43 kD and 26 kD.6. Through the platelets' physical index in Alzheimer's patients and normal healthy controls. The physical indexes of plasma D- dimer(D- Dim) and PCT plateletocrit(PCT) were significant differences between the two groups, the AD is greater than the HC group. The blood in amyloid related diseases is in a high coagulation state and platelets are pre-activated, fully confirmed that in Alzheimer's disease, the patient's blood state has changed, increasing the platelets' activity. Conclusions:1. By using the Thioflavine T specific identification can test the beta amyloid fibers/aggregates. Exploring a method which has the specificity to detect the amyloidosis in blood cells, realize the detection of platelet amyloid fiber, this method can be used to detect the amyloidosis in blood cell.2. This is the first time we have reported that the beta amyloid fibers were existed on platelets' membrane. Using amyloidosis fluorescent probe(ThT) to determinate the level of platelets' amyloid fibers/aggregates. Found that the content of beta amyloid fibers/aggregates existed on platelets in patients with AD is significantly higher than the healthy controls, with statistical significance. We also found the amyloid fibers deposited in platelets have the different forms in patients with AD and health control group.3 Analysis of the amyloid fibers with negative and positive morphological features and two groups of platelets' physical indicators, We have found that in the plasma, the D-dimer(D-Dim) and plateletocrit(PCT) were significant different between the two groups, platelets in patients with AD, amyloid fibers can lead to abnormal platelets' morphology. |
PDF Full Text Request