CN106074600A - Isolated mixture for memory reinforcing and its preparation method and application in blood plasma - Google Patents
Isolated mixture for memory reinforcing and its preparation method and application in blood plasma Download PDFInfo
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Abstract
The invention discloses a kind of isolated mixture for memory reinforcing and its preparation method and application in blood plasma.This mixture includes multiple proteins and multiple little molecule, the SDS PAGE degeneration gel electrophoresis of described mixture includes at least 30 bands, wherein include being evident that 7 bands more than the band of 48kD at molecular weight, the molecular weight of described 7 bands is 56kD the most successively, 68kD, 77kD, 115kD, 175kD, 250kD and 289kD.The preparation method of this mixture includes plasma collection, density gradient centrifugation, dialysis, once concentration successively, goes high kurtosis albumen, anion exchange and secondary concentration step.This mixture prepared by the present invention is possible not only to play the effect of memory reinforcing, it is also possible to be used for preventing, improving and treat alzheimer's disease.
Description
Technical field
The present invention relates to a kind of blood plasma separator, in particular it relates to one is isolated for hypermnesis in blood plasma
Mixture of power and its preparation method and application, this mixture can be used for preventing, improve and treat alzheimer's disease, and has
The effect of memory reinforcing.
Background technology
Alzheimer syndrome is again alzheimer's disease, is characterized in that disease man memory and cognitive competence gradually lose
Lose.Patient is dead in 3 to 9 years after making a definite diagnosis, and accounts for 50% to the 56% of clinical death case.The current understanding of this disease cause of disease is big
Beta-amyloid and Tau albumen that in brain, the exception of accumulation folds result in alzheimer's disease.
If the drug main Western medicine that alzheimer's disease is in the market, it is characterized in that adhering to for a long time taking, but
But curative effect is small, can only alleviate partial symptoms, it is impossible to the development of symptom management.The another one defect of long-term taking is that medicine is secondary
Effect is big, and patient produces drug dependence simultaneously.For being badly in need of better efficacy, the medicine that side effect is lower on this market.
Current current research shows: can promote recognizing of old mouse 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 materials
Concrete component and separate acquisition methods have not been reported.
Summary of the invention
For the defect of prior art, an object of the present invention is to provide a kind of isolated for increasing in blood plasma
The mixture of strong memory.
The two of the purpose of the present invention are to provide the preparation method of said mixture.
The three of the purpose of the present invention are to provide the application of said mixture.
To achieve these goals, present invention employs techniques below scheme:
A kind of isolated mixture for memory reinforcing in blood plasma, described mixture include multiple proteins and
Multiple little molecule, the SDS-PAGE degeneration gel electrophoresis of described mixture includes at least 30 bands, is wherein more than at molecular weight
The band of 48kD includes: molecular weight is 56kD, 68kD, 77kD, 115kD, 175kD, 250kD and 289kD the most successively
Band.
In said mixture, as a kind of preferred implementation, the FPLC of described mixture identifies have 4 peaks, right
The albumen size answered is respectively: 250kD, 120kD, 100kD and 80kD.
In said mixture, as a kind of preferred implementation, being analyzed by protein spectrum, described mixture at least contains
There are 30 albumen, including albumin, globulin, keratin, Fibrinogen and transport protein；Analyzed by little molecular mass,
Described mixture the most at least contains 20 little molecules.
In said mixture, as a kind of preferred implementation, analyzed by protein spectrum, egg contained by described mixture
White as follows:
In said mixture, as a kind of preferred implementation, analyzed by little molecular mass, contained by described mixture
Little molecule as follows:
In said mixture, as a kind of preferred implementation, described blood plasma derives from mammal；Preferably, institute
State blood plasma and derive from people.
The above-mentioned preparation method of the isolated mixture for memory reinforcing in blood plasma, is preferable to carry out as one
Mode, described preparation method includes successively: plasma collection procedure, density gradient centrifugation step, dialysis step, once 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 implementation, in described plasma collection procedure, utilize anticoagulant tube
Collect blood, then collect supernatant by centrifugal, obtain blood plasma；Preferably, described centrifugal rotational speed is 500-1500g, time centrifugal
Between be 10-30 minute.
In above-mentioned preparation method, as a kind of preferred implementation, in described density gradient centrifugation step, by described
The blood plasma that plasma collection procedure obtains mixes with Percoll solution, Forcol solution or sucrose solution, set up density gradient and from
The heart, removes the superiors' RED sector in centrifuge tube afterwards, obtains density gradient centrifugation afterproduct, wherein said centrifugal condition
For: rotating speed is 10000-30000g, and the time is 2-20h；Preferably, described Percoll solution, Forcol solution or sucrose solution
Concentration be 40-60%, the volume ratio of described Percoll solution, Forcol solution or sucrose solution and described blood plasma is (10-
15):(1-3)。
In above-mentioned preparation method, as a kind of preferred implementation, in described dialysis step, by described density gradient
Product after Li Xin is put in bag filter or pipe and is dialysed, and collects the product in bag filter or pipe, produce as dialysis after dialysis
Thing；The dialysis buffer liquid used during wherein said dialysis is Tris-hydrochloride buffer, phosphate buffer or HBS buffer；Preferably
Ground, the concentration of described Tris-hydrochloride buffer is 0.2-200mM, pH value is 7.0-9.2, and the concentration of described phosphate buffer is
1.0-500mM, pH value are 6.0-9.2, and the concentration of described HBS buffer is 0.5-500mM, pH value is 6.4-8.4；More preferably
Ground, the Tris hydrochloride buffer that described dialysis buffer liquid 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 implementation, in described once concentration step, by described dialysis
Product concentrates, and obtains once concentration afterproduct, wherein condensate precursor amass be concentrate after more than 10 times of volume；Preferably,
Described concentration is that the mode using concentration tube centrifugal is carried out, and described concentration tube allows the material of 1.5-2.5KD to pass through, described from
During the heart, rotating speed is 2000-4000g.
In above-mentioned preparation method, as a kind of preferred implementation, going in high kurtosis albumen step described, employing is gone
High kurtosis albumen in once concentration afterproduct is removed by high kurtosis protein reagent box, obtains high kurtosis protein product；Preferably
Ground, described in go the elution buffer in high kurtosis protein reagent box to going the once concentration afterproduct on high kurtosis albumen pillar to enter
The eluent obtained during row eluting, removes high kurtosis protein product for described；Preferably, the content of the described high kurtosis albumen removed
It is 5mg/ml.
In above-mentioned preparation method, as a kind of preferred implementation, in described anion exchange step, go described
High kurtosis protein product is added in anion-exchange column, carries out ladder with the Tris-hydrochloride buffer containing 20-500mM sodium chloride
Degree eluting, starts to collect eluent when elution volume is 15-35ml, stops when elution volume is 40-85ml collecting eluting
Liquid, collecting the eluent obtained is anion exchange afterproduct；Preferably, the concentration of described Tris-hydrochloride buffer is 0.2-
200mM, pH value are 7.0-9.2.
In above-mentioned preparation method, as a kind of preferred implementation, in described secondary concentration step, by described the moon from
Son exchange afterproduct concentrates, and obtains the most described mixture of secondary concentration afterproduct, and wherein condensate precursor is long-pending is body after concentration
Long-pending more than 10 times；Preferably, affiliated concentration is that the mode using concentration tube centrifugal is carried out, and described concentration tube allows 1.5-
The material of 2.5KD passes through, described centrifugal time rotating speed be 2000-4000g.
A kind of pharmaceutical composition, comprises said mixture and pharmaceutically acceptable carrier；Preferably, described pharmaceutically may be used
The carrier accepted is: pharmaceutically acceptable buffer, protein, gelatin, monosaccharide, polysaccharide, aminoacid, chelating agen, sugar alcohol, poly-
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, 8.5wt%NaCl or 1.5M of 9 times of volumes, the PBS of pH7.0；Further, described drug regimen
Thing also includes albumin, glucose and glutamine, wherein, described albumin mass body in described pharmaceutical composition
Long-pending percentage ratio is 2%, and described glucose quality percent by volume in described pharmaceutical composition is 1%, described glutamine
Quality percent by volume in described pharmaceutical composition is 3%.
A kind of slow releasing preparation, comprises said mixture or aforementioned pharmaceutical compositions and pharmaceutically acceptable biology
Compatible substances；Preferably, the dosage form of described slow releasing preparation is liposome, microsphere, hydrogel, Osmotic minipumps or microcapsule.
Said mixture, aforementioned pharmaceutical compositions and above-mentioned slow releasing preparation are preventing, are improving or/and treatment memory subtracts
Move back the application in the medicine of class disease, it is preferable that described hypomnesis class disease is alzheimer's disease.
Said mixture, aforementioned pharmaceutical compositions and the application in memory reinforcing medicine of the above-mentioned slow releasing preparation.
Comprise said mixture, aforementioned pharmaceutical compositions or the test kit of above-mentioned slow releasing preparation.
Compared to prior art, there is advantages that
1, this plasma component prepared by the present invention may be used for prevention, improves and treat alzheimer's disease, this blood plasma group
Lease making checking can delay the disease process of alzheimer's disease and likely reverse the serious journey of pathologic of alzheimer's disease
Degree, it may also operate as the effect of memory reinforcing in addition.
2, the isolated mixture from blood plasma that the present invention provides more effectively can promote than blood plasma and current medicine
The human-subject test of Alzheimer's.Meanwhile, this plasma component of long-term taking can be substantially reduced long-term taking
Side effect that Western medicine brings and drug dependence.
Accompanying drawing explanation
Fig. 1: cocktail components I (i.e. the isolated mixture from blood plasma of the present invention) prepares Anionic column chromatography knot
Really.Mice plasma through density gradient centrifugation, dialyse, concentrate and go high kurtosis albumen after, carry out Anionic column chromatography.Use the moon
Ion column SourceQ adhesion protein, then carry out gradient elution with the Tris buffer containing 500mM sodium chloride, obtain elution curve.
Start to collect eluent when elution volume is 35 milliliters, stop when elution volume is 45 milliliters collecting eluent.Collect
Eluent is cocktail components I after concentrating.
Fig. 2: cocktail components I SDS-degeneration gel electrophoresis qualification result.Swimming lane M: Protein Marker；Swimming lane 1-3: chicken
Tail wine component.
Fig. 3: cocktail components I FPLC qualification result.After cocktail component prepares, its protein component FPLC detects,
Protein peak is the absorbance at ultraviolet 280nm.
Fig. 4: cocktail components I promotes primary hippocampal cells value-added activity figure.In the culture medium of primary hippocampal cells
Being separately added into cocktail components I, other plasma component A and B, and untreated blood plasma, as experimental group.Matched group Hippocampus is thin
Born of the same parents only add DMEM culture medium.Counting hippocampal cell under the microscope after one day, experiment with computing group cell number accounts for comparison
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
Being separately added into cocktail components I in culture medium, other plasma component A and B, and untreated blood plasma, as experimental group.Comparison
Group hippocampal cell only adds DMEM culture medium.Under the microscope synapse number is counted after one day, experiment with computing group number of synapses
Mesh accounts for matched group number of synapses purpose ratio.
Fig. 6: cocktail components I promotes the memory animal model datagram of alzheimer's disease mice.By cocktail group
I is divided systematically to be expelled in alzheimer disease mouse model.By water maze laboratory, evaluate administration little to alzheimer's disease
The castering action of Mus memory.
Fig. 7: the primary hippocampal cells figure after different disposal.It is separately added into cocktail in the culture medium of primary hippocampal cells
Components I, other plasma component A and B, and untreated blood plasma, as experimental group.Matched group hippocampal cell only adds DMEM and cultivates
Base.Under the microscope hippocampal cell is carried out imaging after one day.In figure (a), (b), (c), (d), (e) is cocktail component respectively
I, the primary hippocampal cells after other plasma component A, B, untreated blood plasma, DMEM culture medium (i.e. matched group) process.
Detailed description of the invention
In order to preferably technical characteristic and effect to the present invention illustrates, use detailed description of the invention to this below
Bright it is described in detail, but the present invention is not limited to this.
One isolated mixture from blood plasma that the present invention provides, described mixture includes multiple proteins and multiple
Little molecule, the SDS-PAGE degeneration gel electrophoresis of described mixture includes at least 30 bands, wherein at molecular weight more than 48kD's
Band includes being evident that 7 bands, the molecular weight of described 7 bands are 56kD, 68kD, 77kD the most successively,
115kD, 175kD, 250kD and 289kD.
The FPLC of described mixture identifies have 4 obvious peaks.The preparation process of described mixture comprises the steps:
Analysis, once concentration, go high kurtosis albumen, anion exchange, secondary concentration.The mixture prepared through above step is through FPLC
Purification has 4 obvious peaks (referring to Fig. 3), i.e. abscissa 4 peaks between 8-18, and from left to right 4 significantly
Albumen size corresponding to peak be respectively: 250kD, 120kD, 100kD and 80kD.
Being analyzed by protein spectrum, described mixture at least contains 30 albumen, including albumin, and globulin, keratin,
Fibrinogen and transport protein；Being analyzed by little molecular mass, described mixture the most at least contains 20 little molecules.
Specifically, by protein spectrum (such as MS/MS) Analysis and Identification, albumen such as table 1 below contained by described mixture.
Albumen result contained by table 1 protein spectrum (such as MS/MS) Analysis and Identification mixture
Identified by little molecular mass analysis (such as UPLC-MS/MS), the little molecule such as table 2 below contained by described mixture.
Table 2 MS2 confirms little molecule present in mixture
Described blood plasma derives from mammal, such as people, Mus etc.；Preferably, described blood plasma derives from people.
A kind of above-mentioned preparation method of isolated mixture from blood plasma, this mixture is to be by blood plasma is carried out one
Obtain after column processing, process step and include plasma collection, density gradient centrifugation, dialysis, once concentration successively, remove high kurtosis egg
In vain, anion exchange and secondary concentration step.Specific as follows:
The various culture medium and the common agents that use in the present invention all use conventional method to prepare, and relate in embodiment divides
The most unreceipted concrete experimental condition of sub-biologic operation and method, refer to SambrookJ etc. and edit, Science Press, and 2002,
Molecular Cloning: A Laboratory guide (third edition) or the description of corresponding product.
In described plasma collection procedure, utilize anticoagulant tube to collect blood, collect supernatant by centrifugal, thus obtain blood
Slurry；Preferably, described centrifugal rotating speed be 500-1500g (such as 510g, 600g, 700g, 800g, 900g, 1000g, 1100g,
1200g, 1300g, 1400g, 1480g), centrifugation time be 10-30 minute (such as 12min, 15min, 20min, 25min,
28min)。
In described density gradient centrifugation step, the blood plasma that described plasma collection procedure is obtained and Percoll solution or
Ficoll solution or sucrose solution mixing are set up density gradient and are centrifuged, and are removed by the superiors' RED sector in centrifuge tube afterwards,
Obtaining density gradient centrifugation afterproduct, wherein said centrifugal condition is: rotating speed be 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 be 2-20h (such as
3min、5min、10min、15min、18min)；Preferably, described Percoll solution or Ficoll solution or sucrose solution is dense
Degree for 40-60% (such as 41%, 45%, 50%, 52%, 55%, 58%), described Percoll solution or Ficoll solution or
The volume ratio of sucrose solution and described 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).Can by density gradient centrifugation
To remove the little molecule of the red pigments with stronger toxicity a small amount of in blood plasma.
The compound method of the Percoll solution of variable concentrations: first (Pharmacia is public by 9 (volume) part Percoll stock solution
Department's product) mix with 1 (volume) part 8.5wt%NaCl or 1.5M PBS and to reach physiological osmotic pressure, now obtain for 100%
Percoll solution, then with physiological solution (0.85wt%NaCl or 0.15M PBS) dilution 100%Percoll solution to required
Concentration.
The compound method of the Ficoll solution of variable concentrations: first with 9 (volume) part Ficoll stock solution (Pharmacia company
Product) mix with 1 (volume) part 8.5wt%NaCl or 1.5M PBS and to reach physiological osmotic pressure, now obtain for 100%
Ficoll solution, then with physiological solution (0.85wt%NaCl or 0.15M PBS) dilution 100%Ficoll solution to required dense
Degree.
The compound method of the sucrose solution of variable concentrations: solid for sucrose block (Pharmacia Products) is added in room temperature
To saturated to 8.5wt%NaCl or 1.5M PBS solution, stirring while adding.Saturated sucrose solution concentration is 2.3M, is
100% sucrose solution.Then 100% sucrose solution is diluted to required with physiological solution (0.85wt%NaCl or 0.15M PBS)
Concentration.
In described dialysis step, described density gradient centrifugation afterproduct is put in bag filter or pipe and dialyses, thoroughly
The product in bag filter or pipe is collected, as dialysis product, for once concentration step afterwards, during wherein said dialysis after analysis
The Tris-hydrochloride buffer that dialysis buffer liquid is 0.2-200mM, pH7.0-9.2 used is (such as: 0.3mM, pH7.0's
Tris-hydrochloride buffer, the Tris-hydrochloride buffer of 1mM, pH7.0, the Tris-hydrochloride buffer of 20mM, pH7.0,50mM,
The Tris-hydrochloride buffer of pH7.0, the Tris-hydrochloride buffer of 100mM, pH7.0, the Tris-hydrochloric acid of 150mM, pH7.0 delays
Rush liquid, the Tris-hydrochloride buffer of 180mM, pH7.0, the Tris-hydrochloride buffer of 195mM, pH7.0,0.3mM, pH8.0's
Tris-hydrochloride buffer, the Tris-hydrochloride buffer of 1mM, pH8.0, the Tris-hydrochloride buffer of 30mM, pH8.0,60mM,
The Tris-hydrochloride buffer of pH8.0, the Tris-hydrochloride buffer of 90mM, pH8.0, the Tris-hydrochloride buffer of 150mM, pH8.0
Liquid, the Tris-hydrochloride buffer of 180mM, pH8.0, the Tris-hydrochloride buffer of 195mM, pH8.0,0.3mM, pH9.0's
Tris-hydrochloride buffer, the Tris-hydrochloride buffer of 1mM, pH9.0, the Tris-hydrochloride buffer of 30mM, pH9.0,60mM,
The Tris-hydrochloride buffer of pH9.0, the Tris-hydrochloride buffer of 90mM, pH9.0, the Tris-hydrochloride buffer of 150mM, pH9.0
Liquid, the Tris-hydrochloride buffer of 180mM, pH9.0, the Tris-hydrochloride buffer of 195mM, pH9.0), 1.0-500mM,
The phosphate buffer of pH6.0-9.2 (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, 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
Rush liquid (such as: the buffer of 1mM, pH6.5HBS, the buffer of 50mM, pH6.5HBS, the buffer of 100mM, pH6.5HBS,
The buffer of 150mM, pH6.5HBS, the buffer of 200mM, pH6.5HBS, the buffer of 250mM, pH6.5HBS, 300mM,
The buffer of pH6.5HBS, the buffer of 350mM, pH6.5HBS, the buffer of 450mM, pH6.5HBS, 495mM, pH6.5HBS
Buffer, the buffer of 1mM, pH7.5HBS, the buffer of 50mM, pH7.5HBS, the buffer of 100mM, pH7.5HBS,
The buffer of 150mM, pH7.5HBS, the buffer of 200mM, pH7.5HBS, the buffer of 250mM, pH7.5HBS, 300mM,
The buffer of pH7.5HBS, the buffer of 350mM, pH7.5HBS, the buffer of 450mM, pH7.5HBS, 495mM, pH7.5HBS
Buffer, the buffer of 150mM, pH8.2HBS, the buffer of 200mM, pH8.2HBS, the buffering of 250mM, pH8.2HBS
Liquid, the buffer of 300mM, pH8.2HBS, the buffer of 350mM, pH8.2HBS, the buffer of 450mM, pH8.2HBS,
The buffer of 495mM, pH8.2HBS)；The aperture of described bag filter or pipe is 0.2-0.3nm；Preferably, described dialysis buffer liquid
Tris hydrochloride buffer for 1mM, pH=8；It is highly preferred that the time of described dialysis be 20-72h (such as 21h, 25h, 30h,
35h, 40h, 45h, 55h, 65h, 71h), volume ratio (i.e. material and the dialysis buffer being in outside bag filter in bag filter of dialysing
The volume ratio of liquid) be 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 dialysis step, previous step can be removed close
Medium i.e. sucrose in degree gradient centrifugation, Percoll or Ficoll, to prevent this medium from downstream product preparation is produced bad shadow
Ring.
In described once concentration step, the product that described dialysis step obtains is concentrated, after obtaining once concentration
Product, wherein condensate precursor amass be concentrate after volume at least 10 times (such as: 15 times, 30 times, 40 times, 50 times, 70 times, 90 times,
100 times)；The concrete grammar of described concentration includes that filter membrane concentrates and concentration tube concentrates.Preferably, described concentration is to use concentration tube
Centrifugal mode is carried out, described concentration tube allow 1.5-2.5KD (such as 1.6KD, 1.8KD, 1.9KD, 2.0KD, 2.2KD,
Material 2.4KD) passes through, described centrifugal time rotating speed be 2000-4000g (such as 2100g, 2300g, 2500g, 3000g,
3200g、3500g、3800g).The purpose of this step is to allow plasma component volume diminish, thus more efficient realizing in next step
The removal of high kurtosis albumen.
Go in high kurtosis albumen step described, use and go high kurtosis protein reagent box by the height in once concentration afterproduct
Kurtosis albumen removes, thus obtains high kurtosis protein product；Preferably, the eluting in high kurtosis protein reagent box is gone to delay described in
Rush the eluent that the once concentration afterproduct gone on high kurtosis albumen pillar is carried out obtaining during first time eluting by liquid, be described
Remove high kurtosis protein product.The purpose of this step is to remove the high kurtosis albumen that content in blood plasma is 5mg/ml, in case hemostasis slurry
In active component cannot play effect.
In described anion exchange step, high kurtosis protein product is gone to be added in anion-exchange column, with containing by described
The Tris hydrochloride buffer (0.2-200mM, pH7.0-9.2) having sodium chloride (20-500mM) carries out gradient elution, when eluting body
Amass and start to collect eluent for (such as 16ml, 20ml, 25ml, 30ml, 34ml) during 15-35ml, when elution volume is 40-85ml
Time (such as 57ml, 60ml, 65ml, 75ml, 83ml) stop collecting eluent, collect the eluent that obtains and be anion exchange
Afterproduct.Wherein, the Tris hydrochloride buffer (0.2-200mM, pH7.0-9.2) containing sodium chloride (20-500mM) may is that
The Tris hydrochloride buffer of 1mM, the pH7.2 containing 25mM sodium chloride, the Tris hydrochloric acid of 1mM, the pH8.2 containing 25mM sodium chloride
Buffer, the Tris hydrochloride buffer of 1mM, the pH9.0 containing 25mM sodium chloride, 1mM, the pH7.2's containing 100mM sodium chloride
Tris hydrochloride buffer, the Tris hydrochloride buffer of 1mM, the pH8.2 containing 100mM sodium chloride, containing 100mM sodium chloride
The Tris hydrochloride buffer of 1mM, pH9.0, the Tris hydrochloride buffer of 1mM, the pH7.2 containing 200mM sodium chloride, contains
The Tris hydrochloride buffer of 1mM, pH8.2 of 200mM sodium chloride, the Tris hydrochloric acid of 1mM, the pH9.0 containing 200mM sodium chloride delays
Rush liquid, the Tris hydrochloride buffer of 1mM, the pH7.2 containing 400mM sodium chloride, 1mM, the pH8.2's containing 400mM sodium chloride
Tris hydrochloride buffer, the Tris hydrochloride buffer of 1mM, the pH9.0 containing 400mM sodium chloride, containing 490mM sodium chloride
The Tris hydrochloride buffer of 1mM, pH7.2, the Tris hydrochloride buffer of 1mM, the pH8.2 containing 490mM sodium chloride, contains
The Tris hydrochloride buffer of 1mM, pH9.0 of 490mM sodium chloride, the Tris hydrochloric acid of 100mM, the pH7.2 containing 30mM sodium chloride
Buffer, the Tris hydrochloride buffer of 100mM, the pH7.2 containing 150mM sodium chloride, 100mM containing 400mM sodium chloride,
The Tris hydrochloride buffer of pH7.2, the Tris hydrochloride buffer of 100mM, the pH7.2 containing 480mM sodium chloride, containing 30mM chlorine
Change the Tris hydrochloride buffer of 190mM, pH7.2 of sodium, the Tris hydrochloride buffer of 190mM, the pH7.2 containing 150mM sodium chloride
Liquid, the Tris hydrochloride buffer of 190mM, the pH8.2 containing 400mM sodium chloride, containing 190mM, pH9.0 of 480mM sodium chloride
Tris hydrochloride buffer.The purpose of this step is to remove composition electronegative in plasma component, to prevent these compositions pair
The toxic action of disease, the existence of electronegative composition almost can cause plasma component to be of no curative effect.It is upper for collecting elution volume
Eluent in the range of stating can obtain plasma fraction evident in efficacy.
In described secondary concentration step, described anion exchange afterproduct is concentrated, produce after obtaining secondary concentration
The most described mixture of thing, wherein condensate precursor amass be concentrate after volume at least 10 times (such as: 15 times, 30 times, 40 times, 50 times,
70 times, 90 times, 100 times)；The concrete grammar of described secondary concentration includes that filter membrane concentrates and concentration tube concentrates.Preferably, described dense
Contracting is that the mode using concentration tube centrifugal is carried out, described concentration tube allow 1.5-2.5KD (such as 1.6KD, 1.8KD, 1.9KD,
2.0KD, 2.2KD, 2.4KD) material pass through, described centrifugal time rotating speed be 2000-4000g (such as 2100g, 2300g,
2500g、3000g、3200g、3500g、3800g).The purpose of secondary concentration is to improve concentration and the viscosity of cocktail component, from
And improve the curative effect of unit composition.
A kind of pharmaceutical composition, comprises said mixture and pharmaceutically acceptable carrier.Described pharmaceutically acceptable
Carrier can be: pharmaceutically acceptable various conventional buffer (such as phosphate, citrate and other organic acid buffer
Liquid), protein (such as albumin and immunoglobulin, it can be greatly promoted the stability of product), gelatin, monosaccharide, polysaccharide, amino
In acid, chelating agen (such as EDTA), sugar alcohol (such as mannitol), Polyethylene Glycol and surfactant such as TWEEN and PLURONICS
One or more.
Preferably, described pharmaceutical composition includes following component: the said mixture of 1 times of volume (i.e. produces after secondary concentration
Thing), 8.5wt%NaCl or the 1.5M PBS (pH7.0) of 9 times of volumes.It is highly preferred that described pharmaceutical composition also includes white egg
In vain, glucose and glutamine, wherein, described albumin quality percent by volume in described pharmaceutical composition is 2%
(w/v), described glucose quality percent by volume in described pharmaceutical composition is 1% (w/v), and described glutamine is in institute
Stating the quality percent by volume in pharmaceutical composition is 3% (w/v).
The dosage form of described pharmaceutical composition may is that solution, injection, intravenous injection emulsion, syrup, mucilage,
Suspensoid, microspheres agent, microcapsule, tablet, powder, granule, pill, lyophilized powder, aerosol, spray.
Described pharmaceutical composition can be administered as follows: intravenous drip, intravenous injection, intramuscular injection, abdominal cavity note
Penetrate, Hepatic artery 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 slow releasing preparation, comprises: said mixture or aforementioned pharmaceutical compositions and pharmaceutically acceptable biology
Compatible substances；Preferably, the dosage form of described slow releasing preparation is liposome, microsphere, hydrogel, Osmotic minipumps or microcapsule.
Said mixture, aforementioned pharmaceutical compositions and/or above-mentioned slow releasing preparation are preventing, are improving or/and treat memory
Application in the medicine of the class that goes down disease；Preferably, described hypomnesis class disease is alzheimer's disease.
Said mixture, aforementioned pharmaceutical compositions and/or the application in memory reinforcing medicine of the above-mentioned slow releasing preparation.
A kind of comprise said mixture, aforementioned pharmaceutical compositions and/or the test kit of above-mentioned slow releasing preparation.Described test kit
In can also contain negative control or positive control etc..
Below by embodiment to the preparation of inventive mixture, identify and application illustrates.Following example make
Primary hippocampal cells cultivate according to following list of references: 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) take a blood sample from adult mice, collect blood plasma
By the mice of 100 adult C75BL/6 strains (this mice can also use other strain laboratory animals to replace,
Such as: the mice of BALB/cA strain, the mice of DBA/2 strain, SD rat, Wistar rat) with the carbon dioxide of 20vol%
(i.e. carbon dioxide content is the gas of 20vol%) anaesthetizes, and then uses blood taking needle from mice eye blood-letting to blood taking tube (blood taking tube
For anticoagulant tube) in, blood taking tube is rocked on blood-letting limit, limit, prevents blood coagulation.Blood taking tube containing blood is put into centrifuge, sets
Rotating speed is 1000g, centrifugal 30 minutes.Then carefully draw supernatant with pipettor, be the blood plasma collected.
(2) from mice plasma, the cocktail component i.e. mixture of the present invention is isolated
(a) density gradient centrifugation
First blood plasma is carried out density gradient centrifugation: toward the interior 12 milliliters of concentration of addition of the centrifuge tube that cumulative volume is 13 milliliters be
The Percoll of 50%, then adds the mice plasma of 1 milliliter；Centrifuge tube is put into supercentrifuge, and setting speed is
12000g, design temperature is 4 degrees Celsius, centrifugal 5 hours.After density gradient centrifugation, remove in centrifuge tube with pipettor and go up most
Layer RED sector, dialyses plasma component remaining in centrifuge tube together with Percoll.
B () is dialysed
General bag filter is selected in dialysis, and bag filter aperture is about 0.25 nanometer.The blood that will obtain after density gradient centrifugation
Slurry component together joins in bag filter together with Percoll, is then put into by bag filter in the beaker of 1L, in beaker outside bag filter
Add 1mM Tris hydrochloride buffer (pH8.0) of 1L, dialyse while stirring.Dialysis temperature is 4 DEG C, and dialysis time is 24 little
Time.After dialysis terminates, using the remaining plasma component in bag filter as dialysis product, pending once concentration.
(c) once concentration
Joining in the concentration tube that volume is 2 milliliters by the plasma component after dialysis, concentration tube allows size to be 2,000 dongle
The material paused passes through.Being put into by concentration tube in centrifuge, setting speed is 3000g, and design temperature is 4 DEG C, starts centrifuge, opens
Begin to concentrate, until final volume is 500 microlitres.Afterwards, the blood plasma after remaining dialysis is added again in this centrifuge tube so that
The volume of plasma component reaches 2mL, is centrifuged with identical parameter, and being again concentrated to final volume is 500 microlitres.So follow
Ring concentrates, until the plasma component after dialysis is finally concentrated to volume is 500 microlitres.Using this 500 microlitre plasma component as
Once concentration product, is designated as plasma component A, pending goes high kurtosis albumen to process.
D () removes high kurtosis albumen
Go high kurtosis albumen use BIO-RAD company produce model be 163-3006 remove high kurtosis protein reagent box,
The 500 microlitre blood plasma obtained after concentrating join in the pillar of high kurtosis albumen, and then this pillar is put into the ice of 4 DEG C
Case, places 5 hours.Afterwards toward the dcq buffer liquid of addition 2 milliliters in pillar, 4 degrees Celsius are centrifuged 5 minutes, and centrifugal rotational speed is
10000g.The most again toward pillar adds the elution buffer of 500 microlitres, place 30 minutes for 4 DEG C, then 4 DEG C centrifugal 5 minutes,
Centrifugal rotational speed is 10000g, and the plasma component eluted afterwards is the plasma component after high kurtosis albumen, is designated as blood plasma
Component B, is further purified stand-by for this plasma component B anion-exchange column.
(e) anion exchange
500 microlitre plasma component after removing high kurtosis albumen are added in anion-exchange column Source Q, with containing
The Tris hydrochloride buffer (200mM, pH8.0) having 500mM sodium chloride carries out gradient elution, the wherein filler of anion-exchange column
For Q Sepharose (purchased from GE Healthcare), particle mean size is 3 microns, and anion exchange column volume is 25 milliliters, washes
De-speed is 4ml/min, and loading speed is 3ml/ml.Start to collect eluent when elution volume is 35 milliliters, when eluting body
Amass and stop when being 45 milliliters collecting eluent；This elution fractions has healing activity.The eluent collected is handed over as anion
Change product, after pending secondary concentration.Seeing Fig. 1, it is Anionic column chromatography result, uses anion column Source Q absorption
Albumen, then use NaCl gradient eluant solution, this figure is elution curve.
(f) secondary concentration
Plasma component after anion exchange being joined in the concentration tube that volume is 2 milliliters, concentration tube allows size to be 2
The material of kilodalton passes through, and is put into by concentration tube in centrifuge, and setting speed is 3000g, and design temperature is 4 DEG C, start from
Scheming, starts to concentrate, until final volume is 500 microlitres.Afterwards, the blood plasma after remaining anion exchange is added this again
In centrifuge tube so that in concentration tank the volume of solution be 2 milliliters, be centrifuged with identical parameter, be again concentrated to final volume
It is 500 microlitres.So circulate concentration, until the plasma component after anion exchange is finally concentrated to volume is 500 microlitres.Should
The plasma component of 500 microlitres, is the cocktail component of isolated from mice plasma, by its named cocktail components I.
Embodiment 2: the cocktail components I SDS-PAGE degeneration gel electrophoresis of embodiment 1 preparation is identified
From above-mentioned cocktail component, take out 2 microlitres, under ultraviolet 280nm, measure its absorbance, thus calculate cocktail
The protein concentration of component.Take the cocktail component of certain volume, with 1 microlitre 5 × albumen sample-loading buffer (purchased from Beijing Lan Boli
Moral Bioisystech Co., Ltd, article No. D621) mixing, it is the sample needing to carry out electrophoresis, inside this sample, has 10 micrograms
Protein.Electrophoresis Sample is warming up to 100 DEG C, heats and make protein denaturation in 20 minutes, immediately after sample is put on ice, etc.
(compound method of described SDS degeneration also virgin rubber is as follows: 30 (w/v) %'s to start to run SDS-PAGE degeneration also virgin rubber after 5 minutes
Acrylamide Acr-Bis (purchased from GE Healthcare) takes 1.3ml, 1.5M Tris-hydrochloride buffer, and (pH8.8, purchased from GE
Healthcare) take 1.3ml, the SDS of 10 (w/v) % takes 0.05ml, 10% (w/v) Ammonium persulfate. is (purchased from GE
Healthcare) take 0.05ml, TEMED (purchased from GE Healthcare) and take 0.003ml and water takes 2.3ml, 5ml altogether,
Plastic can be solidified in room temperature) after mixing.Run glue time, the albumen applied sample amount of each swimming lane is 10 micrograms, sets run glue voltage as
100V, starts to run glue, and the race glue time is 1 hour.After running through glue, by the coomassie brilliant blue staining liquid (preparation method of this dyeing liquor
For: 2.5g coomassie brilliant blue R_250 is dissolved in 500ml 95% ethanol solution, adds the acetic acid solution of 100ml 85%, then,
Being supplemented to 1000ml with distilled water, this dye liquor is put 4 DEG C and within least 6 months, is kept stable) glue is dyeed, it appeared that cocktail
At least containing polypeptide (Fig. 2) seen from 10 naked eyes in component, it is 31kD, 56kD the most successively that its molecular weight is respectively,
68kD, 77kD, 115kD, 128kD, 165kD, 175kD, 250kD and 289kD.Wherein wrap in the molecular weight band more than 48kD
Include and be evident that 7 bands, the molecular weight of described 7 bands are 56kD, 68kD, 77kD, 115kD the most successively,
175kD, 250kD and 289kD.
Embodiment 3: the cocktail components I FPLC of embodiment 1 preparation identifies
From cocktail component, take out 2 microlitres, under ultraviolet 280nm, measure its absorbance, thus calculate cocktail component
Protein concentration.First with 20mM Tris-hydrochloride buffer (pH 8.0) balance FPLC pillar (Superdex 200), afterwards will
The cocktail component of certain volume is expelled in FPLC pillar, the cocktail component of injection microgram in protein 300.Then chicken is allowed
Tail wine component flows through FPLC pillar with the speed of 1 milliliter per minute, finds containing being evident that 4 peaks in cocktail component,
See Fig. 3, albumen size corresponding to peak from left to right 4 obvious peaks (abscissa 4 peaks between 8-18) respectively:
250kD, 120kD, 100kD and 80kD.
Embodiment 4: the cocktail Fraction Ⅰ protein matter Mass Spectrometric Identification of embodiment 1 preparation
Cocktail component is transferred in FALCON pipe, add two volumes sample buffer (formula of buffer is:
7.5M carbamide UREA, 1.5M thiourea THIOUREA, 4 (w/v) %3-[3-(gallbladder amido propyl) dimethylamino] propane sulfonic acid inner salt
CHAPS, 0.05 (w/v) % sodium lauryl sulphate SDS, 100mM dithiothreitol, DTT DDT, the shown concentration before each component is it
Respective components concentration in buffer), and by 3kDa molecular weight cut-off spin columns
(Pall GmbH, Austria) centrifuge tube centrifugal concentrating.Concentrated solution dithiothreitol, DTT carries out reduction reaction, is also originated in
Thing；Wherein, concentrated solution is 1:3 with the volume ratio of dithiothreitol, DTT, and the response time is 15 minutes, and temperature is room temperature.Again by this also
Originate in thing to react with iodoacetamide, obtain alkylate；Wherein, this reduzate and volume ratio 1:1 of iodoacetamide,
Response time is 15 minutes, and temperature is room temperature；Carrying out digestion reaction overnight with trypsin at 37 DEG C subsequently, wherein alkylation is produced
Thing and tryptic volume ratio 1:1, obtain postdigestive peptide fragment.Trypsinization gained peptide fragment is pure by C18 chromatographic column
Change, obtain sample.Gained sample traditional vacuum is dried and is saved in subsequently in-20 DEG C of refrigerators to be analyzed for MS/MS.MS/MS divides
Analyse specific as follows: HPLC's is Ultimate 3000 system, is wherein 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 be the peptide fragment process range of 400-1400m/z, MS/MS be 100-2800m/
z.Subsequently, each MS data can search for matched three top-quality CID MS/MS peak spectrum automatically.Jet hole voltage sets
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 quantitatively
(LFQ) data analysis uses open-source software MaxQuant1.3.0.5.By search SwissProt data base's (version
10/2003 20354) protein to be identified, qualification result sees table 1.
Embodiment 5: the little molecular mass of cocktail components I of embodiment 1 preparation is identified
Vial put into by the cocktail component sample taking 200 microlitres, is inserted in by vial in ice 15 minutes.Again by glass
Bottle is added thereto to 800 microlitre methane/dichloromethane mixed liquor (methane and dichloromethane with 1 milliliter of syringe in fume hood
Volume ratio be 2:1).The most again vial is inserted in Hui Bing, Ultrasonic Pulverization 2 minutes.Vial is placed on after terminating by pulverizing
Stand 20-30 minute on ice, be placed on concussion mixing 1 minute on concussion instrument afterwards.Then stand 5 minutes in being placed on refrigerator, see it
It is placed on concussion mixing on concussion instrument after layering again, repeats 3-5 time altogether.Room temperature 2000rpm is centrifuged 20 minutes afterwards, can divide after being centrifuged
Three layers, upper strata is metabolite (marking with M), and centre is protein, and lower floor is lipid (marking with LP), with the note of 250 microlitres
Upper solution in emitter gentle aspiration vial is interior to a new 1.5 milliliter EP pipe；Glass is drawn with the syringe of 250ul
Lower floor's solution in Ping is interior to another 1.5 milliliters of new EP pipes.Lower floor's liquid of sucking-off is placed in 4 DEG C of centrifuges simultaneously
13000rpm is centrifuged 10 minutes, and Aspirate supernatant divides by UPLC-MS/MS high-resolution LC-MS to 1.5 milliliters of new EP pipes
Small molecule metabolite is identified by analyzer, and Q Exactive Orbitrap mass spectrograph is for the qualification of metabolite, and it is heavy
Wanting parameter such as table 3 below, qualification result sees table 2.
Table 3 Q Exactive Orbitrap mass spectrograph identifies parameter
Embodiment 6: the cocktail components I of embodiment 1 preparation is external promotes the value-added activity of primary hippocampal cells
From cocktail component, take out 2 microlitres, under ultraviolet 280nm, measure its absorbance, thus calculate cocktail component
Protein concentration.Primary hippocampal cells (the training of the primary hippocampal cells used in the present invention in rat source is cultivated with 24 orifice plates
Breeding method is all with reference to the method 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.), cultivation used medium is DMEM, and condition of culture is 37 DEG C, 5vol% carbon dioxide.The thinnest
Born of the same parents' density reaches every hole 4 × 105During individual cell, in culture medium, add cocktail component, the cocktail group added in each hole
Divide the microgram Han protein 10 00, as experimental group 1-cocktail component.Experimental group also includes following three groups simultaneously: i.e. toward different holes
Culture medium in be separately added into mice plasma ((1st) step of embodiment 1 prepares), the egg that protein content is 1000 micrograms
Bai Hanliang is plasma component A (preparing in (2nd) step (c) of embodiment 1) of 1000 micrograms and protein content is 1000
Plasma component B (preparing in (2nd) step (d) of embodiment 1) of microgram, be divided into named experimental group 2-mice plasma,
Experimental group 3-plasma component A, experimental group 4-plasma component B.Arranging matched group, in matched group, cell density reaches by the time simultaneously
To every hole 4 × 105During individual cell, in the culture medium in hole, add the DMEM culture medium of 100 microlitres.Afterwards under conditions of original
Continue to cultivate cell 24 hours, the most under an optical microscope experimental group and matched group are carried out cell counting, calculate each experiment
Group cell number accounts for the ratio of cellular control unit number.See Fig. 4, it appeared that for comparing blood plasma, plasma component A and B, chicken
Tail wine component can be obviously promoted the increment of primary hippocampal cells, and cell number accounts for cellular control unit number and is about 150%.
Embodiment 7: the external activity promoting primary hippocampal cells Synaptic formation of cocktail components I of embodiment 1 preparation
From cocktail component, take out 2 microlitres, under ultraviolet 280nm, measure its absorbance, thus calculate cocktail component
Protein concentration.Cultivating primary hippocampal cells with 24 orifice plates, cultivation used medium is DMEM, and condition of culture is 37 DEG C,
5vol% carbon dioxide.By the time cell density reaches every hole 4 × 105During individual cell, in culture medium, add cocktail component, often
The cocktail component microgram Han protein 10 00 added in individual hole, as experimental group 1-cocktail component.Experimental group also wraps simultaneously
Include following three groups: i.e. toward be separately added in the culture medium in different holes mice plasma that protein content is 1000 micrograms (embodiment 1
(1st) step prepares), protein content be that plasma component A of 1000 micrograms (is prepared in (2nd) step (c) of embodiment 1
To) and plasma component B that protein content is 1000 micrograms (in (2) (d) of embodiment 1, step prepares), it is divided into life
Entitled experimental group 2-mice plasma, experimental group 3-plasma component A, experimental group 4-plasma component B.Matched group is set simultaneously, right
According in group, cell density reaches every hole 4 × 10 by the time5During individual cell, the DMEM adding 100 microlitres in the culture medium in hole cultivates
Base.Continue afterwards to cultivate cell 24 hours, the most under an optical microscope to each experimental group and matched group under conditions of original
Carry out synapse counting, calculate each experimental group synapse number and account for matched group number of synapses purpose ratio.See Fig. 5, it appeared that compare
For blood plasma, plasma component A and B, cocktail component can be obviously promoted primary hippocampal cells and form synapse, and it is right that synapse number accounts for
It is about 210% according to group synapse number.Continue to cultivate cell and under the microscope hippocampal cell is carried out imaging after 24 hours, see figure
7, as can be seen from Figure 7: for comparing 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, Synaptic formation number accounts for cellular control unit number and is about 210%.
Embodiment 8: the cocktail components I of embodiment 1 preparation effectively promotes the memory of alzheimer's disease mice
The present embodiment uses 5XFAD alzheimer disease mouse model, orders in U.S. jackson laboratory, and
Breed according to zoopery standard and raise.Each experimental model mice all carries out gene identification by rat-tail, it is ensured that
App gene and the stable sudden change of PS1 gene.Cocktail component is entered in Mice Body by tail vein injection, administration group guarantee 30
Micrograms of protein/time dosage, within every three days in 24 days, be administered once, altogether be administered 8 times.Water maze laboratory is used for detecting mice
Learning capacity and memory, without the 5XFAD male mice that administration group is 20 14 week old, within first 24 days, accept at Behaviors survey
8 Saline (i.e. normal saline) injections, administration group is 20 14 week old 5XFAD male mices, before Behaviors survey 24
It accepts 8 times and is administered, and negative control group is the c57BL/6 hero Mus of 20 14 week old, accepts Saline injection equally.
Water maze laboratory is carried out between at 1 in afternoon at 8 a.m..Water maze spatial memory training period is 4 days, four times a day,
Train interval time is 10 minutes every time.In an experiment, every four mices are randomly divided into a training group.For each training
Group, the position of platform of water maze is probabilistically assigned, and keeps constant in whole training.In training, mice is released from optional position
Put in water maze, and allow it to search for hiding platform in 120 seconds.If mice did not found platform in 120 seconds, it
Platform will be directed into.Training is found every time the time used by platform and the distance of process by the automatic record of intelligent video camera head
Get off.Water maze test training the last time was carried out after 48 hours, and every mice is released into the water fan not having placement platform
Gong Zhong, and allow it the most movable 60 seconds.Its travelling route is automatically recorded by intelligent video camera head.When the test phase is than training period
Between short one times, with avoid mice produce Depressive behavior.Mice is spent at three quadrants of the time that target quadrant is spent and other
The time of expense is recorded, for the assessment to mouse memory power.Its result sees Fig. 6, from fig. 6 it can be seen that be administered
The time that the group i.e. mice target quadrant of injection cocktail component is spent is essentially identical with Normal group, and cocktail group is described
Divide and alzheimer's disease mouse memory power is obviously improved effect.
The preparation of embodiment 9 cocktail composition-
In addition to step (2)-(e) is different from embodiment 1, other preparation processes are the most same as in Example 1, at the present embodiment
In, (2)-(e) is specific as follows for step:
Anion exchange:
500 microlitre plasma component after removing high kurtosis albumen are added in anion-exchange column Source Q, with containing
The Tris hydrochloride buffer (100mM, pH8.0) having 200mM sodium chloride carries out gradient elution, the wherein filler of anion-exchange column
For Q Sepharose (purchased from GE Healthcare), particle mean size is 3 microns, and anion exchange column volume is 25 milliliters, washes
De-speed is 4ml/min, and loading speed is 3ml/ml.Start to collect eluent when elution volume is 20 milliliters, when eluting body
Amass and stop when being 80 milliliters collecting eluent.The eluent collected is as anion exchanged product, after pending secondary concentration.
The cocktail component that this embodiment obtains, named cocktail composition-.
The preparation of embodiment 10 cocktail component III
In addition to step (2)-(a) is different from embodiment 1, other preparation processes are the most same as in Example 1, at the present embodiment
In, (2)-(a) is specific as follows for step:
Density gradient centrifugation
First blood plasma is carried out density gradient centrifugation: toward the interior 10 milliliters of concentration of addition of the centrifuge tube that cumulative volume is 13 milliliters be
The sucrose of 40%, then adds the mice plasma of 3 milliliters；Centrifuge tube is put into supercentrifuge, and setting speed is 30000g,
Design temperature is 4 degrees Celsius (DEG C), centrifugal 20 hours.After density gradient centrifugation, remove the superiors in centrifuge tube with pipettor
RED sector, dialyses plasma component remaining in centrifuge tube together with sucrose.
The preparation of embodiment 11 cocktail component IV
In addition to step (2)-(a) is different from embodiment 1, other preparation processes are the most same as in Example 1, at the present embodiment
In, (2)-(a) is specific as follows for step:
Density gradient centrifugation
First blood plasma is carried out density gradient centrifugation: toward the interior 11 milliliters of concentration of addition of the centrifuge tube that cumulative volume is 13 milliliters be
The Ficoll of 60%, then adds the mice plasma of 2 milliliters；Centrifuge tube is put into supercentrifuge, and setting speed is
15000g, design temperature is 4 degrees Celsius, centrifugal 10 hours.After density gradient centrifugation, remove in centrifuge tube with pipettor and go up most
Layer RED sector, dialyses plasma component remaining in centrifuge tube together with Ficoll.
Embodiment 12
Respectively cocktail composition--IV is identified according to the authentication method of embodiment 2-5, cocktail composition--IV
SDS-PAGE protein electrophoresis result show, all containing molecular weight in cocktail composition--IV is 31kD the most successively,
Polypeptide seen from 10 naked eyes of 56kD, 68kD, 77kD, 115kD, 128kD, 165kD, 175kD, 250kD and 289kD, and dividing
The son amount 7 bands more than 48kD are very clear, and its molecular weight is 56kD, 68kD, 77kD, 115kD the most successively,
175kD, 250kD and 289kD.FPLC qualification result shows, cocktail composition--IV all have albumen size be respectively 250kD,
The peak of 120kD, 100kD and 80kD.Proteomic image and little molecular mass qualification result show, in cocktail composition--IV all
Containing the protein shown in Tables 1 and 2 and little molecule.
Embodiment 13
Detect according to the method for embodiment 6 that cocktail composition--IV is external promotes the value-added work of primary hippocampal cells respectively
Property.
Testing result shows, cocktail composition--IV can also be obviously promoted the increment of primary hippocampal cells, wherein, chicken
The cell number of tail wine composition- group is 5 times of cellular control unit number；The cell number that cocktail component is III group is matched group
6 times of cell number；The cell number of cocktail component IV group is 5.5 times of cellular control unit number.
Embodiment 14
Detect according to the method for embodiment 7 that cocktail component II-IV is external promotes primary hippocampal cells Synaptic formation respectively
Activity.
Testing result shows, 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 group is 7 times of cellular control unit number；The Synaptic formation number of cocktail component III group is
6.5 times of cellular control unit number；The Synaptic formation number of cocktail component IV group is 8 times of cellular control unit number.
Claims (21)
1. an isolated mixture for memory reinforcing in blood plasma, it is characterised in that described mixture includes many
Planting protein and multiple little molecule, the SDS-PAGE degeneration gel electrophoresis of described mixture includes at least 30 bands, is wherein dividing
The son amount band more than 48kD includes: molecular weight is 56kD the most successively, 68kD, 77kD, 115kD, 175kD, 250kD
Band with 289kD.
The isolated mixture for memory reinforcing in blood plasma the most according to claim 1, it is characterised in that institute
The FPLC stating mixture identifies have 4 peaks, corresponding albumen size respectively: 250kD, 120kD, 100kD and 80kD.
The isolated mixture for memory reinforcing in blood plasma the most according to claim 1, it is characterised in that logical
Crossing protein spectrum analysis, described mixture at least contains 30 albumen, including albumin, globulin, keratin, Fibrinogen
And transport protein；Being analyzed by little molecular mass, described mixture the most at least contains 20 little molecules.
The isolated mixture for memory reinforcing in blood plasma the most according to claim 3, it is characterised in that logical
Crossing protein spectrum analysis, albumen contained by described mixture is as follows:
The isolated mixture for memory reinforcing in blood plasma the most according to claim 3, it is characterised in that
Being analyzed by little molecular mass, the little molecule contained by described mixture is as follows:
The isolated mixture for memory reinforcing in blood plasma the most according to claim 1, it is characterised in that institute
State blood plasma and derive from mammal；Preferably, described blood plasma derives from people.
7. the arbitrary described preparation side of the isolated mixture for memory reinforcing in blood plasma of claim 1-6
Method, it is characterised in that include successively: plasma collection procedure, density gradient centrifugation step, dialysis step, once concentration step, go
High kurtosis albumen step, anion exchange step and secondary concentration step.
Preparation method the most according to claim 7, it is characterised in that
In described plasma collection procedure, utilize anticoagulant tube to collect blood, then collect supernatant by centrifugal, obtain blood plasma；Excellent
Selection of land, described centrifugal rotational speed is 500-1500g, and centrifugation time is 10-30 minute.
Preparation method the most according to claim 7, it is characterised in that
In described density gradient centrifugation step, the blood plasma that described plasma collection procedure is obtained and Percoll solution, Forcol
Solution or sucrose solution mixing, set up density gradient and be centrifuged, being removed by the superiors' RED sector in centrifuge tube afterwards, obtain close
Degree gradient centrifugation afterproduct, wherein said centrifugal condition is: rotating speed is 10000-30000g, and the time is 2-20h；Preferably,
The concentration of described Percoll solution, Forcol solution or sucrose solution is 40-60%, and described Percoll solution, Forcol are molten
The volume ratio of liquid or sucrose solution and described blood plasma is (10-15): (1-3).
Preparation method the most according to claim 7, it is characterised in that
In described dialysis step, the product after described density gradient centrifugation is put in bag filter or pipe and dialyses, dialysis
Product in rear collection bag filter or pipe, as dialysis product；The dialysis buffer liquid used during wherein said dialysis is Tris-salt
Acid buffer, phosphate buffer or HBS buffer；Preferably, the concentration of described Tris-hydrochloride buffer is 0.2-200mM, pH
Value is 7.0-9.2, and the concentration of described phosphate buffer is 1.0-500mM, pH value is 6.0-9.2, the concentration of described HBS buffer
It is 6.4-8.4 for 0.5-500mM, pH value；It is highly preferred that described dialysis buffer liquid be 1mM, pH value be 8 Tris hydrochloride buffer
Liquid；Dialysis time is 20-72h, and dialysis volume ratio is 1:100-1:10000.
11. preparation methoies according to claim 7, it is characterised in that
In described once concentration step, described dialysis product is concentrated, obtain once concentration afterproduct, before wherein concentrating
Volume be concentrate after more than 10 times of volume；Preferably, described concentration is that the mode using concentration tube centrifugal is carried out, described dense
The draw allow 1.5-2.5KD material pass through, described centrifugal time rotating speed be 2000-4000g.
12. preparation methoies according to claim 7, it is characterised in that
Go in high kurtosis albumen step described, use and go high kurtosis protein reagent box by the high kurtosis in once concentration afterproduct
Albumen removes, and obtains high kurtosis protein product；Preferably, go the elution buffer in high kurtosis protein reagent box to going described in
Once concentration afterproduct on high kurtosis albumen pillar carries out the eluent obtained during eluting, goes high kurtosis albumen to produce for described
Thing；Preferably, the content of the described high kurtosis albumen removed is 5mg/ml.
13. preparation methoies according to claim 7, it is characterised in that
In described anion exchange step, high kurtosis protein product is gone to be added in anion-exchange column, with containing 20-by described
The Tris-hydrochloride buffer of 500mM sodium chloride carries out gradient elution, starts to collect eluent when elution volume is 15-35ml,
Stopping when elution volume is 40-85ml collecting eluent, collecting the eluent obtained is anion exchange afterproduct；Preferably
Ground, the concentration of described Tris-hydrochloride buffer is 0.2-200mM, pH value is 7.0-9.2.
14. preparation methoies according to claim 7, it is characterised in that in described secondary concentration step, by described the moon from
Son exchange afterproduct concentrates, and obtains the most described mixture of secondary concentration afterproduct, and wherein condensate precursor is long-pending is body after concentration
Long-pending more than 10 times；Preferably, affiliated concentration is that the mode using concentration tube centrifugal is carried out, and described concentration tube allows 1.5-
The material of 2.5KD passes through, described centrifugal time rotating speed be 2000-4000g.
15. 1 kinds of pharmaceutical compositions, comprise the arbitrary described mixture of claim 1-6 and pharmaceutically acceptable carrier.
16. pharmaceutical compositions according to claim 15, it is characterised in that described pharmaceutically acceptable carrier is: medicine
Acceptable buffer, protein, gelatin, monosaccharide, polysaccharide, aminoacid, chelating agen, sugar alcohol, Polyethylene Glycol and surface on
One or more in activating agent.
17. pharmaceutical compositions according to claim 15, it is characterised in that described pharmaceutical composition includes following component: 1
The arbitrary described mixture of claim 1-5 of times volume, 8.5wt%NaCl or 1.5M of 9 times of volumes, the PBS of pH7.0；Excellent
Selection of land, also includes albumin, glucose and glutamine in described pharmaceutical composition, wherein, described albumin is at described medicine
Quality percent by volume in compositions is 2%, described glucose quality percent by volume in described pharmaceutical composition
Being 1%, described glutamine quality percent by volume in described pharmaceutical composition is 3%.
18. 1 kinds of slow releasing preparation, comprise the arbitrary described mixture of claim 1-6 or claim 15-17 is arbitrary described
Pharmaceutical composition and pharmaceutically acceptable biocompatible substance；Preferably, the dosage form of described slow releasing preparation is lipid
Body, microsphere, hydrogel, Osmotic minipumps or microcapsule.
The arbitrary described mixture of 19. claim 1-6, the arbitrary described pharmaceutical composition of claim 15-17 and right
Require slow releasing preparation described in 18 in prevention, improve or/and application in the medicine for the treatment of hypomnesis class disease, it is preferable that
Described hypomnesis class disease is alzheimer's disease.
The arbitrary described mixture of 20. claim 1-6, the arbitrary described pharmaceutical composition of claim 15-17 and right
Require slow releasing preparation application in memory reinforcing medicine described in 18.
21. 1 kinds comprise the arbitrary described mixture of claim 1-6, the arbitrary described pharmaceutical composition of claim 15-17
Or the test kit of slow releasing preparation described in claim 18.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106581062A (en) * | 2016-12-29 | 2017-04-26 | 田野 | Mixture for improving memory and preparation method and application thereof |
| CN106581063A (en) * | 2016-12-29 | 2017-04-26 | 田野 | Blood plasma isolate for enhancing memory, and preparation method and application thereof |
| CN106692192A (en) * | 2016-12-29 | 2017-05-24 | 田野 | Memory-enhancing mixture separated from blood plasma and preparation method and application of memory-enhancing mixture |
| CN106890192A (en) * | 2016-12-29 | 2017-06-27 | 田野 | A kind of mixture from blood plasma for strengthening memory and its preparation method and application |
| CN110724176A (en) * | 2018-07-16 | 2020-01-24 | 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 |
|---|
| SAUL VILLEDA等: "Young blood reverses age-related impairments in cognitive function and synaptic plastictity in mice", 《NAT MED.》 * |
| 杨月欣: "人类必需的营养素极其功能(二)", 《中国健康教育》 * |
| 王子佳等: "蛋白质分离纯化方法研究进展", 《化学与生物工程》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106581062A (en) * | 2016-12-29 | 2017-04-26 | 田野 | Mixture for improving memory and preparation method and application thereof |
| CN106581063A (en) * | 2016-12-29 | 2017-04-26 | 田野 | Blood plasma isolate for enhancing memory, and preparation method and application thereof |
| CN106692192A (en) * | 2016-12-29 | 2017-05-24 | 田野 | Memory-enhancing mixture separated from blood plasma and preparation method and application of memory-enhancing mixture |
| CN106890192A (en) * | 2016-12-29 | 2017-06-27 | 田野 | A kind of mixture from blood plasma for strengthening memory and its preparation method and application |
| 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 |
| 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 |
| CN110724176A (en) * | 2018-07-16 | 2020-01-24 | 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 |
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| CN106074600B (en) | 2018-06-19 |
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