CN104293734B - A kind of preparation method of human gamma delta t cells - Google Patents
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Abstract
The invention provides a kind of preparation method of human gamma delta t cells, category cellular immunology field；The preparation of the human gamma delta t cells comprises the following steps：Step 1: prepare tubercle bacillus heat resistance antigen with the tubercle bacillus of culture；Step 2: stimulating PMNC with purifying gained tubercle bacillus heat resistance antigenic components, while add sulindac and licorice polysaccharide；Step 3: 3 days later half amounts of cell culture change the nutrient solution containing sulindac, licorice polysaccharide, cell density is kept；Step 4: according to cell growth state, the 7th day harvesting.The defects of for prior art, the present invention provide the gamma delta T cells that a kind of short preparation period, purity are high, fragmentation effect is good, cell proliferation is fast, preparation cost is low, it is ensured that finished product meets the requirements.
Description
Technical field
The invention belongs to cellular immunology field, and in particular to a kind of preparation method of human gamma delta t cells.
Background technology
T lymphocytes are divided into two classes according to the difference of expression T cell antigen acceptor (T cell receptor, TCR)：TCR
α β T cells and TCR gamma delta T cells, are briefly referred to as α β T cells and gamma delta T cells.Wherein gamma delta T cells are between specific immunity
A kind of cell of specific type between nospecific immunity, most of double-negative for CD4 and CD8 molecules, minority can be expressed
CD8 molecules.Gamma delta T cells are distributed mainly on skin and mucous membrane tissue, are usually no more than the 5% of T cell sum.Gamma delta T cells have
There is specific recognition antigen and restricted without MHC, there is the functions such as anti-infective, antitumor and immunological regulation, be immunity of organism prison
Depending on the first line of defence.But content is not high in peripheral blood and tumor tissues due to gamma delta T cells, a large amount of high cell toxicants are obtained
The gamma delta T cells of activity are very difficult.Although prior art is obtained big by anti-tcr γ anti-δs or non-peptide phosphoric acid class antigen
Gamma delta T cells are measured, this can undoubtedly increase the cost of its preparation.Therefore a kind of practical, efficient, a large amount of advantages of rapid in-vitro are developed to expand
Increase the method for human peripheral gamma delta T cells, the research for gamma delta T cells immunologic function, and patient's immunity, resistance are improved with it
Tumour and anti-infection ability are very important, and malignant neoplastic disease this problem solved for capturing the mankind to thirst for is also
It is very valuable.
Found through retrieval, though there are the cultural method that some technical schemes disclose gamma delta T cells, manufacturing cycle now
Longer, purity is not high enough, and lethality is not strong enough.
For prior art defect, a kind of short preparation period of present invention offer, purity is high, fragmentation effect is good, cell proliferation
It hurry up, prepare the low gamma delta T cells of cost, it is ensured that finished product meets the requirements.
The content of the invention
For in the prior art the defects of, it is an object of the invention to provide a kind of preparation method of human gamma delta t cells.
The present invention is achieved by the following technical solutions：
The present invention provides a kind of preparation method of human gamma delta t cells, and methods described comprises the following steps：
Step 1: prepare tubercle bacillus heat resistance antigen with the tubercle bacillus of culture；
Step 2: PMNC is stimulated with purifying gained tubercle bacillus heat resistance antigenic components, simultaneously
The RPMI1640 nutrient solution Cytokines in Peripheral Blood Mononuclear containing sulindac and licorice polysaccharide is added to be cultivated；
Step 3: 2~3 days later half amounts of culture change the nutrient solution；
Step 4: according to cell growth state, the 7th day harvesting.
Preferably, in step 1, it is described with culture tubercle bacillus prepare tubercle bacillus heat resistance antigen, specifically include as
Lower step：
A, by pretreated tubercle bacillus culture, it is inoculated in Soviet Union's formula fluid nutrient medium, in 37 DEG C of incubators
Culture 1~2 week；Then it is dispensed into Soviet Union's formula fluid nutrient medium containing NBCS；It is further continued in 37 DEG C of cultures
Static gas wave refrigerator 1~2 week in case；The bacterial suspension of culture is collected, centrifuges to obtain core bacillus thalline；
B, by the core bacillus thalline of collection through brine, then with distilled water resuspension thalline, high steam processs,
Supernatant is taken to obtain tubercle bacillus heat resistance antigen through 0.22 μm of micropore miillpore filter.
Preferably, in step A, the time of the pretreatment is 5~15 minutes.
Preferably, described pre-process is specially：Standby tubercle bacillus culture is added to sulfuric acid solution or hydroxide
In sodium solution, concussion mixes, and thalline is collected by centrifugation.
It is highly preferred that the volumetric concentration of the sulfuric acid solution is 0.5%；The concentration of the sodium hydroxide solution is 0.2mg/
ml。
It is highly preferred that the volume ratio of the standby tubercle bacillus culture and sulfuric acid solution is 1：(1~3)；It is described standby
The volume ratio of tubercle bacillus culture and sodium hydroxide solution is 1:(2~6).
Preferably, in step B, the condition of the high steam processs is 120 DEG C, 15 minutes.
Preferably, in step 2, the purifying concrete operations are：Core bacillus heat resistance antigen obtained by step 1 is quickly led to
Cross the molecular sieve type S-100 chromatographic columns and ion-exchange type Mono Q chromatographic columns of protein liquid chromatography instrument.
Preferably, in step 2, the active component includes the high Mr protein components (Mtb-HW-Ag) of Mtb-Ag, the low Mr of Mtb
The single main peptide (Mtb-Ag-Pk3) of polypeptide fractions (Mtb-LW-Ag) or Mtb-Ag peaks 3.
Preferably, it is described to stimulate peripheral blood list with purifying gained tubercle bacillus heat resistance antigenic components in step 2
Individual nucleus, while sulindac and licorice polysaccharide are added, specifically include：Peripheric venous blood is gathered, through ficoll-general shadow aminoglucose
Density gradient centrifugation obtains mononuclearcell；By mononuclearcell be placed in containing zoledronic acid, sulindac, licorice polysaccharide,
The RPMI1640 nutrient solutions of 0.05mg/ml autologous plasmas, 50~100IU/ml tubercle bacillus heat resistance purifying antigen active components
Interior, it is 1~2 × 10 to adjust cell density5/ ml, is transferred to Tissue Culture Plate, in 37 DEG C, 5%CO2Trained with the incubator of saturated humidity
Support.
Preferably, the concentration of the zoledronic acid is 0.01~1ng/ml.
Preferably, in step 2 and step 3, the sulindac, the concentration of licorice polysaccharide are 0.1~0.5ng/ml.
Preferably, in step 4, the cell growth state is embodied in：Visible gamma delta T cells are sunken within 20~28 hours
The bottom of culture vessel, and tend to be colonization；30~40 hours colonies start to become big；Culture can see big collection in 5~7 days
Fall with irregular mononuclearcell, the auspicious Ji's Albert'stain Albert of cell progress to cultivating 7 days, the volume increase of discovery most cells,
Oval or irregular shape, nucleus are mostly ellipse or circle, and nuclear staining is loose, and nuclear membrane is irregular, has projection, each
Visible 1 little nucleolar of cell, in navy blue；Kytoplasm enriches, and dyes dusty blue, and form is irregular, has pseudopodium, there is light dye at nearly core
Phenomenon, also have in irregular shape；Trypan Blue liquid, living cells do not dye, and dead cell dyes blueness, is counted under microscope.
Compared with prior art, the present invention has following beneficial effect：
(1) preparation method of the present invention is simple, and cell culture period is short, substantially reduces toxigenic capacity；
(2) for preparation method of the present invention by the pretreatment to tubercle bacillus culture, the speed for obtaining Mtb-Hag is fast, pure
Degree is high, and binding ability is strong；
(3) present invention carries out cell culture by using the nutrient solution containing zoledronic acid, sulindac, licorice polysaccharide so that
Cell proliferation rate is fast, and quality is good, and purity is high, and activity is strong.
Embodiment
With reference to specific embodiment, the present invention is described in detail.Following examples will be helpful to the technology of this area
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill to this area
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection domain.
Embodiment 1
The present embodiment is related to a kind of human gamma delta t cells, and the preparation of the human gamma delta t cells comprises the following steps：
Step 1: preparing tubercle bacillus heat resistance antigen with the tubercle bacillus of culture, comprise the following steps：
A, tubercle bacillus culture (the about bacterium number 1 × 10 that 1ml is pretreated is taken3), it is inoculated in 200ml Soviet Union's formula liquid
In culture medium, in static gas wave refrigerator 1 week in 37 DEG C of incubators, after taking out concussion, continue culture 1 week；Then be dispensed into containing
In the 150ml of 15% NBCS Soviet Union's formula fluid nutrient medium, every bottle of 50ml, 4 bottles are distributed into；It is further continued in 37 DEG C of cultures
Static gas wave refrigerator 2 weeks in case；The bacterial suspension of culture is collected, through 4000 revs/min, centrifuges 30 minutes, obtains core bacillus thalline；
B, by the thalline of collection through brine 2 times, then the distilled water resuspension thalline with 2 times of volumes, 120 DEG C of height
Press steam treatment 15 minutes, take supernatant to obtain Mtb-Hag through 0.22 μm of micropore miillpore filter；
Step 2: stimulating PBMCs with purifying gained Mtb-Hag active components, while sulindac and licorice polysaccharide are added, had
Body step includes：
20ml detection in peripheral blood of patients underwent is gathered, mononuclearcell is obtained through ficoll-general shadow aminoglucose density gradient centrifugation,
Concretely comprise the following steps：1500 revs/min, centrifuge 10 minutes, draw upper plasma layer, 56 DEG C inactivation 15 minutes after centrifuge it is standby, it is sterile
The haemocyte of water two-fold dilution precipitation, human lymphocyte separating liquid press 1 with dilute blood:4 mixing, 2000 revs/min, centrifugation 20
Minute, tunica albuginea layer is drawn, sterile water washing 2 times, rotating speed is respectively 1500 revs/min, centrifuges 5 minutes, that is, obtains peripheral blood list
Individual nucleus；
By the PMNC of above-mentioned separation be placed in containing 0.01ng/ml zoledronic acids, 0.1ng/ml sulindacs,
0.1ng/ml licorice polysaccharides, 0.05mg/ml autologous plasmas, 50IU/ml tubercle bacillus heat resistance purifying antigen active components
In RPMI1640 nutrient solutions, it is 1~2 × 10 to adjust cell density5/ ml, is transferred to Tissue Culture Plate, in 37 DEG C, 5%CO2It is wet with saturation
Cultivated in the incubator of degree；
Step 3: 3 days later half amounts of cell culture change the nutrient solution containing sulindac, licorice polysaccharide, cell density is kept
For 1~1.5 × 106/ml；
Step 4: according to cell growth state, the 7th day harvesting；By cell culture, 24 hours are visible gamma delta T cells
The bottom of culture vessel is sunken to, and tends to be colonization, 30~40 hours colonies start to become big, and culture can be seen big for 5~7 days
Colony and irregular mononuclearcell, auspicious Ji's Albert'stain Albert is carried out to the cell of culture 7 days, it is found that most cells volume increases
Greatly, oval or irregular shape, nucleus are mostly ellipse or circle, and nuclear staining is loose, and nuclear membrane is irregular, there is projection,
Visible 1 little nucleolar of each cell, in navy blue；Kytoplasm enriches, and dyes dusty blue, and form is irregular, has pseudopodium, has at nearly core
Light dye phenomenon, also has in irregular shape；The μ l of cell 100 of culture 10 days are taken, add 100 μ l0.4% Trypan Blue liquid, it is living thin
Born of the same parents do not dye, and dead cell dyes blueness, is counted under microscope；
The cell of the 0th, 3,5,7 day is taken respectively, with the phosphate buffer received containing 0.5% NBCS and 0.01% nitrine
Washing 2 times, adjustment cell density are 1 × 106/ ml, each detection pipe is interior to add 50 μ l cell suspensions, adds fluorescent labeled antibody
(AntiCD3 McAb-FITC, anti-tcr γ δ-PE) is dyed, and 4 DEG C of lucifuges are incubated 30 minutes, are washed 2 times with above-mentioned PBS, are added and are contained more than 1%
After the PBS solution of polyformaldehyde is fixed, detected with flow cytometer, data file uses WinMDI software analysis.
In step 1, the time of the pretreatment in the A is 5 minutes；The pretreatment is specially：By standby tubercle bacillus
Culture is added in sulfuric acid solution or sodium hydroxide solution, and concussion mixes, 4000 revs/min, is centrifuged 30 minutes and is collected thalline；
The concentration of the sulfuric acid solution is 0.5% (volume ratio)；The concentration of the sodium hydroxide solution is 0.2mg/ml；The standby knot
The volume ratio of core alphacterium culture and sulfuric acid solution is 1：1；The body of the standby tubercle bacillus culture and sodium hydroxide solution
Product is than being 1:2.
Preferably, in step 2, the purifying is specially：By Mtb-Hag obtained by step 1 quickly through albumen liquid phase color
The molecular sieve type S-100 chromatographic columns and ion-exchange type Mono Q chromatographic columns of spectrometer；The active component includes the high Mr of Mtb-Ag
The low Mr polypeptide fractions (Mtb-LW-Ag) of protein component (Mtb-HW-Ag), Mtb and the single main peptide (Mtb-Ag- in Mtb-Ag peaks 3
Pk3)。
Embodiment 2
The present invention provides a kind of human gamma delta t cells, and the preparation of the human gamma delta t cells comprises the following steps：
Step 1: preparing tubercle bacillus heat resistance antigen with the tubercle bacillus of culture, comprise the following steps：
A, the tubercle bacillus culture (bacterium number 1 × 10 that 1ml is pretreated is taken3), it is inoculated in 200ml Soviet Union's formula liquid training
Support in base, in static gas wave refrigerator 1 week in 37 DEG C of incubators, after taking out concussion, continue culture 1 week；Then be dispensed into containing
In the 150ml of 15% NBCS Soviet Union's formula fluid nutrient medium, every bottle of 50ml, 4 bottles are distributed into；It is further continued in 37 DEG C of cultures
Static gas wave refrigerator 2 weeks in case；The bacterial suspension of culture is collected, through 4000 revs/min, centrifuges 30 minutes, obtains core bacillus thalline；
B, by the thalline of collection through brine 2 times, then the distilled water resuspension thalline with 2 times of volumes, 120 DEG C of height
Press steam treatment 15 minutes, take supernatant to obtain Mtb-Hag through 0.22 μm of micropore miillpore filter；
Step 2: stimulating PBMCs with purifying gained Mtb-Hag active components, while sulindac and licorice polysaccharide are added, had
Body step includes：
20ml detection in peripheral blood of patients underwent is gathered, mononuclearcell is obtained through ficoll-general shadow aminoglucose density gradient centrifugation,
Concretely comprise the following steps：1500 revs/min, centrifuge 10 minutes, draw upper plasma layer, 56 DEG C inactivation 15 minutes after centrifuge it is standby, it is sterile
The haemocyte of water two-fold dilution precipitation, human lymphocyte separating liquid press 1 with dilute blood:4 mixing, 2000 revs/min, centrifugation 20
Minute, tunica albuginea layer is drawn, sterile water washing 2 times, rotating speed is respectively 1500 revs/min, centrifuges 5 minutes, that is, obtains peripheral blood list
Individual nucleus；
By the PMNC of above-mentioned separation be placed in containing 1ng/ml zoledronic acids, 0.5ng/ml sulindacs,
0.5ng/ml licorice polysaccharides, 0.05mg/ml autologous plasmas, 100IU/ml tubercle bacillus heat resistance purifying antigen active components
In RPMI1640 nutrient solutions, it is 1~2 × 10 to adjust cell density5/ ml, is transferred to Tissue Culture Plate, in 37 DEG C, 5%CO2It is wet with saturation
Cultivated in the incubator of degree；
Step 3: 3 days later half amounts of cell culture change the nutrient solution containing sulindac, licorice polysaccharide, cell density is kept
For 1~1.5 × 106/ml；
Step 4: according to cell growth state, the 7th day harvesting；By cell culture, 24 hours are visible gamma delta T cells
The bottom of culture vessel is sunken to, and tends to be colonization, 40~50 hours colonies start to become big, and culture can be seen big for 7 days
Colony and irregular mononuclearcell, auspicious Ji's Albert'stain Albert is carried out to the cell of culture 7 days, it is found that most cells volume increases
Greatly, oval or irregular shape, nucleus are mostly ellipse or circle, and nuclear staining is loose, and nuclear membrane is irregular, there is projection,
Visible 1 little nucleolar of each cell, in navy blue；Kytoplasm enriches, and dyes dusty blue, and form is irregular, has pseudopodium, has at nearly core
Light dye phenomenon, also has in irregular shape；The μ l of cell 100 of culture 7 days are taken, add 100 μ l0.4% Trypan Blue liquid, it is living thin
Born of the same parents do not dye, and dead cell dyes blueness, is counted under microscope；
The cell of the 0th, 3,5,7 day is taken respectively, with the phosphate buffer received containing 0.5% NBCS and 0.01% nitrine
Washing 2 times, adjustment cell density are 1 × 106/ ml, each detection pipe is interior to add 50 μ l cell suspensions, adds fluorescent labeled antibody
(AntiCD3 McAb-FITC, anti-tcr γ δ-PE) is dyed, and 4 DEG C of lucifuges are incubated 30 minutes, are washed 2 times with above-mentioned PBS, are added and are contained more than 1%
After the PBS solution of polyformaldehyde is fixed, detected with flow cytometer, data file uses WinMDI software analysis.
In step 1, the time of the pretreatment in the A is 15 minutes；The pretreatment is specially：By standby tuberculosis bar
Bacterium culture is added in sulfuric acid solution or sodium hydroxide solution, and concussion mixes, 4000 revs/min, is centrifuged 30 minutes and is collected bacterium
Body；The concentration of the sulfuric acid solution is 0.5% (volume ratio)；The concentration of the sodium hydroxide solution is 0.2mg/ml；It is described standby
It is 1 with the volume ratio of tubercle bacillus culture and sulfuric acid solution：3；The standby tubercle bacillus culture and sodium hydroxide solution
Volume ratio be 1:6.
Preferably, in step 2, the purifying is specially：By Mtb-Hag obtained by step 1 quickly through albumen liquid phase color
The molecular sieve type S-100 chromatographic columns and ion-exchange type Mono Q chromatographic columns of spectrometer；The active component includes the high Mr of Mtb-Ag
The low Mr polypeptide fractions (Mtb-LW-Ag) of protein component (Mtb-HW-Ag), Mtb and the single main peptide (Mtb-Ag- in Mtb-Ag peaks 3
Pk3)。
Killing activity detects
The killing activity of the cell prepared to embodiment 1, embodiment 2 is tested, and concrete operations and result are as follows：
Take the logarithm growth period K562 cell lines as target cell, and adjust cell density as 2 × 105/ml、1×105/
ml、5×104/ ml, take 100 μ l to be laid in 96 well culture plates per hole, cultivate 24 hours, the cell for taking the present invention to prepare adjusts density
For 1 × 106/ ml, add in 96 well culture plates, make effect target ratio respectively 10:1、20:1 and 40:1, each density sets 3 multiple holes simultaneously
Blank control is set respectively, and culture adds the MTT20 μ l that concentration is 5mg/ml per hole after 48 hours, abandoned after continuing culture 4 hours
Supernatant, DMSO100 μ l are added per hole, absorbance (A) value is surveyed at A570nm and A630nm, calculates absolute A values (A=A570-
A630), killing activity is calculated as follows：Killing activity (%)=(A target cells+A effector cells-A effect targets cell mixing)/A targets
Cell × 100%；
As a result show：Gamma delta T cells prepared by embodiment 1 have higher killing activity to K562 cell lines, and killing rate is put down
It is 95% (n=8)；Gamma delta T cells prepared by embodiment 2 have higher killing activity to K562 cell lines, and killing rate is averaged
For 98% (n=8).
Purity detecting
The purity of the gamma delta T cells prepared to embodiment 1, embodiment 2 is tested：Take respectively culture 7 days embodiment 1,
Gamma delta T cells prepared by embodiment 2, the gamma delta T cells prepared by gamma delta T cells monoclonal antibody and flow cytometry analysis are marked with PE
Purity；
As a result show：The gamma delta T cells purity of embodiment 1 is 92.8%；The gamma delta T cells purity of embodiment 1 is 93.5%.
Cell proliferation rate detects
Cell proliferation rate detects during preparing human gamma delta t cells to embodiment 1, and specific method is as follows：Exist respectively
Sample within the 1st, 3,5,7 day during gamma delta T cells, ELIASA measure wavelength is 450nm absorbance (A), and uses below equation
Calculate appreciation rate：
Appreciation rate=[(A3rd day-A1st day)/A1st day+(A5th day-A3rd day)/A3rd day+(A7th day-A5th day)/A5th day]/3 × 100%；Weight
Test 3 times again；
As a result show：During the inventive method prepares human gamma delta t cells, cell proliferation rate is close to 20%.
The specific embodiment of the present invention is described above.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring the substantive content of the present invention.
Claims (1)
1. a kind of preparation method of human gamma delta t cells, it is characterised in that methods described comprises the following steps：
Step 1: prepare tubercle bacillus heat resistance antigen with the tubercle bacillus of culture；
Step 2: stimulating PMNC with purifying gained tubercle bacillus heat resistance antigenic components, add simultaneously
RPMI1640 nutrient solution Cytokines in Peripheral Blood Mononuclear containing sulindac and licorice polysaccharide is cultivated；
Step 3: 2~3 days later half amounts of culture change the nutrient solution；
Step 4: according to cell growth state, harvest within the 6th~8 day, produce human gamma delta t cells；
Specifically comprise the following steps in the step 1：
A, by pretreated tubercle bacillus culture, it is inoculated in Soviet Union's formula fluid nutrient medium, in cultivating 1 in 37 DEG C of incubators
~2 weeks；Then it is dispensed into Soviet Union's formula fluid nutrient medium containing NBCS；It is further continued in quiet in 37 DEG C of incubators
Only cultivate 1~2 week；The bacterial suspension of culture is collected, centrifuges to obtain core bacillus thalline；
B, by the core bacillus thalline of collection through brine, then with distilled water resuspension thalline, high steam processs, take
Clearly tubercle bacillus heat resistance antigen is obtained through 0.22 μm of micropore miillpore filter；
In step A, the time of the pretreatment is 5~15 minutes；
In step A, the pretreatment is specially：Standby tubercle bacillus culture is added to sulfuric acid solution or sodium hydroxide is molten
In liquid, concussion mixes, and thalline is collected by centrifugation；
In step B, the condition of the high steam processs is 120 DEG C, 15 minutes；
The step 2 specifically includes：
Peripheric venous blood is gathered, mononuclearcell is obtained through ficoll-general shadow aminoglucose density gradient centrifugation；By mononuclearcell
It is placed in containing zoledronic acid, sulindac, licorice polysaccharide, 0.05mg/ml autologous plasmas, 50~100IU/ml tubercle bacillus heat resistances
In the RPMI1640 nutrient solutions of purifying antigen active component, it is 1~2 × 10 to adjust cell density5/ ml, is transferred to Tissue Culture Plate, in
37 DEG C, 5%CO2Cultivated with the incubator of saturated humidity；
The concentration of the zoledronic acid is 0.01~1ng/ml；
In step 2, the purifying specifically includes：By core bacillus heat resistance antigen obtained by step 1 quickly through albumen liquid phase color
The molecular sieve type S-100 chromatographic columns and ion-exchange type Mono Q chromatographic columns of spectrometer；
In step 2, it is single that the active component includes the high Mr protein components of Mtb-Ag, the low Mr polypeptide fractions of Mtb or Mtb-Ag peaks 3
One or more in one main peptide；
The sulindac, the concentration of licorice polysaccharide are 0.1~0.5ng/ml.
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