CN106074600B - Isolated from blood plasma for enhancing mixture of memory and its preparation method and application - Google Patents
Isolated from blood plasma for enhancing mixture of memory and its preparation method and application Download PDFInfo
- Publication number
- CN106074600B CN106074600B CN201610474296.XA CN201610474296A CN106074600B CN 106074600 B CN106074600 B CN 106074600B CN 201610474296 A CN201610474296 A CN 201610474296A CN 106074600 B CN106074600 B CN 106074600B
- Authority
- CN
- China
- Prior art keywords
- concentration
- mixture
- blood plasma
- product
- volume
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/16—Blood plasma; Blood serum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Hematology (AREA)
- Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Virology (AREA)
- Immunology (AREA)
- Developmental Biology & Embryology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of mixtures for being used to enhance memory isolated from blood plasma and its preparation method and application.The mixture includes multiple proteins and a variety of small molecules, the SDS PAGE denaturation gel electrophoresis of the mixture includes at least 30 bands, wherein include being evident that 7 bands in band of the molecular weight more than 48kD, the molecular weight of 7 bands is 56kD successively from small to large, 68kD, 77kD, 115kD, 175kD, 250kD and 289kD.The preparation method of the mixture includes plasma collection, density gradient centrifugation, dialysis, once concentrates, goes high kurtosis albumen, anion exchange and secondary concentration step successively.The mixture prepared by the present invention can not only play the role of enhancing memory, can be also used for preventing, improve and treating alzheimer's disease.
Description
Technical field
The present invention relates to a kind of blood plasma isolates, and in particular, to a kind of enhancing that is used for isolated from blood plasma is remembered
Mixture of power and its preparation method and application, the mixture can be used for preventing, improve and treat alzheimer's disease, and have
Enhance the effect of memory.
Background technology
Alzheimer syndrome is called alzheimer's disease, its main feature is that sick man memory and cognitive ability are gradually lost
It loses.Patient is dead in 3 to 9 years after making a definite diagnosis, and accounts for 50% to the 56% of clinically dead case.The current understanding of the disease cause of disease is big
The beta- amyloid proteins folded extremely and Tau albumen of intracerebral accumulation result in alzheimer's disease.
The drug of alzheimer's disease currently on the market is mainly Western medicine, it is characterized in that must adhere to taking for a long time, however
But curative effect is small, can only alleviate partial symptoms, it is impossible to control the development of the state of an illness.Another defect taken for a long time is drug pair
Effect is big, while patient generates drug dependence.It is badly in need of better efficacy, the lower drug of side effect in the market thus.
Current recent studies have shown that：Recognizing for old mouse can be promoted to the blood plasma of old mouse continuous injection youth mouse
Know level (Villeda S.A.Young blood reverses age-related impairments in cognitive
function and synaptic plasticity in mice.Nature Medicine 2014；20:659-663).For
This, can may effectively promote the human-subject test of Alzheimer's containing Cucumber in blood plasma.But these substances
Concrete component and separation acquisition methods have not been reported.
Invention content
In view of the drawbacks of the prior art, one of the objects of the present invention is to provide it is a kind of isolated from blood plasma for increasing
The mixture of strong memory.
The second object of the present invention is to provide the preparation method of said mixture.
The third object of the present invention is to provide the application of said mixture.
To achieve these goals, present invention employs following technical schemes：
It is a kind of isolated from blood plasma for enhancing the mixture of memory, the mixture include multiple proteins and
A variety of small molecules, the SDS-PAGE denaturation gel electrophoresis of the mixture includes at least 30 bands, wherein being more than in molecular weight
The band of 48kD includes：Molecular weight is 56kD, 68kD, 77kD, 115kD, 175kD, 250kD and 289kD successively from small to large
Band.
In said mixture, as a kind of preferred embodiment, the FPLC identifications of the mixture have 4 peaks, right
The albumen size answered is respectively：250kD, 120kD, 100kD and 80kD.
It in said mixture, as a kind of preferred embodiment, is analyzed by protein spectrum, the mixture at least contains
There are 30 albumen, including albumin, globulin, keratin, fibrinogen and transport protein；By small molecule mass spectral analysis,
The mixture also at least contains 20 small molecules.
It in said mixture, as a kind of preferred embodiment, is analyzed by protein spectrum, egg contained by the mixture
It is white as follows：
In said mixture, as a kind of preferred embodiment, by small molecule mass spectral analysis, contained by the mixture
Small molecule it is as follows：
In said mixture, as a kind of preferred embodiment, the blood plasma derives from mammal；Preferably, institute
Blood plasma is stated from people.
The above-mentioned preparation method for being used to enhance the mixture of memory isolated from blood plasma, as a kind of preferred implementation
Mode, the preparation method include successively：Plasma collection procedure, density gradient centrifugation step, dialysis step, primary concentration step
Suddenly high kurtosis albumen step, anion exchange step and secondary concentration step, are gone.
In above-mentioned preparation method, as a kind of preferred embodiment, in the plasma collection procedure, anticoagulant tube is utilized
Blood is collected, then by the way that supernatant is collected by centrifugation, obtains blood plasma；Preferably, the centrifugal rotational speed is 500-1500g, during centrifugation
Between be 10-30 minutes.
In above-mentioned preparation method, as a kind of preferred embodiment, in the density gradient centrifugation step, by described in
The blood plasma that plasma collection procedure obtains is mixed with Percoll solution, Forcol solution or sucrose solution, establish density gradient and from
Top layer's RED sector in centrifuge tube is removed, obtains product after density gradient centrifugation by the heart later, wherein the condition of the centrifugation
For：Rotating speed is 10000-30000g, time 2-20h；Preferably, the Percoll solution, Forcol solution or sucrose solution
A concentration of 40-60%, the volume ratio of the Percoll solution, Forcol solution or sucrose solution and the blood plasma is (10-
15):(1-3)。
In above-mentioned preparation method, as a kind of preferred embodiment, in the dialysis step, by the density gradient
Product after centrifugation is put into bag filter or pipe and dialyses, and the product in bag filter or pipe is collected after dialysis, is produced as dialysis
Object；Wherein the dialysis when elution buffer that uses be Tris- hydrochloride buffers, phosphate buffer or HBS buffer solutions；It is preferred that
Ground, a concentration of 0.2-200mM, the pH value 7.0-9.2 of the Tris- hydrochloride buffers, the phosphate buffer it is a concentration of
1.0-500mM, pH value 6.0-9.2, a concentration of 0.5-500mM, the pH value 6.4-8.4 of the HBS buffer solutions；More preferably
Ground, the Tris hydrochloride buffers that the elution buffer is 1mM, pH value is 8；Dialysis time is 20-72h, and dialysis volume ratio is
1:100-1:10000。
In above-mentioned preparation method, as a kind of preferred embodiment, in a concentration step, by the dialysis
Product is concentrated, and product after once being concentrated, wherein condensate precursor product are 10 times or more of volume after concentration；Preferably,
The concentration is carried out using by the way of concentration tube centrifugation, and the substance of the concentration tube permission 1.5-2.5KD passes through, it is described from
Rotating speed is 2000-4000g during the heart.
It in above-mentioned preparation method, as a kind of preferred embodiment, is gone in high kurtosis albumen step described, using going
High kurtosis protein reagent box removes the high kurtosis albumen in product after primary concentration, obtains high kurtosis protein product；It is preferred that
Ground, the elution buffer gone in high kurtosis protein reagent box to go after the primary concentration on high kurtosis albumen pillar product into
The eluent obtained during row elution removes high kurtosis protein product to be described；Preferably, the content of the high kurtosis albumen removed
It is 5mg/ml.
In above-mentioned preparation method, as a kind of preferred embodiment, in the anion exchange step, gone described
High kurtosis protein product is added in anion-exchange column, and ladder is carried out with the Tris- hydrochloride buffers containing 20-500mM sodium chloride
Degree elution starts to collect eluent when elution volume is 15-35ml, stops collecting elution when elution volume is 40-85ml
Liquid, the eluent collected are product after anion exchange；Preferably, a concentration of 0.2- of the Tris- hydrochloride buffers
200mM, pH value 7.0-9.2.
In above-mentioned preparation method, as a kind of preferred embodiment, in the secondary concentration step, by it is described it is cloudy from
Product is concentrated after son exchanges, and obtains the i.e. described mixture of product after secondary concentration, and wherein condensate precursor product is body after concentration
Long-pending 10 times or more；Preferably, affiliated concentration is carried out by the way of concentration tube centrifugation, and the concentration tube allows 1.5-
The substance of 2.5KD passes through, and rotating speed is 2000-4000g during the centrifugation.
A kind of pharmaceutical composition, includes said mixture and pharmaceutically acceptable carrier；Preferably, it is described pharmaceutically
The carrier of receiving is：Pharmaceutically acceptable buffer solution, gelatin, monosaccharide, polysaccharide, amino acid, chelating agent, sugar alcohol, gathers protein
It is one or more in ethylene glycol and surfactant.It is further preferred that described pharmaceutical composition includes following component：1 times
The said mixture of volume, the 8.5wt%NaCl of 9 times of volumes or the PBS of 1.5M, pH7.0；Further, the pharmaceutical composition
Albumin, glucose and glutamine are further included in object, wherein, mass body of the albumin in described pharmaceutical composition
Product percentage is 2%, and quality percent by volume of the glucose in described pharmaceutical composition is 1%, the glutamine
Quality percent by volume in described pharmaceutical composition is 3%.
A kind of sustained release preparation includes said mixture or aforementioned pharmaceutical compositions and pharmaceutically acceptable biology
Compatible substances；Preferably, the dosage form of the sustained release preparation is liposome, microballoon, hydrogel, Osmotic minipumps or microcapsules.
Said mixture, aforementioned pharmaceutical compositions and above-mentioned sustained release preparation subtract in prevention, improvement or/and treatment memory
Move back the application in the drug of class disease, it is preferable that the failure of memory class disease is alzheimer's disease.
The application of said mixture, aforementioned pharmaceutical compositions and above-mentioned sustained release preparation in memory drug is enhanced.
Include the kit of said mixture, aforementioned pharmaceutical compositions or above-mentioned sustained release preparation.
Compared with the prior art, the present invention has the advantages that：
1st, the plasma component prepared by the present invention can be used for preventing, improve and treating alzheimer's disease, the blood plasma group
Lease making verification can delay the disease process of alzheimer's disease and it is possible that reverse the serious journey of pathologic of alzheimer's disease
It spends, it can also play the role of enhancing memory in addition.
2nd, the mixture provided by the invention isolated from blood plasma can promotion more more effective than blood plasma and current drug
The human-subject test of Alzheimer's.At the same time, long-term use can be substantially reduced by taking this plasma component for a long time
The side effect and pharmacological dependence that Western medicine is brought.
Description of the drawings
Fig. 1：Cocktail components I (mixture isolated in slave blood plasma of the invention) prepares Anionic column chromatography knot
Fruit.Mice plasma carries out Anionic column chromatography after density gradient centrifugation, dialysis, concentrating and removing high kurtosis albumen.Use the moon
Ion column SourceQ adhesion proteins, then gradient elution is carried out with the Tris buffer solutions of the sodium chloride containing 500mM, obtain elution curve.
Start to collect eluent when elution volume is 35 milliliters, stop collecting eluent when elution volume is 45 milliliters.It collects
Eluent is cocktail components I after concentration.
Fig. 2：Cocktail components I SDS- is denaturalized gel electrophoresis qualification result.Swimming lane M：Protein Marker；Swimming lane 1-3：Chicken
Tail wine component.
Fig. 3：Cocktail components I FPLC qualification results.After cocktail component prepares, protein component is detected with FPLC,
Protein peak is the absorbance at ultraviolet 280nm.
Fig. 4：Cocktail components I promotes the value-added activity figure of primary hippocampal cells.Toward in the culture medium of primary hippocampal cells
Cocktail components I is separately added into, other plasma components A and B and untreated blood plasma, as experimental group.Control group hippocampus is thin
Born of the same parents only add DMEM culture mediums.Hippocampal cell is counted under the microscope after one day, experiment with computing group cell number accounts for control
The ratio of group cell number.
Fig. 5：Cocktail components I promotes the activity figure of Synaptic formation between primary hippocampal cells.Toward primary hippocampal cells
Cocktail components I is separately added into culture medium, other plasma components A and B and untreated blood plasma, as experimental group.Control
Group hippocampal cell only adds DMEM culture mediums.Cynapse number is counted under the microscope after one day, experiment with computing group number of synapses
Mesh accounts for control group number of synapses purpose ratio.
Fig. 6：Cocktail components I promotes the memory animal model datagram of alzheimer's disease mouse.By cocktail group
I is divided systematically to be injected into alzheimer disease mouse model.By water maze laboratory, evaluation administration is small to alzheimer's disease
The castering action of mouse memory.
Fig. 7：Primary hippocampal cells figure after different disposal.Cocktail is separately added into toward the culture medium of primary hippocampal cells
Components I, other plasma components A and B and untreated blood plasma, as experimental group.Control group hippocampal cell only adds DMEM to cultivate
Base.Hippocampal cell is imaged under the microscope after one day.In figure (a), (b), (c), (d), (e) is cocktail component respectively
Ith, other plasma components A, B, untreated blood plasma, DMEM culture mediums (i.e. control group) treated primary hippocampal cells.
Specific embodiment
In order to preferably be illustrated to the technical characteristic and effect of the present invention, below using specific embodiment to this hair
It is bright to be described in detail, but the present invention is not limited thereto.
A kind of mixture isolated from blood plasma provided by the invention, the mixture include multiple proteins and a variety of
Small molecule, the SDS-PAGE denaturation gel electrophoresis of the mixture includes at least 30 bands, wherein in molecular weight more than 48kD's
Band includes being evident that 7 bands, and the molecular weight of 7 bands is 56kD, 68kD, 77kD successively from small to large,
115kD, 175kD, 250kD and 289kD.
The FPLC identifications of the mixture have 4 apparent peaks.The preparation process of the mixture includes the following steps：Thoroughly
It analyses, once concentrates, goes high kurtosis albumen, anion exchange, secondary concentration.Pass through FPLC by mixture prepared by above step
Purification has 4 apparent peaks (referring to Fig. 3), i.e. 4 peaks of the abscissa between 8-18, and from left to right 4 significantly
The corresponding albumen size in peak is respectively：250kD, 120kD, 100kD and 80kD.
It is analyzed by protein spectrum, the mixture is at least containing 30 albumen, including albumin, globulin, keratin,
Fibrinogen and transport protein；By small molecule mass spectral analysis, the mixture also at least contains 20 small molecules.
Specifically, it is analyzed and identified by protein spectrum (such as MS/MS), albumen such as the following table 1 contained by the mixture.
1 protein spectrum of table (such as MS/MS) analyzes and identifies albumen result contained by mixture
It is identified by small molecule mass spectral analysis (such as UPLC-MS/MS), small molecule such as the following table 2 contained by the mixture.
2 MS2 of table confirms small molecule present in mixture
The blood plasma derives from mammal, such as people, mouse etc.；Preferably, the blood plasma derives from people.
A kind of preparation method of the above-mentioned mixture isolated from blood plasma, the mixture are by carrying out a system to blood plasma
It is obtained after column processing, processing step includes plasma collection, density gradient centrifugation, dialysis, once concentrates, removes high kurtosis egg successively
In vain, anion exchange and secondary concentration step.It is specific as follows：
The various culture mediums and common agents used in the present invention are prepared using conventional method, point involved in embodiment
Specific experimental condition and method is such as not specified in sub- biologic operation, please refers to the chief editors such as SambrookJ, Science Press, and 2002,
The specification of Molecular Cloning:A Laboratory guide (third edition) or corresponding product.
In the plasma collection procedure, blood is collected using anticoagulant tube, supernatant is collected by centrifugation, so as to obtain blood
Slurry；Preferably, the rotating speed of the centrifugation for 500-1500g (such as 510g, 600g, 700g, 800g, 900g, 1000g, 1100g,
1200g, 1300g, 1400g, 1480g), centrifugation time for 10-30 minutes (such as 12min, 15min, 20min, 25min,
28min)。
In the density gradient centrifugation step, blood plasma that the plasma collection procedure is obtained and Percoll solution or
Ficoll solution or sucrose solution mixing are established density gradient and are centrifuged, and later remove top layer's RED sector in centrifuge tube,
Product after density gradient centrifugation is obtained, wherein the condition of the centrifugation is：Rotating speed for 10000-30000g (such as 10010g,
10500g、11000g、12000g、13000g、14000g、15000g、16000g、17000g、18000g、19000g、20000g、
21000g, 22000g, 23000g, 24000g, 25000g, 26000g, 27000g, 29000g), centrifugation time for 2-20h (such as
3min、5min、10min、15min、18min)；Preferably, the Percoll solution or Ficoll solution or sucrose solution is dense
Spend for 40-60% (such as 41%, 45%, 50%, 52%, 55%, 58%), the Percoll solution or Ficoll solution or
The volume ratio of sucrose solution and the blood plasma is (10-15):(1-3) (such as 10.5:1、12:1、13:1、14:1、14.8:1、
10.5:2、12:2、13:2、14:2、14.8:2、10.5:3、12:3、13:3、14:3、14.8:3).It can by density gradient centrifugation
To remove the red pigments small molecule with stronger toxicity a small amount of in blood plasma.
The preparation method of the Percoll solution of various concentration：First with 9 (volume) part Percoll stostes, (Pharmacia is public
Department's product) it is mixed with 1 (volume) part 8.5wt%NaCl or 1.5M PBS and reaches physiological osmotic pressure, it obtains being 100% at this time
Then Percoll solution dilutes 100%Percoll solution needed for physiological solution (0.85wt%NaCl or 0.15M PBS)
Concentration.
The preparation method of the Ficoll solution of various concentration：First with 9 (volume) part Ficoll stostes (Pharmacia companies
Product) it is mixed with 1 (volume) part 8.5wt%NaCl or 1.5M PBS and reaches physiological osmotic pressure, it obtains being 100% at this time
Then Ficoll solution dilutes 100%Ficoll solution to required dense with physiological solution (0.85wt%NaCl or 0.15M PBS)
Degree.
The preparation method of the sucrose solution of various concentration：Sucrose is consolidated block (Pharmacia Products) in room temperature to add in
It is stirring while adding into 8.5wt%NaCl or 1.5M PBS solutions to saturation.A concentration of 2.3M of saturation sucrose solution, as
100% sucrose solution.Then 100% sucrose solution is diluted needed for physiological solution (0.85wt%NaCl or 0.15M PBS)
Concentration.
In the dialysis step, product after the density gradient centrifugation is put into bag filter or pipe and is dialysed, thoroughly
The product in bag filter or pipe is collected after analysis, as dialysis product, for a concentration step later, wherein during the dialysis
The elution buffer used be 0.2-200mM, pH7.0-9.2 Tris- hydrochloride buffers (such as：0.3mM, pH7.0's
Tris- hydrochloride buffers, the Tris- hydrochloride buffers of 1mM, pH7.0, the Tris- hydrochloride buffers of 20mM, pH7.0,50mM,
The Tris- hydrochloride buffers of pH7.0, the Tris- hydrochloride buffers of 100mM, pH7.0, the Tris- hydrochloric acid of 150mM, pH7.0 delay
Fliud flushing, the Tris- hydrochloride buffers of 180mM, pH7.0, the Tris- hydrochloride buffers of 195mM, pH7.0,0.3mM, pH8.0's
Tris- hydrochloride buffers, the Tris- hydrochloride buffers of 1mM, pH8.0, the Tris- hydrochloride buffers of 30mM, pH8.0,60mM,
The Tris- hydrochloride buffers of pH8.0, the Tris- hydrochloride buffers of 90mM, pH8.0, the Tris- hydrochloride buffers of 150mM, pH8.0
Liquid, the Tris- hydrochloride buffers of 180mM, pH8.0, the Tris- hydrochloride buffers of 195mM, pH8.0,0.3mM, pH9.0's
Tris- hydrochloride buffers, the Tris- hydrochloride buffers of 1mM, pH9.0, the Tris- hydrochloride buffers of 30mM, pH9.0,60mM,
The Tris- hydrochloride buffers of pH9.0, the Tris- hydrochloride buffers of 90mM, pH9.0, the Tris- hydrochloride buffers of 150mM, pH9.0
Liquid, the Tris- hydrochloride buffers of 180mM, pH9.0, the Tris- hydrochloride buffers of 195mM, pH9.0), 1.0-500mM,
PH6.0-9.2 phosphate buffer (such as：The phosphate buffer of 1.5mM, pH6.5, the phosphate buffer of 30mM, pH6.5,
The phosphate buffer of 90mM, pH6.5, the phosphate buffer of 160mM, pH6.5, the phosphate buffer of 200mM, pH6.5,300mM,
The phosphate buffer of pH6.5, the phosphate buffer of 400mM, pH6.5, the phosphate buffer of 460mM, pH6.5,490mM, pH6.5
Phosphate buffer, the phosphate buffer of 1.5mM, pH7.5, the phosphate buffer of 30mM, pH7.5, the phosphoric acid of 90mM, pH7.5
Buffer solution, the phosphate buffer of 160mM, pH7.5, the phosphate buffer of 200mM, pH7.5, the phosphoric acid buffer of 300mM, pH7.5
Liquid, the phosphate buffer of 400mM, pH7.5, the phosphate buffer of 460mM, pH7.5, the phosphate buffer of 490mM, pH7.5,
The phosphate buffer of 1.5mM, pH8.5, the phosphate buffer of 30mM, pH8.5, the phosphate buffer of 90mM, pH8.5,160mM,
The phosphate buffer of pH8.5, the phosphate buffer of 200mM, pH8.5, the phosphate buffer of 300mM, pH8.5,400mM, pH8.5
Phosphate buffer, the phosphate buffer of 460mM, pH8.5, the phosphate buffer of 490mM, pH8.5, the phosphorus of 1.5mM, pH9.0
Acid buffer, the phosphate buffer of 30mM, pH9.0, the phosphate buffer of 90mM, pH9.0, the phosphoric acid buffer of 160mM, pH9.0
Liquid, the phosphate buffer of 200mM, pH9.0, the phosphate buffer of 300mM, pH9.0, the phosphate buffer of 400mM, pH9.0,
The phosphate buffer of 460mM, pH9.0, the phosphate buffer of 490mM, pH9.0) or 0.5-500mM, pH6.4-8.4HBS's is slow
Fliud flushing (such as：The buffer solution of 1mM, pH6.5HBS, the buffer solution of 50mM, pH6.5HBS, the buffer solution of 100mM, pH6.5HBS,
The buffer solution of 150mM, pH6.5HBS, the buffer solution of 200mM, pH6.5HBS, the buffer solution of 250mM, pH6.5HBS, 300mM,
The buffer solution of pH6.5HBS, the buffer solution of 350mM, pH6.5HBS, the buffer solution of 450mM, pH6.5HBS, 495mM, pH6.5HBS
Buffer solution, the buffer solution of 1mM, pH7.5HBS, the buffer solution of 50mM, pH7.5HBS, the buffer solution of 100mM, pH7.5HBS,
The buffer solution of 150mM, pH7.5HBS, the buffer solution of 200mM, pH7.5HBS, the buffer solution of 250mM, pH7.5HBS, 300mM,
The buffer solution of pH7.5HBS, the buffer solution of 350mM, pH7.5HBS, the buffer solution of 450mM, pH7.5HBS, 495mM, pH7.5HBS
Buffer solution, the buffer solution of 150mM, pH8.2HBS, the buffer solution of 200mM, pH8.2HBS, the buffering of 250mM, pH8.2HBS
Liquid, the buffer solution of 300mM, pH8.2HBS, the buffer solution of 350mM, pH8.2HBS, the buffer solution of 450mM, pH8.2HBS,
The buffer solution of 495mM, pH8.2HBS)；The aperture of the bag filter or pipe is 0.2-0.3nm；Preferably, the elution buffer
Tris hydrochloride buffers for 1mM, pH=8；It is highly preferred that the time of the dialysis for 20-72h (such as 21h, 25h, 30h,
35h, 40h, 45h, 55h, 65h, 71h), dialysis volume ratio (i.e. in bag filter substance be in bag filter outside dialysis buffer
The volume ratio of liquid) it is 1:100-1:10000 (such as 1:150、1:500、1:1000、1:1500、1:2500、1:3500、1:
4000、1:5000、1:6000、1:7000、1:8000、1:9000、1:9500).By step of dialysing, it is close previous step can be removed
Medium, that is, sucrose, Percoll or the Ficoll in gradient centrifugation are spent, bad shadow is generated to prevent the medium from being prepared to downstream product
It rings.
In a concentration step, the product that the dialysis step obtains is concentrated, after once being concentrated
Product, wherein condensate precursor product be concentration after volume at least 10 times (such as：15 times, 30 times, 40 times, 50 times, 70 times, 90 times,
100 times)；The specific method of the concentration includes filter membrane concentration and concentration tube concentration.Preferably, the concentration is using concentration tube
What the mode of centrifugation carried out, the concentration tube allow 1.5-2.5KD (such as 1.6KD, 1.8KD, 1.9KD, 2.0KD, 2.2KD,
Substance 2.4KD) passes through, during the centrifugation rotating speed for 2000-4000g (such as 2100g, 2300g, 2500g, 3000g,
3200g、3500g、3800g).The purpose of the step is that plasma component volume is allowed to become smaller, so as in more efficient realization next step
The removal of high kurtosis albumen.
It is gone in high kurtosis albumen step described, using the height in product after going high kurtosis protein reagent box that will once concentrate
Kurtosis albumen removes, so as to obtain high kurtosis protein product；Preferably, the elution gone in high kurtosis protein reagent box is delayed
Fliud flushing carries out the eluent obtained during elution for the first time to product after going the primary concentration on high kurtosis albumen pillar, as described
Remove high kurtosis protein product.The purpose of the step is to remove the high kurtosis albumen that content in blood plasma is 5mg/ml, is starched to prevent hemostasis
In active principle can not play effect.
In the anion exchange step, high kurtosis protein product is gone to be added in anion-exchange column by described, with containing
The Tris hydrochloride buffers (0.2-200mM, pH7.0-9.2) for having sodium chloride (20-500mM) carry out gradient elution, when elution body
(such as 16ml, 20ml, 25ml, 30ml, 34ml) starts to collect eluent when product is 15-35ml, when elution volume is 40-85ml
When (such as 57ml, 60ml, 65ml, 75ml, 83ml) stop collect eluent, the eluent collected is anion exchange
Product afterwards.Wherein, the Tris hydrochloride buffers (0.2-200mM, pH7.0-9.2) containing sodium chloride (20-500mM) can be：
The Tris hydrochloride buffers of 1mM, pH7.2 containing 25mM sodium chloride, the Tris hydrochloric acid of 1mM, pH8.2 containing 25mM sodium chloride
Buffer solution, the Tris hydrochloride buffers of 1mM, pH9.0 containing 25mM sodium chloride, 1mM, pH7.2's containing 100mM sodium chloride
Tris hydrochloride buffers, the Tris hydrochloride buffers of 1mM, pH8.2 containing 100mM sodium chloride, contain 100mM sodium chloride
The Tris hydrochloride buffers of 1mM, pH9.0, the Tris hydrochloride buffers of 1mM, pH7.2 containing 200mM sodium chloride, contain
The Tris hydrochloride buffers of 1mM, pH8.2 of 200mM sodium chloride, the Tris hydrochloric acid of 1mM, pH9.0 containing 200mM sodium chloride delay
Fliud flushing, the Tris hydrochloride buffers of 1mM, pH7.2 containing 400mM sodium chloride, 1mM, pH8.2's containing 400mM sodium chloride
Tris hydrochloride buffers, the Tris hydrochloride buffers of 1mM, pH9.0 containing 400mM sodium chloride, contain 490mM sodium chloride
The Tris hydrochloride buffers of 1mM, pH7.2, the Tris hydrochloride buffers of 1mM, pH8.2 containing 490mM sodium chloride, contain
The Tris hydrochloride buffers of 1mM, pH9.0 of 490mM sodium chloride, the Tris hydrochloric acid of 100mM, pH7.2 containing 30mM sodium chloride
Buffer solution, the Tris hydrochloride buffers of 100mM, pH7.2 containing 150mM sodium chloride, the 100mM containing 400mM sodium chloride,
The Tris hydrochloride buffers of pH7.2, the Tris hydrochloride buffers of 100mM, pH7.2 containing 480mM sodium chloride, contain 30mM chlorine
Change the Tris hydrochloride buffers of 190mM, pH7.2 of sodium, the Tris hydrochloride buffers of 190mM, pH7.2 containing 150mM sodium chloride
Liquid, the Tris hydrochloride buffers of 190mM, pH8.2 containing 400mM sodium chloride, 190mM, pH9.0 containing 480mM sodium chloride
Tris hydrochloride buffers.The purpose of the step is to remove ingredient negatively charged in plasma component, to prevent these ingredients pair
The toxic action of disease, the presence of negatively charged ingredient can almost cause plasma component to be of no curative effect.It is upper to collect elution volume
Eluent in the range of stating can obtain plasma composition significant in efficacy.
In the secondary concentration step, product after the anion exchange is concentrated, is produced after obtaining secondary concentration
Object, that is, the mixture, wherein condensate precursor product be concentration after volume at least 10 times (such as：15 times, 30 times, 40 times, 50 times,
70 times, 90 times, 100 times)；The specific method of the secondary concentration includes filter membrane concentration and concentration tube concentration.Preferably, it is described dense
Contracting is carried out by the way of concentration tube centrifugation, the concentration tube allow 1.5-2.5KD (such as 1.6KD, 1.8KD, 1.9KD,
2.0KD, 2.2KD, 2.4KD) substance pass through, during the centrifugation rotating speed for 2000-4000g (such as 2100g, 2300g,
2500g、3000g、3200g、3500g、3800g).The purpose of secondary concentration is to improve the concentration and viscosity of cocktail component, from
And the effect of improving unit ingredient.
A kind of pharmaceutical composition, includes said mixture and pharmaceutically acceptable carrier.It is described pharmaceutically acceptable
Carrier can be：Pharmaceutically acceptable various common buffer solutions (such as phosphate, citrate and other organic acid buffers
Liquid), protein (such as albumin and immunoglobulin can greatly promote the stability of product), gelatin, monosaccharide, polysaccharide, amino
In acid, chelating agent (such as EDTA), sugar alcohol (such as mannitol), polyethylene glycol and surfactant such as TWEEN and PLURONICS
It is one or more.
Preferably, described pharmaceutical composition includes following component：The said mixture of 1 times of volume (produces after secondary concentration
Object), 8.5wt%NaCl the or 1.5M PBS (pH7.0) of 9 times of volumes.It is highly preferred that white egg is further included in described pharmaceutical composition
In vain, glucose and glutamine, wherein, quality percent by volume of the albumin in described pharmaceutical composition is 2%
(w/v), quality percent by volume of the glucose in described pharmaceutical composition is 1% (w/v), and the glutamine is in institute
It is 3% (w/v) to state the quality percent by volume in pharmaceutical composition.
The dosage form of described pharmaceutical composition can be：Solution, injection, intravenous injection emulsion, syrup, mucilage,
Suspension, microspheres agent, micro-capsule, tablet, powder, particle, pill, freeze-dried powder, aerosol, spray.
Described pharmaceutical composition can be administered as follows：Intravenous drip, intravenous injection, intramuscular injection, abdominal cavity note
Penetrate, arteria hepatica injection, subcutaneous embedding, nasal mucosa medicine administration and oral.Dosage be 0.01-1ml/kg (such as：0.02ml/kg、
0.05ml/kg、0.1ml/kg、0.4ml/kg、0.6ml/kg、0.8ml/kg、0.9ml/kg)。
A kind of sustained release preparation, comprising：Said mixture or aforementioned pharmaceutical compositions and pharmaceutically acceptable biology
Compatible substances；Preferably, the dosage form of the sustained release preparation is liposome, microballoon, hydrogel, Osmotic minipumps or microcapsules.
Said mixture, aforementioned pharmaceutical compositions and/or above-mentioned sustained release preparation are in prevention, improvement or/and treatment memory
Application in the drug for the class disease that declines；Preferably, the failure of memory class disease is alzheimer's disease.
The application of said mixture, aforementioned pharmaceutical compositions and/or above-mentioned sustained release preparation in memory drug is enhanced.
A kind of kit for including said mixture, aforementioned pharmaceutical compositions and/or above-mentioned sustained release preparation.The kit
In can also contain negative control or positive control etc..
The preparation of inventive mixture, identification and application are illustrated below by embodiment.Make in following embodiment
Primary hippocampal cells are according to following bibliography culture：Guo,W.,Y.Ji,et al.(2014)."Neuronal
activity alters BDNF-TrkB signaling kinetics and downstream functions."J Cell
Sci 127(Pt 10):2249-2260。
Embodiment 1：The preparation of mixture
(1) it takes a blood sample from adult mice, collects blood plasma
By 100 adult C75BL/6 strains mouse (mouse can also use other strain experimental animals replace,
Such as：The mouse of BALB/cA strains, the mouse of DBA/2 strains, SD rats, Wistar rats) with the carbon dioxide of 20vol%
(gas that i.e. carbon dioxide content is 20vol%) anesthesia, then with blood taking needle from mice eye bloodletting to heparin tube (heparin tube
For anticoagulant tube) in, heparin tube is rocked in bloodletting, prevents blood clotting.Heparin tube containing blood is put into centrifuge, is set
Rotating speed is 1000g, is centrifuged 30 minutes.Then supernatant is carefully drawn with pipettor, the blood plasma being as collected into.
(2) cocktail component mixture i.e. of the invention is isolated from mice plasma
(a) density gradient centrifugation
Blood plasma is subjected to density gradient centrifugation first：12 milliliters of addition is a concentration of in the centrifuge tube that total volume is 13 milliliters
Then 50% Percoll adds 1 milliliter of mice plasma；Centrifuge tube is put into ultracentrifuge, setting speed is
12000g, set temperature are 4 degrees Celsius, centrifuge 5 hours.After density gradient centrifugation, removed with pipettor most upper in centrifuge tube
Layer RED sector, plasma component remaining in centrifuge tube is dialysed together with Percoll.
(b) it dialyses
General bag filter is selected in dialysis, and bag filter aperture is about 0.25 nanometer.The blood that will be obtained after density gradient centrifugation
Slurry component is added in bag filter together together with Percoll, then bag filter is put into the beaker of 1L, in beaker outside bag filter
The 1mM Tris hydrochloride buffers (pH8.0) of 1L are added in, are dialysed while stirring.Temperature of dialysing is 4 DEG C, and dialysis time is small for 24
When.After dialysis, using the remaining plasma component in bag filter as dialysis product, pending primary concentration.
(c) primary concentration
Plasma component after dialysis is added in the concentration tube that volume is 2 milliliters, concentration tube allows size to be 2,000 dongles
The substance to pause passes through.Concentration tube is put into centrifuge, setting speed 3000g, set temperature is 4 DEG C, starts centrifuge, opens
Begin to concentrate, until final volume is 500 microlitres.Later, the blood plasma after remaining dialysis is added in again in the centrifuge tube so that
The volume of plasma component reaches 2mL, is centrifuged with identical parameter, and it is 500 microlitres to be again concentrated to final volume.So follow
Ring concentrates, and is 500 microlitres until the plasma component after dialysing finally is concentrated to volume.Using 500 microlitres of plasma components as
One concentration product is denoted as plasma component A, pending that high kurtosis albumen is gone to handle.
(d) high kurtosis albumen is removed
High kurtosis albumen is gone to remove high kurtosis protein reagent box using the model 163-3006 of BIO-RAD companies production,
Obtained after concentration 500 microlitres of blood plasma are added in the pillar of high kurtosis albumen, then which is put into 4 DEG C of ice
Case places 5 hours.2 milliliters of dcq buffer liquid is added in into pillar later, 4 degrees Celsius centrifuge 5 minutes, and centrifugal rotational speed is
10000g.Then 500 microlitres of elution buffer is added in into pillar again, 4 DEG C are placed 30 minutes, are then centrifuged 5 minutes for 4 DEG C,
Centrifugal rotational speed is 10000g, and the plasma component eluted later is to go the plasma component after high kurtosis albumen, is denoted as blood plasma
The for use anion-exchange columns of plasma component B are further purified component B.
(e) anion exchange
500 microlitres of plasma components after high kurtosis albumen will be removed to be added in anion-exchange column Source Q, with containing
The Tris hydrochloride buffers (200mM, pH8.0) for having 500mM sodium chloride carry out the filler of gradient elution, wherein anion-exchange column
For Q Sepharose (being purchased from GE Healthcare), average particle size is 3 microns, and anion exchange column volume is 25 milliliters, is washed
De- speed is 4ml/min, and loading speed is 3ml/ml.Start to collect eluent when elution volume is 35 milliliters, when elution body
Stop collecting eluent when product is 45 milliliters；This elution fractions has healing activity.The eluent of collection is handed over as anion
Product is changed, after pending secondary concentration.It is Anionic column chromatography as a result, being adsorbed using anion column Source Q referring to Fig. 1
Albumen, then eluted with NaCl gradient solution, this figure is elution curve.
(f) secondary concentration
Plasma component after anion exchange is added in the concentration tube that volume is 2 milliliters, concentration tube allows size to be 2
The substance of kilodalton passes through, and concentration tube is put into centrifuge, setting speed 3000g, set temperature be 4 DEG C, start from
Scheming starts to concentrate, until final volume is 500 microlitres.Later, the blood plasma after remaining anion exchange is added in this again
So that the volume of solution is 2 milliliters in concentration tank in centrifuge tube, centrifuged with identical parameter, be again concentrated to final volume
It is 500 microlitres.So cycle concentration is 500 microlitres until the plasma component after anion exchange is finally concentrated to volume.It should
500 microlitres of plasma component, isolated cocktail component, is named as cocktail components I as from mice plasma.
Embodiment 2：Cocktail components I SDS-PAGE denaturation gel electrophoresis identifications prepared by embodiment 1
2 microlitres are taken out in above-mentioned cocktail component, its absorbance is measured under ultraviolet 280nm, so as to calculate cocktail
The protein concentration of component.The cocktail component of certain volume is taken, (Beijing Lan Boli is purchased from 1 microlitre of 5 × albumen sample-loading buffer
Moral Bioisystech Co., Ltd, article No. D621) mixing, it as needs to carry out the sample of electrophoresis, has 10 micrograms inside the sample
Protein.Electrophoresis Sample is warming up to 100 DEG C, heating makes protein denaturation in 20 minutes, immediately after by sample be put on ice, etc.
It treats to start after five minutes to run SDS-PAGE denaturation to go back virgin rubber (preparation method that virgin rubber is gone back in the SDS denaturation is as follows：30 (w/v) %'s
Acrylamide Acr-Bis (being purchased from GE Healthcare) takes 1.3ml, 1.5M Tris- hydrochloride buffers (pH8.8, purchased from GE
Healthcare 1.3ml) is taken, the SDS of 10 (w/v) % takes 0.05ml, 10% (w/v) ammonium persulfate is (purchased from GE
Healthcare 0.05ml, TEMED) is taken 0.003ml and water to be taken to take 2.3ml (purchased from GE Healthcare), altogether 5ml,
Plastic can be solidified in room temperature) after mixing.When running glue, the albumen applied sample amount of each swimming lane is 10 micrograms, set race glue voltage as
100V starts to run glue, and it is 1 hour to run the glue time.After running through glue, with the coomassie brilliant blue staining liquid (preparation method of the dyeing liquor
For：2.5g coomassie brilliant blue R_250s are dissolved in 95% ethanol solutions of 500ml, add in the acetic acid solution of 100ml 85%, then,
1000ml is supplemented to distilled water, this dye liquor, which is put, to be kept 4 DEG C of at least six moons stablizing) glue is dyed, it can be found that cocktail
At least containing the visible polypeptide (Fig. 2) of 10 naked eyes in component, it is 31kD, 56kD successively from small to large that molecular weight, which is respectively,
68kD, 77kD, 115kD, 128kD, 165kD, 175kD, 250kD and 289kD.Wherein wrapped in band of the molecular weight more than 48kD
It including and is evident that 7 bands, the molecular weight of 7 bands is 56kD, 68kD, 77kD, 115kD successively from small to large,
175kD, 250kD and 289kD.
Embodiment 3：Cocktail components I FPLC identifications prepared by embodiment 1
2 microlitres are taken out in cocktail component, its absorbance is measured under ultraviolet 280nm, so as to calculate cocktail component
Protein concentration.First with 20mM Tris- hydrochloride buffers (pH 8.0) balance FPLC pillars (Superdex 200), later will
The cocktail component of certain volume is injected into FPLC pillars, cocktail component 300 microgram containing protein of injection.Then chicken is allowed
Tail wine component flows through FPLC pillars with 1 milliliter per minute of speed, contains in discovery cocktail component and is evident that 4 peaks,
Referring to Fig. 3, from left to right the corresponding albumen size of 4 apparent peaks (4 peaks of the abscissa between 8-18) is respectively at peak：
250kD, 120kD, 100kD and 80kD.
Embodiment 4：Cocktail Fraction Ⅰ protein matter Mass Spectrometric Identification prepared by embodiment 1
Cocktail component is transferred in FALCON pipes, adding in the sample buffers of two volumes, (formula of buffer solution is：
7.5M urea UREA, 1.5M thiocarbamide THIOUREA, 4 (w/v) %3- [3- (courage amido propyl) dimethylamino] propane sulfonic acid inner salt
CHAPS, 0.05 (w/v) % lauryl sodium sulfate SDS, 100mM dithiothreitol (DTT) DDT, the shown concentration before each component is it
Concentration of the respective components in buffer solution), and pass through 3kDa molecular weight cut-off spin columns
(Pall GmbH, Austria) centrifuge tube centrifugal concentrating.Concentrate carries out reduction reaction with dithiothreitol (DTT), is also originated in
Object；Wherein, the volume ratio of concentrate and dithiothreitol (DTT) is 1：3, the reaction time is 15 minutes, and temperature is room temperature.Again by this also
It originates in object to be reacted with iodoacetamide, obtains alkylate；Wherein, the volume ratio 1 of the reduzate and iodoacetamide：1,
Reaction time is 15 minutes, and temperature is room temperature；It then carries out digestion reaction at 37 DEG C with trypsase to stay overnight, wherein alkylation production
The volume ratio 1 of object and trypsase:1, obtain postdigestive peptide fragment.Peptide fragment obtained by trypsin digestion is pure by C18 chromatographic columns
Change, obtain sample.Gained sample traditional vacuum is dried and is then stored in -20 DEG C of refrigerators and analyzed for MS/MS.MS/MS points
It analyses specific as follows：HPLC's is 3000 systems of Ultimate, wherein being equipped with PepMap100C-18trap column (300
μ m 5mm) and two pillars of PepMap100C-18analytical column (75 μ m 250mm).Mass spectrograph uses
Amazon speed ETD, MS data acquisition range are 400-1400m/z, and the peptide fragment process range of MS/MS is 100-2800m/
z.Then, each MS data can search for the top-quality CID MS/MS peaks spectrum of matched three automatically.Jet hole voltage is set
It is set to 1400 volts.The temperature of nitrogen protection is 150 DEG C, and flow velocity is 3 liters/min.The protein identification of MS and unmarked quantitative
(LFQ) data analysis uses open-source software MaxQuant1.3.0.5.By searching for SwissProt database (versions
10/2003 20354) protein to be identified, qualification result is referring to table 1.
Embodiment 5：Cocktail components I small molecule Mass Spectrometric Identification prepared by embodiment 1
200 microlitres of cocktail component sample is taken to be put into vial, vial is inserted in ice 15 minutes.Again by glass
Bottle in adding in 800 microlitres of methane/dichloromethane mixed liquor (methane and dichloromethane thereto in draught cupboard with 1 milliliter of syringe
Volume ratio be 2：1).Then vial is inserted in Hui Bing again, Ultrasonic Pulverization 2 minutes.Vial is placed on after crushing
20-30 minutes are stood on ice, is placed on later in concussion instrument and shakes mixing 1 minute.Then it is placed in refrigerator and stands 5 minutes, see it
It is placed in concussion instrument again after layering and shakes mixing, repeatedly 3-5 times altogether.Room temperature 2000rpm is centrifuged 20 minutes later, can be divided after centrifugation
Three layers, upper strata is metabolite (being marked with M), and centre is protein, and lower floor is lipid (being marked with LP), with 250 microlitres of note
In upper solution to 1.5 milliliters of new EP pipes in emitter gentle aspiration vial；Glass is drawn with the syringe of 250ul
In lower floor's solution to another 1.5 milliliters of new EP pipe in bottle.Lower floor's liquid of suction is placed on simultaneously in 4 DEG C of centrifuges
13000rpm is centrifuged 10 minutes, with UPLC-MS/MS high-resolution LC-MS point in Aspirate supernatant to new 1.5 milliliters of EP pipes
Analyzer identifies small molecule metabolite Q Exactive Orbitrap mass spectrographs are used for the identification of metabolite, heavy
Parameter such as the following table 3 is wanted, qualification result is referring to table 2.
3 Q Exactive Orbitrap mass spectrographs of table identify parameter
Embodiment 6：Cocktail components I prepared by embodiment 1 promotes the value-added activity of primary hippocampal cells in vitro
2 microlitres are taken out in cocktail component, its absorbance is measured under ultraviolet 280nm, so as to calculate cocktail component
Protein concentration.With the primary hippocampal cells (training of primary hippocampal cells used in the present invention in 24 orifice plate culture rat sources
Method of the method for supporting with reference to described in documents below：Guo,W.,Y.Ji,et al.(2014)."Neuronal activity
alters BDNF-TrkB signaling kinetics and downstream functions."J Cell Sci 127
(Pt 10):2249-2260.), culture used medium is DMEM, and condition of culture is 37 DEG C, 5vol% carbon dioxide.Until thin
Born of the same parents' density reaches every hole 4 × 105During a cell, toward culture medium in add in cocktail component, the cocktail group added in each hole
Divide 00 microgram containing protein 10, as experimental group 1- cocktail components.Simultaneously following three groups are further included in experimental group：I.e. toward different holes
Culture medium in be separately added into protein content be the mice plasmas ((1) step of embodiment 1 is prepared) of 1000 micrograms, egg
Bai Hanliang is the plasma component A (being prepared in (2) step (c) of embodiment 1) of 1000 micrograms and protein content is 1000
The plasma component B (being prepared in (2) step (d) of embodiment 1) of microgram, be divided into be named as experimental group 2- mice plasmas,
Experimental group 3- plasma components A, experimental group 4- plasma components B.Control group is set simultaneously, in control group, until cell density reaches
To every hole 4 × 105During a cell, 100 microlitres of DMEM culture mediums are added into the culture medium in hole.Later under conditions of original
Continue to cultivate cell 24 hours, cell count then is carried out to experimental group and control group under an optical microscope, calculates each experiment
Group cell number accounts for the ratio of cellular control unit number.Referring to Fig. 4, it can be found that for compared to blood plasma, plasma component A and B, chicken
Tail wine component can be obviously promoted the increment of primary hippocampal cells, and it is about 150% that cell number, which accounts for cellular control unit number,.
Embodiment 7：Cocktail components I prepared by embodiment 1 promotes the activity of primary hippocampal cells Synaptic formation in vitro
2 microlitres are taken out in cocktail component, its absorbance is measured under ultraviolet 280nm, so as to calculate cocktail component
Protein concentration.With 24 orifice plate culture primary hippocampal cells, culture used medium is DMEM, and condition of culture is 37 DEG C,
5vol% carbon dioxide.Until cell density reaches every hole 4 × 105During a cell, toward culture medium in add in cocktail component, often
The cocktail component added in a hole 00 microgram containing protein 10, as experimental group 1- cocktail components.It is also wrapped in experimental group simultaneously
Include following three groups：I.e. toward be separately added into the culture medium in different holes mice plasma that protein content is 1000 micrograms (embodiment 1
What (1) step was prepared), protein content (is prepared into for the plasma component A of 1000 micrograms in (2) step (c) of embodiment 1
To) and plasma component B ((2) (d) of embodiment 1 in step be prepared) of the protein content for 1000 micrograms, it is divided into life
Entitled experimental group 2- mice plasmas, experimental group 3- plasma components A, experimental group 4- plasma components B.Control group is set simultaneously, right
According in group, until cell density reaches every hole 4 × 105During a cell, 100 microlitres of DMEM cultures are added into the culture medium in hole
Base.Continue to cultivate cell 24 hours under conditions of original later, then under an optical microscope to each experimental group and control group
Cynapse counting is carried out, each experimental group cynapse number is calculated and accounts for control group number of synapses purpose ratio.Referring to Fig. 5, it can be found that comparing
For blood plasma, plasma component A and B, cocktail component can be obviously promoted primary hippocampal cells and form cynapse, and cynapse number accounts for pair
It is about 210% according to a group cynapse number.Continue culture cell to be under the microscope imaged hippocampal cell after 24 hours, referring to figure
7, as can be seen from Figure 7：For blood plasma, plasma component A and B, it is thin that cocktail component can be obviously promoted primary hippocampal
The increment of born of the same parents, it is about 210% that Synaptic formation number, which accounts for cellular control unit number,.
Embodiment 8：Cocktail components I prepared by embodiment 1 effectively promotes the memory of alzheimer's disease mouse
The present embodiment uses 5XFAD alzheimer disease mouse models, orders in U.S. jackson laboratory, and
It is bred and is raised according to zoopery standard.Each experimental model mouse all carries out identified for genes by rat-tail, it is ensured that
App gene and stablizing for PS1 genes are mutated.For cocktail component by tail vein injection into Mice Body, administration group ensures 30
Micrograms of protein/time dosage, be administered once every three days in 24 days, altogether be administered 8 times.Water maze laboratory is used to detect mouse
Learning ability and memory, 5XFAD male mice of the no administration group for 20 14 week old receive for 24 days before Behaviors survey
8 Saline (i.e. physiological saline) injections, administration group is 20 14 week old 5XFAD male mices, 24 before Behaviors survey
It receives 8 administrations, and c57BL/6 hero mouse of the negative control group for 20 14 week old equally receives Saline injections.
Water maze laboratory carries out between 8 a.m. at 1 point in afternoon.Water maze spatial memory training period is 4 days, 4 times a day,
Training interval time is 10 minutes every time.In an experiment, every four mouse are randomly divided into a training group.For each training
Group, the position of platform of water maze are probabilistically assigned, and are remained unchanged in entire training.In training, mouse is released from any position
It is put into water maze, and it is allowed to search for hiding platform in 120 seconds.If mouse did not find platform in 120 seconds, it
Platform will be directed into.The distance for finding the time used in platform in training every time and being passed through is recorded automatically by intelligent video camera head
Get off.Water maze test carries out after last time is trained 48 hours, and every mouse is released into the water fan of no placement platform
Gong Zhong, and allow its freely activity 60 second.Its route that moves about is automatically recorded by intelligent video camera head.When the test phase is than training period
Between it is one times short, to avoid mouse generate Depressive behavior.Mouse is spent in three quadrants of target quadrant the time it takes and other
The time of expense is recorded, for the assessment to mouse memory power.Its result is referring to Fig. 6, from fig. 6 it can be seen that administration
Mouse target quadrant the time it takes of the i.e. injection cocktail component of group is essentially identical with Normal group, illustrates cocktail group
Divide and effect is obviously improved to alzheimer's disease mouse memory power.
The preparation of 9 cocktail compositionⅱ of embodiment
In addition to step (2)-(e) is different from embodiment 1, other preparation processes are same as Example 1, in the present embodiment
In, step (2)-(e) is specific as follows：
Anion exchange：
500 microlitres of plasma components after high kurtosis albumen will be removed to be added in anion-exchange column Source Q, with containing
The Tris hydrochloride buffers (100mM, pH8.0) for having 200mM sodium chloride carry out the filler of gradient elution, wherein anion-exchange column
For Q Sepharose (being purchased from GE Healthcare), average particle size is 3 microns, and anion exchange column volume is 25 milliliters, is washed
De- speed is 4ml/min, and loading speed is 3ml/ml.Start to collect eluent when elution volume is 20 milliliters, when elution body
Stop collecting eluent when product is 80 milliliters.The eluent of collection is as anion exchanged product, after pending secondary concentration.
The cocktail component that the embodiment obtains, is named as cocktail compositionⅱ.
The preparation of 10 cocktail component III of embodiment
In addition to step (2)-(a) is different from embodiment 1, other preparation processes are same as Example 1, in the present embodiment
In, step (2)-(a) is specific as follows：
Density gradient centrifugation
Blood plasma is subjected to density gradient centrifugation first：10 milliliters of addition is a concentration of in the centrifuge tube that total volume is 13 milliliters
Then 40% sucrose adds 3 milliliters of mice plasma；Centrifuge tube is put into ultracentrifuge, setting speed 30000g,
Set temperature is 4 degrees Celsius (DEG C), centrifuges 20 hours.After density gradient centrifugation, top layer in centrifuge tube is removed with pipettor
RED sector dialyses plasma component remaining in centrifuge tube together with sucrose.
The preparation of 11 cocktail component IV of embodiment
In addition to step (2)-(a) is different from embodiment 1, other preparation processes are same as Example 1, in the present embodiment
In, step (2)-(a) is specific as follows：
Density gradient centrifugation
Blood plasma is subjected to density gradient centrifugation first：11 milliliters of addition is a concentration of in the centrifuge tube that total volume is 13 milliliters
Then 60% Ficoll adds 2 milliliters of mice plasma；Centrifuge tube is put into ultracentrifuge, setting speed is
15000g, set temperature are 4 degrees Celsius, centrifuge 10 hours.After density gradient centrifugation, removed with pipettor most upper in centrifuge tube
Layer RED sector, plasma component remaining in centrifuge tube is dialysed together with Ficoll.
Embodiment 12
Cocktail compositionⅱ-IV is identified respectively according to the identification method of embodiment 2-5, cocktail compositionⅱ-IV
SDS-PAGE protein electrophoresis the result shows that, in cocktail compositionⅱ-IV containing molecular weight be 31kD successively from small to large,
The visible polypeptide of 10 naked eyes of 56kD, 68kD, 77kD, 115kD, 128kD, 165kD, 175kD, 250kD and 289kD, and dividing
Son amount is very clear more than 7 bands of 48kD, and molecular weight is 56kD, 68kD, 77kD, 115kD successively from small to large,
175kD, 250kD and 289kD.FPLC qualification results show cocktail compositionⅱ-IV have albumen size be respectively 250kD,
The peak of 120kD, 100kD and 80kD.Proteomic image and small molecule Mass Spectrometric Identification the result shows that, in cocktail compositionⅱ-IV
Contain the protein and small molecule shown in Tables 1 and 2.
Embodiment 13
Method according to embodiment 6 detects cocktail compositionⅱ-IV and promotes the value-added work of primary hippocampal cells in vitro respectively
Property.
Testing result shows that cocktail compositionⅱ-IV can also be obviously promoted the increment of primary hippocampal cells, wherein, chicken
The cell number of tail wine compositionⅱ group is 5 times of cellular control unit number；The cell number that III group of cocktail component is control group
6 times of cell number；The cell number of cocktail component IV groups is 5.5 times of cellular control unit number.
Embodiment 14
Method according to embodiment 7 detects cocktail component II-IV and promotes primary hippocampal cells Synaptic formation in vitro respectively
Activity.
Testing result shows that cocktail component II-IV can also be obviously promoted the increment of primary hippocampal cells, wherein chicken tail
The Synaptic formation number of wine component II groups is 7 times of cellular control unit number；The Synaptic formation number of cocktail component III groups is
6.5 times of cellular control unit number；The Synaptic formation number of cocktail component IV groups is 8 times of cellular control unit number.
Claims (26)
1. a kind of mixture for being used to enhance memory isolated from blood plasma, which is characterized in that the mixture includes more
Kind protein and a variety of small molecules, the SDS-PAGE denaturation gel electrophoresis of the mixture includes at least 30 bands, wherein dividing
Band of the son amount more than 48kD includes：Molecular weight is 56kD, 68kD, 77kD, 115kD, 175kD, 250kD successively from small to large
With the band of 289kD；The FPLC identifications of the mixture have 4 peaks, and corresponding albumen size is respectively：250kD, 120kD,
100kD and 80kD；
The preparation method of the mixture includes the following steps：Plasma collection procedure, density gradient centrifugation step, dialysis step,
Concentration step goes high kurtosis albumen step, anion exchange step and secondary concentration step；
In the density gradient centrifugation step, blood plasma and Percoll solution, Forcol that the plasma collection procedure is obtained
Solution or sucrose solution mixing, establish density gradient and centrifuge, later remove top layer's RED sector in centrifuge tube, obtain close
Product after degree gradient centrifugation, wherein the condition of the centrifugation is：Rotating speed is 10000-30000g, time 2-20h；It is described
A concentration of 40-60% of Percoll solution, Forcol solution or sucrose solution, the Percoll solution, Forcol solution or
The volume ratio of sucrose solution and the blood plasma is (10-15):(1-3)；
In the dialysis step, the product after the density gradient centrifugation is put into bag filter or pipe and is dialysed, is dialysed
The product in bag filter or pipe is collected afterwards, as dialysis product；Wherein the dialysis when elution buffer that uses be 1mM, pH
It is worth the Tris hydrochloride buffers for 8；The aperture of the bag filter or pipe is 0.2-0.3nm；
In a concentration step, the dialysis product is concentrated, product after once being concentrated, wherein before concentration
Volume is 10 times or more of volume after concentration, and the concentration is carried out by the way of concentration tube centrifugation, and the concentration tube is permitted
Perhaps the substance of 1.5-2.5KD passes through, and rotating speed is 2000-4000g during the centrifugation；
It is gone in high kurtosis albumen step described, using the high kurtosis in product after going high kurtosis protein reagent box that will once concentrate
Albumen removes, and obtains high kurtosis protein product, wherein, the content of the high kurtosis albumen removed is 5mg/ml；
In the anion exchange step, high kurtosis protein product is gone to be added in anion-exchange column by described, with containing
The Tris- hydrochloride buffers of 200-500mM sodium chloride carry out gradient elution, start to collect when elution volume is 15-35ml and wash
De- liquid stops collecting eluent when elution volume is 40-85ml, and the eluent collected is product after anion exchange；
A concentration of 100-200mM of the Tris- hydrochloride buffers, pH value 8.0；
In the secondary concentration step, product after the anion exchange is concentrated, product is i.e. after obtaining secondary concentration
The mixture, wherein condensate precursor product are 10 times or more of volume after concentration, and the concentration is the side using concentration tube centrifugation
What formula carried out, the concentration tube allows the substance of 1.5-2.5KD to pass through, and rotating speed is 2000-4000g during the centrifugation.
2. the mixture according to claim 1 that be used to enhance memory isolated from blood plasma, which is characterized in that logical
Protein spectrum analysis is crossed, the mixture is at least containing 30 albumen, including albumin, globulin, keratin, fibrinogen
And transport protein；By small molecule mass spectral analysis, the mixture also at least contains 20 small molecules.
3. the mixture according to claim 2 that be used to enhance memory isolated from blood plasma, which is characterized in that logical
Protein spectrum analysis is crossed, albumen contained by the mixture is as follows：
4. the mixture according to claim 2 that be used to enhance memory isolated from blood plasma, which is characterized in that
By small molecule mass spectral analysis, the small molecule contained by the mixture is as follows：
5. the mixture according to claim 1 that be used to enhance memory isolated from blood plasma, which is characterized in that institute
Blood plasma is stated from mammal.
6. the mixture according to claim 1 that be used to enhance memory isolated from blood plasma, which is characterized in that institute
Blood plasma is stated from people.
7. the mixture according to claim 1 that be used to enhance memory isolated from blood plasma, which is characterized in that
In the plasma collection procedure, blood is collected, then by the way that supernatant is collected by centrifugation, obtain blood plasma using anticoagulant tube.
8. the mixture according to claim 7 that be used to enhance memory isolated from blood plasma, which is characterized in that
In the plasma collection procedure, the centrifugal rotational speed is 500-1500g, and centrifugation time is 10-30 minutes.
9. the mixture according to claim 1 that be used to enhance memory isolated from blood plasma, which is characterized in that
In the dialysis step, dialysis time 20-72h, dialysis volume ratio is 1:100-1:10000.
10. the mixture according to claim 1 that be used to enhance memory isolated from blood plasma, which is characterized in that
The elution buffer gone in high kurtosis protein reagent box is to removing product after the primary concentration on high kurtosis albumen pillar
The eluent obtained when being eluted removes high kurtosis protein product to be described.
11. a kind of pharmaceutical composition includes any mixtures of claim 1-10 and pharmaceutically acceptable carrier.
12. pharmaceutical composition according to claim 11, which is characterized in that the pharmaceutically acceptable carrier is：Medicine
Acceptable buffer solution, protein, gelatin, monosaccharide, polysaccharide, amino acid, chelating agent, sugar alcohol, polyethylene glycol and surface on
It is one or more in activating agent.
13. pharmaceutical composition according to claim 11, which is characterized in that described pharmaceutical composition includes following component：1
Any mixtures of claim 1-10 of times volume, the 8.5wt%NaCl of 9 times of volumes or the PBS of 1.5M, pH7.0.
14. pharmaceutical composition according to claim 11, which is characterized in that
Albumin, glucose and glutamine are further included in described pharmaceutical composition, wherein, the albumin is in the drug
Quality percent by volume in composition is 2%, and quality percent by volume of the glucose in described pharmaceutical composition is
1%, quality percent by volume of the glutamine in described pharmaceutical composition is 3%.
15. a kind of sustained release preparation, any described comprising any mixtures of claim 1-10 or claim 11-14
Pharmaceutical composition and pharmaceutically acceptable biocompatible substance.
16. the sustained release preparation according to claim 15, which is characterized in that
The dosage form of the sustained release preparation is liposome, microballoon, hydrogel, Osmotic minipumps or microcapsules.
17. any mixtures of claim 1-10 are preparing prevention, improving or/and are treating failure of memory class disease
Application in drug.
18. it is applied according to claim 17, which is characterized in that
The failure of memory class disease is alzheimer's disease.
19. any pharmaceutical compositions of claim 11-14 are preparing prevention, improving or/and are treating failure of memory class
Application in the drug of disease.
20. it is applied according to claim 19, which is characterized in that
The failure of memory class disease is alzheimer's disease.
21. the sustained release preparation of claim 15 or 16 is in the medicine for preparing prevention, improving or/and treat failure of memory class disease
Application in object.
22. it is applied according to claim 21, which is characterized in that
The failure of memory class disease is alzheimer's disease.
23. any mixtures of claim 1-10 are preparing the application in enhancing memory drug.
24. any pharmaceutical compositions of claim 11-14 are preparing the application in enhancing memory drug.
25. the sustained release preparation of claim 15 or 16 is preparing the application in enhancing memory drug.
26. a kind of include any mixtures of claim 1-10, any pharmaceutical compositions of claim 11-14
Or the kit of the sustained release preparation of claim 15 or 16.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610474296.XA CN106074600B (en) | 2016-06-24 | 2016-06-24 | Isolated from blood plasma for enhancing mixture of memory and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610474296.XA CN106074600B (en) | 2016-06-24 | 2016-06-24 | Isolated from blood plasma for enhancing mixture of memory and its preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106074600A CN106074600A (en) | 2016-11-09 |
| CN106074600B true CN106074600B (en) | 2018-06-19 |
Family
ID=57252772
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610474296.XA Active CN106074600B (en) | 2016-06-24 | 2016-06-24 | Isolated from blood plasma for enhancing mixture of memory and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106074600B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106581063B (en) * | 2016-12-29 | 2018-11-06 | 北京豪思生物科技有限公司 | It is a kind of to be used to enhance blood plasma isolate of memory and its preparation method and application |
| CN106890192B (en) * | 2016-12-29 | 2019-05-21 | 四川豪思睦可医学检验有限公司 | A kind of mixture and its preparation method and application from blood plasma of enhancing memory |
| CN106692192B (en) * | 2016-12-29 | 2019-04-30 | 四川豪思睦可医学检验有限公司 | A kind of mixture and its preparation method and application for the slave blood plasma separation improving memory |
| CN106581062B (en) * | 2016-12-29 | 2020-09-18 | 江苏豪思睦可生物科技有限公司 | Mixture for improving memory and preparation method and application thereof |
| CN110724176B (en) * | 2018-07-16 | 2021-10-26 | 北京豪思生物科技有限公司 | Plasma protein isolate for treating Alzheimer's disease and preparation method and application thereof |
| CN113940286A (en) * | 2021-09-30 | 2022-01-18 | 南通大学 | Method for training and improving space learning capacity of mouse |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1569031A (en) * | 2004-04-26 | 2005-01-26 | 巫山 | Plasma component series preparation by inactivating virus and sieving sectionally by hyperfiltration membrane |
-
2016
- 2016-06-24 CN CN201610474296.XA patent/CN106074600B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1569031A (en) * | 2004-04-26 | 2005-01-26 | 巫山 | Plasma component series preparation by inactivating virus and sieving sectionally by hyperfiltration membrane |
Non-Patent Citations (3)
| Title |
|---|
| Young blood reverses age-related impairments in cognitive function and synaptic plastictity in mice;Saul Villeda等;《Nat Med.》;20140630;第20卷(第6期);2-3 * |
| 人类必需的营养素极其功能(二);杨月欣;《中国健康教育》;20041020;第20卷(第10期);894-896 * |
| 蛋白质分离纯化方法研究进展;王子佳等;《化学与生物工程》;20090825;第26卷(第08期);8-11 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106074600A (en) | 2016-11-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106074600B (en) | Isolated from blood plasma for enhancing mixture of memory and its preparation method and application | |
| CN106138097B (en) | It detaches to treat the method for mixture of alzheimer's disease and mixture and application from blood plasma | |
| Glowa et al. | Learning impairment following intracerebral administration of the HIV envelope protein gp120 or a VIP antagonist | |
| CN102625712A (en) | Unit dose formulation of antidotes for factor Xa inhibitors and methods of using the same | |
| Wan et al. | Identification of nose-to-brain homing peptide through phage display | |
| BR122020017577B1 (en) | METHOD TO PURIFY INTER-ALFA INHIBITOR PROTEINS | |
| US10597426B2 (en) | Polypeptide compound and preparation method and use thereof | |
| CN110604821A (en) | Brain targeting delivery system mediated by amyloid beta short peptide | |
| CN107760661A (en) | PEG trims of medicinal kininogenase and its preparation method and application | |
| EP3323825A1 (en) | Polypeptide compound and preparation method and use thereof | |
| CN106511378B (en) | It is a kind of to be used to enhance blood plasma extract of memory and its preparation method and application | |
| Dalgėdienė et al. | Activation of macrophages by oligomeric proteins of different size and origin | |
| CN106890192B (en) | A kind of mixture and its preparation method and application from blood plasma of enhancing memory | |
| US20230151077A1 (en) | Soluble ace2 for treatment of covid-19 | |
| CN106692192B (en) | A kind of mixture and its preparation method and application for the slave blood plasma separation improving memory | |
| US20240024459A1 (en) | Method for producing an antigen corresponding to the inactivated sars-cov-2 virus, antigen corresponding to the inactivated sars-cov-2 virus, antigenic composition, kits, and uses thereof | |
| CN107353325A (en) | Folacin receptor alpha specific WHWTNWGKTSPA and its application | |
| CN106581062B (en) | Mixture for improving memory and preparation method and application thereof | |
| CN106581063B (en) | It is a kind of to be used to enhance blood plasma isolate of memory and its preparation method and application | |
| CN105586330B (en) | A kind of Halase and preparation method thereof | |
| US20180303998A1 (en) | Novel Treatment Method for Cockayne Syndrome | |
| Yang et al. | Xiao-Xv-Ming decoction extract ameliorates brain injury in rats with thrombotic focal ischemic stroke and understanding possible therapeutic targets using proteomics. | |
| CN107753953A (en) | The preparation of Pegylation kininogenase and its application | |
| JP3719958B2 (en) | In vitro immunosorbent device | |
| CN107793471A (en) | Folacin receptor alpha specific binding peptide 4 and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Li Lin Inventor after: Cui Wenhong Inventor before: Cui Wenhong |
|
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20180511 Address after: 100094 1, 115, 117, 123, 125 rooms, 1 building, 538 Yongfeng Tun, Haidian District, Beijing. Applicant after: Beijing HAOSI Biotechnology Co. Ltd. Address before: 100000 Beijing Haidian District Renji villa D502 Applicant before: Tian Ye |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: 102208 room 102-2, 1f, building 1, courtyard 7, kekekeyuan Road, Huilongguan town, Changping District, Beijing Patentee after: Beijing HAOSI Biotechnology Co.,Ltd. Address before: 100094 1, 115, 117, 123, 125 rooms, 1 building, 538 Yongfeng Tun, Haidian District, Beijing. Patentee before: Beijing HAOSI Biotechnology Co.,Ltd. |