Study On The Fluorescence Imaging Of APP And Aβ Proteins Of Alzheimer's Disease With QDs-SA Quantum Dot

Objectives:Alzheimer's disease is a degenerative disease of the nervous system which is characterized by deficits in memory and cognition.The clinical diagnosis of AD depends on the clinical findings, scale measuring and image examination.However,because of low sensitivity and specificity,the conventional structure or function imaging techniques have not been widespread used yet in the field of clinical diagnosis of AD.Senile plaques is the characteristic pathological features. Amyloidβ-peptide as the chief component of senile plaques,has currently been identified that it existed in the Pathogenesis of early acute AD and its deposit areas and quantity were closely related to cognitive-assessing.Therefore,detection of Amyloidβ-peptide is meaningful for diagnosis of AD.Quantum Dot is a new fluorescence probe that has significant advantages in fluorescent intensity and ray stability when compared to traditional fluorescent dye.Thus,Quantum Dot may be used as a powerful method for Aβprotein detection and early diagnosis of AD.This study was designed to evaluate the biocompatibility and get the preliminary data of Quantum Dot used as a molecular probe of APP,Aβproteins.The pcDNA3.1/APP595/596 plasmid with coordinate bas-mutation was constructed and stably transfected into HEK293 cells to set up the cell model of AD.Then,Quantum Dots were targeted into APP,Aβproteins respectively and the biocompatibility as well as fluorescence intensity and duration of Quantum Dot were compared with that of traditional fluorochromes.Methods:1) The pcDNA3.1/APP plasmid was gifted by Pro.Perez, University of Pittsburgh,USA.The Coordinate bas-mutations(G→T,A→C) and a accidental mutation(C→G) had been confirmed by DNA sequencing.In this experiment,APP was PCR-amplified,digested by HindⅢand XbaⅠ,and ligated into purified pcDNA3.1 vector.This recombinant plasmid was transfected into E.colo SH5αby means of heat shock.The recombinant plasmid was identified by PCR,and the positive cloning was identified by DNA sequence analysis.2) The construct was transfected into HEK293 cell lines,screened by G418.The interest protein(including APP and Aβ) were identified by immunofluorescence and Western blot.3) The toxicity of QDs-SA to HEK293 cells stably transfected pcDNA3.1/APP595/596 plasmid was investigated by cell morphology,MTT analysis and flow cytometry technology.4) With the help of Laser Scanning Confocal Microscope and Flow Cytometry,this kind of flurescence probe based on the QDs-SA had been used to detect APP and Aβproteins in HEK293 cells stably transfected pcDNA3.1/APP595/596 plasmid,and compared with conventional fluroimmunoassay.Results:1)The positive cloning gene sequence were aligned to the complete gene sequence of APP by DNAStar software.The result confirmed that the accidental mutation(C→G) had been mutated back(G→C).The recombinant pcDNA3.1/APP595/596 plasmid was constructed successfully.2)The detection verified that recombinant plasmid group expressed APP protein,the specific protein bands was only detected in HEK293 Cells stably transfected pcDNA3.1/APP595/596 plasmid,but no expression was found in control groups.The immunofluorescence staining detection indicated APP expression in the plasma membrane,but the Aβmainly located in the endochylema near the cell membrane,and slight Aβexpression also located in the plasma membrane.3) The HEK293 cells were incubated with QDs-SA on different concentrations between 2.5nm and 25nm and lasted for 24 hours, there was no evident morphological deviations.The QDs-SA group's cell activity assayed by MTT had no any apparente difference compared to control group.Meanwhile,the difference of absorbance intensity among the different concentration groups had no any statistical significance(P＞0.05 ).We used AnnexinV/FITC staining flow cytometry to observe the percentage of apoptosis.The percentage of apoptosis had no any significant difference among groups(P＞0.05 ).4) under the stimulation by 388nm excitation light,The maximal Emission of the QDs-SA was 605±10nm,and the full width at half maximum＜35nm.The mean fluorescence intensity of QDs-SA was greater than the Cy3's under confocal fluorescence microscopy(P＜0.05) There was no notable quench of exciting quantum dots for 12 minutes,and the APP and Aβproteins stained by QDs-SA showed intensive orange red fluorescence under confocal fluorescence microscopy.The fluorescence intensity decreased nearly 27.87%,29.25%respectively.The other stained by Cy3 decreased 79.60%and 76.82%.The positive rate of APP staining had no significant difference between the QDs-SA and Cy3 by flow cytometry, but the Mean fluorescence intensity had statistical significance(P＜0.05).Conclusions:1) The recombinant plasmid containing the APP 595/596 Coordinate bas-mutations was successfully constructed.Then the recombinant plasmid was stably transfected into HEK293 cells to construct the transgenic cellular model of Alzheimer's disease.2) The QDs-SA were biocompatible with HEK293 cells in appropriate concentration rang,which provided basis for further biological applications of QDs.3) The QDs-SA fluorescence probes can effectively recognize APP and Aβproteins and exhibited good sensitivity and exceptional photostability,suggesting that QDs-SA fluorescence probes could be a potential method in Aβdetection and offer a novel way for the diagnosis of Alzheimer's disease.