Method of screening for RAGE-amyloid-beta peptide interaction inhibiting materials
An amyloid, RAGE-based technology, used in compound screening, biological testing, biomaterial analysis, etc.
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Embodiment 1
[0034] Example 1: Using mouse bEND.3 cells to establish a simulated blood-brain barrier system
[0035] 1-1. Selection of cell lines and culture conditions
[0036] The bEND.3 cell line (ATCC CRL2299) was used to construct a simulated blood-brain barrier. Cells that were subcultured early after 5 to 10 passages were used. In order to find culture conditions suitable for the formation of the same tight junctions as in the blood-brain barrier, bEND.3 cells were cultured at different cell densities for different culture times in 24-well Transwell plates (Corning).
[0037] At this point, cells were plated at 1 × 10 per well in a 24-well Transwell plate. 5 , 5×10 4 and 2.5×10 4 Cells were cultured to monitor cell status. When using 1×10 per well 5 When cultured at a density of 300 cells, the cells are too dense to adhere satisfactorily to the bottom of the well, and thus undergo differentiation. Under a light microscope, the cells were observed to be in poor condition. 2...
Embodiment 2
[0063] Example 2: Establishment of mouse bEND.3 cell line overexpressing human RAGE
[0064] The human RAGE gene (SEQ ID NO.5) was introduced into bEND.3 cells via a hygromycin selective vector.
[0065] First, the hygromycin selective vector is pcDNA3.1 / Hygro(+) vector (Invitrogen, Cat no.V870-20), such as Figure 7 shown. The human RAGE gene and the pcDNA3.1 / Hygro(+) vector were digested with two restriction enzymes Xho1 and Kpn1, respectively, followed by ligation to each other in the presence of ligase. The recombinant human RAGE-pcDNA3.1 / Hygro(+) vector thus obtained in Lipofectamin TMLTX reagent (Invitrogen, Cat. No. 15338-100) combined with Plus TM Reagent (Invitrogen, Cat. No. 11514-015) was used to transfect into bEND.3 cells. bEND.3 cells were cultured on plates containing hygromycin (200 μg / ml, AMRESCO, Cat. No. K547). Colonies formed after 2-3 weeks of incubation were picked and cultured in broth to establish a cell line expressing the human RAGE gene. Afte...
Embodiment 3
[0066] Example 3: Determination of the efficiency of antagonism of the transport of amyloid beta peptide using the simulated BBB
[0067] Fluorescent (FITC)-labeled amyloid β-peptide (β-α-amyloid β-protein, aa 1-42, FITC-conjugated / traced, Cat. No.M-2585.1000, Bachem) fluorescence to analyze the inhibitory activity of the candidate on RAGE.
[0068] Human RAGE-overexpressing cell lines were cultured in the upper inserts of Transwell plates at concentrations that formed tight junctions, after which the cells were divided into two groups. One was a negative control, to which only FITC-conjugated amyloid [beta] peptide was added. FITC-conjugated amyloid [beta] peptides and candidates were included in other panels. The fluorescence of the lower chamber was measured at regular intervals of 20 min over 120 min using a spectrofluorometer. The two groups were compared with respect to the mean values of the measurements ( Figure 4 ). Such as Figure 4 As shown, candidates 6,...
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