CN102516357B - Polypeptide combined with A-beta amyloid and application thereof - Google Patents
Polypeptide combined with A-beta amyloid and application thereof Download PDFInfo
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Abstract
The invention discloses a polypeptide combined with A-beta amyloid and an application thereof. The polypeptide is a polypeptide shown as (1) or (2), wherein (1) is constituted by an amino acid sequence shown as a sequence 1 in a sequence table, or (2) is obtained by performing substitution and/or deletion and/or addition of one or more amino acid residues on an amino acid residue sequence shown as a sequence 1 in the sequence table, has the same function, and is derived from the sequence 1. As proved by an experiment, the polypeptide passes through a blood brain barrier easily, has lower antigenicity than other large molecular proteins such as antigens and the like, has a small side effect, and has a wide application prospect on the aspect of treatment of an Alzheimer's disease.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of polypeptide and application of combination A-beta amyloid.
Background technology
Alzheimer's disease Alzheimer ' s Disease, hereinafter to be referred as AD, be commonly called as senile dementia, that the elderly who is caused by the aggregation of amyloid-beta monomer molecule (β-Amyloid 40/42 (A β 40/42, hereinafter to be referred as A β)) gathering formation tool toxic action mainly forms the nerve degenerative diseases of senile plaque as feature take hypomnesis and brain.Medical statistics shows, in China and American-European countries, more than 60 years old the elderly has 5～6% to suffer from A Zihaimo disease.This disease has been listed in and has caused dead the fourth-largest disease, is only second to heart trouble, cancer and apoplexy.The existing more than 60 years old population approximately 100,000,000 of China, is 5% estimation according to more than 60 years old population person in middle and old age dementia morbidity, and may there be patients with Alzheimer disease approximately 5,000,000 in the whole nation.Gai Bingyigei family and society bring huge burden.Researchist's estimation, if prophylactico-therapeutic measures can not get improving, the former senile dementia in the 4th of Death causes, by becoming 21 century senior health and fitness's No.1 killer, is suffered from senile dementia by the over-65s old man who has 1/3rd.At present, the clinical treatment of AD is just applied to some neurotrophies and anti-inflammatory type medicine carries out symptomatic treatment, still emerge without any the medicine of specific treatment AD.
Based on the pathogenesis of A β, at present the research of AD treatment preparation is mainly started with from three aspects: reduce A β generation, the gathering that suppresses A β and the removing of cytotoxicity and quickening A β by suppressing enzymatic reaction.A β is mainly made up of 39-42 amino-acid residue, and wherein A β 42 gatherings are very fast, cytotoxicity is larger, is main AD paathogenic factor.It comes from the precursor protein that molecular weight is 110-135KD (Amyloid Precursor Protein, APP).APP produces the aminoterminal of A β through the effect of beta-secretase, in after birth, gamma secretase effect produces the carboxyl terminal of A β.Under normal circumstances, before beta-secretase effect, the Lys16-Leu17 place of α Secretases in the middle of A β is cracked into APP APPs α and is embedded in carboxyl fragment C83 shorter on film.APPs α has growth regulating and neurotrophic effect, can extend neuronic survival, stablizes and strengthen the structure of cynapse, strengthens neuronic function etc.In the situation that body is abnormal, β, gamma secretase increased activity, or α secretase activity reduces, and will produce the A β of more amount.Already reported, many materials comprise that gathering and the cytotoxicity to A β such as mineral compound, organic compound, polypeptide, albumen and antibody has restraining effect in various degree.The polypeptide of report is the hydrophobic fragment in centre of A β or the derivative of C-terminal mostly, the region that they contain with A β is combined and the region that stops A beta peptide aggregation.Separately there is external gathering and the cytotoxicity that can suppress A β of several polypeptide that screened by phage display technology, but there is not yet the report playing a role in vivo.
In cerebral tissue, A β can have several approach to discharge or degraded: degrade A β through insulin-degrading enzyme (IDE), enkephalinase (NEP), endothelin converting enzyme (ECE), Zinc metallopeptidase Zace1 (ACE) effect (1); (2) be expelled to blood circulation through LRP acceptor; (3) in brain, microglia and astroglia cell are engulfed degraded; (4) discharge neural system through other approach.Under normal circumstances, in cerebral tissue, the generation of A β and discharge or degraded are keeping running balance.When A β in cerebral tissue produces too much, or removed slowly, will cause the rising of A β level, brought out the generation of AD.Thereby the preparation that can increase A β removing also has potential AD therapeutic action.
Summary of the invention
An object of the present invention is to provide a kind of polypeptide of combination A-beta amyloid.
Polypeptide provided by the invention is following 1) or 2):
1) polypeptide being formed by the aminoacid sequence shown in sequence in sequence table 1;
2) by aminoacid sequence shown in sequence in sequence table 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and with the polypeptide being derived by sequence 1 with identical function.
In above-mentioned albumen, the replacement of one or several amino-acid residue and/or disappearance and/or interpolation refer to replacement and/or disappearance and/or the interpolation of no more than ten amino-acid residues.
Above-mentioned 2) polypeptide shown in is any one in following a, b, c:
A, the polypeptide being formed by the aminoacid sequence shown in sequence in sequence table 2;
B, the polypeptide being formed by the aminoacid sequence shown in sequence in sequence table 3;
C, the polypeptide being formed by the aminoacid sequence shown in sequence in sequence table 4.
Another object of the present invention is to provide a kind of A of inhibition β cytotoxicity preparation.
Inhibition A β cytotoxicity preparation provided by the invention, its activeconstituents is aforementioned polypeptides.
In above-mentioned inhibition A β cytotoxicity preparation, described cell is specially SH-SY5Y cell, and described A β is specially A β 42.
Suppressing A β cytotoxicity is to suppress the toxicity of A β to cell.
The 3rd object of the present invention is to provide a kind of preparation that promotes microglia removing A β.
Promotion microglia provided by the invention is removed the preparation of A β, and its activeconstituents is aforementioned polypeptides.
Remove in the preparation of A β above-mentioned promotion microglia, described microglia is specially BV-2 cell; Described A β is specially A β 42.
The 4th object of the present invention is to provide a kind of product for the treatment of alzheimer's disease.
Product provided by the invention, its activeconstituents is aforementioned polypeptides; Described product is specially medicine.
The application of aforementioned polypeptides in the cytotoxicity and/or the preparation inhibition A β cytotoxicity preparation that suppress A β is also the scope of protection of the invention; Described cell is specially SH-SY5Y cell, and described A β is specially A β 42.
Aforementioned polypeptides is also the scope of protection of the invention in the application that promotes microglia to engulf A β or to prepare in the preparation that promotes microglia to remove A β; Described A β is specially A β 42.
The application of aforementioned polypeptides in the product of preparation treatment alzheimer's disease is also the scope of protection of the invention; Described product is specially medicine.
Aforementioned polypeptides is embodied in the quantity that improves spatial memory capacity, reduces senile plaque in brain and reduces A β content in the effect of the product of preparation treatment alzheimer's disease; Described A β is specially A β 40 or A β 42.
Of the present inventionly experiment showed, that polypeptide molecular weight of the present invention is little, can specific binding A β 42, and suppress the cytotoxicity of A β 42, protection SH-SY5Y neuroblastoma cell is avoided the impact of A β 42 toxicity.Animal memory experiment is carried out in the brain side room that this polypeptide is expelled to AD transgenic mice (APPswe/PS1dE9 double transgenic), result shows, it can obviously improve the spatial memory capacity of mouse, reduces the quantity of senile plaque in brain and reduces A β 40 and the level of A β 42.This polypeptide is easily through hemato encephalic barrier, and it is as poor in antigenicities such as antibody to compare other high molecular weight protein, and side effect is less, aspect the prevention of alzheimer's disease and treatment, is being with a wide range of applications.
Accompanying drawing explanation
Fig. 1 is that the ELISA of XD4 and A β 42 combinations measures
Fig. 2 is through displacement, lacks or insert the ELISA mensuration that amino acid whose XD4 is combined with A β
Fig. 3 is the cytotoxicity that XD4 suppresses A β 42
Fig. 4 is the generation that XD4 suppresses the ROS being caused by A β 42
Fig. 5 is the generation that XD4 suppresses the NO being caused by A β 42
Fig. 6 is the rising that XD4 suppresses the intracellular calcium concentration being caused by A β 42
Fig. 7 is the water maze laboratory of the AD transgenic mice of injection XD4 polypeptide
Fig. 8 is the variation of the AD transgenic mice brain senile plaque of injection XD4 polypeptide
Fig. 9 is the variation of the AD transgenic mice brain senile plaque area of injection XD4 polypeptide
Figure 10 is solubility A β 40 levels in the AD Transgenic Mice Brain of injection XD4 polypeptide
Figure 11 is solubility A β 42 levels in the AD Transgenic Mice Brain of injection XD4 polypeptide
Figure 12 is insoluble A β 40 and A β 42 levels in the AD Transgenic Mice Brain of injection XD4 polypeptide
Figure 13 is that XD4 promotes cell engulfing A β
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
(formula is the damping fluid that PBS ph value of buffer solution in following embodiment is 7.2: 5.84g NaCl, 4.72g Na
2hPO
4, 2.64g NaH
2pO
4.2H
2o, water is made into 1 liter, adjusting pH is 7.2);
All experimental datas in following embodiment are all by 3 independently experiment acquisitions except water maze memory experiment, and experimental data is expressed as mean number plus-minus standard deviation.The statistical study of data is that application One-way ANOVA software carries out, and applies the Two-way ANOVA of repeating data for the comparative analysis of many groups repetitive memory power determination data.
The acquisition of embodiment 1, polypeptide XD4 and derivative thereof
1, the acquisition of polypeptide XD4
According to the nosogenesis of alzheimer's disease AD, the basic method for the treatment of AD should be the generation that reduces amyloid beta (A β), the gathering that suppresses A β, cytotoxicity or the removing of accelerating A β.The present invention's application phage display technology, take A β 42 monomers as screening substrate, by 1 × 10
8kind of seven peptides have carried out four-wheel elutriation, application Phage-ELISA therefrom filtered out many can with the polypeptide of A β 42 obvious combinations, by comparing the binding characteristic of many peptide chains and A β 42 and on A beta peptide aggregation and Cytotoxic impact, finally selecting polypeptide XD4.
The sequence of polypeptide XD4 is: Pro-Ile-Lys-Thr-Leu-Pro-Met (sequence 1).
Polypeptide XD4 is seven peptides, (preparation method is referring to Weng C C to synthesize according to a conventional method preparation by the biochemical company limited of Shanghai gill, Peter D W.Fmoc Solid Phase Peptide Synthesis:A Practical Approach.Oxford:University Press, 2000.1-8; Atherton E, Sheppard R C.Solid Phase Peptide Synthesis:A Practical Approach.Oxford:IRL Press, 1989); XD4 polypeptide purity is that 95%, XD4 is stored in-20 ℃, avoids multigelation.
The synthetic polypeptide XD4 of 6 Histidines as label that be connected with simultaneously.
2, the acquisition of polypeptide XD4 derivative
The derivative of XD4 be by the amino acid of XD4 through displacement, lack or insert the polypeptide that other amino acid obtains, specific as follows:
XD4-Δ M: be the polypeptide that the polypeptide disappearance methionine(Met) of XD4 (sequence 1) is obtained, aminoacid sequence is: Pro-Ile-Lys-Thr-Leu-Pro (sequence 2, PIKTLP), also can synthetic;
XD4-PM/TQ: for the proline(Pro) of XD4 (sequence 1) and methionine(Met) being replaced as respectively to the polypeptide that Threonine and glutamine obtain, aminoacid sequence is: Thr-Ile-Lys-Thr-Leu-Pro-Gln (sequence 3, TIKTLPQ), also can synthetic;
XD4-N: for inserting the polypeptide that l-asparagine obtains in XD4 (sequence 1), aminoacid sequence is: Pro-Ile-Lys-Thr-Asn-Leu-Pro-Met (sequence 4, PIKTNLPM), also can synthetic.
Synthetic polypeptide XD4-Δ M, XD4-PM/TQ and the XD4-N of 6 Histidines as label that be connected with simultaneously.
The application of embodiment 2, polypeptide XD4
One, the specific binding of XD4 and derivative thereof and A β 42
1, the specific binding of XD4 and A β 42
Respectively by A β 42 (the American Peptide Co. of PBS damping fluid preparation, 62-0-80B) and negative control polypeptide (sequence is: SMSARQL is synthetic by the biochemical (Shanghai) Co., Ltd. of gill) with the coated 96 hole Aminated ELISA Plates in 1 μ g/ hole, 4 ℃ are spent the night.With the blank site of not being combined with polypeptide on BSA sealase target, then add respectively and be connected with the XD4 that by embodiment 1 obtained 1,0.5,0.25,0.125, the 0 μ g/ hole of 6 Histidines as label, room temperature (25 ℃) effect 2h.After washing, add can with the antibody of the coupling HRP of histidine-tagged combination, room temperature effect 1h.After washing, add substrate TMB, develop the color after 20 minutes, measure the absorbancy at 450nm place with multi-functional microplate reader.
Under identical condition, in triplicate, the results are shown in Figure 1, wherein, the column diagram A β 42 in left side is the specific binding of XD4 and A β 42, and the column diagram contrast polypeptide on right side is the specific binding of XD4 and negative control polypeptide; In figure, data are the mean value of three times, and error bar is SD.
Can find out from the column diagram in left side, 1,0.5,0.25,0.125,0 μ g/ hole XD4 respectively with A β 42 in conjunction with after OD450nm be 0.37,0.35,0.25,0.14,0.08;
Can find out from the column diagram on right side, the OD450nm after 1,0.5,0.25,0.125,0 μ g/ hole XD4 is combined with negative control polypeptide is respectively 0.08,0.09,0.06,0.07,0.06;
Can find out, compared with negative control polypeptide, XD4 can with A β 42 specific bindings.
2, the derivative of XD4 and A β 42 specific combination
Respectively the above-mentioned polypeptide XD4-Δ M being obtained by embodiment 1, XD4-PM/TQ, XD4-N are connected histidine-taggedly, then detect and the binding characteristic of A β 42 according to above-mentioned 1 test method, the every hole of each polypeptide adds 1 μ g/ hole, measures OD value by ELISA method.
The results are shown in Figure 2, wherein, the derivative that the column diagram A β 42 in left side is XD4 and the specific binding of A β 42, the derivative that the column diagram contrast polypeptide on right side is XD4 and the specific binding of negative control polypeptide;
Can find out from the column diagram in left side, XD4-Δ M, XD4-PM/TQ, XD4-N respectively with A β 42 in conjunction with after OD450nm be 0.30,0.25,0.23;
Can find out from the column diagram on right side, the OD450nm after XD4-Δ M, XD4-PM/TQ, XD4-N are combined with negative control polypeptide is respectively 0.04,0.03,0.04;
Show that certain amino acid of XD4 still retains and the ability (compared with negative control, *, P < 0.01) of A β 42 combinations through displacement, disappearance or increase amino acid in polypeptide after.
Two, XD4 suppresses the cytotoxicity of A β 42
1, the cytotoxicity of XD4 vitro inhibition A β 42
With the substratum (MEM containing 10% foetal calf serum, Invotrigen, 12561-056) by SH-SY5Y cell (Chinese Academy of Medical Sciences's tumour cell storehouse, production number is SH-SY5Y) be made into individual cells suspension, be inoculated into 96 porocyte culture plates with 10000, every hole cell, every pore volume 100 μ L.Cell is in 37 ℃ of cultivations 24 hours, incubator CO
2concentration is 5%.Every hole adds following sample:
Do not hatch A β 42 (A β): the final concentration of A β 42 albumen is 1 μ M;
Do not hatch A β 42 and XD4 mixture (A β: XD4=1: 1): the final concentration of A β 42 albumen is 1 μ M, the final concentration of XD4 is 1 μ M;
Do not hatch A β 42 and XD4 mixture (A β: XD4=1: 10): the final concentration of A β 42 albumen is 1 μ M, the final concentration of XD4 is 10 μ M;
Hatch A β 42 (hatching A β for 24 hours): the final concentration of A β 42 albumen is 1 μ M;
Hatch A β 42 and (within 24 hours, hatch A β: XD4=1: 1): the final concentration of A β 42 albumen is 1 μ M, the final concentration of XD4 is 1 μ M with the mixture of XD4;
Hatch A β 42 and (within 24 hours, hatch A β: XD4=1: 10): the final concentration of A β 42 albumen is 1 μ M, the final concentration of XD4 is 10 μ M with the mixture of XD4;
Above-mentioned hatching as A β 42 is hatched to 24h at 37 ℃.
Take PBS as contrast;
Above-mentioned 7 groups of cells were continued to cultivate after 48 hours, every hole adds MTT solution (Sigma, catalog number is that M5655, solvent are PBS, 5mg/mL) 10 μ L, hatch 3 hours for 37 ℃, stop cultivating, every hole adds 100 μ L dissolution of crystals liquid (10%SDS and 5% isopropylcarbinol are dissolved in 0.01M HCL), 37 ℃ of overnight incubation (12h), fully dissolve crystallisate.On multi-functional enzyme-linked immunosorbent assay instrument, measure each hole absorbance value with 570nm wavelength.Deduct after background, using the absorbancy of sample divided by the absorbancy of cell contrast that does not add sample as the activity index of cell, and significant difference situation between analytic sample.
Result is (*, P < 0.05) as shown in Figure 3,
The cytoactive of not hatching A β 42 (A β) group is 70.88%;
Do not hatch mixture (the A β: XD4=1: 1) cytoactive of group is 82.03% of A β 42 and XD4;
Do not hatch mixture (the A β: XD4=1: 10) cytoactive of group is 87.79% of A β 42 and XD4;
The cytoactive of hatching A β 42 (hatching A β for 24 hours) group is 76.46%;
Hatch A β 42 and (within 24 hours, hatch A β: XD4=1: 1) cytoactive of group is 79.84% with the mixture of XD4;
Hatch A β 42 and (within 24 hours, hatch A β: XD4=1: 10) cytoactive of group is 81.24% with the mixture of XD4;
As can be seen from the above, A β 42 cytotoxicities after hatching reduce, and XD4 does not show the effect of remarkable reduction A β 42 toxicity.Do not hatch compared with A β 42 samples with independent, XD4 adds the activity that can significantly improve cell, shows that XD4 can suppress the cytotoxicity of A β 42.
2, XD4 suppresses the generation of the ROS being caused by A β 42
According to the cultivation SH-SY5Y cell 24h in above-mentioned 1, then apply following three groups of culture medium culturing cell 12h:
42 groups of A β: the serum free medium (Hyclone, catalog number: SH30022.01B) that contains 10 μ M A β 42;
A β 42: XD4 (mol ratio)=1: 1 group: the serum free medium (Hyclone, catalog number: SH30022.01B) that contains 10 μ M A β 42 and 10 μ M XD4;
A β 42: XD4 (mol ratio)=1: 10 group: the serum free medium (Hyclone, catalog number: SH30022.01B) that contains 10 μ M A β 42 and 100 μ M XD4;
Remove substratum, application PBS washed cell three times, add 2 of 10mM ', 7 '-dichlorofluorescein diacetate (DCFH-DA, green skies biotechnology research institute, catalog number: S0063), hatch 20min for 37 ℃, application PBS washed cell is removed unnecessary DCFH-DA three times.On multi-functional enzyme-linked immunosorbent assay instrument, measure fluorescence intensity take exciting light as 488nm utilizing emitted light as 525nm.The generation of ROS is to represent with respect to the per-cent of cell blank contrast.
As shown in Figure 4, the relative intensity of fluorescence that A β is 42 groups is 142.46% to result;
The relative intensity of fluorescence of A β 42: XD4 (mol ratio)=1: 1 group is 139.7%;
The relative intensity of fluorescence of A β 42: XD4 (mol ratio)=1: 10 group is 111.99%;
Can find out, A β 42 can make the ROS of cell produce to raise, can make ROS level significantly decline (compared with adding separately the sample of A β 42, *, P < 0.05) after adding XD4.
3, XD4 suppresses the generation of the NO being caused by A β 42
According to the cultivation SH-SY5Y cell 24h in above-mentioned 1, then apply following three groups of culture medium culturing cell 12h:
42 groups of A β: the serum free medium (Hyclone, catalog number: SH30022.01B) that contains 10 μ M A β 42;
A β 42: XD4 (mol ratio)=1: 1 group: the serum free medium (Hyclone, catalog number: SH30022.01B) that contains 10 μ M A β 42 and 10 μ M XD4;
A β 42: XD4 (mol ratio)=1: 10 group: the serum free medium (Hyclone, catalog number: SH30022.01B) that contains 10 μ M A β 42 and 100 μ M XD4;
Remove 60 μ L substratum, add isopyknic Griess reagent (1% sulfanilamide, 0.1% naphthylethylene diamine is dissolved in 2.5% phosphoric acid solution), room temperature (25 ℃) effect 10min.Measure the photoabsorption (the OD value contrasting using cell, as 100%, is calculated the OD relative value of each sample) at 545nm place.
The OD relative value that the results are shown in Figure 42 groups of 5, A β is 204%;
The OD relative value of A β 42: XD4 (mol ratio)=1: 1 group is 180.63%;
The OD relative value of A β 42: XD4 (mol ratio)=1: 10 group is 140.65%;
Can find out, A β 42 can make the NO of cell produce to raise, can make NO level significantly decline (compared with adding separately the sample of A β 42, *, P < 0.05) after adding XD4.
4, XD4 suppresses the rising of the intracellular calcium concentration being caused by A β 42
According to the cultivation SH-SY5Y cell 24h in above-mentioned 1, then apply following three groups of culture medium culturing cell 12h:
42 groups of A β: the serum free medium (Hyclone, catalog number: SH30022.01B) that contains 10 μ M A β 42;
A β 42: XD4 (mol ratio)=1: 1 group: the serum free medium (Hyclone, catalog number: SH30022.01B) that contains 10 μ M A β 42 and 10 μ M XD4;
A β 42: XD4 (mol ratio)=1: 10 group: the serum free medium (Hyclone, catalog number: SH30022.01B) that contains 10 μ M A μ M β 42 and 100 μ M XD4;
Add fluorochrome Fluo-3/acetoxymethyl ester (catalog number is: S1056 for Fluo-3/AM, the life of green skies biotechnology research), put 37 ℃ of dark places 30min.On multi-functional enzyme-linked immunosorbent assay instrument, take exciting light as 485nm, utilizing emitted light is that 538nm measures fluorescence intensity.
The relative intensity of fluorescence that the results are shown in Figure 42 groups of 6, A β is 247.25%;
The relative intensity of fluorescence of A β 42: XD4 (mol ratio)=1: 1 group is 165.07%;
The relative intensity of fluorescence of A β 42: XD4 (mol ratio)=1: 10 group is 156.70%;
Can find out, A β 42 can make intracellular calcium concentration raise, and can make intracellular calcium concentration significantly decline (compared with adding separately the sample of A β 42, *, P < 0.05) after adding XD4.
Because the generation of AD also with ROS, NO, that metabolic defect in cellular calcium ion concentration is unbalance etc. is relevant, these experiments simultaneously also can explain that from some aspect XD4 suppresses the cytotoxic effect of A β 42.
Three, XD4 improves the effect embodiment of AD disease
1, improve the memory capability of AD transgenic mice
Experimental technique:
1) brain side room injection: the AD female mice that turns APP and PS1 gene (APPswe/PS1dE9) large 8 monthly ages (is turned to APP and PS1 gene (APPswe/PS1dE9) mouse purchased from Jackson Lab (Bar Harbor, ME, the U.S., catalog number is: 004462), after breeding through identifying that the mouse that obtains containing APP and PS1 gene is for turning the AD mouse of APP and PS1 gene (APPswe/PS1dE9)), be divided at random injection polypeptide XD4 (AD+XD4) and injection PBS group (AD+con), 8 every group.(turn APP and PS1 gene (APPswe/PS1dE9) mouse purchased from Jackson Lab with 8 identical brood wild-type mices of age as control group simultaneously, Bar Harbor, ME, the U.S., catalog number is: 004462, after breeding through identifying that the mouse that obtains not containing APP and PS1 gene is wild-type mice; WT+con).
Above-mentioned three groups of mouse are handled as follows:
Before intracerebral injection, 12h can't help water to mouse fasting.First by mouse anesthesia, anaesthesia dosage is: the Chloral Hydrate that 25g mouse peritoneal injection 0.1mL concentration is 10%.Mouse is fixed on stereotaxic instrument, and reference standard collection of illustrative plates (Academic Publish) carries out side room, the three-dimensional location of brain drug administration by injection.Mouse intracerebroventricular positional parameter is: 1.8mm after anterior fontanelle, the other open ± 1.8mm of center line, 2.5mm under dura mater.With sterilized water dissolving XD4 to 1mg/ml, injection speed 0.2 μ l/min, injected dose is 5 μ L.The 5min that injected that let the acupuncture needle remain at a certain point.Every injection in 7 days once, inject altogether 4 times.In last injection latter 5 days, mouse is carried out to the detection of water maze memory capability.
2) memory training: be placed on 25 ℃ of room temperatures, the environmental adaptation of humidity 46% 3 days before Mice water maze training.All study of behaviour detect the mode that all adopts randomized, double-blind in test.Before training, remove platform, at tank, central authorities put into gently, allow its 60s that freely moves about.Measure every group of mouse swimming quadrant preference, select the wall of opposite tank, as this mouse initial release position.Before training, allow for the first time mouse stand in the upper 15s of platform (10 centimetres of platform diameter), allow it remember the locus of pond (1.1 meters of diameters) middle platform.Platform is placed on the second quadrant middle part, and the upper surface of platform is apart from 1.5 centimetres of the waters surface.Chi Shuizhong adds milk powder, to increase the visual contrast of animal, is beneficial to image recording.Mouse is faced to pool wall, put into gently water, be allowed to condition at tank went swimming.The mouse 2s that stands on platform just stops timing, thinks and appears on the stage, and the training time is at every turn the longest is 90s.Application software (purchased from medicine institute of Chinese medical courses in general institute) records its track and from entering water to the time of climbing up platform, i.e. latent period during this time.If mouse is found platform in 90s, be allowed to condition at and on platform, stop 10s.If mouse can not find platform in 90s, after 90s, guide its upper mounting plate by experimenter, and be allowed to condition at and on platform, stop 10s.Continuously training 5 days, trains 2 every day, between twice, is spaced apart 3-4h.Then interval 24h, removes platform, allows mouse in water, find platform 90s.With the result of video recording and the training in 5 days of software system record and exploration experiment, and process experimental result with the amount of repetition number two-factor analysis of variance (two-way ANOVA, repeated measures).
As shown in Figure 7, along with the growth of training time, control group mice finds shorten gradually the latent period of platform to experimental result; Specific as follows: the AD mouse of injection XD4 finds the latent period of platform in the time training by the 3rd, 4,5 days, the AD mouse contrasting with injection PBS is compared has significant difference (compared with injection PBS AD mouse group, *, P < 0.05), show to inject AD mouse spatial memory capacity after the XD4 control group (Fig. 7 A) significantly better than injection PBS.Removing the AD mouse of injecting XD4 after platform finds and is also significantly less than AD control group (Fig. 7 B) latent period of platform.The AD mouse of injection XD4 is significantly higher than AD control group (Fig. 7 C) through the number of times of platform position, and the time at AD mouse place in target quadrant after injection XD4 is significantly higher than AD control mice (Fig. 7 D).These results show to inject AD mouse spatial memory capacity after XD4 significantly better than the AD control group of injection not.
2, XD4 reduces the quantity of senile plaque in AD Transgenic Mice Brain
The impact of application ThS fluorescent dye measuring XD4 on senile plaque in AD Transgenic Mice Brain:
1) by above-mentioned 1 injection polypeptide XD4 (AD+XD4) group, injection PBS group (AD+con), wild-type (WT+con) group mouse heart perfusion, get cerebral tissue, application organizes refrigerating fulid and liquid nitrogen to carry out freezing treatment.And be stored in-80 ℃.Used time cuts into slices with freezing-microtome, and slice thickness 16 μ m get the observation of dyeing every 9 sections.
2) application 1mg/ml ThS (Sigma, catalog number: T1892-25G, with 70% ethanol preparation) contaminates section 10min, then with 70% alcohol flushing 3 times.
3) application fluorescent microscope gathers image (Olympus BX60) (excitation wavelength 488nm, 4 × object lens).
Detected result is shown in Fig. 8, and wherein Fig. 8 A is the AD mouse brain cortex of injection PBS, and Fig. 8 D is the AD hippocampus of mice of injection PBS, and Fig. 8 B is injection polypeptide XD4 AD mouse brain cortex; Fig. 8 E is injection polypeptide XD4 AD hippocampus of mice; Fig. 8 C is the cortex of wild-type mice, the hippocampus that Fig. 8 F is wild-type mice; Can find out, the senile plaque quantity in AD mouse brain cortex (Fig. 8 A) and the hippocampus (Fig. 8 D) of injection PBS is obviously more than (Fig. 8 E) senile plaque quantity in injection polypeptide XD4 AD mouse brain cortex (Fig. 8 B) and hippocampus.
Application software system (visiopharm) is measured injection polypeptide XD4 (AD+XD4) group (n=8), injection PBS group (AD+con or control) hippocampus (n=8) and the senile plaque area of cortex (every each location detection 10-12 of animal opens section).As shown in Figure 9, statistical analysis shows result, injects the hippocampus of polypeptide XD4 (AD+XD4) group and the senile plaque area proportion (visual field person in middle and old age's spot area accounts for the ratio in the whole visual field) of cortex and is respectively 3.3% and 2.1%; The hippocampus of injection PBS group (AD+con) and the senile plaque area proportion of cortex are respectively 12.2% and 10.9%;
Can find out, the ratio that the mouse senile plaque area of injection polypeptide XD4 accounts for section area significantly reduces (compared with injection PBS group, *, P < 0.01).
3, XD4 reduces A β 40 and A β 42 levels in AD Transgenic Mice Brain
PH7.2PBS (the proteinase inhibitor that above-mentioned 1 injection polypeptide XD4 (AD+XD4) group, the cerebral tissue of injection PBS group (AD+con) experiment mice are containing proteinase inhibitor, Calbiochem company, catalog number is: 539131,100 times of dilutions) middle homogenate.Then with the centrifugal 30min of 15000rpm, collect supernatant.Precipitation adds the Guanidinium hydrochloride (with the preparation of tris-HCl damping fluid, pH8.0) of 5M.Then centrifugal treating, collects supernatant.
Be dissolved in and be insoluble to the A β 40 of PBS and A β 42 levels are measured OD value and A β typical curve by test kit (catalog number is respectively for Invotrigen, USA: KHB3441 and KHB3482) with ELISA method.The A β amount (ng or mg) that A β level contains according to every Borneo camphor tissue represents.The function of the typical curve of A β 40 and A β 42 is respectively
y＝625x-14.8；y＝636.62x-7.15。X is OD value, and y is the quality of A β 40 or A β 42.
The results are shown in Figure 10-12, in Figure 10, in AD transgenic mice (AD+con) brain of the AD transgenic mice (AD+XD4) of injection XD4 polypeptide, injection PBS polypeptide, the content of solubility A β 40 is respectively 28.2ng/g brain albumen and 119ng/g cell protein;
The content of injecting solubility A β 42 in AD transgenic mice (AD+con) brain of AD transgenic mice (AD+XD4), injection PBS polypeptide of XD4 polypeptide in Figure 11 is respectively 0.91ng/g brain albumen and 2.35ng/g brain albumen;
In Figure 12, for insoluble A β 40 content in AD transgenic mice (AD+con) brain of the AD transgenic mice (AD+XD4) of injection XD4 polypeptide, injection PBS polypeptide are respectively 5.8mg/g brain albumen and 9.5mg/g brain albumen, insoluble A β 42 content are respectively 7.5mg/g brain albumen and 11.2mg/g brain albumen;
Result shows in AD injected in mice XD4 hindbrain that solubility A β 40 (Figure 10) and A β 42 (Figure 11) level and insoluble A β 40 and A β 42 levels (Figure 12) are starkly lower than the AD mouse of not injecting XD4.
Four, XD4 promotes cell engulfing A β
With the DMEM substratum (DMEM containing 10% foetal calf serum, Invotrigen, catalog number is: 21969) by BV-2 cell (Chinese Academy of Medical Sciences's tumour cell storehouse, catalog number is: BV-2) be made into individual cells suspension, be inoculated into 96 porocyte culture plates with 10000, every hole cell, every pore volume 100 μ L.Cell is in 37 ℃ of cultivations 24 hours, incubator CO
2concentration is 5%, then adds respectively following three kinds of materials:
A β 42: the final concentration of A β 42 albumen is 0.1 μ M;
A β 42: XD4 (mol ratio)=1: the final concentration of 20:A β 42 albumen is 0.1 μ M; The final concentration of XD4 is 2 μ M;
A β 42: XD4 (mol ratio)=1: the final concentration of 50:A β 42 albumen is 0.1 μ M; The final concentration of XD4 is 5 μ M;
A β 42: XD4 (mol ratio)=1: the final concentration of 200:A β 42 albumen is 0.1 μ M; The final concentration of XD4 is 20 μ M;
Contrast polypeptide (sequence is: SMSARQL is synthetic by the biochemical (Shanghai) Co., Ltd. of gill): the final concentration of A β 42 albumen is 0.1 μ M; The final concentration of contrast polypeptide is 20 μ M;
Cell continued to cultivate after 4 hours, collecting cell.(catalog number is the cell pyrolysis liquid that application contains proteinase inhibitor for proteinase inhibitor, Calbiochem company: 539131,100 times of dilutions.Cell pyrolysis liquid, Beijing Bo Aosen Bioisystech Co., Ltd, catalog number is: C-0013) and processing cell, collecting cell extracting solution.Application A β detects box (catalog number is respectively for Invotrigen, USA: KHB3441 and KHB3482) and measures intracellular A β 42 levels.
The results are shown in Figure 13,
Adding A β 42 content in the cell of A β 42 is 54.7ng/g cell protein;
Adding A β 42 content in the cell of A β 42: XD4 (mol ratio)=1: 20 is 69.1ng/g cell protein;
Adding A β 42 content in the cell of A β 42: XD4 (mol ratio)=1: 50 is 75.3ng/g cell protein;
Adding A β 42 content in the cell of A β 42: XD4 (mol ratio)=1: 200 is 82.6ng/g cell protein;
Adding A β 42 content in the cell of polypeptide contrast (negative control polypeptide) is 61.2ng/g cell protein;
Along with the XD4 concentration that adds raises, intracellular A β 42 levels of BV-2 raise gradually, show XD4 have the effect that promotes microglia to engulf A β (compared with only adding the cell sample of A β 42, *, P < 0.05; #, P < 0.01).
Studies show that, XD4 can obviously suppress in vitro the cytotoxicity of A β and increase the phagolysis of microglia to A β.In the body that application AD transgenic animal carry out, experimental result shows, XD4 can obviously improve the spatial memory capacity of mouse, reduces the quantity of senile plaque in brain, and reduces the level of A β 40 and A β 42.XD4 molecular weight is very little, and easily through hemato encephalic barrier performance curative effect, and antigenicity is poor, and side effect is less, is a polypeptide that has AD treatment potentiality.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (6)
1. a peptide species, the polypeptide being formed by the aminoacid sequence shown in sequence in sequence table 1.
2. suppress an A β cytotoxicity preparation, its activeconstituents is polypeptide described in claim 1; Described cell is specially SH-SY5Y cell, and described A β is specially A β 42.
3. treat a product for alzheimer's disease, its activeconstituents is polypeptide described in claim 1; Described product is specially medicine.
4. described in claim 1, polypeptide suppresses the application in A β cytotoxicity preparation in preparation; Described cell is specially SH-SY5Y cell, and described A β is concrete A β 42.
5. described in claim 1, polypeptide promotes microglia to remove the application in the preparation of A β in preparation; Described A β is specially A β 42.
6. the application of polypeptide in the product of preparation treatment alzheimer's disease described in claim 1; Described product is specially medicine.
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| CN104277092B (en) * | 2013-07-12 | 2017-10-31 | 天津医科大学 | Beta lamellar blocking peptide for preventing and/or treating senile dementia |
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