CN105200010B - The extracorporeal culturing method and its special culture media of People living with HIV HLA-A2 Specific CTL Cells - Google Patents
The extracorporeal culturing method and its special culture media of People living with HIV HLA-A2 Specific CTL Cells Download PDFInfo
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- CN105200010B CN105200010B CN201510666770.4A CN201510666770A CN105200010B CN 105200010 B CN105200010 B CN 105200010B CN 201510666770 A CN201510666770 A CN 201510666770A CN 105200010 B CN105200010 B CN 105200010B
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Abstract
The invention discloses the extracorporeal culturing methods and its special culture media of People living with HIV HLA-A2 Specific CTL Cells.The complete set of culture medium of amplification in vitro People living with HIV HLA-A2 Specific CTL Cells provided by the invention is made of culture medium 1 and culture medium 2；Culture medium 1 and culture medium 2 are serum-free medium for mammalian cell, and containing amino acid sequence, 9 kinds of polypeptides as shown in SEQ ID No.1-SEQ ID No.9, culture medium 2 contain interleukin 2 to culture medium 1 respectively.The different People living with HIV HLA-A2 Specific CTL Cells of separation can successfully be carried out by amplification cultivation using complete set of culture medium and extracorporeal culturing method of the invention, the cell activity of acquisition is more stable, total number of cells dramatically increase, can efficient identification and dissolution autologous patient HIV infection after CD4+T cell, have great importance for the treatment of AIDS.
Description
Technical field
The present invention relates to the extracorporeal culturing methods of People living with HIV HLA-A2 Specific CTL Cells in immunotherapy field
And its special culture media.
Background technique
AIDS is the global disease for seriously threatening human life and health, although antiviral therapy significantly reduces
The morbidity and mortality of AIDS patient however, antiviral therapy requires lifelong medication, and need to reach 95% compliance
Property, it is a heavy burden in medical resource undeveloped country and area.Therefore, find radical cure AIDS step from
Do not stop.
Adoptive cellular immunotherapy is since the 1980s, the application of adoptive cellular immunotherapy clinically
Extension rapidly.In recent years, the adoptive cellular immunotherapy technology based on T cell is widely used in malignant tumour and tumour
The treatment of patient is shifted, there is preferable safety and validity.
Cytotoxic T cell (cytotoxic T-lymphocyte, CTL) is a kind of specific T cell, is specially secreted various
Cell factor participates in immunization, has lethal effect to antigenic substances such as certain viruses, tumour cells, constitutes with nature cell
The important defence line of body disease-resistant poison, antineoplastic immune.Its action character: can continuously kill target cell, have high efficiency, antigen
Specificity and itself MHC are restricted.HIV specific C D8+T cell is directly related with the decline of inhibition of HIV carrying capacity, especially acute
In the later period of infection phase, during virus load declines rapidly, CD8+T cell plays the role of most important, passes through in experiment in vitro
The CD8+T cell that IFN-γ secretion experiment and tetramer dye visible HIV infection person has special antivirus action.From essence
Virus load can be controlled the patient in set point level by English controller (elite controller) and autoimmunity for a long time
Peripheral blood obtain cytotoxic T lymphocyte stimulated in advance without HIV antigen in vitro experiment, can efficient identification and
CD4+T cell after dissolving its self HIV infection.
CTL cellular immunity transmission therapy is the lymphocyte using own venous blood, in vitro by target cell antigen and
The induction of lymphokine, differentiation amplification is at the CTL cell with powerful lethality, then through in venous re-transfusion body, thus effectively
Immunological effect is played, has the function that remove virus and killing tumor cell.CTL cell therapy is mainly used in hepatitis B, hepatitis
With the treatment of tumour.
CD3 molecular distribution is at least made of 5 kinds of polypeptide chains of γ, δ, ε, ζ and η, in mature T lymphocytic cell surface with T cell
Antigen receptor non-covalent linking.CD3 monoclonal antibody can induce CD3 polypeptide and TCR is total to cap and forms (co-capping), and induce
T lymphocyte activation.TCR identifies that the compound that exotic antigen and own MHC molecules are formed, CD3 have the transmitting of signal
Important function.
CD8 molecular distribution wears film sugar egg in part T lymphocyte and thymocyte, by what two polypeptide chains of α and β formed
It is white.It is the ligand of CD8 molecule, CD8 molecule and mhc class i antigen knot as cell and intercellular attached viscoelastic element mhc class i antigen
It closes the T cell (mainly CTL) that can stablize the limitation of mhc class i antigen and the target with mhc class i antigen and antigenic compound is thin
Born of the same parents combine.CD8 positive cell is suppressive lymphocyte T/cytotoxic T lymphocyte (suppressor T lymphocyte/
cytotoxic T lymphocyte,Ts/Tc).CD8 molecule plays an important role in the signal transduction that T cell is proliferated and is broken up.
Interferon (IFN) is a kind of broad-spectrum disease resistance toxic agent, not direct killing or inhibition virus, and mainly passes through cell
Surface receptor effect makes cell generate antiviral protein, to inhibit the duplication of virus, type is divided into three classes, α-(leucocyte)
Type, β-(fibroblast) type and γ-(lymphocyte) type；It is thin that natural killer cells (NK cell), macrophage can also be enhanced simultaneously
The vigor of born of the same parents and T lymphocyte to play immunoregulation effect, and enhance anti-virus ability.Interferon is one group with more
The reactive protein (mainly glycoprotein) of kind function is a kind of cell factor generated by monocyte and lymphocyte, it
On allogenic cell with wide spectrum it is antiviral, influence cell Growth and Differentiation and adjust the multiple biological activities such as immune function.
Human leukocyte antigen (human leukocyte antigen, HLA) is that the ajor histocompatibility of the mankind is compound
Body is located on No. 6 chromosomes, is divided into HLA-I class molecule and HLA- class Ⅱmolecule, the close phase of function of immune system with the mankind
It closes.Research thinks that the HLA molecule on antigen presenting cell surface is the key factor for influencing HIV specific CTL, and different HLA divides
Son is significant related to progression of disease.
Summary of the invention
The technical problem to be solved by the present invention is to how carry out the body of People living with HIV HLA-A2 Specific CTL Cells
Outer amplification cultivation.
In order to solve the above technical problems, present invention firstly provides amplification in vitro People living with HIV HLA-A2 specificity
The complete set of culture medium of CTL cell.
The complete set of culture medium of amplification in vitro People living with HIV HLA-A2 Specific CTL Cells provided by the present invention, by
The complete culture medium 1 used and culture medium 2 form；The culture medium 1 and the culture medium 2 are mammalian cell serum-free
Culture medium, the culture medium 1 contain amino acid sequence 9 kinds of polypeptides as shown in SEQ ID No.1-SEQ ID No.9 respectively, institute
It states culture medium 2 and contains interleukin 2.
In above-mentioned complete set of culture medium, the amino acid sequence of polypeptide 1 is as shown in SEQ ID No.1；The amino acid sequence of polypeptide 2
As shown in SEQ ID No.2；The amino acid sequence of polypeptide 3 is as shown in SEQ ID No.3；The amino acid sequence of polypeptide 4 such as SEQ
Shown in ID No.4；The amino acid sequence of polypeptide 5 is as shown in SEQ ID No.5；The amino acid sequence of polypeptide 6 such as SEQ ID No.6
It is shown；The amino acid sequence of polypeptide 7 is as shown in SEQ ID No.7；The amino acid sequence of polypeptide 8 is as shown in SEQ ID No.8；It is more
The amino acid sequence of peptide 9 is as shown in SEQ ID No.9.
The complete set of culture medium of amplification in vitro People living with HIV HLA-A2 Specific CTL Cells provided by the present invention, point
For the storage type culture medium convenient for storage and the instant culture medium being made by the storage type culture medium and water.
The storage type culture medium in above-mentioned complete set of culture medium can are as follows: the content of 9 kinds of polypeptides described in the culture medium 1
The use concentration for meeting every kind of polypeptide in 9 kinds of polypeptides is 2 μ g/mL；Interleukin 2 contains in the culture medium 2
The use concentration that amount meets interleukin 2 is 500U/mL.
Instant culture medium in above-mentioned complete set of culture medium can are as follows: every kind of polypeptide of 9 kinds of polypeptides described in the culture medium 1
Content be 2 μ g/mL；The content of interleukin 2 is 500U/mL in the culture medium 2.
In above-mentioned complete set of culture medium, the culture medium 1 is to add 9 kinds of polypeptides in MD-CM-STM-N culture medium to obtain
The fluid nutrient medium arrived, the culture medium 2 are the fluid nutrient mediums for adding interleukin 2 in IMSF100 culture medium and obtaining.
In order to solve the above technical problems, the present invention also provides amplification in vitro People living with HIV HLA-A2 specific CTLs
The product of cell.
The product of amplification in vitro People living with HIV HLA-A2 Specific CTL Cells provided by the present invention, including it is above-mentioned
The in vitro peripheral blood mononuclear cells of the People living with HIV of complete set of culture medium and the HLA-A2 positive.
In order to solve the above technical problems, the present invention also provides following A 1 or the preparation methods of A2:
The preparation method of A1, above-mentioned complete set of culture medium include the following steps: to prepare the culture medium 1 and the training respectively
Base 2 is supported, then the culture medium 1 and the culture medium 2 are subjected to independent packaging respectively, obtains the complete set of culture medium；
The preparation method of A2, the said goods include the following steps: that above-mentioned complete set of culture medium and the HLA-A2 is positive
The in vitro peripheral blood mononuclear cells of People living with HIV is individually packed respectively, obtains the product.
In order to solve the above technical problems, the present invention also provides amplification in vitro People living with HIV HLA-A2 specific CTLs
The composition of cell.
The composition of amplification in vitro People living with HIV HLA-A2 Specific CTL Cells provided by the present invention, by SEQ
9 kinds of polypeptide compositions, the No.9 of SEQ ID No.1-SEQ ID described in the composition shown in ID No.1-SEQ ID No.9
Shown in 9 kinds of polypeptides mass ratio be 1:1:1:1:1:1:1:1:1.
In order to solve the above technical problems, the present invention also provides amplification in vitro People living with HIV HLA-A2 specific CTLs
The reagent set of cell.
The reagent set of amplification in vitro People living with HIV HLA-A2 Specific CTL Cells provided by the present invention, by upper
Composition and interleukin 2 composition are stated, the composition and interleukin 2 match.
In above-mentioned reagent set, the polypeptide 1, the polypeptide 2, the polypeptide 3, the polypeptide 4 in the composition,
The polypeptide 5, the polypeptide 6, the polypeptide 7, the polypeptide 8, the polypeptide 9 and matching for interleukin 2 can expire
2 μ g:2 μ g:2 μ g:2 μ g:2 μ g:2 μ g:2 μ g:2 μ g:2 μ g:(4000U-5500U of the following ratio of foot), it can specifically meet 2 μ g:2 μ
G:2 μ g:2 μ g:2 μ g:2 μ g:2 μ g:2 μ g:2 μ g:4000U or 2 μ g:2 μ g:2 μ g:2 μ g:2 μ g:2 μ g:2 μ g:2 μ g:2 μ g:
5500U。
People living with HIV HLA-A2 Specific CTL Cells described herein are the People living with HIV of the HLA-A2 positive
In vitro CD3+CD8+T lymphocyte.
The present invention also provides following 1) -7) in any application:
1) complete set of culture medium is in preparing amplification in vitro People living with HIV HLA-A2 Specific CTL Cells product
Using；
2) complete set of culture medium expands the application in People living with HIV HLA-A2 Specific CTL Cells in vitro；
3) product expands the application in People living with HIV HLA-A2 Specific CTL Cells in vitro；
4) composition is preparing answering in amplification in vitro People living with HIV HLA-A2 Specific CTL Cells product
With；
5) composition expands the application in People living with HIV HLA-A2 Specific CTL Cells in vitro；
6) reagent set is preparing answering in amplification in vitro People living with HIV HLA-A2 Specific CTL Cells product
With；
7) reagent set expands the application in People living with HIV HLA-A2 Specific CTL Cells in vitro.
In order to solve the above technical problems, the present invention also provides amplification in vitro People living with HIV HLA-A2 specific CTLs
The method of cell.
The method of amplification in vitro People living with HIV HLA-A2 Specific CTL Cells provided by the present invention, including it is as follows
Step: after the in vitro peripheral blood mononuclear cells of the People living with HIV of the HLA-A2 positive and the culture medium 1 are mixed, training
After supporting 2 days, the culture medium 2 identical with 1 volume of culture medium is added, it is 1 body of culture medium that every 3-4 days, which are added, later
The culture medium 2 of 1.5 times of product is cultivated, and from the culture medium 2 is added for the first time, is further cultured for 10-16 days, and AIDS sense is completed
The amplification of dye person's HLA-A2 Specific CTL Cells.It is 0 at the time of entire amplification in vitro incubation time is to be added culture medium 1
Moment amounts to 12-18 days (such as 12 or 18 days).
The methods of amplification in vitro People living with HIV HLA-A2 Specific CTL Cells provided by the present invention specifically as 1) or
2) the step of:
1) the in vitro peripheral blood mononuclear cells of the People living with HIV of the HLA-A2 positive and the culture medium 1 are mixed
Afterwards, after cultivating 2 days, cultivating system 1 is obtained；The training identical with 1 volume of culture medium is added into the cultivating system 1
It supports base 2 to cultivate 3 days, obtains cultivating system 2；Being added into the cultivating system 2 is described in described 1.5 times of 1 volume of culture medium
Culture medium 2 is cultivated 3 days, and cultivating system 3 is obtained；Being added into the cultivating system 3 is 1.5 times of 1 volume of culture medium of the institute
It states culture medium 2 to cultivate 4 days, completes the amplification of People living with HIV HLA-A2 Specific CTL Cells.When entire amplification in vitro culture
Between culture medium 1 is added at the time of be 0 moment, amount to 12 days.
2) the in vitro peripheral blood mononuclear cells of the People living with HIV of the HLA-A2 positive and the culture medium 1 are mixed
Afterwards, after cultivating 2 days, cultivating system 1 is obtained；The training identical with 1 volume of culture medium is added into the cultivating system 1
It supports base 2 to cultivate 4 days, obtains cultivating system 2；Being added into the cultivating system 2 is described in described 1.5 times of 1 volume of culture medium
Culture medium 2 is cultivated 4 days, and cultivating system 3 is obtained；Being added into the cultivating system 3 is 1.5 times of 1 volume of culture medium of the institute
It states culture medium 2 to cultivate 4 days, obtains cultivating system 4；Being added into the cultivating system 4 is described 1.5 times of 1 volume of culture medium
The culture medium 2 is cultivated 4 days, and the amplification of People living with HIV HLA-A2 Specific CTL Cells is completed.Entire amplification in vitro culture
It was 0 moment at the time of time is to be added culture medium 1, amounts to 18 days.
The method of above-mentioned amplification in vitro People living with HIV HLA-A2 Specific CTL Cells, further includes from cultivating system
The step of collecting cell.
In the method for above-mentioned amplification in vitro People living with HIV HLA-A2 Specific CTL Cells, the culture is as follows
Under the conditions of carry out: temperature can be for 35 DEG C -37 DEG C, concretely 37 DEG C；CO2Volume content can be 4.0%-10.0%, specifically may be used
It is 5.0%.
The complete set of culture medium of external preparation People living with HIV HLA-A2 Specific CTL Cells of the invention is serum-free training
Support base, the methods of external preparation People living with HIV HLA-A2 Specific CTL Cells of the invention using serum free medium into
Row culture is avoided the unfavorable factor of the sex pheromone pollution and the growth of other pairs of cells that may cause using serum, also kept away
Possible safety factor is exempted from.
It is demonstrated experimentally that can be by the different AIDS senses of separation using complete set of culture medium and extracorporeal culturing method of the invention
Dye person's HLA-A2 Specific CTL Cells-CD3+CD8+T cell successfully carries out amplification cultivation, and the cell activity of acquisition is more stable,
It is maintained at 90% or more, and the CTL with HLA-A2 specificity of the different People living with HIV obtained when cultivating 12 days
Cell-CD3+CD8+T cell number is significantly higher than CD3+CD8+T cell number at the 0th day, and increased multiple range is 1.97-
21.32 times, the percentage range that different People living with HIV CD3+CD8+IFN- γ+T cell numbers account for CD3+CD8+T cell number is
0.90%-11.70%.CD3+CD8+T cell is directly related with the decline of inhibition of HIV carrying capacity, can efficient identification and dissolution patient from
CD4+T cell after body HIV infection, has great importance for the treatment of AIDS.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
In following embodiments peripheral blood mononuclear cells (Peripheral blood mononuclear cell,
PBMC blood sample) is come from, the acquisition of blood sample is the anticoagulant whole blood sample of EDTA, room temperature preservation.Blood specimen collection 6 is small
When interior separation PBMC.
" MD-CM-STM-N " culture medium in following embodiments is the product of Tianjin MD Pacific Technology Co., Ltd.；
IMSF100 culture medium is the product that applicating technology Co., Ltd is immunized in Beijing Yongtai.
Interleukin 2 (IL-2) in following embodiments is the production of Weixing Biological Product Inst. (Co., Ltd.), Liaoning
Product, product number are sadie grace, and specification is 2,000,000 IU/ branch；Interleukin-4 (IL-4) (catalog number 554605, rule
Lattice are 5 μ g), interleukin-17 (IL-7) (catalog number 554608, specification are 5 μ g), the CD3 antibody of FITC label (produces
Product catalog number (Cat.No.) is that 555339), the CD8 of the CD4 antibody (catalog number 560650) of PerCP-Cy5.5 label, APC label is anti-
The IFN-γ antibody (catalog number 559326) of body (catalog number 340584), PE label is the production of BD company
Product.
Ficoll in following embodiments is the product in General Electric's Medical Group life science portion (GEHC), catalogue
Number be 17-5442-03.
Embodiment 1, amplification in vitro People living with HIV HLA-A2 Specific CTL Cells
One, the complete set of culture medium of amplification in vitro People living with HIV HLA-A2 Specific CTL Cells
Cell culture medium such as RPMI-1640 or DMEM etc. must add fetal calf serum or human serum when cultivating cell,
However addition serum will bring some problems, such as introduce serum origin pathogeny body pollution, between different batches serum there may be
Difference, immunological rejection risk etc..It is serum free medium suitable for what is used in the method for the invention.
Complete set of culture medium includes culture medium 1 and culture medium 2.
Culture medium 1 is the liquid for adding 9 kinds of polypeptides of entitled polypeptide 1- polypeptide 9 in MD-CM-STM-N culture medium and obtaining
Body culture medium；The amino acid sequence of polypeptide 1 is as shown in SEQ ID No.1；The amino acid sequence of polypeptide 2 such as SEQ ID No.2 institute
Show；The amino acid sequence of polypeptide 3 is as shown in SEQ ID No.3；The amino acid sequence of polypeptide 4 is as shown in SEQ ID No.4；Polypeptide
5 amino acid sequence is as shown in SEQ ID No.5；The amino acid sequence of polypeptide 6 is as shown in SEQ ID No.6；The amino of polypeptide 7
Acid sequence is as shown in SEQ ID No.7；The amino acid sequence of polypeptide 8 is as shown in SEQ ID No.8；The amino acid sequence of polypeptide 9
As shown in SEQ ID No.9.
The content of 9 kinds of polypeptides is 2 μ g/mL in culture medium 1.
Culture medium 2 is the fluid nutrient medium for adding interleukin 2 in IMSF100 culture medium and obtaining；Interleukin 2
Content in culture medium 2 is 500U/mL.
Two, using different cytokines and different incubation time to People living with HIV HLA-A2 Specific CTL Cells into
Row amplification in vitro culture
1, the separation of peripheral blood mononuclear cells (PBMC)
By the in vitro of People living with HIV that collected HLA parting is HLA-A2 (being identified using serological typing method)
Peripheral blood (blood sample sample) is put into 50mL centrifuge tube, addition and the isometric injection physiological saline of blood sample sample, after mixing
It is added slowly in the 50mL centrifuge tube for having dispensed every pipe 15mL Ficoll lymphocyte separation medium, 2000rpm is centrifuged 20min.
After centrifugation, it is raw to add injection according to the volume ratio of 1:1 into a new 50mL centrifuge tube for cellular layer between drawing interface
Salt water is managed, is mixed, 1200rpm is centrifuged 10min, and incline supernatant, collects cell precipitation.It is resuspended with 50mL injection physiological saline thin
Born of the same parents' precipitating, 1200rpm are centrifuged 10min, and incline supernatant, collect cell precipitation.Add 5mL injection physiological saline that cell is resuspended heavy
It forms sediment, 10 μ L cell suspensions is taken to carry out cell count after mixing, then supplement injection physiological saline to 50mL, 1200rpm centrifugation
10min, incline supernatant, collects cell precipitation, as peripheral blood mononuclear cells (PBMC).
2, People living with HIV HLA-A2 Specific CTL Cells are carried out using different cytokines and different incubation times
Amplification in vitro culture
The method of amplification in vitro culture includes method A and B, the specific steps are as follows:
Method A is inoculated into 6 orifice plates after the peripheral blood mononuclear cells (PBMC) that step 1 obtains is resuspended with 12mL culture medium 1
(Corning company, the U.S., article No. 3516 grow area 9.5cm2) in, every hole cell density is 2.0 × 106, every hole 2mL puts
Enter cell incubator in 37 DEG C, 5%CO2In start to cultivate, be denoted as 0 moment.After inoculated and cultured 2 days, cultivating system 1 is obtained；From
Culture plate is taken out in cell incubator, and 2mL culture medium 2 is added to every hole of cultivating system 1, is put into cell incubator and continues
37 DEG C, 5%CO2Middle culture obtains cultivating system 2 in 3 days；Culture plate is taken out from cell incubator, to every hole of cultivating system 2
3mL culture medium 2 is added, is put into cell incubator and continues in 37 DEG C, 5%CO2Middle culture obtains cultivating system 3 in 3 days；To culture
3mL culture medium 2 is added in every hole in system 3, is put into cell incubator and continues in 37 DEG C, 5%CO2Middle culture 4 days, completes Chinese mugwort
The amplification for growing sick the infected's HLA-A2 Specific CTL Cells, amounts to 12 days, collects cell suspension, obtains cell culture 12 days
Cell suspension.
Method A is inoculated into 6 orifice plates after the peripheral blood mononuclear cells (PBMC) that step 1 obtains is resuspended with 12mL culture medium 1
(Corning company, the U.S., article No. 3516 grow area 9.5cm2) in, every hole cell density is 2.0 × 106, every hole 2mL puts
Enter cell incubator in 37 DEG C, 5%CO2In start to cultivate, be denoted as 0 moment.After inoculated and cultured 2 days, cultivating system 1 is obtained；From
Culture plate is taken out in cell incubator, and 2mL culture medium 2 is added to every hole of cultivating system 1, is put into cell incubator and continues
37 DEG C, 5%CO2Middle culture obtains cultivating system 2 in 4 days；Culture plate is taken out from cell incubator, to every hole of cultivating system 2
3mL culture medium 2 is added, is put into cell incubator and continues in 37 DEG C, 5%CO2Middle culture obtains cultivating system 3 in 4 days；To culture
3mL culture medium 2 is added in every hole in system 3, is put into cell incubator and continues in 37 DEG C, 5%CO2Middle culture 4 days, is trained
Support system 4；3mL culture medium 2 is added in every hole into cultivating system 4, is put into cell incubator and continues in 37 DEG C, 5%CO2In
The amplification of People living with HIV HLA-A2 Specific CTL Cells is completed in culture 4 days, is amounted to 18 days, is collected cell suspension, is obtained
The cell suspension of cell culture 18 days.
The cell suspension of the cell suspension of cell culture 12 days and cell culture 18 days is collected into 50mL centrifuge tube respectively
In, 1200rpm is centrifuged 10min, and incline and supernatant and shakes and open precipitating, supplement injection physiological saline to 50mL, 1200rpm centrifugation
10min, incline again supernatant and shake open precipitating, then with 50mL contain 1% human serum albumin injection physiological saline resuspension
Cell, the cell suspension and 18 days cell suspensions of the culture 12 days of preparation method A.
In method B in addition to the culture medium 2 in method A is replaced with control medium 2, other operating procedures are constant, point
The cell suspension and 18 days cell suspensions of the culture 12 days of other preparation method B；Control medium 2 is in IMSF100 culture medium
The fluid nutrient medium that middle addition interleukin-4 and interleukin-17 obtain；Interleukin-4 containing in control medium 2
Amount is 400U/mL；Interleukin-17 is 10ng/mL in 2 content of control medium.
Expect that blue dyeing microscopic examination determines the number of living cells and dead cell in the cell suspension obtained by cell counting board and platform
Amount, and calculate living cells percentage: living cells percentage (%)=viable count/(viable count+dead cell number) × 100%.
The CD4 antibody of CD3 antibody, PerCP-Cy5.5 label that the Phenotypic examination of the cell of amplification is marked using FITC,
The IFN-γ antibody that CD8 antibody, the PE of APC label are marked, is measured by flow cytometry.
CD3+CD8+ cell increases multiple=(the 12nd day or the 18th day-the 0 day CD3+ of CD3+CD8+T total number of cells
CD8+T total number of cells)/the 0th day CD3+CD8+T total number of cells.
Experimental result is as shown in table 1, and CD3+CD8+T cell is the CTL cell with HLA-A2 specificity, using method A
To total number of cells and CD3+CD8+T cell number after the progress amplification in vitro culture of People living with HIV HLA-A2 Specific CTL Cells
Be above using method B to People living with HIV HLA-A2 Specific CTL Cells carry out amplification in vitro culture total number of cells and
CD3+CD8+T cell number.Amplification in vitro culture 12 is carried out to People living with HIV HLA-A2 Specific CTL Cells using method A
It and after 18 days, cell survival rate after 90% or more, amplification in vitro culture 12 days CD3+CD8+T total number of cells than the 0th day
CD3+CD8+T total number of cells increased by 10.07 times than the 0th day after increasing by 8.31 times, amplification in vitro culture 18 days.To HIV infection
Person's HLA-A2 Specific CTL Cells carry out amplification in vitro culture 12 days, the CD3+CD8+IFN- γ+T expanded using method A
Cell number (8.30 × 104It is a), CD3+CD8+IFN- γ+T cell number account for CD3+CD8+T cell number percentage (1.50%) it is equal
It is apparently higher than the CD3+CD8+IFN- γ+T cell number (5.34 × 10 expanded using method B2It is a), CD3+CD8+IFN- γ
+ T cell number accounts for the percentage (0.20%) of CD3+CD8+T cell number.To People living with HIV HLA-A2 Specific CTL Cells into
Row amplification in vitro culture 18 days, the CD3+CD8+IFN- γ+T cell number (2.83 × 10 expanded using method A5It is a), CD3
The percentage (4.30%) that+CD8+IFN- γ+T cell number accounts for CD3+CD8+T cell number is expanded obviously higher than using method B
CD3+CD8+IFN- γ+T cell the number (1.23 × 10 arrived3It is a), CD3+CD8+IFN- γ+T cell number account for CD3+CD8+T cell
Several percentage (0.30%).CD3+CD8+IFN- γ+T cell is the CD3+CD8+T cell of secretion of gamma-IFN.The above results are aobvious
Can effectively People living with HIV HLA-A2 Specific CTL Cells-CD3+CD8+T cell be carried out in vitro using method A by showing
Amplification cultivation.
Table 1, under different cytokines and different incubation time HLA-A2 Specific CTL Cells amplification in vitro result
Note: being 0 moment at the time of incubation time is to access culture medium 1.
Three, in vitro culture is carried out to different People living with HIV HLA-A2 Specific CTL Cells using interleukin 2
1, the separation of peripheral blood mononuclear cells (PBMC)
Same step 2.
2, it carries out carrying out in vitro culture to People living with HIV HLA-A2 Specific CTL Cells using interleukin 2
According to the method culture in amplification in vitro culture 12 days of method A in step 2, People living with HIV HLA-A2 is completed
Cell suspension is collected in the amplification of Specific CTL Cells-CD3+CD8+T cell, obtains the different AIDS senses cultivated 12 days respectively
The cell suspension of dye person.
Expect that blue dyeing microscopic examination determines the number of living cells and dead cell in the cell suspension obtained by cell counting board and platform
Amount, and calculate living cells percentage: living cells percentage (%)=viable count/(viable count+dead cell number) × 100%.
The CD4 antibody of CD3 antibody, PerCP-Cy5.5 label that the Phenotypic examination of the cell of amplification is marked using FITC,
The IFN-γ antibody that CD8 antibody, the PE of APC label are marked, is measured by flow cytometry.
CD3+CD8+ cell increases multiple=(the 12nd day-the 0 day CD3+CD8+T cell of CD3+CD8+T cell number
Number)/the 0th day CD3+CD8+T cell number.
The results are shown in Table 2, more stable using all cell activity after method A amplification in vitro culture 12 days, keeps
90% or more；HLA-A2 Specific CTL Cells-CD3+CD8+T the cell number of different People living with HIV is significantly higher than culture
The CD3+CD8+T cell number of 0d, increased multiple range is 1.97-21.32 times.Using method A amplification in vitro culture 12 days
Afterwards, the percentage range that different People living with HIV CD3+CD8+IFN- γ+T cell numbers account for CD3+CD8+T cell number is
0.90%-11.70%, CD3+CD8+IFN- γ+T cell are the CD3+CD8+T cell (table 3) of secretion of gamma-IFN.
Table 2, the result that using IL-2 different People living with HIV HLA-A2 Specific CTL Cells are carried out with amplification in vitro
Note: being 0 moment at the time of incubation time is to access culture medium 1.
Table 3, CD3+CD8+IFN- γ+T cell number account for the percentage of CD3+CD8+T cell number
CD3+% in table 1- table 3 is the percentage that CD3+T cell number accounts for total number of cells, CD3+CD8+% CD3+CD8+
T cell number accounts for the percentage of CD3+T cell number, and CD3+CD4+% is the percentage that CD3+CD4+T cell number accounts for CD3+T cell number
Than CD3+CD8+IFN- γ+% is the percentage that CD3+CD8+IFN- γ+T cell number accounts for CD3+CD8+ cell number.
Claims (7)
1. the reagent set of amplification in vitro People living with HIV HLA-A2 Specific CTL Cells, by SEQ ID No.1-SEQ ID
9 kinds of polypeptides shown in No.9 and interleukin 2 composition, 9 kinds of polypeptides and interleukin 2 match；The AIDS
Sick the infected HLA-A2 Specific CTL Cells are that the in vitro CD3+CD8+T lymph of the People living with HIV of the HLA-A2 positive is thin
Born of the same parents.
2. the complete set of culture medium of amplification in vitro People living with HIV HLA-A2 Specific CTL Cells, by the complete culture medium 1 used
It is formed with culture medium 2；The culture medium 1 and the culture medium 2 are serum-free medium for mammalian cell, the culture medium
1 contains amino acid sequence 9 kinds of polypeptides as shown in SEQ ID No.1-SEQ ID No.9 respectively, and the culture medium 2 is containing white thin
Born of the same parents' interleukin 2；The People living with HIV HLA-A2 Specific CTL Cells are the in vitro of the People living with HIV of the HLA-A2 positive
CD3+CD8+T lymphocyte.
3. complete set of culture medium according to claim 2, it is characterised in that: every in 9 kinds of polypeptides described in the culture medium 1
The content of kind polypeptide is 2 μ g/mL；The content of interleukin 2 is 500U/mL in the culture medium 2.
4. complete set of culture medium according to claim 2 or 3, it is characterised in that: the culture medium 1 is in MD-CM-STM-N
Add the fluid nutrient medium that 9 kinds of polypeptides obtain in culture medium, the culture medium 2 be added in IMSF100 culture medium it is white
The fluid nutrient medium that cytokine 2 obtains.
5. the product of amplification in vitro People living with HIV HLA-A2 Specific CTL Cells, including any right in claim 2-4
It is required that the in vitro peripheral blood mononuclear cells of the People living with HIV of the complete set of culture medium and the HLA-A2 positive；The Chinese mugwort
Grow the in vitro CD3+CD8+T lymph for the People living with HIV that sick the infected's HLA-A2 Specific CTL Cells are the HLA-A2 positive
Cell.
6. complete set of culture medium described in any claim is preparing amplification in vitro People living with HIV HLA- in claim 2-4
Application in A2 Specific CTL Cells product；The People living with HIV HLA-A2 Specific CTL Cells are HLA-A2 positive
The in vitro CD3+CD8+T lymphocyte of People living with HIV.
7. reagent set described in claim 1 is preparing the production of amplification in vitro People living with HIV HLA-A2 Specific CTL Cells
Application in product；The People living with HIV HLA-A2 Specific CTL Cells be the HLA-A2 positive People living with HIV from
The CD3+CD8+T lymphocyte of body.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997034621A1 (en) * | 1996-03-21 | 1997-09-25 | Cytel Corporation | Hla-a2.1 binding peptides and their uses |
| WO2010037402A1 (en) * | 2008-10-02 | 2010-04-08 | Dako Denmark A/S | Molecular vaccines for infectious disease |
| CN103073620A (en) * | 2002-09-12 | 2013-05-01 | 肿瘤疗法科学股份有限公司 | KDR peptides and vaccines comprising the same |
| CN103370333A (en) * | 2010-11-10 | 2013-10-23 | 埃斯特韦实验室有限公司 | Highly immunogenic HIV P24 sequences |
-
2015
- 2015-10-15 CN CN201510666770.4A patent/CN105200010B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997034621A1 (en) * | 1996-03-21 | 1997-09-25 | Cytel Corporation | Hla-a2.1 binding peptides and their uses |
| CN103073620A (en) * | 2002-09-12 | 2013-05-01 | 肿瘤疗法科学股份有限公司 | KDR peptides and vaccines comprising the same |
| WO2010037402A1 (en) * | 2008-10-02 | 2010-04-08 | Dako Denmark A/S | Molecular vaccines for infectious disease |
| CN103370333A (en) * | 2010-11-10 | 2013-10-23 | 埃斯特韦实验室有限公司 | Highly immunogenic HIV P24 sequences |
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