CN104293734A - Preparation method of human gamma delta T cell - Google Patents
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Abstract
The invention provides a preparation method of a human gamma delta T cell and belongs to the field of cell immunology. The preparation method of the human gamma delta T cell comprises the following steps: firstly, preparing a mycobacterium tuberculosis heat stable antigen from cultured mycobacterium tuberculosis; secondly, stimulating a peripheral blood mononuclear cell by virtue of the active components of the mycobacterium tuberculosis heat stable antigen obtained by purification, and simultaneously adding sulindac and licorice polysaccharide; thirdly, after culturing the cell for three days, replacing the culture solution containing sulindac and licorice polysaccharide in a half amount and maintaining the density of the cell; and fourthly, harvesting the cell seven days later according to the growth state of the cell. With respect to the defects of the prior art, the invention provides the gamma delta T cell with the advantages of short preparation period, high purity, good killing and damage effects, rapid cell reproduction and low preparation cost and can ensure that the product meets requirements.
Description
Technical field
The invention belongs to cellular immunology field, be specifically related to a kind of preparation method of human gamma delta t cells.
Background technology
T lymphocyte is divided into two classes according to the difference expressing T cell antigen acceptor (T cell receptor, TCR): TCR α β T cell and TCR gamma delta T cells, respectively referred to as α β T cell and gamma delta T cells.Wherein gamma delta T cells is the cell of a kind of specific type between specific immunity and non-specific immunity, and great majority are that CD4 and CD8 molecule is double-negative, and minority can express CD8 molecule.Gamma delta T cells is mainly distributed in skin and mucous membrane tissue, is generally no more than 5% of T cell sum.Gamma delta T cells has specific recognition antigen and restricted without MHC, has the functions such as anti-infective, antitumor and immunomodulatory, is the first line of defence that immunity of organism monitors.But because gamma delta T cells content in peripheral blood and tumor tissues is not high, the gamma delta T cells that obtain a large amount of high cytotoxic activity is very difficult.Although prior art obtains a large amount of gamma delta T cells by anti-tcr γ anti-δ or non-peptide phosphoric acid class antigen, this can increase its cost prepared undoubtedly.Therefore a kind of method of practicality, efficient, rapid in-vitro a large amount of advantage pcr human peripheral gamma delta T cells is developed, for the research of gamma delta T cells immunologic function, and to improve patient immunizing power, opposing tumour and anti-infection ability with it be very important, be also very valuable for capturing this difficult problem of malignant neoplastic disease that the mankind thirst for solving.
Find through retrieval, though have some technical schemes to disclose the cultural method of gamma delta T cells now, but preparation cycle is longer, and purity is not high enough, and lethality is strong not.
For prior art defect, the invention provides the gamma delta T cells that a kind of preparation cycle is short, purity is high, fragmentation effect is good, cell proliferation is fast, preparation cost is low, guarantee that finished product meets the requirements.
Summary of the invention
For defect of the prior art, the object of this invention is to provide a kind of preparation method of human gamma delta t cells.
The present invention is achieved by the following technical solutions:
The invention provides a kind of preparation method of human gamma delta t cells, described method comprises the steps:
Step one, with cultivate tubercule bacillus prepare tubercule bacillus thermotolerance antigen;
Step 2, use purifying gained tubercule bacillus thermotolerance antigenic components stimulate peripheral blood mononuclear cell, and the RPMI1640 nutrient solution Cytokines in Peripheral Blood Mononuclear simultaneously added containing sulindac and licorice polysaccharide is cultivated;
Step 3, cultivation 2 ~ 3 days later half amounts change described nutrient solution;
Step 4, according to cell growth state, the 7th day harvested cell.
Preferably, in step one, the tubercule bacillus of described cultivation prepares tubercule bacillus thermotolerance antigen, specifically comprises the steps:
A, by pretreated tubercule bacillus culture, be inoculated in Soviet Union's general formula liquid nutrient medium, cultivate 1 ~ 2 week in 37 DEG C of incubators; Then be dispensed in the Soviet Union's general formula liquid nutrient medium containing new-born calf serum; To continue in 37 DEG C of incubators static gas wave refrigerator again 1 ~ 2 week; Collect the bacterial suspension cultivated, centrifugal core bacillus thalline;
B, by the core bacillus thalline collected through brine, then use distilled water resuspension thalline, autoclaved, get supernatant and obtain tubercule bacillus thermotolerance antigen through 0.22 μm of micropore millipore filtration.
Preferably, in steps A, the described pretreated time is 5 ~ 15 minutes.
Preferably, described pre-treatment is specially: join in sulphuric acid soln or sodium hydroxide solution by tubercule bacillus culture for subsequent use, concussion mixing, collected by centrifugation thalline.
More preferably, the volumetric concentration of described sulphuric acid soln is 0.5%; The concentration of described sodium hydroxide solution is 0.2mg/ml.
More preferably, the volume ratio of described tubercule bacillus culture for subsequent use and sulphuric acid soln is 1:(1 ~ 3); The volume ratio of described tubercule bacillus culture for subsequent use and sodium hydroxide solution is 1:(2 ~ 6).
Preferably, in step B, the condition of described autoclaved is 120 DEG C, 15 minutes.
Preferably, in step 2, described purifying concrete operations are: by step one gained core bacillus thermotolerance antigen quickly through the molecular sieve type S-100 chromatography column of protein liquid chromatography instrument and ion-exchange type Mono Q chromatography column.
Preferably, in step 2, described active ingredient comprises the single main peptide (Mtb-Ag-Pk3) in Mtb-Ag height Mr protein ingredient (Mtb-HW-Ag), Mtb low Mr polypeptide fraction (Mtb-LW-Ag) or Mtb-Ag peak 3.
Preferably, in step 2, described purifying gained tubercule bacillus thermotolerance antigenic components stimulates peripheral blood mononuclear cell, adds sulindac and licorice polysaccharide simultaneously, specifically comprise: gather peripheric venous blood, obtain mononuclearcell through ficoll-general shadow glycosamine density gradient centrifugation; Mononuclearcell is placed in the RPMI1640 nutrient solution containing Zoledronic acid, sulindac, licorice polysaccharide, 0.05mg/ml autologous plasma, 50 ~ 100IU/ml tubercule bacillus thermotolerance purifying antigen active ingredient, adjusts cell density to be 1 ~ 2 × 10
5/ ml, proceeds to Tissue Culture Plate, in 37 DEG C, 5%CO
2cultivate with in the incubator of saturated humidity.
Preferably, the concentration of described Zoledronic acid is 0.01 ~ 1ng/ml.
Preferably, in step 2 and step 3, the concentration of described sulindac, licorice polysaccharide is 0.1 ~ 0.5ng/ml.
Preferably, in step 4, described cell growth state is embodied in: within 20 ~ 28 hours, visible gamma delta T cells is sunken to the bottom of culture vessel, and is tending towards colonization; 30 ~ 40 hours colonies start to become large; Cultivate and namely can see large colony and irregular mononuclearcell in 5 ~ 7 days, auspicious Ji's Albert'stain Albert is carried out to the cultivation cell of 7 days, find that most cells volume increases, ovalize or irregular shape, nucleus is mostly oval or circular, and nuclear staining is loosened, nuclear membrane is irregular, have projection, visible 1 the little kernel of each cell, in mazarine; Kytoplasm enriches, and dye dusty blue, form is irregular, has pseudopodium, and there is light dye phenomenon at nearly core place, also has in irregular shape; Trypan Blue liquid, viable cell does not dye, and dead cell dyes blueness, 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, greatly reduces toxigenic capacity;
(2) preparation method of the present invention is by the pre-treatment to tubercule bacillus culture, and the speed of acquisition Mtb-Hag is fast, purity is high, and binding ability is strong;
(3) the present invention is by carrying out cell cultures with the nutrient solution containing Zoledronic acid, sulindac, licorice polysaccharide, and make cell proliferation rate fast, quality is good, and purity is high, active strong.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.
embodiment 1
The present embodiment relates to a kind of human gamma delta t cells, and the preparation of described human gamma delta t cells comprises the steps:
Step one, with cultivate tubercule bacillus prepare tubercule bacillus thermotolerance antigen, comprise the steps:
A, get the pretreated tubercule bacillus culture of 1ml (about bacterium number 1 × 10
3), be inoculated in Soviet Union's general formula liquid nutrient medium of 200ml, static gas wave refrigerator 1 week in 37 DEG C of incubators, after taking out concussion, continue cultivation 1 week; Then be dispensed in Soviet Union's general formula liquid nutrient medium of the 150ml containing 15% new-born calf serum, every bottle of 50ml, is distributed into 4 bottles; To continue in 37 DEG C of incubators static gas wave refrigerator again 2 weeks; Collect the bacterial suspension cultivated, through 4000 revs/min, centrifugal 30 minutes, obtain core bacillus thalline;
B, by the thalline collected through brine 2 times, then use the distilled water resuspension thalline of 2 times of volumes, 120 DEG C of autoclaved 15 minutes, get supernatant and obtain Mtb-Hag through 0.22 μm of micropore millipore filtration;
Step 2, use purifying gained Mtb-Hag active ingredient stimulate PBMCs, and add sulindac and licorice polysaccharide, concrete steps comprise simultaneously:
Gather 20ml detection in peripheral blood of patients underwent, mononuclearcell is obtained through ficoll-general shadow glycosamine density gradient centrifugation, concrete steps are: 1500 revs/min, centrifugal 10 minutes, draw upper plasma layer, 56 DEG C of deactivations are centrifugal for subsequent use after 15 minutes, the hemocyte of sterilized water two-fold dilution precipitation, human lymphocyte parting liquid and dilute blood mixing by 1:4,2000 revs/min, centrifugal 20 minutes, draw tunica albuginea layer, sterilized water washs 2 times, and rotating speed is respectively 1500 revs/min, all centrifugal 5 minutes, namely obtain peripheral blood mononuclear cell;
The peripheral blood mononuclear cell of above-mentioned separation is placed in the RPMI1640 nutrient solution containing 0.01ng/ml Zoledronic acid, 0.1ng/ml sulindac, 0.1ng/ml licorice polysaccharide, 0.05mg/ml autologous plasma, 50IU/ml tubercule bacillus thermotolerance purifying antigen active ingredient, adjusts cell density to be 1 ~ 2 × 10
5/ ml, proceeds to Tissue Culture Plate, in 37 DEG C, 5%CO
2cultivate with in the incubator of saturated humidity;
Step 3, cell cultures 3 days later half amounts change the nutrient solution containing sulindac, licorice polysaccharide, keep cell density to be 1 ~ 1.5 × 10
6/ ml;
Step 4, according to cell growth state, the 7th day harvested cell; By the bottom that cell cultures is visible gamma delta T cells and is sunken to culture vessel for 24 hours, and be tending towards colonization, 30 ~ 40 hours colonies start to become large, cultivate and namely can see large colony and irregular mononuclearcell in 5 ~ 7 days, auspicious Ji's Albert'stain Albert is carried out to the cultivation cell of 7 days, finds that most cells volume increases, ovalize or irregular shape, nucleus is mostly oval or circular, nuclear staining is loosened, and nuclear membrane is irregular, has projection, visible 1 the little kernel of each cell, in mazarine; Kytoplasm enriches, and dye dusty blue, form is irregular, has pseudopodium, and there is light dye phenomenon at nearly core place, also has in irregular shape; Get the cultivation cell of 10 days 100 μ l, add 100 μ l0.4% Trypan Blue liquid, viable cell does not dye, and dead cell dyes blueness, counted under microscope;
Get the cell of the 0th, 3,5,7 day respectively, wash 2 times with the phosphoric acid buffer received containing 0.5% new-born calf serum and 0.01% nitrine, adjustment cell density is 1 × 10
6/ ml, 50 μ l cell suspensions are added in each detector tube, add fluorescent-labeled antibody (AntiCD3 McAb-FITC, anti-tcr γ δ-PE) dyeing, 4 DEG C of lucifuges hatch 30 minutes, 2 times are washed with above-mentioned PBS, after the PBS solution added containing 1% paraformaldehyde is fixed, detect with flow cytometer, data file adopts WinMDI software analysis.
In step one, the pretreated time in described A is 5 minutes; Described pre-treatment is specially: join in sulphuric acid soln or sodium hydroxide solution by tubercule bacillus culture for subsequent use, concussion mixing, 4000 revs/min, within centrifugal 30 minutes, collects thalline; The concentration of described sulphuric acid soln is 0.5% (volume ratio); The concentration of described sodium hydroxide solution is 0.2mg/ml; The volume ratio of described tubercule bacillus culture for subsequent use and sulphuric acid soln is 1:1; The volume ratio of described tubercule bacillus culture for subsequent use and sodium hydroxide solution is 1:2.
Preferably, in step 2, described purifying is specially: by step one gained Mtb-Hag quickly through the molecular sieve type S-100 chromatography column of protein liquid chromatography instrument and ion-exchange type Mono Q chromatography column; Described active ingredient comprises the single main peptide (Mtb-Ag-Pk3) in Mtb-Ag height Mr protein ingredient (Mtb-HW-Ag), Mtb low Mr polypeptide fraction (Mtb-LW-Ag) and Mtb-Ag peak 3.
embodiment 2
The invention provides a kind of human gamma delta t cells, the preparation of described human gamma delta t cells comprises the steps:
Step one, with cultivate tubercule bacillus prepare tubercule bacillus thermotolerance antigen, comprise the steps:
A, get pretreated tubercule bacillus culture (the bacterium number 1 × 10 of 1ml
3), be inoculated in Soviet Union's general formula liquid nutrient medium of 200ml, static gas wave refrigerator 1 week in 37 DEG C of incubators, after taking out concussion, continue cultivation 1 week; Then be dispensed in Soviet Union's general formula liquid nutrient medium of the 150ml containing 15% new-born calf serum, every bottle of 50ml, is distributed into 4 bottles; To continue in 37 DEG C of incubators static gas wave refrigerator again 2 weeks; Collect the bacterial suspension cultivated, through 4000 revs/min, centrifugal 30 minutes, obtain core bacillus thalline;
B, by the thalline collected through brine 2 times, then use the distilled water resuspension thalline of 2 times of volumes, 120 DEG C of autoclaved 15 minutes, get supernatant and obtain Mtb-Hag through 0.22 μm of micropore millipore filtration;
Step 2, use purifying gained Mtb-Hag active ingredient stimulate PBMCs, and add sulindac and licorice polysaccharide, concrete steps comprise simultaneously:
Gather 20ml detection in peripheral blood of patients underwent, mononuclearcell is obtained through ficoll-general shadow glycosamine density gradient centrifugation, concrete steps are: 1500 revs/min, centrifugal 10 minutes, draw upper plasma layer, 56 DEG C of deactivations are centrifugal for subsequent use after 15 minutes, the hemocyte of sterilized water two-fold dilution precipitation, human lymphocyte parting liquid and dilute blood mixing by 1:4,2000 revs/min, centrifugal 20 minutes, draw tunica albuginea layer, sterilized water washs 2 times, and rotating speed is respectively 1500 revs/min, all centrifugal 5 minutes, namely obtain peripheral blood mononuclear cell;
The peripheral blood mononuclear cell of above-mentioned separation is placed in the RPMI1640 nutrient solution containing 1ng/ml Zoledronic acid, 0.5ng/ml sulindac, 0.5ng/ml licorice polysaccharide, 0.05mg/ml autologous plasma, 100IU/ml tubercule bacillus thermotolerance purifying antigen active ingredient, adjusts cell density to be 1 ~ 2 × 10
5/ ml, proceeds to Tissue Culture Plate, in 37 DEG C, 5%CO
2cultivate with in the incubator of saturated humidity;
Step 3, cell cultures 3 days later half amounts change the nutrient solution containing sulindac, licorice polysaccharide, keep cell density to be 1 ~ 1.5 × 10
6/ ml;
Step 4, according to cell growth state, the 7th day harvested cell; By the bottom that cell cultures is visible gamma delta T cells and is sunken to culture vessel for 24 hours, and be tending towards colonization, 40 ~ 50 hours colonies start to become large, cultivate and namely can see large colony and irregular mononuclearcell in 7 days, auspicious Ji's Albert'stain Albert is carried out to the cultivation cell of 7 days, finds that most cells volume increases, ovalize or irregular shape, nucleus is mostly oval or circular, nuclear staining is loosened, and nuclear membrane is irregular, has projection, visible 1 the little kernel of each cell, in mazarine; Kytoplasm enriches, and dye dusty blue, form is irregular, has pseudopodium, and there is light dye phenomenon at nearly core place, also has in irregular shape; Get the cultivation cell of 7 days 100 μ l, add 100 μ l0.4% Trypan Blue liquid, viable cell does not dye, and dead cell dyes blueness, counted under microscope;
Get the cell of the 0th, 3,5,7 day respectively, wash 2 times with the phosphoric acid buffer received containing 0.5% new-born calf serum and 0.01% nitrine, adjustment cell density is 1 × 10
6/ ml, 50 μ l cell suspensions are added in each detector tube, add fluorescent-labeled antibody (AntiCD3 McAb-FITC, anti-tcr γ δ-PE) dyeing, 4 DEG C of lucifuges hatch 30 minutes, 2 times are washed with above-mentioned PBS, after the PBS solution added containing 1% paraformaldehyde is fixed, detect with flow cytometer, data file adopts WinMDI software analysis.
In step one, the pretreated time in described A is 15 minutes; Described pre-treatment is specially: join in sulphuric acid soln or sodium hydroxide solution by tubercule bacillus culture for subsequent use, concussion mixing, 4000 revs/min, within centrifugal 30 minutes, collects thalline; The concentration of described sulphuric acid soln is 0.5% (volume ratio); The concentration of described sodium hydroxide solution is 0.2mg/ml; The volume ratio of described tubercule bacillus culture for subsequent use and sulphuric acid soln is 1:3; The volume ratio of described tubercule bacillus culture for subsequent use and sodium hydroxide solution is 1:6.
Preferably, in step 2, described purifying is specially: by step one gained Mtb-Hag quickly through the molecular sieve type S-100 chromatography column of protein liquid chromatography instrument and ion-exchange type Mono Q chromatography column; Described active ingredient comprises the single main peptide (Mtb-Ag-Pk3) in Mtb-Ag height Mr protein ingredient (Mtb-HW-Ag), Mtb low Mr polypeptide fraction (Mtb-LW-Ag) and Mtb-Ag peak 3.
killing activity detects
The killing activity of cell prepared by embodiment 1, embodiment 2 is tested, concrete operations and result as follows:
The K562 cell strain of taking the logarithm vegetative period is as target cell, and to adjust cell density be 2 × 10
5/ ml, 1 × 10
5/ ml, 5 × 10
4/ ml, every hole is got 100 μ l and is laid in 96 well culture plates, cultivates 24 hours, gets cell prepared by the present invention and adjusts density to be 1 × 10
6/ ml, add in 96 well culture plates, effect target ratio is made to be respectively 10:1,20:1 and 40:1, each density is established 3 multiple holes and is arranged blank respectively, cultivate every hole after 48 hours and add the MTT20 μ l that concentration is 5mg/ml, continue cultivation and abandon supernatant after 4 hours, every hole adds DMSO100 μ l, absorbancy (A) value is surveyed in A570nm and A630nm place, calculate absolute A value (A=A570-A630), be calculated as follows killing activity: killing activity (%)=(A target cell+A effector cell-A imitates target cell mixing)/A target cell × 100%;
Result shows: gamma delta T cells prepared by embodiment 1 has higher killing activity to K562 cell strain, kill rate average out to 95% (n=8); Gamma delta T cells prepared by embodiment 2 has higher killing activity to K562 cell strain, kill rate average out to 98% (n=8).
purity detecting
The purity of gamma delta T cells prepared by embodiment 1, embodiment 2 is tested: the gamma delta T cells getting the cultivation embodiment of 7 days 1, embodiment 2 preparation respectively, marks the purity of the gamma delta T cells prepared by gamma delta T cells monoclonal antibody and flow cytometry analysis with PE;
Result shows: 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
Prepare cell proliferation rate in human gamma delta t cells process to embodiment 1 to detect, concrete grammar is as follows: the sampling in the 1st, 3,5,7 day respectively in gamma delta T cells process, it is the absorbancy (A) of 450nm that microplate reader measures wavelength, and with following formulae discovery appreciation rate:
Appreciation rate=[(A
3rd day-A
1st day)/A
1st day+ (A
5th day-A
3rd day)/A
3rd day+ (A
7th day-A
5th day)/A
5th day]/3 × 100%; Repeat experiment 3 times;
Result shows: the inventive method is prepared in human gamma delta t cells process, and cell proliferation rate is close to 20%.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (10)
1. a preparation method for human gamma delta t cells, is characterized in that, described method comprises the steps:
Step one, with cultivate tubercule bacillus prepare tubercule bacillus thermotolerance antigen;
Step 2, use purifying gained tubercule bacillus thermotolerance antigenic components stimulate peripheral blood mononuclear cell, and the RPMI1640 nutrient solution Cytokines in Peripheral Blood Mononuclear simultaneously added containing sulindac and licorice polysaccharide is cultivated;
Step 3, cultivation 2 ~ 3 days later half amounts change described nutrient solution;
Step 4, according to cell growth state, 6th ~ 8 days results, obtain human gamma delta t cells.
2. the preparation method of human gamma delta t cells according to claim 1, is characterized in that, specifically comprises the steps: in described step one
A, by pretreated tubercule bacillus culture, be inoculated in Soviet Union's general formula liquid nutrient medium, cultivate 1 ~ 2 week in 37 DEG C of incubators; Then be dispensed in the Soviet Union's general formula liquid nutrient medium containing new-born calf serum; To continue in 37 DEG C of incubators static gas wave refrigerator again 1 ~ 2 week; Collect the bacterial suspension cultivated, centrifugal core bacillus thalline;
B, by the core bacillus thalline collected through brine, then use distilled water resuspension thalline, autoclaved, get supernatant and obtain tubercule bacillus thermotolerance antigen through 0.22 μm of micropore millipore filtration.
3. the preparation method of human gamma delta t cells according to claim 2, is characterized in that, in steps A, the described pretreated time is 5 ~ 15 minutes.
4. the preparation method of human gamma delta t cells according to claim 2, is characterized in that, in steps A, described pre-treatment is specially: join in sulphuric acid soln or sodium hydroxide solution by tubercule bacillus culture for subsequent use, concussion mixing, collected by centrifugation thalline.
5. the preparation method of human gamma delta t cells according to claim 2, is characterized in that, in step B, the condition of described autoclaved is 120 DEG C, 15 minutes.
6. the preparation method of human gamma delta t cells according to claim 1, is characterized in that, described step 2 specifically comprises:
Gather peripheric venous blood, obtain mononuclearcell through ficoll-general shadow glycosamine density gradient centrifugation; Mononuclearcell is placed in the RPMI1640 nutrient solution containing Zoledronic acid, sulindac, licorice polysaccharide, 0.05mg/ml autologous plasma, 50 ~ 100IU/ml tubercule bacillus thermotolerance purifying antigen active ingredient, adjusts cell density to be 1 ~ 2 × 10
5/ ml, proceeds to Tissue Culture Plate, in 37 DEG C, 5%CO
2cultivate with in the incubator of saturated humidity.
7. the preparation method of human gamma delta t cells according to claim 6, is characterized in that, the concentration of described Zoledronic acid is 0.01 ~ 1ng/ml.
8. the preparation method of human gamma delta t cells according to claim 1, it is characterized in that, in step 2, described purifying specifically comprises: by step one gained core bacillus thermotolerance antigen quickly through the molecular sieve type S-100 chromatography column of protein liquid chromatography instrument and ion-exchange type Mono Q chromatography column.
9. the preparation method of the human gamma delta t cells according to claim 1 or 6, is characterized in that, in step 2, described active ingredient comprises one or more in Mtb-Ag height Mr protein ingredient, the single main peptide of Mtb low Mr polypeptide fraction or Mtb-Ag peak 3.
10. the preparation method of the human gamma delta t cells according to claim 1 or 6, is characterized in that, the concentration of described sulindac, licorice polysaccharide is 0.1 ~ 0.5ng/ml.
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| CN110184240A (en) * | 2019-06-13 | 2019-08-30 | 上海市肺科医院 | A kind of method that efficient amplification activation 2 T cell of V γ 2V δ enhances its anti-tubercular |
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