A kind of blood plasma extract for memory reinforcing and its preparation method and application
Technical field
The invention belongs to biology field, is related to a kind of blood plasma extract, in particular it relates to one kind is divided from blood plasma
From, for the mixture and preparation method thereof of memory reinforcing, the mixture can be used to prevent, improve and treat senile dementia
Disease, and the effect with memory reinforcing.
Background technology
Alzheimer syndrome (Alzheimer ' s disease, AD) is called senile dementia, is that the geratic period is common
One class is chronic, progressive neurocyte moves back the change of type sexually transmitted disease (STD), is characterized in that disease man memory and cognitive competence are gradually lost.Patient exists
It is dead in 3 to 9 years after making a definite diagnosis, account for 50% to the 56% of clinical death case.The current understanding of the cause of disease is accumulation in brain
The abnormal beta- amyloids for folding and Tau albumen result in senile dementia.
Senile dementia medicine in the market is mainly Western medicine, it is characterized in that to adhere 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.Another defect of long-term taking is that medicine is secondary to be made
With big, while patient produces drug dependence.For example, " class has improves alzheimer disease work to Chinese invention patent application
β1-adrenergicreceptor agonist " (application number:201210543597.5), disclose a class and there is β 1- epinephrines to receive
The compound of body agonist activity or its pharmaceutically acceptable salt, for treating Alzheimer.But the compound for Ah
The curative effect of Alzheimer's disease is still not clear.For this purpose, being badly in need of better efficacy on market, the lower medicine of side effect is used for the disease.
Current recent studies have shown that:Recognizing for old mouse can be lifted 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 lift the human-subject test of patients of senile dementia containing Cucumber in blood plasma.But, these materials
Concrete component and separation acquisition methods have not been reported.
The content of the invention
For the defect of prior art, an object of the present invention is to provide a kind of blood plasma for memory reinforcing to carry
Take thing.
The second object of the present invention is the preparation method for providing above-mentioned blood plasma extract.
The third object of the present invention is to provide the application of above-mentioned blood plasma extract.
To achieve these goals, present invention employs technical scheme below:
A kind of blood plasma extract for memory reinforcing, including multiple proteins and various small molecules;The blood plasma point
At least including the apparent band of 5 naked eyes, the molecular weight of the band is SDS-PAGE degeneration gel electrophoresis from thing:
25kD、35kD、50kD、69kD、105kD。
In the preferred implementation of blood plasma extract, analyzed by protein spectrum, at least contained in the blood plasma extract
There are 57 kinds of albumen, shown in side's table specific as follows 1.Preferably, various small molecules at least include appointing with 57 kinds of albumen
A kind of what protein bound small molecule.
In the preferred implementation of blood plasma extract, the blood plasma derives from mammal;Preferably, the blood plasma comes
Come from the mankind.
To achieve these goals, present invention employs following another technical scheme:
The preparation method of the above-mentioned blood plasma extract for memory reinforcing is comprised the following steps:Plasma collection, molecular weight
Retention, SD inactivations, centrifugation, anion exchange, dialysis, concentration;Preferably, the preparation method is in plasma collection procedure and divides
Also include between son amount retention step:Filter at low temperature step and low temperature ultrafiltration step.
In the preferred implementation of preparation method, in the plasma collection procedure, using anticoagulant tube collect blood, then
By supernatant is collected by centrifugation, blood plasma is obtained;Preferably, in the centrifugation, centrifugal force is 500-1000g, and the time is that 10-20 divides
Clock, centrifuging temperature are 0-15 DEG C.
In the preferred implementation of preparation method, in the filter at low temperature step, under stress by the blood
Slurry is filtered, and obtains filtrate;Preferably, the aperture of filter membrane is that, not less than 0.22 micron, the aperture of more preferably described filter membrane is
0.22 micron and 0.45 micron;Preferably, the temperature of the filtration is 0-5 DEG C, and pressure is 1-20MPa.
In the preferred implementation of preparation method, in the low temperature ultrafiltration step, by the filtrate by applying pressure
Power carries out film bag ultrafiltration, obtains low temperature ultrafiltration product;Preferably, the film bag molecular cut off is 3kD-10kD;Preferably, institute
The temperature for stating ultrafiltration is 0-8 DEG C, and pressure is 1-20MPa;Preferably, the buffer that the ultrafiltration is used be phosphate buffer,
One kind in Tris- hydrochloride buffers, HBS buffer;It is highly preferred that the concentration of the Tris- hydrochloride buffers is 0.2-
200mM, pH value are 7.0-8.8, and it is 6.0-9.2 that the concentration of the phosphate buffer is 1.0-500mM, pH value, the HBS bufferings
The concentration of liquid is 0.5-350mM, pH value is 6.4-8.4;It is highly preferred that the buffer is 100mM, the Tris- that pH value is 8.0
Hydrochloride buffer.
In the preferred implementation of preparation method, in molecular weight retention step, by the blood plasma or described low
Warm ultrafiltration product carries out molecular weight retention by applying pressure, obtains molecular weight retention product;Preferably, the molecular weight retention
The molecular weight of product is that more preferably higher than 10kD is to less than or equal to 50kD more than 10kD;Preferably, it is described to apply stressed side
Formula is:The low temperature ultrafiltration product is placed in concentration tube and is centrifuged;It is highly preferred that the concentration tube allows 10-50kD's
Material passes through;The centrifugal force of the centrifugation is 500-3000g, and the time is 10-120 minutes, and temperature is 0-15 DEG C;Preferably, institute
State and apply stressed mode and be:The low temperature ultrafiltration product is placed in concentration cup and is pressurizeed by liquid nitrogen;It is highly preferred that institute
Stating concentration cup allows the material of 10-50kD to pass through;The pressure of the pressurization is 1-20MPa, and pressing time is 4-8 hours.
In the preferred implementation of preparation method, in the SD inactivation steps, molecular weight retention product is used
SD inactivators are stood after suspending again, obtain inactivating product;Preferably, the dwell temperature is 4~28 DEG C, and the time is 6~24
Hour;More preferably described dwell temperature is 20 DEG C, and the time is 8 hours;Preferably, the SD inactivators contain 0.3~2%
N- butyl triphosphates, 0.5~2% tween.
In the preferred implementation of preparation method, in the step with centrifugal separation, after the inactivation product centrifugation
Retain supernatant, obtain centrifugation product;Preferably, in the centrifugation, centrifugal force is 2000-5000g, and the time is 10-20
Minute, temperature is 0-6 DEG C.
In the preferred implementation of preparation method, in the anion exchange step, by the centrifugation product
Carry out walking level eluting in adding anion-exchange column, collect eluent, after mixing, obtain anion exchanged product;Preferably, institute
State in step level eluting, first with first time elution buffer eluting, obtain first time eluent;Second elution is used again,
Obtain waste liquid;Third time elution is used again, obtains third time eluent;Merge the first time eluent and third time is washed
De- liquid, obtains anion exchanged product;It is highly preferred that the chlorination containing 100-150mM in the first time eluent buffer
Sodium, second eluent buffer contain the Sodium Chloride of 250-300mM, and the third time eluent buffer contains 450-
The Sodium Chloride of 500mM;It is further preferred that the first time elution buffer, second elution buffer, third time eluting delay
Liquid is rushed, and concentration is for 0.2-200mM, the Tris- hydrochloride buffers that pH value is 7.0-9.2.
In the preferred implementation of preparation method, in the dialysis step, the anion exchanged product is carried out
Dialysis, collects the product in Dialysis container and obtains product of dialysing;Preferably, the elution buffer for using of dialysing is Tris-
One kind in hydrochloride buffer, phosphate buffer, HBS buffer;It is highly preferred that the concentration of the Tris- hydrochloride buffers is
0.2-200mM, pH value are 7.0-8.8, and it is 6.0-9.2, the HBS that the concentration of the phosphate buffer is 1.0-500mM, pH value
The concentration of buffer is 0.5-350mM, pH value is 6.4-8.4;It is further preferred that the elution buffer is 1mM, pH value being
8 Tris hydrochloride buffers;Preferably, the time of the dialysis is 20-72h;Preferably, the volume ratio of the dialysis is 1:
(100-10000)。
In the preferred implementation of preparation method, in the concentration step, the dialysis product is concentrated, is obtained
To enriched product, as described blood plasma extract;Preferably, the volume of the dialysis product is being not less than for the enriched product
5 times, more preferably 5-100 times;Preferably, the concentration is centrifuged using concentration tube, and the concentration tube allows 1.5-3.5KD's
Material passes through, and in the centrifugation, centrifugal force is 1000-1500g, and temperature is 2-8 DEG C;Preferably, the concentration is using concentration
Cup pressurization, the concentration cup allow the material of 1.5-3.5KD to pass through, and the pressure of the pressurization is 1-20MPa.
To achieve these goals, present invention employs following another technical scheme:
A kind of pharmaceutical composition, comprising above-mentioned blood plasma extract and pharmaceutically acceptable carrier.
In the preferred implementation of pharmaceutical composition, the pharmaceutically acceptable carrier is:It is pharmaceutically acceptable
In buffer, protein, gelatin, monosaccharide, polysaccharide, aminoacid, chelating agen, sugar alcohol, Polyethylene Glycol and surfactant one
Plant or various.
In the preferred implementation of pharmaceutical composition, described pharmaceutical composition includes following component:1 times of volume it is above-mentioned
Blood plasma extract, the PBS of the 8.5wt%NaCl or 1.5M, pH7.0 of 9 times of volumes;Preferably, also wrap in described pharmaceutical composition
Include albumin, glucose and glutamine;It is highly preferred that quality volume hundred of the albumin in described pharmaceutical composition
Divide than being 2%, quality percent by volume of the glucose in described pharmaceutical composition is 1%, and the glutamine is in institute
It is 3% to state the quality percent by volume in pharmaceutical composition.
To achieve these goals, present invention employs following another technical scheme:
A kind of slow releasing preparation, comprising above-mentioned blood plasma extract or aforementioned pharmaceutical compositions and pharmaceutically acceptable
Biocompatible substance;Preferably, the dosage form of the slow releasing preparation is liposome, microsphere, hydrogel, Osmotic minipumps or micro- glue
Capsule.
To achieve these goals, present invention employs following another technical scheme:
A kind of test kit, comprising above-mentioned blood plasma extract, aforementioned pharmaceutical compositions, or above-mentioned slow releasing preparation.
To achieve these goals, present invention employs following another technical scheme:
Above-mentioned blood plasma extract, aforementioned pharmaceutical compositions, above-mentioned slow releasing preparation or mentioned reagent box, prevention, improve or/
With the application in treatment senile dementia.
Above-mentioned blood plasma extract, aforementioned pharmaceutical compositions, above-mentioned slow releasing preparation, mentioned reagent box, in memory reinforcing medicine
Application in thing.
Compared to prior art, the present invention has the advantages that:
What the 1st, the present invention was provided can more effectively lift senile dementia than blood plasma and current medicine from blood plasma extract
The human-subject test of patient.At the same time, this mixture of long-term taking can substantially reduce the secondary work that long-term taking Western medicine brings
With and drug dependence.
2nd, blood plasma extract prepared by the present invention can be used for preventing, improve and treating senile dementia, and which may be used also in addition
To play a part of memory reinforcing.
3rd, the preparation method of blood plasma extract of the invention is simple, directly industrialization can amplify, produce blood in a large number
Slurry extract supplies clinical practice.
4th, synergism between each step and its parameter of the preparation method of blood plasma extract of the invention, common to improve
The curative effect of the blood plasma extract.
Description of the drawings
Fig. 1:In detection example 1, HC4201614 blood plasma extract SDS- degeneration gel electrophoresis qualification results prepared by embodiment 1
Electrophoretogram.Swimming lane M:Protein Marker;Swimming lane 1-4:HC4201614 blood plasma extracts.
Fig. 2:In detection example 1, the microphotograph of the primary hippocampal cells after different disposal.There is primary sea toward different cultures
HC4201614 blood plasma extracts, and untreated human plasma are separately added in the DMEM culture medium of horse cell, as experimental group;
Normal saline is added in matched group hippocampal cell.Under the microscope hippocampal cell is imaged after one day;(a) in figure,
(b), (c) be respectively HC4201614 blood plasma extracts, untreated human plasma, normal saline (i.e. matched group) process after original
For hippocampal cell.
Fig. 3:In detection example 1, HC4201614 blood plasma extract prepared by embodiment 1 suppresses primary hippocampal cells apoptosis
The block diagram of activity.HC4201614 blood plasma is separately added in the DMEM culture medium for there are primary hippocampal cells toward different cultures to carry
Thing, and untreated human plasma are taken, as experimental group;Only add normal saline in matched group hippocampal cell;In microscope after one day
Under hippocampal cell is counted, experiment with computing group cell number and cellular control unit number.
Fig. 4:In detection example 1, HC4201614 blood plasma extract prepared by embodiment 1 promotes to dash forward between primary hippocampal cells
Touch the scatterplot of the activity for being formed;It is separately added in the DMEM culture medium for there are primary hippocampal cells toward different cultures
HC4201614 blood plasma extracts, and untreated human plasma, as experimental group;Only add normal saline in matched group hippocampal cell;
Under the microscope synapse number is counted after one day, experiment with computing group synapse number and matched group synapse number.
Fig. 5:In detection example 1, it is (old silly that HC4201614 blood plasma extract prepared by embodiment 1 lifts alzheimer's disease
Slow-witted disease) mice memory animal model data block diagram.HC4201614 blood plasma extract is systematically expelled to into A Erzi
In the silent disease mouse model in sea;By water maze laboratory, castering action of the administration to alzheimer's disease mouse memory power is evaluated.
Specific embodiment
In order to preferably illustrate to the technical characteristic and effect of the present invention, below using specific embodiment to this
It is bright to be described in detail, but the present invention is not limited to this.
First, the present invention provides a kind of blood plasma extract for memory reinforcing, and which derives from blood plasma, i.e., from blood plasma
The blood plasma extract HC4201614 for isolating, the mixture include multiple proteins and various small molecules, the mixture
SDS-PAGE degeneration gel electrophoresis with the apparent band of 5 naked eyes, the molecular weight of the band are:25kD、35kD、
50kD、69kD、105kD。
By protein spectrum (such as MS/MS) Analysis and Identification, the blood plasma extract at least contains 57 kinds of albumen, specifically such as
Shown in table 1 below;Also contain various small molecules in the blood plasma extract, wherein at least includes any with 57 kinds of albumen
A kind of protein bound small molecule.
Table 1:The albumen that blood plasma extract of the present invention contains
The blood plasma derives from mammal, and such as mankind, muroid etc. are preferably derived from the mankind.
Secondly, the present invention provides the preparation method for stating blood plasma extract blood plasma extract HC4201614, and the blood plasma is extracted
Thing is extracted from blood plasma and is prepared from, and the preparation method is comprised the following steps successively:Plasma collection, molecular weight are retained, SD is inactivated,
Centrifugation, anion exchange, dialysis, concentration step;Preferably, after plasma collection procedure, molecular weight retention step it
Before, the preparation method also includes filter at low temperature step and low temperature ultrafiltration step.It is specific as follows:
(1) plasma collection:
Using anticoagulant tube collect blood, then by supernatant is collected by centrifugation, obtain blood plasma;Preferably, the centrifugal force is
500-1000g, centrifugation time are 10-20min, and centrifuging temperature is 0-15 DEG C.
In an embodiment of the present invention, above-mentioned centrifugal force can be the middle arbitrary value such as 500g, 750g, 800g, 1000g or appoint
Meaning numerical range between the two, above-mentioned centrifugation time can for arbitrary value in 10min, 15min, 20min etc. or arbitrarily both
Between numerical range, above-mentioned centrifuging temperature can for arbitrary value in 0 DEG C, 5 DEG C, 7 DEG C, 10 DEG C, 15 DEG C etc. or arbitrarily both it
Between numerical range.
The quality of the blood plasma separator further to control finally to prepare, it is preferable that the plasma collection procedure it
After also need to carry out filter at low temperature step and low temperature ultrafiltration step, carry out the steps such as molecular weight retention afterwards again.
(2) filter at low temperature:
During the blood plasma that the plasma collection procedure is obtained adds filter, apply pressure using peristaltic pump and filtered (pressure
Power scope is 1-20MPa), obtain filtrate;The filter sizes of the filter are not less than 0.22 micron, and preferably 0.22 or 0.45 is micro-
Rice, the temperature control of whole defecator at 0-5 DEG C, preferably 0-4 DEG C.
Above-mentioned filter sizes can not be less than 0.22 micron, and not so hemocyte can not be filtered to remove;The temperature for filtering simultaneously
Degree not above 5 DEG C, preferably 0-4 DEG C, not so the albumen inside final product can variability so as to losing activity.This step is
In order to remove the hemocyte that may be remained in blood plasma.
In an embodiment of the present invention, above-mentioned pressure can be any in 1MPa, 5MPa, 10MPa, 15MPa, 20MPa etc.
Value or numerical range arbitrarily between the two, said temperature can for arbitrary value in 0 DEG C, 2 DEG C, 45 DEG C, 6 DEG C, 8 DEG C etc. or arbitrarily
Numerical range between the two.
(3) low temperature ultrafiltration:
The filtrate that the filter at low temperature is obtained carries out film bag ultrafiltration, using peristaltic pump apply pressure (scope be 1~
20MPa), obtain low temperature ultrafiltration product;In film bag ultra-filtration process, effective ingredient can be entered in buffer;The film bag retention
Molecular weight be 3kD-10kD, the buffer that ultrafiltration is used be phosphate buffer, Tris- hydrochloride buffers and HBS buffer
Etc. conventional buffer, the temperature control of whole ultrafiltration apparatus is at 0-8 DEG C, it is preferable that the concentration of the Tris- hydrochloride buffers is
0.2-200mM, pH value are 7.0-8.8, and it is 6.0-9.2 that the concentration of the phosphate buffer is 1-500mM, pH value, and the HBS delays
The concentration for rushing liquid is 0.5-350mM, pH value is 6.4-8.4;It is highly preferred that it is 8.0 that the buffer is 100mM, pH value
Tris- hydrochloride buffers.
Above-mentioned film bag aperture can not be more than 10kD, and not so in ultra-filtration process, some effective albumen are lost from;On
The temperature for stating ultrafiltration should be controlled at 0-8 DEG C, not so the albumen inside final product can variability so as to losing activity.This step
Material inside blood plasma is replaced in the buffer determined in pH, so as to the pH of convenient control solution system.
In an embodiment of the present invention, above-mentioned pressure can be any in 1MPa, 5MPa, 10MPa, 15MPa, 20MPa etc.
Value or numerical range arbitrarily between the two;Above-mentioned film bag molecular cut off can be arbitrary value in 3kD, 5kD, 7kD, 10kD etc.
Or numerical range arbitrarily between the two;The concentration of above-mentioned Tris- hydrochloride buffers can for 0.2mM, 1mM, 5mM, 10mM,
50mM, 100, arbitrary value or numerical range arbitrarily between the two in 125mM, 150mM, 200mM etc., pH value can for 7.0,
8.0th, the middle arbitrary values such as 8.5,8.8 or numerical range arbitrarily between the two;The concentration of above-mentioned phosphate buffer can for 1mM,
Arbitrary value or numerical range arbitrarily between the two, pH in 10mM, 50mM, 100mM, 200mM, 350mM, 400mM, 500mM etc.
Value can be arbitrary value or numerical range arbitrarily between the two in 6.0,7.0,8.0,9.2 etc.;The concentration of above-mentioned HBS buffer
Can be arbitrary value or number arbitrarily between the two in 0.5mM, 1mM, 10mM, 50mM, 100mM, 200mM, 300mM, 350mM etc.
Value scope, pH value can be arbitrary value or numerical range arbitrarily between the two in 6.4,7.0,7.5,8.0,8.4 etc..
(4) molecular weight retention:
In the molecular weight retention step, by the low temperature ultrafiltration product (if not carrying out filter at low temperature and low temperature ultrafiltration
Step, the then blood plasma for step one being obtained) molecular weight retention is carried out by concentration tube or concentration cup applying pressure, obtain molecular weight
Retention product, the concentration tube or concentration cup allow the material of 10-50kD to pass through;For example:When the concentration tube with 10kD or concentration
During cup, the molecular weight being trapped in concentration tube or concentration cup is retained into product as molecular weight more than the material of 10kD;When with
When the concentration tube or concentration cup of 50kD, the molecular weight being trapped in concentration tube or concentration cup is more than into the material of 50kD as molecule
Amount retention product;
Wherein, the first is applied stressed mode and is:The low temperature ultrafiltration product (or blood plasma) is placed in concentration tube
Row centrifugation, collects supernatant and retains product as molecular weight;The concentration tube allows the material of 10-50kD to pass through, the centrifugation
Centrifugal force be 500-3000g, the time be 10-120 minutes, temperature be 0-15 DEG C;Applying stressed mode for second is:By institute
State low temperature ultrafiltration product (or blood plasma) be placed in concentration cup in pressurizeed by liquid nitrogen;The concentration cup allows the thing of 10-50kD
Matter passes through, and in collection cups, remaining material retains product, pressing time 4-8 hour as molecular weight;The pressure of the pressurization is
1-20MPa。
In this step, when concentration tube or concentration cup allow the material for passing through to be more than 50kD, number molecular weight is less than 50kD
Effective ingredient will pass in operation, when concentration tube or concentration cup allow the material that passes through to be less than 10kD, the behaviour
Make to take in the extreme again.Temperature is set as 0 to 15 DEG C, higher than 15 degree DEG C, the effective protein groups of some in final product
Point may degeneration lose activity, less than 0 degree DEG C, requirement of the operation to temperature control instrument can be significantly increased again.This step
Purpose is powerful protein component and these protein bound small molecules in the isolated present invention.
In an embodiment of the present invention, above-mentioned centrifugal force can be in 500g, 1000g, 1500g, 2000g, 3000g etc.
Meaning value or numerical range arbitrarily between the two, time can be any in 10min, 30min, 60min, 90min, 120min etc.
Value or numerical range arbitrarily between the two, temperature can for arbitrary value in 0 DEG C, 5 DEG C, 10 DEG C, 15 DEG C etc. or arbitrarily both it
Between numerical range, above-mentioned pressure can for arbitrary value in 1MPa, 5MPa, 10MPa, 15MPa, 20MPa etc. or arbitrarily both it
Between numerical range.
(5) SD inactivations:
By the molecular weight retention product suspended with SD inactivators again, be put in 4~28 DEG C standing 6~24h, preferably 20 DEG C
8h is stood, obtains inactivating product;SD inactivators contain 0.3~2%TnBP (N- butyl triphosphates) and 0.5~2%
Tween80, preferably containing 0.3%TnBP and 1%Tween80.SD inactivators are formulated as follows:Gone out with the SD for preparing 100mL
As a example by agent living, 0.3-2ml TnBP are dissolved in suitable quantity of water, 0.5~2mLTween80 are subsequently adding, are mended with water after mix homogeneously
To 100mL.
The temperature of above-mentioned standing cannot be below 4 DEG C, and the time can not be shorter than 6 hours, and not so the effect of inactivation of virus can compare
Typically, the inactivation time of different virus is also different.The potential virus that the purpose of this step is so that in initial feed human blood lose
Activity, so as to improve the safety of final products.
In an embodiment of the present invention, the time of above-mentioned standing can be in 6h, 8h, 12h, 18h, 20h, 24h, 28h etc.
Arbitrary value or numerical range arbitrarily between the two, temperature can in 4 DEG C, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 28 DEG C etc. arbitrarily
Value or numerical range arbitrarily between the two, the percent concentration of above-mentioned TnBP can for 0.3%, 0.5%, 1%, 1.5%,
Arbitrary value or numerical range arbitrarily between the two in 2% grade, the percent concentration of above-mentioned Tween80 can for 0.5%, 1%,
1.5%th, the middle arbitrary value such as 2% or numerical range arbitrarily between the two,.
(5) centrifugation:
The inactivation product is centrifuged, retains supernatant, as centrifugation product;Preferably, it is described from
Mental and physical efforts are 2000-5000g, and centrifugation time is 10-20min, and centrifuging temperature is 0-6 DEG C.
Above-mentioned centrifugal force can not be less than 2000g, and centrifugation time can not be less than 10min, not so the miscellaneous egg of degeneration aggregate and precipitate
The unreal situation of sedimentation occurs in vain.This step eliminates some because the miscellaneous egg of SD viral inaction steps and degeneration aggregate and precipitate
In vain, not so the foreign protein of these degeneration coagulations can disturb the anion exchange step in downstream.
In an embodiment of the present invention, above-mentioned centrifugal force can be in 2000g, 2500g, 3000g, 4000g, 5000g etc.
Arbitrary value or numerical range arbitrarily between the two, the time can for arbitrary value in 10min, 12min, 15min, 20min etc. or
Numerical range arbitrarily between the two, temperature can for arbitrary value in 0 DEG C, 1 DEG C, 2 DEG C, 3 DEG C, 4 DEG C, 6 DEG C etc. or arbitrarily both
Between numerical range.
(7) anion exchange:
The centrifugation product is added in anion-exchange column (such as SourceQ), carries out walking level eluting, collection is washed
De- liquid, obtains anion exchanged product after mixing;
Wherein, in the step level eluting, first with first time elution buffer eluting, obtain first time eluent;Again with
Secondary elution buffer eluting, obtains waste liquid;Third time elution buffer eluting is used again, obtains third time eluent;Merge institute
First time eluent and third time eluent are stated, anion exchanged product is obtained;The purpose that selection eluting is 3 times is to remove second
The waste liquid for affording;Because the liquid of second eluting, toxic to neuronal cell, if not removing, it will substantially reduce
The curative effect of final whole mixture;In above-mentioned each eluting, start to collect when showing and albumen occur etc. UV-detector, egg
Stop collecting when having gone out in vain, that is, obtain the eluent of eluting each time;If using the parameter beyond these scopes, prepared
Product, its albumen and small molecule composition are above and HC4201614 differences can be larger, and at the same time curative effect can be remarkably decreased.
Sodium Chloride containing 100-150mM in the first time elution buffer, second elution buffer contain
The Sodium Chloride of 250-300mM, the third time elution buffer contain the Sodium Chloride of 450-500mM;The eluting of three eluting delays
Rush liquid concentration is for 0.2-200mM, the Tris- hydrochloride buffers that pH value is 7.0-9.2;Each elution speed is controlled in instrument
In the range of device is allowed, such as 2-5ml/min.
In an embodiment of the present invention, in above-mentioned first time elution buffer Sodium Chloride concentration can for 100mM,
Arbitrary value or numerical range arbitrarily between the two, above-mentioned second elution buffer in 110mM, 125mM, 140mM, 150mM etc.
In liquid the concentration of Sodium Chloride can for arbitrary value in 250mM, 260mM, 270mM, 280mM, 290mM, 300mM etc. or arbitrarily both
Between numerical range, in above-mentioned third time elution buffer the concentration of Sodium Chloride can for 450mM, 460mM, 470mM,
Arbitrary value or numerical range arbitrarily between the two, the concentration of above-mentioned Tris- hydrochloride buffers in 480mM, 490mM, 500mM etc.
Can be arbitrary value or number arbitrarily between the two in 0.2mM, 0.5mM, 1mM, 10mM, 50mM, 100mM, 150mM, 200mM etc.
Value scope, pH value can be arbitrary value or numerical range arbitrarily between the two in 7.0,8.0,8.5,9.2 etc..
(8) dialyse:
Dialysed during product after the anion exchange is put into bag filter or pipe, after dialysis, collected bag filter or pipe
In product, as dialysis product;Wherein, the elution buffer for using during the dialysis be Tris- hydrochloride buffers, phosphoric acid delay
Rush liquid or HBS buffer;Preferably, the concentration of the Tris- hydrochloride buffers be 0.2-200mM, pH value be 7.0-8.8, institute
The concentration for stating phosphate buffer is 1.0-500mM, pH value is 6.0-9.2, and the concentration of the HBS buffer is 0.5-350mM, pH
It is worth for 6.4-8.4;It is highly preferred that the elution buffer is 1mM, the Tris hydrochloride buffers that pH value is 8;Dialysis time is
20-72h, dialysis volume ratio is 1:(100-10000).
Dialysis time in this step is less than 20 hours, it may occur that the incomplete situation of Impurity removal, when dialysis time surpasses
72 hours are spent, the time cost of production can be dramatically increased again.Dialysis operation in this step, it is miscellaneous in step before can removing
Matter, such as inactivator, produce harmful effect to prevent the medium from preparing to downstream product;When dialysis volume ratio is less than 1:When 100, it
Impurity in front step, such as inactivator etc. can not be removed completely;When dialysis volume ratio is more than 1:When 10000, it will using big
The dialysis of amount needs substantial amounts of time dialysis simultaneously, increases time cost.
In an embodiment of the present invention, the concentration of above-mentioned Tris- hydrochloride buffers can for 0.2mM, 1mM, 5mM, 10mM,
50mM, 100mM, 125mM, 150mM, arbitrary value or numerical range arbitrarily between the two in 200 etc., pH value can for 7.0,
7.5th, the middle arbitrary values such as 8.0,8.5,8.8 or numerical range arbitrarily between the two;The concentration of above-mentioned phosphate buffer can be
Arbitrary value or numerical value model arbitrarily between the two in 1mM, 10mM, 50mM, 100mM, 200mM, 350mM, 400mM, 500mM etc.
Enclose, pH value can be arbitrary value or numerical range arbitrarily between the two in 6.4,7.0,7.5,8.0 etc.;Above-mentioned HBS buffer
Concentration can for arbitrary value in 0.5mM, 1mM, 10mM, 50mM, 100mM, 200mM, 300mM, 350mM etc. or arbitrarily both it
Between numerical range, pH value can be arbitrary values or numerical range arbitrarily between the two in 6.0,7.0,8.0,8.4 etc.;It is above-mentioned
Dialysis time can be arbitrary value or numerical range arbitrarily between the two in 20h, 25h, 50h, 60h, 72h etc.;Above-mentioned dialysis
Volume ratio can be 1:100、1:500、1:1000、1:2500、1:5000、1:Arbitrary value or arbitrarily between the two in 10000 grades
Numerical range.
(9) concentrate:
The dialysis product is concentrated, the i.e. described blood plasma extract HC4201614 of the product after being concentrated;Its
In, condensate precursor product be concentration after volume not less than 5 times, preferably 5-100 times;Preferably, the concentration is using concentration
What the mode of pipe centrifugation was carried out, the concentration tube allows the material of 1.0-3.5KD to pass through, and the centrifugal force is 1000-1500g,
Temperature is 2-8 DEG C, and cycles of concentration stops concentration after reaching requirement;Preferably, the concentration is using concentration cup pressurization
What mode was carried out, the concentration cup allows the material of 1.5-3.5KD to pass through, and the pressure limit of the pressurization is 1~20MPa.
When cycles of concentration is less than 5 times, the curative effect of unit composition is general, and osmotic pressure is low, needs separately to supplement
Sodium Chloride adjusts osmotic pressure;When the pressure that concentration is used is less than 1000g less than 1MPa or centrifugal force, concentration speed can be very
Slowly, take long;When concentration tube used or concentration cup size are more than 3.5kD, some inside the final mixture for preparing
Effective ingredient can be lost in, but when concentration tube or concentration cup size are less than 1.5kD, concentration speed also can be very slow, time-consuming to compare
It is long;When thickening temperature is higher than 8 DEG C, in concentration process some effective ingredient may because of temperature drift degeneration so as to losing work
Property, when thickening temperature is less than 2 DEG C, the price of temperature control system again can be of a relatively high, while there is enriched product because temperature
The too low risk that aggregate and precipitate occurs;One of purpose of this step is to improve effective group in the blood plasma extract HC4201614
The concentration divided, so as to improve the curative effect of unit composition;The two of purpose are the osmotic pressuries that solution is adjusted by concentration.
In an embodiment of the present invention, above-mentioned multiple can be 5 times, 10 times, 15 times, 25 times, 50 times, 75 times, 100 times etc.
Middle arbitrary value or numerical range arbitrarily between the two, the concentration tube, concentration cup allow the size of the material for passing through to be
Arbitrary value or numerical range arbitrarily between the two in 1.5KD, 1.75KD, 2KD, 2.25KD, 2.5KD, 3.0kD, 3.5kD etc.,
The centrifugal force is arbitrary value or numerical value arbitrarily between the two in 1000g, 1100g, 1200g, 1300g, 1400g, 1500g etc.
May range from temperature be 2 DEG C, 4 DEG C, 5 DEG C, arbitrary value or numerical range arbitrarily between the two in 8 DEG C etc., the pressure is
Arbitrary value or numerical range arbitrarily between the two in 1MPa, 5MPa, 7.5MPa, 10MPa, 15MPa etc..
Various common agents used in above-mentioned preparation method are prepared using conventional method.
Furthermore, the present invention provides a kind of pharmaceutical composition, comprising the blood plasma extract HC4201614 and can pharmaceutically connect
The carrier received.The pharmaceutically acceptable carrier includes:It is pharmaceutically acceptable buffer, protein, gelatin, monosaccharide, many
One or more in sugar, aminoacid, chelating agen, sugar alcohol, Polyethylene Glycol and surfactant.
Used as preferred embodiment, described pharmaceutical composition includes following component:The blood plasma extract of 1 times of volume
HC4201614, the PBS of the 8.5wt%NaCl or 1.5M, pH7.0 of 9 times of volumes;Preferably, also include in described pharmaceutical composition
Albumin, glucose and glutamine, wherein, quality percent by volume of the albumin in described pharmaceutical composition is
2%, quality percent by volume of the glucose in described pharmaceutical composition is 1%, and the glutamine is in the medicine
Quality percent by volume in compositionss is 3%.
Furthermore, the present invention provides a kind of slow releasing preparation, comprising the blood plasma extract HC4201614 and can pharmaceutically connect
The biocompatible substance received;Preferably, the dosage form of the slow releasing preparation is liposome, microsphere, hydrogel, Osmotic minipumps or micro-
Capsule;The pharmaceutically acceptable biocompatible substance includes:Aqueous pH buffer, including phosphate, citrate or
The buffer of other organic acid, ascorbic acid or other antioxidants, low-molecular-weight (being less than 10 residues) polypeptide, serum are white
Albumen, gelatin, immunoglobulin or other protein, polyvinylpyrrolidone or other hydrophilic polymers, glycine, paddy ammonia
Amide, agedoite, arginine, lysine or other aminoacid, monosaccharide, disaccharide, glucose, mannose, dextrin or other sugar
Class, EDTA or other chelating agen, Mannitol, Sorbitol or other sugar alcohols, sodium ion or other into salt counter ion,
Polyethylene Glycol (PEG),Or other nonionic surfactants.
Furthermore, the present invention provides a kind of test kit, includes:The blood plasma extract HC4201614 or/and include institute
State the aforementioned pharmaceutical compositions of blood plasma extract HC4201614 or/and comprising the above-mentioned of the blood plasma extract HC4201614
Slow releasing agent.
Furthermore, the present invention provides the blood plasma extract HC4201614 or/and includes the blood plasma extract
The aforementioned pharmaceutical compositions of HC4201614 or/and the above-mentioned slow releasing agent comprising the blood plasma extract HC4201614 or/and
Mentioned reagent box comprising the blood plasma extract HC4201614, in the medicine of prevention, improvement or/and treatment senile dementia
In application.
Finally, the present invention provides the blood plasma extract HC4201614 or/and includes the blood plasma extract
The aforementioned pharmaceutical compositions of HC4201614 or/and the above-mentioned slow releasing agent comprising the blood plasma extract HC4201614 or/and
Mentioned reagent box comprising the blood plasma extract HC4201614, the application in the medicine of memory reinforcing.
Illustrate below by preparation, identification and application of the embodiment to inventive mixture.Relate in following examples
And for example unreceipted concrete experimental condition of molecular biology manipulations and method, refer to SambrookJ etc. chief editor, scientific publication
Society, 2002, the description of Molecular Cloning:A Laboratory guide (third edition) or corresponding product.
Illustrate below by preparation, identification and application of the embodiment to inventive mixture.Make in following examples
Primary hippocampal cells are according to following list of references 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 present embodiment is the preparation method of blood plasma extract HC4201614, and the method is comprised the following steps:
(1) collect blood plasma
The blood of Healthy People is donated blood gained by hospital, and blood taking tube is anticoagulant tube, rocks blood sampling in blood collection procedure in blood-letting
Pipe, prevents blood coagulation;Blood taking tube containing blood is put into into centrifuge, centrifugal force is set as 800g, be centrifuged 15 minutes, centrifugation
Temperature is 4 DEG C;Then supernatant is carefully drawn with pipettor, the human plasma as collected.
(2) filter at low temperature:
In being 0.22 micron of filter by the blood plasma collected addition filter sizes, apply pressure 10MPa using peristaltic pump
Filtered, during which, the temperature control of whole defecator obtains filtrate at 0-4 DEG C.
(3) low temperature ultrafiltration:
Molecular weight by the filtrate retention is the film bag ultrafiltration of 8kD, applies pressure 10MPa, its process using peristaltic pump
Used in buffer for concentration 100mM, pH8.0 Tris hydrochloride buffers, the temperature control of whole ultrafiltration apparatus is in 0-4
DEG C, collect the buffer after ultrafiltration and obtain low temperature ultrafiltration product.
(4) molecular weight retention:
The low temperature ultrafiltration product is put in the concentration tube for allowing the material of 30kD to pass through, in 4 DEG C of centrifugal force 2000g centrifugations
60 minutes, collection, supernatant obtained molecular weight retention product.
(5) SD inactivations:
The molecular weight retention shallow lake product is suspended again with SD inactivators (containing 1%TnBP, 1%Tween80), 25 are put in
DEG C 10 hours are stood, the product after standing as inactivates product.
(6) centrifugation:
The inactivation product is centrifuged 15 minutes in 3500g, 0-4 DEG C, retains supernatant, as centrifugation product.
(7) anion exchange:
The centrifugation product is added into anion-exchange column (such as SourceQ, purchased from GE Healthcare, average grain
Spend for 30 microns, anion exchange column volume is 25 milliliters) in, first with the Tris- hydrochloride buffers containing 125mM Sodium Chloride
(100mM, pH8.0) carries out first time eluting, retains eluent;Then with the Tris- hydrochloride buffers containing 275mM Sodium Chloride
(100mM, pH8.0) carries out second eluting, discards eluent;Finally with the Tris- hydrochloride buffers containing 475mM Sodium Chloride
(100mM, pH8.0) carries out third time eluting, retains eluent;Merge above-mentioned first time and third time afford eluent,
As anion exchanged product.
Every time in eluting, it is ultraviolet manifest have albumen to start to collect when occurring, stop collecting when albumen appearance is finished, that is, obtain
The eluent of this eluting;Elution speed is 4ml/min every time, and loading speed is 3ml/ml.
(8) dialyse:
The ion exchange product is added to aperture to be about in 0.25 nanometer of bag filter, the bag filter is put into into 1L's then
In beaker, then to outside bag filter in the beaker add 1L 1mM Tris hydrochloride buffers (pH8.0), dialyse while stirring, thoroughly
Analysis volume ratio is 1:1000;Dialysis temperature is 4 DEG C, and dialysis time is 24 hours, obtains product of dialysing.
(9) concentrate:
Take a small amount of dialysis product to be added in the concentration tube that volume is 2mL, concentration tube allows size for the material of 2.5kD
Pass through;Again concentration tube being put in centrifuge, centrifugal force being set as 1200g, design temperature is 4 DEG C, starts centrifuge, is started dense
Contracting, until final volume is 500 microlitres;Afterwards, by the addition again centrifuge tube of the remaining dialysis product, so that volume
2mL is reached, is centrifuged with identical parameter, final volume is again concentrated to for 500 microlitres;So circulation concentration, until inciting somebody to action
Plasma component after dialysis is finally concentrated to volume for 500 microlitres, as enriched product, as above-mentioned blood plasma extract
HC4201614。
Detection example 1
The present embodiment is to carry out various detections to blood plasma extract HC4201614 prepared by embodiment 1.
Detection 1:SDS-PAGE degeneration gel electrophoresis identify that the authentication method is comprised the following steps:
(1) 2 microlitres are taken out from above-mentioned blood plasma extract HC4201614, its absorbance is determined under ultraviolet 280nm, from
And calculate the protein concentration of blood plasma extract HC4201614.
(2) the blood plasma extract HC4201614 of certain volume is taken, and (Beijing is purchased from 1 microlitre of 5 × albumen sample-loading buffer
Lan Bolide Bioisystech Co., Ltd, article No. D621) mixing, as need to carry out the sample of electrophoresis, inside the sample, have 10
The protein of microgram.
(3) electrophoresis Sample is warming up to into 100 DEG C, heating makes protein denaturation in 20 minutes, and sample is put into ice immediately after
On, (compound method of the SDS degeneration also virgin rubber is as follows to start race SDS-PAGE degeneration also virgin rubber after waiting 5 minutes:30(w/
V) (pH8.8 is purchased from the acrylamide Acr-Bis (being purchased from GEHealthcare) of % to take 1.3ml, 1.5MTris- hydrochloride buffer
GEHealthcare 1.3ml, the SDS of 10 (w/v) %) are taken take 0.05ml, 10% (w/v) Ammonium persulfate. and (be purchased from
GEHealthcare) take that 0.05ml, TEMED (purchased from GEHealthcare) take 0.003ml and water takes 2.3ml, 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 run glue voltage as
100V, starts to run glue, runs the glue time for 1 hour.
(4), after running through glue, with coomassie brilliant blue staining liquid, (preparation method of the dyeing liquor is:By 2.5g Coomassie brilliant blues
R-250 is dissolved in 500ml95% ethanol solution, adds the acetic acid solution of 100ml85%, then, is supplemented to 1000ml with distilled water,
This dye liquor is put 4 DEG C and keeps within least 6 months stable) glue is dyeed.
Testing result is referring to Fig. 1:Wherein:Swimming lane M:Protein Marker;Swimming lane 1-4:HC4201614 mixture;Should
At least containing the visible polypeptide of 5 naked eyes in blood plasma extract HC4201614, its molecular weight is respectively is from small to large successively
25kD、35kD、50kD、69kD、105kD。
Detection 2:Proteomic image identifies that the authentication method comprises the steps:
(1) blood plasma extract HC4201614 is transferred in FALCON pipes, adds the sample buffer of two volumes (slow
The formula for rushing liquid 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 institute before each component
Show that concentration is concentration of its respective components in buffer), and pass through 3kDamolecular weight cut-off spin
Columns (Pall GmbH, Austria) centrifuge tube centrifugal concentrating, obtains concentrated solution.
(2) concentrated solution carries out reduction reaction with dithiothreitol, DTT, obtains reduzate;Wherein, concentrated solution and two sulfur threoses
The volume ratio of alcohol is 1:3, the response time is 15 minutes, and temperature is room temperature.
(3) again the reduzate and iodoacetamide are reacted, is obtained alkylate;Wherein, the reduzate with
The volume ratio 1 of iodoacetamide:1, the response time is 15 minutes, and temperature is room temperature;
(4) subsequently digestion reaction is carried out overnight at 37 DEG C with trypsin, wherein alkylate and tryptic body
Product compares 1:1, obtain postdigestive peptide fragment.
(5) peptide fragment obtained by trypsinization obtains sample by C18 chromatographies.
(6) sample traditional vacuum obtained by is dried and analyzes for MS/MS in being subsequently stored in -20 DEG C of refrigerators.MS/MS is analyzed
It is specific as follows:HPLC's is 3000 systems of Ultimate, wherein being equipped with PepMap100 C-18 trap column (300
μ m 5mm) and two pillars of PepMap100 C-18 analytical column (75 μ m 250mm).Mass spectrograph is adopted
Amazon speed ETD, MS data acquisition ranges are 400-1400m/z, and the peptide fragment process range of MS/MS is 100-2800m/
z.Subsequently, each MS data can search for the top-quality CID MS/MS peaks spectrum of matched three 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 quantitative
(LFQ) data analysiss adopt open-source software MaxQuant 1.3.0.5.By searching for SwissProt data base's (versions
10/2003 20354) protein to be identified, qualification result is referring to table 1.
Detection 3:Detection blood plasma extract HC4201614 has the activity of vitro inhibition primary hippocampal cells apoptosis, promotes
The activity of Synaptic formation between hippocampal cell.The detection method is comprised the following steps:
(1) 2 microlitres are taken out from above-mentioned blood plasma extract HC4201614, its absorbance is determined under ultraviolet 280nm, from
And calculate the protein concentration of above-mentioned blood plasma extract HC4201614.
(2) with 24 orifice plate culture primary hippocampal cells, culture used medium is DMEM, and condition of culture is 37 DEG C,
5vol% carbon dioxide.
(3) when cell density reaches every hole 4 × 105During individual cell, blood plasma extract is added toward in culture medium
HC4201614, blood plasma extract HC4201614 00 micrograms containing protein 10 added in each hole, carries as experimental group 1- blood plasma
Take thing HC4201614.Also include in experimental group simultaneously:It is micro- for 1000 protein content to be separately added into toward in the culture medium in different holes
Gram human plasma (the step of 1 preparation method of embodiment (1) prepares), be named as experimental group 2- human plasmas.Arrange right simultaneously
According to group, in matched group, when cell density reaches every hole 4 × 105During individual cell, add 100 microlitres toward the culture medium in hole
Normal saline.
(4) continue cultured cells 24 hours under conditions of original, then under an optical microscope to each experimental group and right
The counting of cell quantity and synapse quantity is carried out according to group.
Such as Fig. 2, wherein (a), (b), is (c) according to experimental group 1-HC4201614, experimental group 2- human plasmas, control respectively
The primary hippocampal cells that group is obtained after processing.The synapse quantity that either hippocampal cell quantity is still formed is can observe, is tested
Group 1-HC4201614 mixture is far longer than matched group, more than experimental group 2- human plasmas.
Such as Fig. 3, the quantity of the primary hippocampal cells of experimental group 1 reaches about 1250-1700/square centimeter, much larger than reality
Test about 500-750/square centimeter of group 2, and about 400-700/square centimeter of matched group, it was demonstrated that blood plasma extract
HC4201614 has the activity for suppressing primary hippocampal cells apoptosis.
Such as Fig. 4, the Synaptic formation quantity of experimental group 1 reaches about 140/square centimeter, much larger than about the 70 of experimental group 2
Individual/square centimeter, and about 50/square centimeter of matched group, it was demonstrated that separator HC4201614 has between promotion hippocampal cell
The activity of Synaptic formation.
Detection 4:Detection blood plasma extract HC4201614 can effectively lift alzheimer's disease (senile dementia) mice
Memory, the detection method comprises the following steps:
(1) the present embodiment adopts 5XFAD Elderly dementia patients models, orders in U.S. jackson laboratory, and
Bred and raised according to zoopery standard;Each of which experimental model mice all carries out gene identification by rat-tail,
Guarantee the stable mutation of app gene and PS1 genes.
Mice is grouped into:Experimental group 1- blood plasma extract HC4201614:Using 20 14 week old 5XFAD male mices,
Blood plasma extract HC4201614 prepared by injection embodiment 1;Experimental group 2- human plasmas:It is little using 20 14 week old 5XFAD males
The blood plasma that the step of Mus, injection 1 preparation method of embodiment (1) prepares;Matched group:Using 20 14 week old 5XFAD males
Mice, injecting normal saline.
Various administrations are entered in mice body by tail vein injection, experimental group ensure 30 micrograms of protein/time dosage,
Behaviors survey (specially water maze laboratory, for detecting learning capacity and the memory of mice) in first 24 days per three days to
Medicine 1 time, is administered 8 times altogether.
(2) water maze laboratory:Carry out between 8 points of every morning at 1 point in afternoon.Water maze spatial memory training period is 4 days,
Four times a day, training interval time is 10 minutes every time;In an experiment, a training group is randomly divided into per four mices.For
Each training group, the position of platform of water maze are probabilistically assigned, and in whole training keep constant.In training, mice from appoint
Meaning position is released in water maze, and allows it that hiding platform was searched in 120 seconds.If mice is no looked in 120 seconds
To platform, it will be directed into platform.The time used by platform is found in training every time with the distance passed through by intelligent camera
Head automatically records.
Water maze test is carried out after last time is trained 48 hours.In testing every time, every mice is released into not to be had
In the water maze of placement platform, and allow its freely activity 60 second.Its travelling route is automatically recorded by intelligent video camera head.Survey
The examination phase is shorter than time training period one times, to avoid mice from producing Depressive behavior.Time that mice is spent in target quadrant and its
The time spent by his three quadrants is recorded, for the assessment to mouse memory power.Test result such as Fig. 5, experimental group 1
Target quadrant stop memory time about 90 seconds, considerably longer than about 5 seconds of about the 10 of experimental group 2 second and matched group.
Embodiment 2
In the present embodiment, in addition to step (9) is different from embodiment 1, other preparation processes are same as Example 1,
In the present embodiment, step (9) is specific as follows:
Dialysis product prepared by 1 step of embodiment (8) is added in the concentration cup for allowing 2.5kD materials to pass through, and is covered dense
Then concentration cup is connected liquid nitrogen bottle, opens liquid nitrogen bottle air valve, set pressure by contracting bowl cover, the chromatography cabinet that concentration cup is put into
For 10MPa;Under this air pressure, concentration cup starts product after concentration dialysis, before after observing concentration, the volume of product is concentration
When 1/50, stop concentration, obtain enriched product, as blood plasma extract HC4201614.
Detection example 2
Various detections are carried out to blood plasma extract HC4201614 prepared by embodiment 2.
Detection 1-3:Carried out using the blood plasma extract HC4201614 obtained to embodiment 2 with detection 1 identical method of example
Detection, it is as a result identical with the mixture that embodiment 1 is obtained.
Detection 4:There are blood plasma extract HC4201614 prepared by detection embodiment 2 vitro inhibition primary hippocampal cells to wither
The activity died, the activity for promoting Synaptic formation between hippocampal cell.The detection method is identical with detection example 1.Testing result table
Bright, the quantity of the primary hippocampal cells of experimental group 1 reaches about 1350-1800/square centimeter, much larger than the pact of experimental group 2
500-750/square centimeter, and about 400-700/square centimeter of matched group, it was demonstrated that blood plasma extract HC4201614 has
Suppress the activity of primary hippocampal cells apoptosis.The Synaptic formation quantity of experimental group 1 reaches about 150/square centimeter, much larger than reality
Test about 70/square centimeter of group 2, and about 50/square centimeter of matched group, it was demonstrated that separator HC4201614 has promotion
The activity of Synaptic formation between hippocampal cell.Prove that HC4201614 mixture has and promote Synaptic formation between hippocampal cell
Activity.
Detection 5:It is (old that blood plasma extract HC4201614 prepared by detection embodiment 2 can effectively lift alzheimer's disease
Dementia disease) mice memory.The detection method is identical with detection example 1.Test result shows, the target of experimental group 1 as
Limit stops memory time about 100 seconds, considerably longer than about 5 seconds of about the 10 of experimental group 2 second and matched group.
Embodiment 3
In the present embodiment, in addition to step (4) is different from embodiment 1, other preparation processes are same as Example 1,
In the present embodiment, step (4) is specific as follows:
During blood plasma prepared by 1 step of embodiment (1) adds the concentration cup for allowing 30kD materials to pass through, concentration cup is covered
Lid, will the concentration chromatography cabinet that is put into of cup, concentration cup is connected into liquid nitrogen bottle then, liquid nitrogen bottle air valve is opened, set pressure as
10MPa, pressurizes 480 minutes, obtains molecular weight retention product.
Detection example 3
Various detections are carried out to blood plasma extract HC4201614 prepared by embodiment 3.
Detection 1-3:Carried out using the blood plasma extract HC4201614 obtained to embodiment 3 with detection 1 identical method of example
Detection, it is as a result identical with the mixture that embodiment 1 is obtained.
Detection 4:There are blood plasma extract HC4201614 prepared by detection embodiment 3 vitro inhibition primary hippocampal cells to wither
The activity died, the activity for promoting Synaptic formation between hippocampal cell.The detection method is identical with detection example 1.Testing result table
Bright, the quantity of the primary hippocampal cells of experimental group 1 reaches about 1200-1500/square centimeter, much larger than the pact of experimental group 2
500-750/square centimeter, and about 400-700/square centimeter of matched group, it was demonstrated that blood plasma extract HC4201614 has
Suppress the activity of primary hippocampal cells apoptosis.The Synaptic formation quantity of experimental group 1 reaches about 150/square centimeter, much larger than reality
Test about 70/square centimeter of group 2, and about 50/square centimeter of matched group, it was demonstrated that separator HC4201614 has promotion
The activity of Synaptic formation between hippocampal cell.Prove that HC4201614 mixture has and promote Synaptic formation between hippocampal cell
Activity.
Detection 5:Blood plasma extract HC4201614 prepared by detection embodiment 10 can effectively lift alzheimer's disease
The memory of (senile dementia) mice.The detection method is in the same manner as in Example 5.Test result shows, the target of experimental group 1
Quadrant stops memory time about 90 seconds, considerably longer than about 5 seconds of about the 10 of experimental group 2 second and matched group.
Embodiment 4
The present embodiment is the preparation method of blood plasma extract HC4201614, and the method is comprised the following steps:
(1) collect blood plasma:It is identical with the method for step (1) in embodiment 1.
(2) filter at low temperature:
In being 0.45 micron of filter by the blood plasma collected addition filter sizes, apply pressure 20MPa using peristaltic pump
Filtered, during which, the temperature control of whole defecator obtains filtrate at 0-4 DEG C.
(3) low temperature ultrafiltration:
Molecular weight by the filtrate retention is the film bag ultrafiltration of 3kD, applies pressure 20MPa, its process using peristaltic pump
Used in buffer for concentration 100mM, pH8.0 Tris hydrochloride buffers, the temperature control of whole ultrafiltration apparatus is in 0-4
DEG C, collect the buffer after ultrafiltration and obtain low temperature ultrafiltration product.
(4) molecular weight retention:
The low temperature ultrafiltration product is put in the concentration tube for allowing the material of 10kD to pass through, in 0 DEG C of centrifugal force 500g centrifugation
120 minutes, supernatant is collected, obtain molecular weight retention product.
(5) SD inactivations:
The molecular weight retention product is suspended again with SD inactivators (containing 1%TnBP, 1%Tween80), 4 DEG C are put in
24 hours are stood, the product after standing as inactivates product.
(6) centrifugation:
The inactivation product is centrifuged 20 minutes in 2000g, 0 DEG C, retains supernatant, as centrifugation product.
(7) anion exchange:
The centrifugation product is added into anion-exchange column (such as SourceQ, purchased from GE Healthcare, average grain
Spend for 30 microns, anion exchange column volume is 25 milliliters) in, first with the Tris- hydrochloride buffers containing 125mM Sodium Chloride
(100mM, pH8.0) carries out first time eluting, retains eluent;Then with the Tris- hydrochloride buffers containing 275mM Sodium Chloride
(100mM, pH8.0) carries out second eluting, discards eluent;Finally with the Tris- hydrochloride buffers containing 475mM Sodium Chloride
(100mM, pH8.0) carries out third time eluting, retains eluent;Merge above-mentioned first time and third time afford eluent,
As anion exchanged product.
Every time in eluting, it is ultraviolet manifest have albumen to start to collect when occurring, stop collecting when albumen appearance is finished, that is, obtain
The eluent of this eluting;Elution speed is 4ml/min every time, and loading speed is 3ml/ml.
(8) dialyse:
The ion exchange product is added to aperture to be about in 0.25 nanometer of bag filter, the bag filter is put into into 1L's then
In beaker, then to outside bag filter in the beaker add 1L 1mM Tris hydrochloride buffers (pH8.0), dialyse while stirring;Thoroughly
Eutectoid temperature is 4 DEG C, and dialysis time is 20 hours, obtains product of dialysing.
(9) concentrate:
Take a small amount of dialysis product to be added in the concentration tube that volume is 2mL, concentration tube allows size for the material of 1.5kD
Pass through;Again concentration tube being put in centrifuge, centrifugal force being set as 1000g, design temperature is 4 DEG C, starts centrifuge, is started dense
Contracting, until final volume is 500 microlitres;Afterwards, by the addition again centrifuge tube of the remaining dialysis product, so that volume
2mL is reached, is centrifuged with identical parameter, final volume is again concentrated to for 500 microlitres;So circulation concentration, until inciting somebody to action
Plasma component after dialysis is finally concentrated to volume for 500 microlitres, as enriched product, as above-mentioned blood plasma extract
HC4201614。
Detection example 4
Various detections are carried out to blood plasma extract HC4201614 prepared by embodiment 4.
Detection 1-3:Entered using the blood plasma extract HC4201614 obtained to the present embodiment with detection 1 identical method of example
Row detection, it is as a result identical with the mixture that embodiment 1 is obtained.
Detection 4:There are blood plasma extract HC4201614 prepared by detection embodiment 4 vitro inhibition primary hippocampal cells to wither
The activity died, the activity for promoting Synaptic formation between hippocampal cell.The detection method is in the same manner as in Example 4.Testing result table
Bright, the quantity of the primary hippocampal cells of experimental group 1 reaches about 1200-1750/square centimeter, much larger than the pact of experimental group 2
500-750/square centimeter, and about 400-700/square centimeter of matched group, it was demonstrated that blood plasma extract HC4201614 has
Suppress the activity of primary hippocampal cells apoptosis.The Synaptic formation quantity of experimental group 1 reaches about 120/square centimeter, much larger than reality
Test about 70/square centimeter of group 2, and about 50/square centimeter of matched group, it was demonstrated that separator HC4201614 has promotion
The activity of Synaptic formation between hippocampal cell.Prove that HC4201614 mixture has and promote Synaptic formation between hippocampal cell
Activity.
Detection 5:It is (old that blood plasma extract HC4201614 prepared by detection embodiment 4 can effectively lift alzheimer's disease
Dementia disease) mice memory.The detection method is in the same manner as in Example 5.Test result shows, the target of experimental group 1 as
Limit stops memory time about 90 seconds, considerably longer than about 5 seconds of about the 10 of experimental group 2 second and matched group.
Embodiment 5
The present embodiment is the preparation method of blood plasma extract HC4201614, and the method is comprised the following steps:
(1) collect blood plasma:It is identical with the method for step (1) in embodiment 1.
(2) filter at low temperature:
In being 0.22 micron of filter by the blood plasma collected addition filter sizes, apply pressure 20MPa using peristaltic pump
Filtered, during which, the temperature control of whole defecator obtains filtrate at 0-4 DEG C.
(3) low temperature ultrafiltration:
Molecular weight by the filtrate retention is the film bag ultrafiltration of 10kD, applies pressure 1MPa, its process using peristaltic pump
Used in buffer for concentration 100mM, pH8.0 Tris hydrochloride buffers, the temperature control of whole ultrafiltration apparatus is in 0-4
DEG C, collect the buffer after ultrafiltration and obtain low temperature ultrafiltration product.
(4) molecular weight retention:
The low temperature ultrafiltration product is put in the concentration tube for allowing the material of 50kD to pass through, in 0 DEG C of centrifugal force 3000g centrifugation
10 minutes, supernatant is collected, obtain molecular weight retention product.
(5) SD inactivations:
The molecular weight retention product is suspended again with SD inactivators (containing 1%TnBP, 1%Tween80), 28 DEG C are put in
6 hours are stood, the product after standing as inactivates product.
(6) centrifugation:
The inactivation product is centrifuged 10 minutes in 5000g, 4 DEG C, retains supernatant, as centrifugation product.
(7) anion exchange:
The centrifugation product is added into anion-exchange column (such as SourceQ, purchased from GE Healthcare, average grain
Spend for 3 microns, anion exchange column volume is 25 milliliters) in, first with the Tris- hydrochloride buffers containing 100mM Sodium Chloride
(100mM, pH8.0) carries out first time eluting, retains eluent;Then with the Tris- hydrochloride buffers containing 250mM Sodium Chloride
(100mM, pH8.0) carries out second eluting, discards eluent;Finally with the Tris- hydrochloride buffers containing 450mM Sodium Chloride
(100mM, pH8.0) carries out third time eluting, retains eluent;Merge above-mentioned first time and third time afford eluent,
As anion exchanged product.
Every time in eluting, it is ultraviolet manifest have albumen to start to collect when occurring, stop collecting when albumen appearance is finished, that is, obtain
The eluent of this eluting;Elution speed is 4ml/min every time, and loading speed is 3ml/ml.
(8) dialyse:
The ion exchange product is added to aperture to be about in 0.25 nanometer of bag filter, the bag filter is put into into 1L's then
In beaker, then to outside bag filter in the beaker add 1L 1mM Tris hydrochloride buffers (pH8.0), dialyse while stirring;Thoroughly
Eutectoid temperature is 4 DEG C, and dialysis time is 72 hours, obtains product of dialysing.
(9) concentrate:
Take a small amount of dialysis product to be added in the concentration tube that volume is 2mL, concentration tube allows size for the material of 3.5kD
Pass through;Again concentration tube being put in centrifuge, centrifugal force being set as 1500g, design temperature is 4 DEG C, starts centrifuge, is started dense
Contracting, until final volume is 500 microlitres;Afterwards, by the addition again centrifuge tube of the remaining dialysis product, so that volume
2mL is reached, is centrifuged with identical parameter, final volume is again concentrated to for 500 microlitres;So circulation concentration, until inciting somebody to action
Plasma component after dialysis is finally concentrated to volume for 500 microlitres, as enriched product, as above-mentioned blood plasma extract
HC4201614。
Detected using the blood plasma extract HC4201614 obtained to the present embodiment with embodiment 2-3 identical method,
As a result it is identical with the mixture that embodiment 1 is obtained.
Detection example 5
Detection 1-3:Carried out using the blood plasma extract HC4201614 obtained to embodiment 5 with detection 1 identical method of example
Detection, it is as a result identical with the mixture that embodiment 1 is obtained.
Detection 4:There are blood plasma extract HC4201614 prepared by detection embodiment 5 vitro inhibition primary hippocampal cells to wither
The activity died, the activity for promoting Synaptic formation between hippocampal cell.The detection method is identical with detection example 1.Testing result table
Bright, the quantity of the primary hippocampal cells of experimental group 1 reaches about 1400-1800/square centimeter, much larger than the pact of experimental group 2
500-750/square centimeter, and about 400-700/square centimeter of matched group, it was demonstrated that blood plasma extract HC4201614 has
Suppress the activity of primary hippocampal cells apoptosis.The Synaptic formation quantity of experimental group 1 reaches about 120/square centimeter, much larger than reality
Test about 70/square centimeter of group 2, and about 50/square centimeter of matched group, it was demonstrated that separator HC4201614 has promotion
The activity of Synaptic formation between hippocampal cell.Prove that HC4201614 mixture has and promote Synaptic formation between hippocampal cell
Activity.
Detection 5:It is (old that blood plasma extract HC4201614 prepared by detection embodiment 5 can effectively lift alzheimer's disease
Dementia disease) mice memory.The detection method is identical with test case 1.Test result shows, the target of experimental group 1 as
Limit stops memory time about 100 seconds, considerably longer than about 5 seconds of about the 10 of experimental group 2 second and matched group.
Embodiment 6
The present embodiment is the preparation method of blood plasma extract HC4201614, and the method is comprised the following steps:
(1) collect blood plasma:It is identical with the method for step (1) in embodiment 1.
(2) filter at low temperature:
In being 0.22 micron of filter by the blood plasma collected addition filter sizes, apply pressure 15MPa using peristaltic pump
Filtered, during which, the temperature control of whole defecator obtains filtrate at 0-4 DEG C.
(3) low temperature ultrafiltration:
Molecular weight by the filtrate retention is the film bag ultrafiltration of 5kD, applies pressure 15MPa, its process using peristaltic pump
Used in buffer for concentration 100mM, pH8.0 Tris hydrochloride buffers, the temperature control of whole ultrafiltration apparatus is in 0-4
DEG C, collect the buffer after ultrafiltration and obtain low temperature ultrafiltration product.
(4) molecular weight retention:
The low temperature ultrafiltration product is put in the concentration tube for allowing the material of 40kD to pass through, in 5 DEG C of centrifugal force 1000g centrifugations
90 minutes, supernatant is collected, obtain molecular weight retention product.
(5) SD inactivations:
The molecular weight retention product is suspended again with SD inactivators (containing 1%TnBP, 1%Tween80), 15 DEG C are put in
15 hours are stood, the product after standing as inactivates product.
(6) centrifugation:
The inactivation product is centrifuged 15 minutes in 4000g, 6 DEG C, retains supernatant, as centrifugation product.
(7) anion exchange:
The centrifugation product is added into anion-exchange column (such as SourceQ, purchased from GE Healthcare, average grain
Spend for 3 microns, anion exchange column volume is 25 milliliters) in, first with the Tris- hydrochloride buffers containing 150mM Sodium Chloride
(100mM, pH8.0) carries out first time eluting, retains eluent;Then with the Tris- hydrochloride buffers containing 300mM Sodium Chloride
(100mM, pH8.0) carries out second eluting, discards eluent;Finally with the Tris- hydrochloride buffers containing 500mM Sodium Chloride
(100mM, pH8.0) carries out third time eluting, retains eluent;Merge above-mentioned first time and third time afford eluent,
As anion exchanged product.
Every time in eluting, it is ultraviolet manifest have albumen to start to collect when occurring, stop collecting when albumen appearance is finished, that is, obtain
The eluent of this eluting;Elution speed is 4ml/min every time, and loading speed is 3ml/ml.
(8) dialyse:
The ion exchange product is added to aperture to be about in 0.25 nanometer of bag filter, the bag filter is put into into 1L's then
In beaker, then to outside bag filter in the beaker add 1L 1mM Tris hydrochloride buffers (pH8.0), dialyse while stirring;Thoroughly
Eutectoid temperature is 4 DEG C, and dialysis time is 50 hours, obtains product of dialysing.
(9) concentrate:
Take a small amount of dialysis product to be added in the concentration tube that volume is 2mL, concentration tube allows the material that size is 2kD to lead to
Cross;Again concentration tube being put in centrifuge, centrifugal force being set as 1200g, design temperature is 4 DEG C, starts centrifuge, is started dense
Contracting, until final volume is 500 microlitres;Afterwards, by the addition again centrifuge tube of the remaining dialysis product, so that volume
2mL is reached, is centrifuged with identical parameter, final volume is again concentrated to for 500 microlitres;So circulation concentration, until inciting somebody to action
Plasma component after dialysis is finally concentrated to volume for 500 microlitres, as enriched product, as above-mentioned blood plasma extract
HC4201614。
Detection example 6
Various detections are carried out to blood plasma extract HC4201614 prepared by embodiment 6.
Detection 1-3:Carried out using the blood plasma extract HC4201614 obtained to embodiment 6 with detection 1 identical method of example
Detection, it is as a result identical with the mixture that embodiment 1 is obtained.
Detection 4:Detection embodiment:There are the 6 blood plasma extract HC4201614 for preparing vitro inhibition primary hippocampal cells to wither
The activity died, the activity for promoting Synaptic formation between hippocampal cell.The detection method is identical with detection example 1.Testing result table
Bright, the quantity of the primary hippocampal cells of experimental group 1 reaches about 1000-1500/square centimeter, much larger than the pact of experimental group 2
500-750/square centimeter, and about 400-700/square centimeter of matched group, it was demonstrated that blood plasma extract HC4201614 has
Suppress the activity of primary hippocampal cells apoptosis.The Synaptic formation quantity of experimental group 1 reaches about 100/square centimeter, much larger than reality
Test about 70/square centimeter of group 2, and about 50/square centimeter of matched group, it was demonstrated that separator HC4201614 has promotion
The activity of Synaptic formation between hippocampal cell.Prove that HC4201614 mixture has and promote Synaptic formation between hippocampal cell
Activity.
Detection 5:It is (old that blood plasma extract HC4201614 prepared by detection embodiment 6 can effectively lift alzheimer's disease
Dementia disease) mice memory.The detection method is identical with detection example 1.Test result shows, the target of experimental group 1 as
Limit stops memory time about 100 seconds, considerably longer than about 5 seconds of about the 10 of experimental group 2 second and matched group.
Embodiment 7
In addition to the molecular weight of the film bag retention used in step (3) low temperature ultrafiltration step is 13kD, other operating procedures
It is same as Example 1.
The final product that the embodiment is obtained is named as C1, and C1 electrophoresis results are as follows:25kD、35kD、50kD、69kD、
105kD;Suppress the activity of primary hippocampal cells apoptosis using product C1 being tested with detection 1 identical method of example, promote Hippocampus
The activity of Synaptic formation between cell, its result are as follows:
The quantity of the primary hippocampal cells of experimental group 1 reaches about 800-950/square centimeter, the about 550- of experimental group 2
780/square centimeter, and about 430-700/square centimeter of matched group.The Synaptic formation quantity of experimental group 1 reaches about 82
Individual/square centimeter, about 68/square centimeter of experimental group 2, about 54/square centimeter of matched group.
Embodiment 8
In addition to step (4) molecular weight retention step is eliminated, other operating procedures are same as Example 1.
The final product that the embodiment is obtained is named as C2, and C2 electrophoresis results are as follows:8kD、25kD、35kD、50kD、
69kD、105kD;Suppress the activity of primary hippocampal cells apoptosis using product C2 being tested with detection 1 identical method of example, promote
The activity of Synaptic formation between hippocampal cell, its result are as follows:
The quantity of the primary hippocampal cells of experimental group 1 reaches about 780-890/square centimeter, the about 500- of experimental group 2
750/square centimeter, and about 450-700/square centimeter of matched group.The Synaptic formation quantity of experimental group 1 reaches about 80
Individual/square centimeter, about 65/square centimeter of experimental group 2, about 53/square centimeter of matched group.
Embodiment 9
In addition to step (5) and (6) are eliminated, other operating procedures are same as Example 1.
The final product that the embodiment is obtained is named as C3, and C3 electrophoresis results are as follows:25kD、35kD、50kD、69kD、
105kD;Suppress the activity of primary hippocampal cells apoptosis using product C3 being tested with detection 1 identical method of example, promote Hippocampus
The activity of Synaptic formation between cell, its result are as follows:
The quantity of the primary hippocampal cells of experimental group 1 reaches about 790-960/square centimeter, the about 560- of experimental group 2
760/square centimeter, and about 440-710/square centimeter of matched group.The Synaptic formation quantity of experimental group 1 reaches about 80
Individual/square centimeter, about 66/square centimeter of experimental group 2, about 53/square centimeter of matched group.