CN110317785A - Improvement, the immunocyte for promoting nerve cell function and the preparation method and application thereof - Google Patents
Improvement, the immunocyte for promoting nerve cell function and the preparation method and application thereof Download PDFInfo
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Abstract
The present invention provides one kind to improve, the immunocyte for promoting nerve cell function and the preparation method and application thereof, and the preparation method of the immunocyte includes: in-vitro separation and purifies peripheral blood mononuclear cells；Peripheral blood mononuclear cells is activated with cell activating agent I；Wherein, cell activating agent I contains IL-2, IL-10, NT3, IL-4, BDNF, is configured with physiological saline；The cell after activation is expanded with cell activating agent II；Wherein, cell activating agent II contains IL-2, IL-4, BDNF, NT3, is configured with physiological saline；Harvest immunocyte.Immunocyte of the invention can secrete the neural factors such as a large amount of NGF, NT3/NT4, BDNF, these neural factors can promote the recovery of nervous system, the immunocyte also has the function of that the neurotransmitter receptor similar with nerve cell can play adjusting nervous function simultaneously, is effectively improved anxiety disorder.
Description
Technical field
The present invention relates to one kind to improve, immunocyte for promoting nerve cell function and preparation method thereof with answer
With specifically, being about a kind of immunocyte NEIC (Nerval for activating and expanding peripheral blood mononuclear cells and be prepared
Enhancement Immune Cells) and preparation method thereof, and preparing the system for improving, promoting nerve cell function
Application in agent (such as: treating the preparation of anxiety disorder), belongs to field of biotechnology.
Background technique
Bipolar disorder (Bipolar Disorder, BD) is also known as manic depression, abbreviation anxiety disorder, abnormal feeling
It is a kind of phrenoblabia characterized by high or low, existing mania and depressed two states have periodicity and palliative, can lead
Fraction is caused, work or learning ability decline, or even suicide, the serious quality of life for reducing patient are influenced.Bipolar disorders disease
Because unknown, biology, psychology participate in its pathogenic process with all many factors of social environment.Biological factor relate generally to heredity,
Neural biochemistry, neuroendocrine, nerve regneration etc.；It is ring gas that psychology in close relations, which is susceptible to suffer from quality, with bipolar disorders
Matter.And stressful life event is important socio-psychological factor.
Many is it is demonstrated experimentally that nervous system, endocrine system and immune system connect each other with high degree of uniformity, altogether
With the homeostasis for maintaining body.Brain is at least connected there are two major avenues of approach with immune system, and one is autonomic nerves system, secondly
For neuroendocrine system.Many principal characteristic psychiatric patients also often occur together immune function change, such as research discovery schizophrenia
Interleukin 2 (IL-2) soluble recepter number increases in disease patient's blood and the percentage of lymphocyte of abnormal activation increases；
The lymphocytosis of the case-finding abnormal activation of Autism children prompts have the phenomenon that immunologic dysfunction increases, with certain
The change of a little autoimmune diseases is similar, this may with foetal period central nervous system infection or central nervous system from
Body immunologic mechanism is related；And major depression usually has HPA (hypothalamic-pituitary-adrenal) s function excessively high to inhibit to exempt from
Epidemic disease function, thus concomitant immunity hypofunction, as T cell lowers the reaction of phytohemagglutin phytolectin and NK cell activity.All T
Lymphocyte can activate the immunocyte as caused by slight chronic infection with immunological tolerance, and restore immunocyte subunit
The balance of group.Different immunocyte subgroups expresses different neurotrophic factor acceptors and generates different cell factors, special
It is not brain-derived neurotrophic factor (Brain Derived Neurotrophic Factor, BDNF) and neurotrophin 3
(Neurotrophin-3, NT-3), both substances are strong nervous function Auto-regulators.
BDNF is a kind of activated protein factor that can promote and maintain neure growth, survival and function, is distributed widely in
The nervous centralis such as hippocampus position is activated by tyrosine kinase receptor B (tyrosine kinase receptor B, TrkB) and is believed
Number conduction.Patients with depression Serum Levels of BDNF is lower than normal person, and Serum Levels of BDNF and depressed severity may be in negative
It is related.
NT-3 is the key regulatory molecule of neuronal cell development, survival and plasticity, their activity is related to largely
The transcription and translation expression of related gene.Bipolar disorder (bipolar disorder, BD) patients serum NT-3 and
NT-4/5 level increases, and this increase exists only under BD depression in patients state, prompts, BD patient's NT-3 and NT-4/5 water-glass
Now increase for course of disease State-dependence formula.Main neurotransmitter such as 5-HT, dopamine (dopamine, DA) He Qujia is adjusted in NT-3
The level of adrenaline (norepinephrine, NE) etc., to play antidepressant effect.
Since 1999, there is many cases immunity of organism related to mental disorder or immune cell therapy mental disease
The report of disease.The polarized T lymphocyte energy secretory nerve factor of Th1/Th2 and nerve growth factor are found for the first time within 1999.
It is proposed schizophrenia in 2002 and other mental disorders need long-term adoptive immunotherapy.It 2005 will be through work
The immunocyte (NEIC cell) of change is applied to anxiety disorder treatment and succeeds.It is proposed mental disorders and Chlamydia within 2011
Extensive occult infection is related.The abnormal tune of discovery mental disorders in 2013 and lymphocyte factor (interferon) after infection
Section is related.NEIC cell was applied to the wake-up of long-term persistent vegetative state in 2017 to succeed.2017 by NEIC cell application
It succeeds in azheimer's disease.Mental disorder and the close phase of immune function of human body it can be seen from numerous relevant reports
It closes, therefore, the rehabilitation of mental disorder may be implemented with cellular immunotherapy.
Summary of the invention
It is an object of the present invention to provide the immunocytes that one kind can improve, promote nerve cell function.
Another object of the present invention is to provide the preparation methods of above-mentioned immunocyte.
Another object of the present invention is to provide the applications of above-mentioned immunocyte.
In order to achieve the object of the present invention, the present invention provides a kind of immunocytes for improving, promoting nerve cell function
Preparation method preparation-obtained immunocyte is known as NEIC cell in the present invention to obtain a kind of immunocyte
(Nerval Enhancement Immune Cells, NEIC).NEIC cell can be by secreting a large amount of NGF, NT3/NT4, BDNF
Equal neural factors promote the recovery of nervous system, and/or play the role of adjusting nervous function.
Specifically, on the one hand, the present invention provides a kind of preparation methods of immunocyte, this method comprises:
In-vitro separation simultaneously purifies peripheral blood mononuclear cells；
With cell activating agent I (being also known as NEIC cell activating agent I in the present invention) activation peripheral blood mononuclear cells
(cell after activation becomes NEIC cell)；Wherein, NEIC cell activating agent I contain IL-2, IL-10, NT3, IL-4,
BDNF is configured with physiological saline；
It is by the cell after activation with cell activating agent II (being also known as NEIC cell activating agent II in the present invention)
NEIC cell is expanded；Wherein, NEIC cell activating agent II contains IL-2, IL-4, BDNF, NT3, is matched with physiological saline
It sets；
It harvests immunocyte (NEIC cell).
Specific embodiment according to the present invention, in the preparation method of immunocyte of the invention, in-vitro separation is simultaneously purified
The process of peripheral blood mononuclear cells includes:
Anticoagulant heparin will be added treated that peripheral blood is dispensed into centrifuge tube, centrifugation is abandoned supernatant, is added into pipe
Lymphocyte suspension is made in RPMI1640 culture medium；
It is then added in the centrifuge tube for preset lymphocyte separation medium and is centrifuged, be transferred to buffy coat after centrifugation
It in another centrifuge tube, is centrifuged after RPMI1640 culture medium is added, abandons supernatant, be centrifuged after adding RPMI1640 culture medium, in abandoning
Clearly, peripheral blood mononuclear cells is obtained.
In some specific embodiments of the invention, the centrifugal condition is 1800rpm, 5~15min.
Add complete medium into peripheral blood mononuclear cells, lymphocyte suspension is made；
Specific embodiment according to the present invention, in the preparation method of immunocyte of the invention, the complete medium
It is prepared in accordance with the following methods:
Whole blood centrifugation, abandons supernatant, and 4 DEG C stand overnight, then be centrifuged, and takes supernatant as adding into RPMI1640 culture medium
Blood plasma；In some specific embodiments of the invention, wherein the centrifugal condition is that 1500g is centrifuged 15~20 minutes；
Above-mentioned blood plasma is added into RPMI1640 culture medium, and gentamicin is added by 100U/ml concentration, is made containing 10v/
The complete medium of v% self blood plasma.
Specific embodiment according to the present invention, in preparation method of the invention, cell activating agent I contains IL-2 concentration
200-5000IU/mL, IL-10 concentration 50-10000IU/mL, NT3 concentration 20-150pg/mL, IL-4 concentration 10-500IU/mL,
BDNF concentration 20-100pg/mL, is configured with physiological saline；
The cell after activation is expanded with cell activating agent II；Wherein, it is dense to contain IL-2 by cell activating agent II
Spend 300-10000IU/mL, IL-4 concentration 300-10000IU/mL, BDNF concentration 10-1000pg/mL, NT3 concentration 20-150pg/
ML is configured with physiological saline.
In some specific embodiments of the invention, NEIC cell activating agent I contains IL-2 concentration 2000-
4000IU/mL, IL-10 concentration 5000-7000IU/mL, NT3 concentration 80-120pg/mL, IL-4 concentration 200-400IU/mL,
BDNF concentration 50-85pg/mL, is configured with physiological saline.NEIC cell activating agent II contains IL-2 concentration 4500-7000IU/
ML, IL-4 concentration 5000-8000IU/mL, BDNF concentration 500-800pg/mL, NT3 concentration 50-100pg/mL, with physiological saline
Configuration.
Specific embodiment according to the present invention, in the preparation method of immunocyte of the invention, with NEIC cell-stimulating
The factor I activation peripheral blood mononuclear cells process include:
NEIC cell activating agent I 0.5ml is added into every 20ml cell suspension, in 38.5 DEG C, 6.5%CO2Constant temperature
It is cultivated 72 hours in incubator.
Specific embodiment according to the present invention, in the preparation method of immunocyte of the invention, with NEIC cell-stimulating
The process that NEIC cell expands includes: by factor II
After inoculating cell 72h, culture is transferred in centrifuge tube, is centrifuged (the centrifugation item in some specific embodiments
Part is 1800rpm, 5~15min)；Supernatant, RPMI1640 culture medium suspension cell are abandoned, and adjusts cell density 0.5 × 106-1
×106NEIC cell activating agent II is added in a/ml；Cell suspension is added in not coated Tissue Culture Flask, in 38.5
DEG C, 6.5%CO2Constant incubator in continue culture 72 hours.
Specific embodiment according to the present invention, in the preparation method of immunocyte of the invention, harvest NEIC cell
Process includes:
By the centrifugation of cell culture product (centrifugal condition in some specific embodiments is 1800rpm, 5~15min), abandon
Supernatant, and with physiological saline or RPMI1640 culture medium suspension cell containing albumin, it freezes.
Specific embodiment according to the present invention, in the preparation method of immunocyte of the invention, NEIC cell is frozen
Step includes:
By the centrifugation of cell culture product (centrifugal condition in some specific embodiments is 1800rpm, 5~15min), abandon
Supernatant is added precooled frozen stock solution A and is resuspended, mixes well postposition on ice, be slowly added to precooled frozen stock solution B, sufficiently mixed
It is transferred in cryopreservation tube immediately after even, -80 DEG C of preservations.
In some specific embodiments of the invention, the frozen stock solution A are as follows: the human seralbumin egg for being 200g/L to concentration
The PBS buffer solution of 100mL pH7.0 is added in the white every 100mL of solution；- 20 DEG C of preservations after mixing；
The frozen stock solution B are as follows: the PBS buffer solution and dimethyl sulfoxide of pH7.0 is mixed according to 4:1 volume ratio to obtain the final product.
The wherein cryopreservation step of NEIC cell are as follows: (1) by cell culture product centrifugation (in some specific embodiments from
Heart condition is 1800rpm, 5~15min), abandon supernatant, be added precooled frozen stock solution A, mix cell, take be placed on a small quantity it is sterile
Physiological saline is used for inspection.(2) it is slowly added to frozen stock solution B, is added on one side, shakes centrifuge tube on one side, is transferred to jelly after mixing immediately
It deposits in pipe.(3) it is put into foam box, and is wrapped up with cotton, is put into -80 DEG C of refrigerators.Note: 1. frozen stock solution A, frozen stock solution B are needed
To configure in advance, be put into 4 DEG C of refrigerators and be pre-chilled, or after preparing packing be put into -20 DEG C of refrigerators freeze it is spare.Frozen stock solution exists
Need to teetertotter mixing when taking-up.2. cryopreservation tube, freeze pipe support, foam box is both needed to be put into advance in -20 DEG C of refrigerators and 30min is pre-chilled
Left and right.
On the other hand, the present invention also provides a kind of immunocytes, pass through immunocyte table described in flow cytomery
Colourless cell differentiation antigen, has the following performance:
CD3-FITC/CD4-PE/CD8-PECy5 identification: CD3+ cell >=85%, CD3+/CD4+ cell >=60%, CD3
+/CD8+ cell >=20%；
CD3-FITC/CD16-PE/CD56-PECy5 identification: CD3+/CD56+ cell >=20%, CD3-/CD56+ and
CD3-/CD16+ cell >=5%；
FoxP3-FITC/CD25-PE/-CD4-PECy5 identification: FoxP3+/CD25+/- CD4+ cell≤1%；
CD3-FITC/Intracelluler IL-10PE identification: CD3+/Intracelluler IL-10+ >=45%.
Specific embodiment according to the present invention, immunocyte of the invention are method systems according to the present invention
For what is obtained.
Immunocyte of the invention, other Quality Identifications and criterion of acceptability: cell culture finished product is required according to " China is raw
Tetramune " in " biological products sterility test regulation " tested, cell finished product should be through Gram-positive and gram-negative
Property bacteria cultivation results be feminine gender；It is feminine gender through fungal culture qualification result；It is feminine gender through detection of mycoplasma result；It is examined through syphilis
Surveying result is feminine gender；It is feminine gender through hepatitis B antigen testing result；Endotoxin testing result is feminine gender.
On the other hand, the present invention also provides the immunocytes to prepare for improving, promoting nerve cell function
Preparation in application.
Some specific embodiments according to the present invention, it is described for improve, promote the preparation of nerve cell function to be logical
Cross intracutaneous injection, intravenous injection, intramuscular injection or inside tumor or around be administered, dosage is every time 500,000-3,000 ten thousand
Immunocyte.
Some specific embodiments according to the present invention, it is described for improve, promote the preparation of nerve cell function to be logical
The recovery for secreting the neural factors such as a large amount of NGF, NT3/NT4, BDNF promotion nervous system is crossed, and/or plays adjusting nervous function
Effect.
Specific embodiment according to the present invention, the immunocyte are waited in specific application, are first passed through recovery and are fed back again.
The recovery step of NEIC cell are as follows: the cell freezing pipe that need to be recovered thaws in 37 DEG C of waters bath with thermostatic control, and moves into rapidly preset
In the centrifuge tube of 10ml cell recovery liquid, centrifugation (centrifugal condition in some specific embodiments be 1800rpm, 5~
15min), and with 10ml physiological saline it is resuspended, then is centrifuged.It is resuspended with 2ml cell recovery liquid, prepares to feed back.Sampling carries out platform and expects
Indigo plant dyeing, and count, calculate motility rate.The wherein preparation method of resuscitation fluid: it is 200g/ that concentration is added in 95ml sterile saline
The human serum albumin solution 5ml of L, after mixing with the filtering of 0.22 μm of filter to get.The wherein feedback step of NEIC cell are as follows:
By the NEIC cell after recovery by intracutaneous injection, intravenous injection, intramuscular injection or the inside tumor or surrounding administration, with
The dosage of 500,000-3,000 ten thousand cell of per injection is administered.It is certain before feeding back to obtain Bacteria Identification and other quality inspections report, to prevent training
Infection caused by the process of supporting.
Beneficial effects of the present invention:
(1) used the combination of cytokines of IL-2, IL-10, NT3, IL-4, BDNF common in the cells in vitro activation stage
Under effect, quickly peripheral blood mononuclear cells is activated to become NEIC cell.
(2) combination of cytokines that IL-2, IL-4, BDNF, NT3 have been used in the cell amplification stage, keeps NEIC cell big
Amount amplification, can secrete the neural factors such as a large amount of NGF, NT3/NT4, BDNF, and nervous system is promoted to restore.
(3) the NEIC cell that method of the invention expands can improve anxiety disorder symptom.
(4) the NEIC cell that method of the invention expands has the neurotransmitter receptor similar with nerve cell can
Play the role of adjusting nervous function.
(5) the NEIC cell that method of the invention expands can improve the inferior healths diseases such as common anxiety and insomnia
Shape.
Detailed description of the invention
Fig. 1 is NEIC cell proliferative conditions figure in embodiment 1.
Fig. 2 is each sample culture when the NEIC cell that the cultural method of Application Example 1 in embodiment 5 obtains is supported the 7th day
The content of BDNF in supernatant.
Fig. 3 is each sample culture when the NEIC cell that the cultural method of Application Example 1 in embodiment 5 obtains is supported the 7th day
The content of NT3 in supernatant.
Specific embodiment
Below by way of the implementation and possessed beneficial effect of specific embodiment the present invention will be described in detail technical solution, but not
It can regard as any restriction to enforceable range of the invention.Unspecified method and operating condition in each embodiment,
Routine techniques according to the field or the operation according to apparatus manufacturer suggestion carry out.
The preparation of embodiment 1.NEIC cell
1. the separation of peripheral blood lymphocytes
(1) primary blood drawing amount: 150ml, anticoagulant heparin.150ml whole blood can obtain 1.5-2 × 10 under normal circumstances8A cell,
A 75cm can be planted2Culture bottle；Treated that peripheral blood is dispensed into centrifuge tube for anticoagulant heparin, and 1800rpm, 15min centrifugation are abandoned
Supernatant；
(2) dilute blood: lymphocyte suspension is made in RPMI1640 culture medium equimultiple dilute blood；
(3) 25ml lymphocyte suspension is carefully placed on 12.5ml lymphocyte separation medium, 1800rpm, 15min centrifugation,
Lifting speed is adjusted to minimum.
(4) top section blood plasma is taken to continue to employ, 4 DEG C of refrigerators save；
(5) buffy coat is taken, is placed in 50ml centrifuge tube, addition RPMI1640 culture medium to total volume 50ml,
1800rpm, 5min centrifugation；
(6) supernatant is abandoned, a certain amount of RPMI1640 culture medium is added, suspension cell takes a small amount of counting.
(7) 1800rpm, 5min centrifugation；
(8) supernatant is abandoned, peripheral blood mononuclear cells is obtained；Add complete medium into peripheral blood mononuclear cells, is made
Lymphocyte suspension.
2. reagent and solution prepare culture medium preparation method
Whole blood is centrifuged 20 minutes in 1500g, abandons supernatant, and 4 DEG C stand overnight, and is centrifuged 15 minutes then at 1500g, supernatant is taken to make
For the blood plasma added into RPMI1640 culture medium；Blood plasma is added into RPMI1640 culture medium, and is added by 100U/ml concentration
The complete medium of the self blood plasma containing 10v/v% is made in gentamicin.
3. Tissue Culture Flask is handled
Coating buffer, and every bottle of addition 30ml physiological saline is sucked out, shakes gently, physiological saline is sucked out.
4.NEIC cell-stimulating
The step of by the culture of NEIC cell activating agent I by NEIC cell-stimulating are as follows: after Tissue Culture Flask processing, stand
20ml cell suspension is added, NEIC cell activating agent I 0.5ml, in 38.5 DEG C, 6.5%CO is added2Constant incubator
Middle culture 72 hours.
Wherein NEIC cell activating agent I contains IL-2 (3000IU/mL), IL-10 (6000IU/mL), NT3 (90pg/
ML), IL-4 (380IU/mL), BDNF (60pg/mL), are configured with physiological saline.
The amplification of 5.NEIC cell
(1) after inoculating cell 72h, culture is transferred in 50ml centrifuge tube, it, can when part attached cell can not blow down
15ml physiological saline is added, flapping bottle or disengages it from bottom of bottle with cell scraper, merges with archaeocyte suspension.1800rpm, 5min
Centrifugation；
(2) supernatant is abandoned, RPMI1640 culture medium suspension cell counts；
(3) RPMI1640 culture medium is added, adjusts cell density 0.5 × 106-1×106A/ml is added NEIC cell and swashs
Factor II0.5ml living.
(4) cell suspension is added to new 75cm2It (is not coated with) in Tissue Culture Flask, 50ml or so/bottle.
(5) by culture bottle in 38.5 DEG C, 6.5%CO2Constant incubator in continue culture 72 hours.
Wherein NEIC cell activating agent II contains IL-2 (6500IU/mL), IL-4 (7000IU/mL), BDNF (800pg/
ML), NT3 (85pg/mL), is configured with physiological saline.
6.NEIC cell is harvested and is frozen
By cell culture product 1800rpm, 5min be centrifuged, abandon supernatant, and with containing albumin physiological saline or
RPMI1640 culture medium suspension cell counts.According to cell quantity, determination freezes quantity.
The wherein cryopreservation step of NEIC cell are as follows:
(1) cell culture product 1800rpm, 5min are centrifuged, abandon supernatant, precooled frozen stock solution A4ml is added, mixed
Cell takes and is placed in sterile saline on a small quantity, is used for inspection.
(2) it is slowly added to frozen stock solution B 4ml, is added on one side, centrifuge tube is shaken on one side, is transferred to cryopreservation tube after mixing immediately
In.
(3) it is put into foam box, and is wrapped up with cotton, is put into -80 DEG C of refrigerators.
The frozen stock solution A are as follows: 100mL is added into the every 100mL of human serum albumin solution that concentration is 200g/L
The PBS buffer solution of pH7.0；- 20 DEG C of preservations after mixing；
The frozen stock solution B are as follows: the PBS buffer solution and dimethyl sulfoxide of pH7.0 is mixed according to 4:1 volume ratio to obtain the final product.
Note: 1. frozen stock solution A, frozen stock solution B need to configure in advance, are put into 4 DEG C of refrigerators and are pre-chilled, or dispense after preparing
Be put into -20 DEG C of refrigerators freeze it is spare.Frozen stock solution need to teetertotter mixing when taking out.2. cryopreservation tube freezes pipe support, foam box
It is both needed to be put into advance and 30min or so is pre-chilled in -20 DEG C of refrigerators.
Above-mentioned inspection step are as follows: by flow cytomery finished product cell surface leukocyte differentiation antigen, be commonly used in
The antibody of fluorescent staining includes following groups:
(1) CD3-FITC/CD4-PE/CD8-PECy5, identification culture cell finished product criterion of acceptability are as follows: CD3+ cell >=
85%, CD3+/CD4+ cell >=60%, CD3+/CD8+ cell >=20%
(2) CD3-FITC/CD16-PE/CD56-PECy5, identification culture cell finished product criterion of acceptability are as follows: CD3+/CD56
+ cell >=20%, CD3-/CD56+ and CD3-/CD16+ cell >=5%；
(3) FoxP3-FITC/CD25-PE/-CD4-PECy5 identification culture cell finished product criterion of acceptability are as follows: FoxP3+/
CD25+/- CD4+ cell≤1%；
(4) CD3-FITC/Intracelluler IL-10PE identification culture cell finished product criterion of acceptability are as follows: CD3+/
Intracelluler IL-10+ >=45%.
Other Quality Identifications and criterion of acceptability: cell culture finished product is required according to " the biology in " Products in China "
Product sterility testing regulations " it is tested, cell finished product should be through Gram-positive and gramnegative bacterium cultivation results
It is negative；It is feminine gender through fungal culture qualification result；It is feminine gender through detection of mycoplasma result；It is feminine gender through Results of Syphilis；Through
Hepatitis B antigen testing result is feminine gender；Endotoxin testing result is feminine gender.
Through detecting, the finished product cell of the present embodiment meets the requirement of the qualified standard of above-mentioned quality inspection.
7.NEIC cell recovery and feedback
The cell freezing pipe that need to be recovered is thawed in 37 DEG C of waters bath with thermostatic control, and moves into preset 10ml cell recovery liquid rapidly
Centrifuge tube in, 1800rpm, 5min centrifugation, and with the resuspension of 10ml physiological saline then is centrifuged.It is resuspended with 2ml cell recovery liquid,
Prepare to feed back.Sampling carries out Trypan Blue, and counts, calculates motility rate.
The wherein preparation method of resuscitation fluid: the human serum albumins that concentration is 200g/L is added in 95ml sterile saline
Solution 5ml, after mixing with the filtering of 0.22 μm of filter to get.
The wherein feedback step of NEIC cell are as follows: the NEIC cell after recovery is passed through into intracutaneous injection, intravenous injection, intramuscular
Injection is administered in the inside tumor or surrounding, with the administration of the dosage of 500,000-3,000 ten thousand cell of per injection.It is certain before feeding back
Bacteria Identification and other quality inspections report are obtained, to prevent infection caused by incubation.
The detection of embodiment 2.NEIC cell amplification ability
Using the method culture of embodiment 1, and taking incubation time is the 0th, 3,5,7 day NEIC cell, and detection cell increases
Situation is grown, experimental result is for example as shown in Figure 1.It is preferable using the NEIC cell proliferative conditions of the method for the present invention culture as shown in Figure 1.
The detection of embodiment 3.NEIC cell viability
Using the method culture of embodiment 1, and the 100 μ l of cell of culture 12 days is taken, 100 μ l, 0.4% trypan blue dye is added
Color liquid, living cells are not colored, and dead cell is dyed to blue, and experimental result is as shown in table 1.
Table 1
| Sample 1 | Sample 2 | Sample 3 | |
| Percent living cells | 95.1% | 98.3% | 97.2% |
As can be seen from Table 1, cell viability prepared by the present invention is greater than 95%.
Embodiment 4.NEIC cell streaming Phenotypic examination
Using the method culture of embodiment 1,0,4,7,10 day 100 μ l of culture are taken respectively, and the cell of concentration about 106/ml divides
It Jia Ru not detect and use 10 μ l of humanized murine antibodies, under room temperature environment, be protected from light and be incubated for 30min.It is washed twice, is flowed with PBS later
Formula cell detection, experimental result are as shown in table 2.
Table 2
| Donor 1 | Donor 2 | Donor 3 | |
| CD3+/CD19-/CD14- (T cell) | 90.2% | 93.3% | 93.7% |
| CD3+/CD8+/CD4- (T kills cell) | 20.4% | 30.3% | 23.6% |
| CD3+/CD4+/CD8- (t helper cell) | 63.1% | 62.6% | 68.1% |
| CD3+/CD56+ or CD16+ (NKT cell) | 28.3% | 27.2% | 27.9% |
| CD3-/CD56+/CD16+ (NK cell) | 5.2% | 6.1% | 7.2% |
| CD3+/CD4+/IL-4+ (Th2 cell) | 49.0% | 50.2% | 48.4% |
As can be seen from Table 2: NEIC culture can obtain a large amount of T cells, and T lymphocyte can be activated by slight chronic
With the immunocyte of immunological tolerance caused by infection, and restore the balance of immunocyte subgroup.Specifically, culture of the present invention
Cell, CD3+/CD19-/CD14- cell (T cell) meets performance >=85% of NEIC cell in 90.2%-93.7%；
CD3-/CD56+/CD16+ (NK cell) meets performance >=5% of NEIC cell between 5.2%-7.2%；CD3+/CD56+
Or CD16+ (NKT cell) meets performance >=20% of NEIC cell between 27.2%-28.3%；CD3+/CD4+/IL-4+
(Th2 cell) meets performance >=45% of NEIC cell between 48.4%-50.2%.
Embodiment 5.NEIC cell toxicant Vitro Experimental Results
BDNF and NT3 is horizontal in the 7th day detection culture supernatant of cell culture.
Illustrate according to common lymphocyte stimulation (IL-2 500IU+IL-4500IU) in the 7th day culture supernatant, through we
The peripheral blood lymphocytes of method culture can largely secrete the neural factor for being conducive to nerve cell function.
Using commercially available interlayer type 148ELISA (enzyme linked immunosorbent assay (ELISA)), BDNF and NT3 is measured in 147 total homogenate
Total concentration.149 development system mankind BDNF DY 248 (150 protein 23.40-1500pg/mL of detection range, specificity:
151 in 50ng/mL and b-NGF, GDNF, NT3 and NT4 no cross reaction) and mankind NT3DY 152 267 (according to manufacturer
Specification detection protein range 31.20-2000pg/mL, specificity: 153 at 50ng/mL with BDNF, CNTF,
GDNF, NT4 and 154b-NGF no cross reaction).
By the peripheral blood lymphocytes through this method culture known to Fig. 2, Fig. 3 can largely secrete BDNF, NT-3 cell because
Sub (BDNF P < 0.001, NT-3P < 0.01), and it is statistically significant.It can be seen that the cell of this method culture can have
Effect improves anxiety disorder.
Claims (10)
1. a kind of preparation method of immunocyte, this method comprises:
In-vitro separation simultaneously purifies peripheral blood mononuclear cells；
Peripheral blood mononuclear cells is activated with cell activating agent I；Wherein, cell activating agent I contain IL-2, IL-10, NT3,
IL-4, BDNF are configured with physiological saline；
The cell after activation is expanded with cell activating agent II；Wherein, cell activating agent II contain IL-2, IL-4,
BDNF, NT3 are configured with physiological saline；
Harvest immunocyte.
2. preparation method according to claim 1, wherein in-vitro separation and the process packet for purifying peripheral blood mononuclear cells
It includes:
Anticoagulant heparin will be added treated that peripheral blood is dispensed into centrifuge tube, centrifugation is abandoned supernatant, is added into pipe
Lymphocyte suspension is made in RPMI1640 culture medium；
It is then added in the centrifuge tube for preset lymphocyte separation medium and is centrifuged, be transferred to buffy coat after centrifugation another
It in centrifuge tube, is centrifuged after RPMI1640 culture medium is added, abandons supernatant, be centrifuged after adding RPMI1640 culture medium, abandoned supernatant, obtain
Obtain peripheral blood mononuclear cells；
Add complete medium into peripheral blood mononuclear cells, cell suspension is made.
3. preparation method according to claim 2, wherein the complete medium is to be operated in accordance with the following methods
:
Whole blood centrifugation, abandons supernatant, 4 DEG C stand overnight, then are centrifuged, and take supernatant as the blood added into RPMI1640 culture medium
Slurry；
Above-mentioned blood plasma is added into RPMI1640 culture medium, and gentamicin is added by 100U/ml concentration, is made containing 10v/v%
The complete medium of self blood plasma.
4. preparation method according to claim 1, wherein cell activating agent I contains IL-2 concentration 200-5000IU/
ML, IL-10 concentration 50-10000IU/mL, NT3 concentration 20-150pg/mL, IL-4 concentration 10-500IU/mL, BDNF concentration 20-
100pg/mL is configured with physiological saline；
The cell after activation is expanded with cell activating agent II；Wherein, cell activating agent II contains IL-2 concentration
300-10000IU/mL, IL-4 concentration 300-10000IU/mL, BDNF concentration 10-1000pg/mL, NT3 concentration 20-150pg/
ML is configured with physiological saline.
5. preparation method according to claim 1, wherein with cell activating agent I activation peripheral blood mononuclear cells
Process includes:
Cell activating agent I0.5ml is added into every 20ml cell suspension, in 38.5 DEG C, 6.5%CO2Constant incubator in train
It supports 72 hours；
Include: by the process that the cell after activation expands with cell activating agent II
After inoculating cell 72h, culture is transferred in centrifuge tube, is centrifuged；Abandoning supernatant, RPMI1640 culture medium suspension cell,
And adjust cell density 0.5 × 106-1×106Cell activating agent II is added in a/ml；Cell suspension is added to not coated
In Tissue Culture Flask, in 38.5 DEG C, 6.5%CO2Constant incubator in continue culture 72 hours.
6. preparation method according to claim 1, wherein the process for harvesting immunocyte includes:
Cell culture product is centrifuged, abandons supernatant, and with physiological saline or RPMI1640 culture medium suspension cell containing albumin,
It freezes.
7. preparation method according to claim 1, wherein the cryopreservation step of immunocyte includes:
Cell culture product is centrifuged, supernatant is abandoned, precooled frozen stock solution A is added and is resuspended, mixes well postposition on ice, slowly adds
Enter precooled frozen stock solution B, is transferred in cryopreservation tube immediately after mixing well, -80 DEG C of preservations；
The frozen stock solution A are as follows: be added 100mL pH7.0's into the every 100mL of human serum albumin solution that concentration is 200g/L
PBS buffer solution；- 20 DEG C of preservations after mixing；
The frozen stock solution B are as follows: the PBS buffer solution and dimethyl sulfoxide of pH7.0 is mixed according to 4:1 volume ratio to obtain the final product.
8. a kind of immunocyte, by immunocyte surface leukocyte differentiation antigen described in flow cytomery, have with
Lower performance:
CD3-FITC/CD4-PE/CD8-PECy5 identification: CD3+ cell >=85%, CD3+/CD4+ cell >=60%, CD3+/CD8
+ cell >=20%；
CD3-FITC/CD16-PE/CD56-PECy5 identification: CD3+/CD56+ cell >=20%, CD3-/CD56+ and CD3-/
CD16+ cell >=5%；
FoxP3-FITC/CD25-PE/-CD4-PECy5 identification: FoxP3+/CD25+/- CD4+ cell≤1%；
CD3-FITC/Intracelluler IL-10PE identification: CD3+/Intracelluler IL-10+ >=45%.
9. immunocyte according to claim 8 is that described in any item methods are prepared into according to claim 1~7
It arrives.
10. immunocyte described in claim 8 or 9 is preparing answering in the preparation for improving, promoting nerve cell function
With；Alternatively, immunocyte described in claim 8 or 9 is in preparation for treating the application in anxiety disorder preparation；
Preferably, described for improve, promote the preparation of nerve cell function to be by intracutaneous injection, intravenous injection, intramuscular note
Penetrate or inside tumor or around be administered, dosage is 500,000-3,000 ten thousand immunocyte every time；
Preferably, the preparation for improving, promoting nerve cell function is by secreting a large amount of NGF, NT3/NT4, BDNF
Neural factor promotes the recovery of nervous system, and/or plays the role of adjusting nervous function.
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| CN1705438A (en) * | 2002-06-14 | 2005-12-07 | 耶达研究及发展有限公司 | Antigen-presenting cells for neuroprotection and nerve regeneration |
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