CN102229651A

CN102229651A - Amyloid protein intra-membrane segment for treating Alzheimer disease and application thereof

Info

Publication number: CN102229651A
Application number: CN2011101518654A
Authority: CN
Inventors: 邬力祥; 张松江; 韩仰
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2011-06-08
Filing date: 2011-06-08
Publication date: 2011-11-02

Abstract

The invention relates to an amyloid protein intra-membrane segment for treating Alzheimer disease and an application thereof, in particular to the application of the amyloid protein intra-membrane segment in the preparation of a vaccine medicament for treating Alzheimer disease. The problem of treating Alzheimer disease can be efficiently solved. The technical scheme provided by the invention is to choose an intra-membrane segment (IF-A beta segment), which is called as AIIGLMVGGVVIA for short, from amino acid sequences of amyloid proteins (A beta42) of 42 amino acids. In a use process, after the synthesized IF-A beta and a Freund adjuvant are mixed and injected to an immunized AD (Alzheimer Disease) model mouse, the A beta42 plaques can be efficiently eliminated, the cognitive function of the AD mouse can be improved and the defect of the sub-acute meningitis caused by using A beta42 as an immunogen can be overcome, thereby developing a therapeutic medicament for efficiently treating AD without side effect.

Description

A kind of interior fragment of amyloid film and application thereof for the treatment of alzheimer's disease

Technical field

The present invention relates to medical field, be specifically related to a kind of interior fragment of amyloid film and application thereof, especially application on the vaccine preparation of preparation treatment alzheimer's disease for the treatment of alzheimer's disease.

Background technology

Alzheimer's disease (Alzheimer ' s disease, important pathological characters AD) is the formation of A beta plaque in the brain.The gathering of A β is the trigger point of a series of pathology cascade reactions in the AD process in the brain.Mainly be from reducing the gathering of amyloid in the brain (amyloid-β, A β) by all kinds of means to the treatment research strategy of AD at present, these comprise and suppress APP and be metabolized to A β, disturb the gathering of A β and the removing that increases A β.Reducing A β deposition and improving the spatial cognition function aspects, A β vaccine presents the most attractive research prospect.The research report of the first routine A β vaccine shows that the active immunity of injecting A β 42 for the PDAPP mouse muscle has suppressed the development of A beta plaque in the mouse brain.But in testing in the clinical II phase, the subacute meningitis symptom has appearred in 6% experimenter, has caused the termination of clinical experiment.The pia mater lymphocytic infiltration is found in postmortem.

For fear of the generation of the such serious side effects of meningitis, many research groups are devoted to the research of immunogenic epi-position structure, the exploitation of adjuvant and different immunization routes, thus improve and innovate imperative.

Summary of the invention

At above-mentioned situation, for overcoming the prior art defective, the present invention's purpose just provides fragment and the application in the preparation of preparation treatment alzheimer's disease, the especially application in preparing the vaccine medicine in a kind of amyloid film, can effectively solve the treatment problem of alzheimer's disease.

A kind of interior segmental aminoacid sequence of amyloid film for the treatment of alzheimer's disease is AIIGLMVGGVVIA.It can be used for preparing the preparation for the treatment of alzheimer's disease; Particularly can be used for preparing the vaccine preparation for the treatment of alzheimer's disease.

The present invention chooses the aminoacid sequence of 42 amino acid whose amyloids (amyloid-β, A β 42):

Asp?Ala?Glu?Phe?Arg?His?Asp?Ser?Gly?Tyr?Glu?Val?His?His?Gln?Lys

1 5 10 15

Leu?Val?Phe?Phe?Ala?Glu?Asp?Val?Gly?Ser?Asn?Lys?Gly?Ala?Ile?Ile

20 25 30

Gly Leu Met Val Gly Gly Val Val Ile Ala (amino acid whose trigram abbreviation)

35 40

Abbreviate as: fragment (Intramembranous fragment of amyloid, IF-A β fragment) in the film among the DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (amino acid whose single-letter abbreviation):

Ala?Ile?Ile?Gly?Leu?Met?Val?Gly?Gly?Val?Val?Ile?Ala

1 5 10

Abbreviate as: AIIGLMVGGVVIA, during use,, can improve the cognitive function of AD mouse with IF-A β behind the synthetic and freund's adjuvant hybrid injection immunity AD model mice comprehensively, side effect does not take place, and can be effective to prepare the medicine for the treatment of alzheimer's disease; The aminoacid sequence of described A β 42 is sold by Beijing Bo Aosen biotech firm.

The present invention can effectively eliminate A β 42 patches, improves the cognitive function of AD mouse, and having overcome with A β 42 is the drawback that immunogen causes subacute meningitis, thus the medicine that finds effectively and have no side effect for the treatment of AD.

Description of drawings

Fig. 1 is for respectively organizing the ELISA OD value after pooled serum dilutes 640 times in the embodiment;

The determined with Morris water that Fig. 2 respectively organizes mouse after for immunotherapy in the embodiment is figure as a result;

Fig. 3 respectively organizes mouse IFN-γ content histogram in the embodiment;

Fig. 4 is for respectively organizing mouse brain section cortical area congo red staining (* 250) figure in the embodiment;

Fig. 5 is for respectively organizing mouse brain section A β 42 immunohistochemical methods results (* 250) figure in the embodiment;

Fig. 6 respectively organizes mouse brain cortex CD3 immunohistochemical methods figure in the embodiment

The positive CD3 lymphocyte of arrow indication (* 100);

Fig. 7 respectively organizes mouse hippocampus CD4 immunohistochemical methods figure in the embodiment

The arrow indication is CD4 positive lymphocyte (* 100);

Fig. 8 is immunity back mice serum and cerebral tissue solubility A β 42 content histograms in the embodiment.

Embodiment

Below in conjunction with embodiment the present invention is elaborated, and can not limit the present invention.

Choose 42 amino acid whose amyloid (amyloid-β, A β 42, sequence derives from Genebank)) aminoacid sequence: fragment (Intramembranous fragment of amyloid in the film among the DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA, IF-A β fragment, be called for short: IF-A β): AIIGLMVGGVVIA, during use, by inferior photo bio company limited of middle section IF-A β fragment is carried out synthetic, the result of treatment of the IF-A β behind the synthetic being carried out the AD model mouse detects, detect its influence to AD model mouse cognitive function, concrete steps are as follows:

1 materials and methods

1.1 key instrument and reagent microplate reader are available from Bio Tek company; The water maze self-control; (A β 31-42 aminoacid sequence: AIIGLMVGGVVIA) (KLH is from the isolating a kind of protein of sea mollusk megathyrid red corpuscle to IF-A β with KLH-IF-A β.It is at present as the most general carrier of proteantigen.) synthetic by the BeiJing ZhongKe Yaguang Biology Science Co., Ltd; BSA; RIPA; PMSF comes precious biotech firm available from Beijing rope; Freund's adjuvant is available from Sigma; Congo red available from Shanghai chemical reagent factory; DMSO, the anti-mouse two anti-IgG of alkali phosphatase enzyme mark horse are available from Beijing ancient cooking vessel state biotechnology limited liability company; A β 42, KLH-A β 42, CD3, CD4, CD8, CD19 and the anti-mouse IgG of A β 42 rabbits polyclonal antibody are available from Beijing Bo Aosen Bioisystech Co., Ltd; Instant SABC immunohistochemical staining test kit is available from Wuhan Boster Biological Technology Co., Ltd.; Mouse INF-γ and A β 42 quantitative elisa kit for detecting are available from the Shanghai scientific and technological cause of gloomy hero company limited; The RPMI1640 substratum is available from U.S. Gibco company.

1.2 method

1.2.1 28 APP transgenic mices of immune model mice are available from Institute of Experimental Animals, Chinese Academy of Medical Sciences (SPF level), at 6 monthly ages, male and female half-and-half are divided into IF50 group, IF100 group, 42 groups of A β and control group, 7 every group at random.Sub-cage rearing, free diet drinking-water.Keyhole limpet hemocyanin-IF-A β (KLH-IF-A β) and keyhole limpet hemocyanin-A β 42 (KLH-A β 42) respectively with the concentration of IF-A β 0.5mg/ml, IF-A β 1mg/ml and A β 421mg/ml with 1: 1 volume and freund's adjuvant thorough mixing.Dosage abdominal injection with 42/ of 50 μ g IF-A β/only, 100 μ g IF-A β/only and 100 μ g A β is inoculated in IF50 group, IF100 group and 42 groups of mouse of A β respectively.Inoculate first and use complete Freund's adjuvant, full freund's adjuvant for the second time and for the third time toos many or too much for use.Do not add freund's adjuvant for the last time.2 weeks were carried out the 2nd booster shots in the inoculation back first, carried out 1 booster shots later on every 1 month, inoculated altogether 4 times, and control group is only inoculated PBS and added the equal-volume freund's adjuvant.In the immunologic process, note to observe the diet of respectively organizing mouse, sleep, the mental status etc.The 10th day vena orbitalis posterior got blood in last inoculation back, and the preparation serum sample is used to detect humoral immune reaction.

1.2.2 the serum anti-amyloid beta antibodies detects utilization enzyme-linked immunosorbent assay (ELISA) and detects anti-A β 42IgG antibody titers in every group of pooled serum.A β 42 is dissolved in (15.1mmol/LNa in the coating buffer with the concentration of 10 μ g/ml ₂CO ₃, 34.7mmol/L NaHCO ₃, pH 9.0), bag is by 96 hole polystyrene Sptting plates (costar), 4 ℃ of overnight incubation.Washings (138mmol/L NaCl, 1.48mmol/L KH ₂PO ₄, 2.7mmol/L KCl, 8.12mmol/L Na ₂HPO ₄, 0.82mmol/LTween-20) wash twice after, with 37 ℃ of 1%BSA sealing 2h.Pooled serum is hatched bag by plate 2h for back 37 ℃ by dilution in 1: 640.2h is hatched for 37 ℃ with the anti-mouse IgG of the horse of alkali phosphatase enzyme mark in the washing back, and the washing back drips PNPP once more, reads the OD value with microplate reader at 405nm.Preimmune serum is done negative control, and the PBS substitute blood serum is done blank.

1.2.3 back one month of the last immunity of mouse cognitive function is carried out water maze laboratory to each group mouse after the determined with Morris water immunity.Circular channels, diameter 1.2m, depth of water 24cm, 22 ℃ ± 1 ℃ of water temperature, platform diameter 8cm, inboard wall of cistern are decorated with the black and white pattern, mark as thing.Swimming time and the surrounding environment of mouse every day remain unchanged.Platform is hidden in the quadrant 1.5cm under water during orientation navigation experiment.Mouse is cooked 4 training (putting into water from 4 quadrants respectively) every day, each 2min at interval.Test it and find the used time of platform, maximum time is 90s, when not finding above 90s, presses 90s and calculates, and manually guides mouse to arrive platform, trains altogether 4 days.The record above-mentioned parameter also carries out statistical analysis.

1.2.4 the preparation of splenocyte suspension and ELISA behind the chloral hydrate anesthesia mouse, get the spleen of mouse after detecting γ-IFN cognitive function detection of splenocyte release, smash to pieces respectively in the RPMI RPMI-1640,100 mesh sieves filter, 1000r.min ^-1Centrifugal 10min removes supernatant liquor; With 0.7% ammonium chloride hypotonic medium broken red blood cell, 1000r.min ^-1Centrifugal 10min removes supernatant liquor; Again with the washing of D-hanks liquid, 1200r.min ^-1Centrifugal 10min removes supernatant liquor, totally 2 times; Cell counting is with 1 * 10 ⁶.L ^-1, be inoculated in 8 well culture plates by group that (splenocyte of cultivation is by the animal grouping: 42 groups of control group, IF50 group, IF100 group and A β.Every group of culture plate, culture hole of every mouse is got 6 mouse, 1 a hole/mouse, 18 well culture plate/group), overnight incubation, treat cell attachment after, every group all adds A β 42 and stimulates, final concentration is 10 μ mol/L.Establish the blank hole for every group: only add nutrient solution RPMI1640; Negative control hole: only adding PBS stimulates.After cultivating 48h, collect nutrient solution.By the quantitative elisa kit for detecting description operation of mouse IFN-γ.

After 1.2.5 the paraffin section of brain prepares the cognitive function detection, mouse is anaesthetized with 10% Chloral Hydrate (0.35mg/kg), open chest and expose heart, puncture needle punctures to aorta ascendens through left ventricle, the about 10min of PBS (pH7.4) liquid 30ml irrigation vescular bed with 0.1mol/L gets the cerebral tissue left hemisphere that comprises hippocampus, through the fixedly routine paraffin wax embedding behind the 48h of 4% Paraformaldehyde 96, serial section (thick about 4 μ m) is attached on the anticreep slide glass, and 60 ℃ of baking box dried overnight are standby.

Routine dewaxes to water 1.2.6 congo red staining detection mouse brain senile plaque (1) will be cut into slices, (2) the Hematorylin dye liquor dyes 5min, (3) wash 30min from the beginning, (4) the Congo red dye liquor of methyl alcohol (Congo red 0.5g methyl alcohol 80ml, glycerine 20ml) dyes 5min, and (5) wash 20min from the beginning, (6) alkaline alcohol differentiation liquid (potassium hydroxide 0.2g, 80% alcohol 100ml differentiation) 10s, (7) wash 20min from the beginning, (8) conventional dehydration, transparent, neutral gum mounting.

Utilization ImageProPlusv6.0 image analysis software is carried out the photodensitometry of positive staining to image.

1.2.7 immunohistochemical methods detects the mouse brain senile plaque and brain lymphocytic infiltration immunohistochemical experiment operation steps is operated according to instant SABC immunohistochemical staining test kit:

When doing the A beta plaque and detecting, one anti-ly is the anti-mouse A of rabbit β 42 polyclonal antibodies (dilution in 1: 200); Two anti-are biotinylated goat anti-rabbit igg.Replace an anti-blank that carries out with PBS.

When doing the inflammation detection, the anti-anti-mouse CD3 of rabbit, CD4, CD8, the CD19 polyclonal antibody (dilution in 1: 200) of being respectively, they represent mature T lymphocyte, helper T lymphocyte, cytotoxic T lymphocyte and bone-marrow-derived lymphocyte respectively.Two anti-are biotinylated goat anti-rabbit igg.Replace an anti-blank that carries out with PBS.

Getting the similar connective tissue's section of each mouse section level observes.Every mouse selects a brain section, every section 5 not overlapping visuals field of picked at random under 20 times of object lens, and every group amounts to 35 visuals field.Calculating or scanning is this index positive cell number or the optical density value in each visual field wherein, carries out statistical analysis.

Inflammation detects with the positive cell counting process, and senile plaque detects utilization ImageProPlusv6.0 image analysis system optical density value calculating is carried out in the positive expression zone.

After 1.2.8ELISA solubility A β cognitive function detects in detection brain and the serum, the chloral hydrate anesthesia mouse, get the right hemisphere cerebral tissue that comprises hippocampus, after cleaning with physiological saline, add 1mLRIPA and 10 μ l PMSF (PMSF is now with now adding) by the 100mg cerebral tissue.Organize homogenate 15min at low temperatures, 4 ℃ of joltings, 60000r/min 30min, centrifugal 30min gets supernatant.Carry out protein quantification with the BCA test kit.

Adopt sandwich ELISA method to detect blood plasma and brain water dissolubility A β level.Trace routine is pressed the test kit description operation.

1.2.9 statistical data analysis SPSS11.0 software processes, experimental data is relatively used variance analysis with mean scholar standard deviation (mean scholar S.D.) expression between group, with P＜0.05 for statistical significance is arranged.Chart production uses the EXCEL7.0 statistical software.

2 results

Through totally 4 times immunization in 2.5 months, each diet, sleep of organizing mouse was normal substantially, and the mental status and fur glossiness are fair.Each organizes mouse body weight no significant difference.

2.1 the anti-A β of serum IgG titre detects the anti-amyloid beta antibodies IgG titre that was produced in the 10th day behind each immunization APP transgenic mice by the ELISA method, estimates the humoral immunization originality of IF-A β.The result shows with after having produced IgG, especially the 4th immunity of higher titre behind the IF-A β immune transgenic mouse.Control group does not produce any immune response.Pointing out this antibody is that IF-A β is specific.Pooled serum index duplicate detection 4 times is seen Fig. 1.Data are averaged ± S.D. (n=4).Compare with control group, ^*P＜0.01.

2.2 water maze with determined with Morris water AD transgenic models mouse through IF-A β and A β 422.5 months the cognitive function after totally 4 times the immunity change.It is the most obvious to find out obviously that from Fig. 2 cognitive function through IF50 group mouse after the immunotherapy improves, and A β takes second place for 42 groups, does not have notable difference between two groups; The IF100 group descends more on the contrary through treatment back cognitive function and control group, does not have notable difference with control group, but 42 groups obvious significant difference is arranged with IF50 group and A β. ^*, the IF50 group compares p＜0.01 with control group; #, the IF50 group compares p＜0.01. with the IF100 group

2.3 splenocyte discharges IFN-γ according to the typical curve formula, calculating is respectively organized mouse boosting cell suspension IFN-γ content and is seen Fig. 3.As can be seen from the figure each mouse IFN-γ content of control group is 0, and each experimental group IFN-γ content obviously raises, and wherein IF100 group content is the highest, and mean value reaches 56.49pg/ml; A β takes second place for 42 groups, and mean value reaches 32.84pg/ml; The IFN-γ of IF50 group is minimum in each experimental group, and mean value is 14.39pg/ml.The IF50 group compares for 42 groups with A β, and p＜0.001 is compared with the IF100 group in p＜0.01.

2.4 congo red staining is respectively organized mouse brain tissue slice congo red staining, as can be seen from Figure 4, senile plaque extensively is present in the interior and intercellular substance of cell in cortex zone.Compare with control group, after 2.5 months, senile plaque reduces the IF50 group to some extent through immunotherapy, 42 groups of IF100 group and A β, and senile plaque obviously reduces.

2.5A β immunohistochemical methods Fig. 5 is each group mouse brain tissue slice A β 42 immunohistochemical methods result.As can be seen from the figure, senile plaque mainly is present in intercellular substance, and the result is consistent with congo red staining.

Use CD3, CD4, CD8 and CD19 polyclonal antibody 2.6 inflammation detects, detect the CD cell, represent mature T lymphocyte, helper T lymphocyte, cytotoxic lymphocyte and bone-marrow-derived lymphocyte respectively.The result shows that the IF100 group detects volume CD3 and CD4 lymphocyte, and A β detects a small amount of CD3 and CD4 lymphocyte for 42 groups, and IF50 group and control group do not detect lymphocyte, see Fig. 6,7.Respectively organize at all and all not detect CD8 and CD19 lymphocyte (figure slightly) in the mouse brain.

2.7 mice serum and cerebral tissue solubility A β 42 content are as can be seen from Figure 8 compared with control group after the immunotherapy, the content that cerebral tissue solubility A β 42 is respectively organized in experiment all descends to some extent, and the content of serum soluble A β 42 all rises to some extent.Compare with control group, the variation of IF50 group and IF100 group solubility A β 42 all has statistical significance, p＜0.05, and the variation of 42 groups of solubility A of A β β 42 does not have statistical significance.

3 conclusions

With A β vaccine AD being carried out immunologic intervention is a very potential methods of treatment that delays even reverse the AD course of disease.For fear of the generation of meningitis side effect, different A β immunotherapy methods is attempted to seek by a lot of research groups.Though obtained certain achievement in research, do not had substantial clinical study report.

Theoretically, fragment is hidden in the cytolemma in the A β film, and the antibody that it produced can only be incorporated into and be secreted into the extracellular and be free on A β peptide in the tissue space, and can not be attached to the APP that is embedded on the cytolemma.So just avoid the attack of immunocyte and factor pair cytolemma, thereby avoided the generation of meningitis side effect.In addition, IF-A β is the little peptide that has only 13 amino-acid residues, and its epitope is limited, and the cross-immune reaction of other oneself protein is few in antibody that is produced and the body, avoids the attack to autologous tissue and cell that brings because of cross reaction.Based on these two theories, we design and synthesize fragment in the amyloid film, are applied to APP transgenic models mouse, the immunogenicity of observing it, result of treatment and side effect with it as immunogen.

Use 2.5 months AD model mices of IF-A β immunity, in serum, detected anti-A β 42IgG.Synthetic subunit I F-A β vaccine and complete the same equal A β 42 antibody that can produce high titre of peptide vaccine are described.The antibody titers content of IF100 group is apparently higher than 42 groups of IF50 group and A β.By the splenocyte release experiment, successfully detected INF-γ, illustrate that IF-A β also has cell immunogenicity.The INF-γ content of IF100 group is apparently higher than 42 groups of IF50 group and A β.From immunogenicity, IF50 group and IF100 group compare, and along with the increasing of immunizing dose, immunogenicity improves.The immunizing dose of three experimental group is by the mole Mass Calculation, and the immunizing dose of IF50 group is 41.2nmol, and the immunizing dose of IF100 group is 82.5nmol, and the immunizing dose that A β is 42 groups is 22.2nmol.42 groups of comparisons of IF50 group and A β, the former immune mole dosage be the latter near 2 times situation under, both body fluid is suitable with the cellular immunization effect, illustrates that the immunogenicity of IF-A β is lower than A β 42.

Showed by immune group result, IF50 group and control group mice brain almost do not detect lymphocyte, and IF100 and A β have tangible CD3 and CD4 lymphocytic infiltration for 42 groups, all each groups all do not detect CD8 and CD19 cell.Illustrate that 42 groups of IF100 and A β have tangible T lymphocytic infiltration.By the mole immunizing dose relatively, IF-A β is along with immunizing dose strengthens, the T lymphocytic infiltration appears in cerebral tissue, and cognitive function obviously descends, supposition is owing to produced lymphocytic infiltration in the brain, has caused inflammatory reaction, causes neurone and spongiocyte apoptosis and necrosis, illustrate that cellular immunization causes the mouse cognitive function to descend, and the immune response of suitable immunizing dose and appropriateness is extremely important for the treatment of AD.42 groups of IF50 group and A β relatively, the former immunizing dose is 2 times of the latter, humoral immunization originality is suitable, but the former the T lymphocytic infiltration do not occur, and cognitive function is better than the latter, and the immunogen for the treatment of as AD is described, IF-A β is superior to A β 42.

The at present relevant mechanism of anti-amyloid beta antibodies in the middle of the scavenging process of senile plaque is not also illustrated fully, but infers that according to limited experimental data and theoretical foundation the mechanism that following three aspects are arranged participates in: opsonization of (1) patch and microglia engulf destruction.(2) transportation of A β between brain and blood plasma and the change of running balance.This result of study shows that by after the immunotherapy solubility A β 42 reduces in the brain, and A β 42 content increase in the serum.Reason may be to pass through immune mouse, the antibody that produces in the recycle system with after A β 42 in the blood combines, the content of A β 42 in the blood reduces the solubility A β 42 that impels in the brain and transports to blood through hemato encephalic barrier, make A β 42 content rising in the blood, and solubility A β 42 content reduce in the brain.This indirect proof as a result the exactness of above-mentioned theory.(3) catalytic degradation effect: broken the depolymerization that the space structure of A β in the patch causes A β thereby anti-amyloid beta antibodies and senile plaque directly interact.(Proc Natl Acad Sci USA, 1997,94 (8): 4109-4112.) the experiment in vitro result shows that monoclonal antibody can suppress the formation of A beta plaque to Solomon etc.(J Neuroimmunol, 1998,88 (1-2): 85-90.) point out that also anti-amyloid beta antibodies can depolymerization A beta plaque and their toxicity of neutralizing such as Frenkel.

In sum, by above experiment, learn:

(1) IF-A β is under the immunizing dose of 50 a μ g/ mouse, and the mouse cognitive function obviously improves, and NIP reaction in the brain; (2) compare with A β 42, under the application dose of identical molar mass, IF-A β is more safe and effective.

These experiment in vitro result for future IF-A β be used for providing solid experimental basis in the AD of body treatment research.IF-A β is self integral part of A β 42, believes that it can overcome the inflammation side effect that A β 42 self brings as immunogen, thereby finds effectively and the good curing medicine of high specificity for the treatment of AD.

Claims

1. the interior fragment of amyloid film for the treatment of alzheimer's disease is characterized in that aminoacid sequence is AIIGLMVGGVVIA.

2. segmental application method in the amyloid film of the described treatment alzheimer's disease of claim 1 is characterized in that, is used to prepare the preparation for the treatment of alzheimer's disease.

3. application method according to claim 2 is characterized in that, is used to prepare the vaccine preparation for the treatment of alzheimer's disease.