CN103571791B - Morphology and purity and immunophenotyping detection method of human (gamma)(delta)T cells - Google Patents
Morphology and purity and immunophenotyping detection method of human (gamma)(delta)T cells Download PDFInfo
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Abstract
The invention belongs to the technical field of cellular immunology, and particularly relates to a morphology and purity and immunophenotyping detection method of human (gamma)(delta)T cells, which is used for detecting the prepared human (gamma)(delta)T cells. The method comprises the following steps: preparing human (gamma)(delta)T cells; and performing morphological observation with a microscope and purity and immunophenotyping analysis with a flow cytometer. The method provided by the invention has the advantages that the (gamma)(delta)T cells obtained by the test have the characteristics of large quantity, high purity, strong cytotoxicity and the like so as to ensure that the prepared human (gamma)(delta)T cells can meet the clinical needs; and moreover, the effects that the culture cost is greatly reduced from the non-peptide phosphate antigens such as IPP and the irritants such as anti-TCR (gamma)(delta) antibody are realized and the problems of small quantity, low toxicity, high cost and the like of in-vitro culture of (gamma)(delta)T cells in the prior art are solved.
Description
The divisional application that the application is on February 10th, 2012, application number is the patent of invention " a kind of method simply efficiently preparing human gamma delta t cells " of 201210030197.4.
Technical field
The invention belongs to technical field of cellular immunology, specifically a kind of morphology of external a large amount of advantage pcr human peripheral gamma delta T cells, purity and immunophenotype detection method.
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.Through extensively consulting domestic and international patent documentation and various publication, up to now, practicality, the invention of efficient, rapid in-vitro a large amount of advantage pcr human peripheral gamma delta T cells or report is showed no.Therefore a kind of method of practicality, efficient, rapid in-vitro a large amount of advantage pcr human peripheral gamma delta T cells is invented, 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.After the preparation of human peripheral gamma delta T cells, need to carry out morphology, purity and immunophenotype to it detect, guarantee that finished product meets the requirements.
Summary of the invention
The object of the invention is the morphology in order to provide a kind of human gamma delta t cells, purity and immunophenotype detection method.For detecting human gamma delta t cells.
The technical solution adopted in the present invention is: a kind of method preparing human gamma delta t cells, comprise the steps: to cultivate tubercule bacillus (Mycobacterium tuberculosis, and prepare tubercule bacillus thermotolerance antigen (Mycobacterium tuberculosisheated antigen Mtb), Mtb-HAg), gather and separating peripheral blood mononuclear cells (Peripheral blood mononuclear cells, PBMCs), activate with Mtb-HAg, add cytokine rhIL-2 and rhIL-21 simultaneously, be placed in 37 DEG C, cultivate in the incubator of 5%CO2 and saturated humidity, carry out half amount after 72 hours and change nutrient solution and the rhIL-2 adding equivalent, within every 2 ~ 3 days, carry out adding the nutrient solution containing equivalent rhIL-2 according to cell growth state later, be (0.5 ~ 2.0) × 10 to maintain cell density
6/ ml.Within 10 ~ 15 days, namely can obtain the gamma delta T cells of a large amount of, high cytotoxic activity.Described Mtb substratum for Soviet Union's general formula liquid nutrient medium or Middlebrook7H9, Mtb are through autoclaved, and is prepared into Mtb-HAg through centrifugal ultrafiltration.Described PBMCs adopts ficoll-general shadow glycosamine density gradient centrifugation separated and collected, and with brine 2 times, obtains after low-speed centrifugal.The final concentration of the final concentration of described Mtb-HAg to be the final concentration of 5 ~ 10 μ g/ml, rhIL-2 be 50 ~ 500IU/ml, rhIL-21 is 5 ~ 20ng/ml.According to the growth conditions of PBMCs, the cell cultures time is about 10 ~ 15 days.
The raw material that the present invention is used and reagent apart from outside specified otherwise, all commercially.
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, 48 hours colonies start to become large, cultivate and namely can see large colony and irregular mononuclearcell in 6 ~ 10 days.Carry out auspicious Ji's Albert'stain Albert to the cultivation cell of 10 days, find that most cells volume increases, ovalize or irregular shape, nucleus is mostly oval or circular, and nuclear staining is loosened, and nuclear membrane is irregular, has projection, and 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, 7,14,21 and 28 day respectively, wash 2 times with the PBS received containing 5% new-born calf serum and 0.1% 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.
The invention has the beneficial effects as follows: the gamma delta T cells that inspection obtains has the features such as quantity is large, purity is high, cytotoxicity is strong, thus guarantee that the human gamma delta t cells made can meet clinical needs, and achieve the stimulants such as non-peptide phosphoric acid class antigen, anti-tcr γ anti-δ such as IPP and compare toxigenic capacity and greatly reduce; Solve that prior art vitro culture gamma delta T cells quantity is low, toxicity is weak, the effect of high in cost of production problem.
Embodiment
Illustrate the present invention with example below, but the present invention is not limited.The experimental technique of all unreceipted actual conditionses in example below, the operation instructions that being the method for observing a usual practice and producer provides performs.
First the present invention prepares tubercule bacillus thermotolerance antigen with the tubercule bacillus cultivated.Comprise the following steps:
1. tubercule bacillus thalline seed weight in wet base 50 ~ 100 grams is inoculated in Soviet Union's general formula liquid nutrient medium of 200ml, static gas wave refrigerator 2 weeks in 37 DEG C of incubators, then be dispensed in Soviet Union's general formula liquid nutrient medium of the 250ml containing 10% ~ 30% new-born calf serum, every bottle of 50ml, be distributed into 4 bottles, then continue in 37 DEG C of incubators static gas wave refrigerator 4 weeks, collect the bacterial suspension cultivated, through 4000 revs/min, centrifugal 30 minutes to gather in the crops tubercule bacillus thalline.
2. by the thalline collected through brine 2 times, then use the distilled water resuspension thalline of 2 times of volumes, 115 DEG C of autoclaved 20 minutes, collect supernatant liquor, are Mtb-HAg for centrifugal 30 minutes by 4000 revs/min.
PBMCs is stimulated, to obtain a large amount of, high purity, high cytotoxic activity gamma delta T cells with the Mtb-HAg of preparation.Concrete operations comprise the following steps:
1. gather detection in peripheral blood of patients underwent with blood cell separator or 50ml syringe under aseptic condition, obtain mononuclearcell 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 30 minutes, and with the hemocyte of physiological saline two-fold dilution precipitation, human lymphocyte parting liquid and dilute blood add in centrifuge tube in the ratio of 1:2,2000 revs/min, centrifugal 20 minutes, careful draw tunica albuginea layer, with brine 2 times, rotating speed is respectively 1600 revs/min, 1300 revs/min, all centrifugal 7 minutes, namely obtain peripheral blood mononuclear cell.
2. the PBMCs of above-mentioned separation is placed in RPMI1640, AIM-V or GT-T551 substratum of Mtb-HAg, 50 ~ 500IU/ml rhIL-2,5 ~ 20ng/ml rhIL-21 and 1% ~ 10% autologous plasma containing 1 ~ 10 μ g/ml, adjusts cell density to be (1 ~ 2) × 10
6/ ml, the concentration of preferred stimulant and cytokine is respectively: 5 μ g/ml Mtb-HAg, 200IU/ml rhIL-2,10ng/ml rhIL-21, and the concentration of autologous plasma is 5%, the initial density 2 × 10 of cell
6/ ml, proceed in Tissue Culture Plate, culturing bottle or culture bag, preferred cell culture bag, in 37 DEG C, cultivate in the incubator of 5%CO2 and saturated humidity, carry out half amount after 72 hours and change nutrient solution and the rhIL-2 adding equivalent, within every 2 ~ 3 days, carry out adding the nutrient solution containing equivalent rhIL-2 according to cell growth state, to maintain cell density for (0.5 ~ 2.0) × 10 later
6/ ml.According to the growth conditions of PBMCs, the cell cultures time is about 10 ~ 15 days.At the 14th day of cell cultures, cultivate the absolute amplifying cells multiple of human gamma delta t cells of propagation on average up to 500 times (n=8).
Morphology, purity and immunophenotype and cytotoxicity assay are carried out to the gamma delta T cells of above-mentioned cultivation.Concrete operations comprise the following steps:
1. cell cultures 24 hours i.e. visible gamma delta T cells is sunken to the bottom of culture vessel, and is tending towards colonization, and 48 hours colonies start to become large, cultivate and namely can see large colony and irregular mononuclearcell in 6 ~ 10 days.Carry out auspicious Ji's Albert'stain Albert to the cultivation cell of 10 days, find that most cells volume increases, ovalize or irregular shape, nucleus is mostly oval or circular, and nuclear staining is loosened, and nuclear membrane is irregular, has projection, and 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.The cell viability can being observed preparation by the present invention is greater than 95%.
2. get the cell of the 0th, 7,14,21 and 28 day respectively, wash 2 times with the PBS received containing 5% new-born calf serum and 0.1% 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.The cell preparation of preparation can be detected by the present invention, CD3+T high enrichment purity reaches 98%, and gamma delta T cells reached peak averaging from 10th ~ 14 days be 80%(n=8).
3. the K562 cell strain of taking the logarithm vegetative period is as target cell, and to adjust cell density be 1 × 10
5/ ml, 5 × 10
4/ ml, 2.5 × 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 the present invention and has higher killing activity to K562 cell strain, kill rate average out to 82%(n=8).
Claims (1)
1. the morphology of human gamma delta t cells, purity and an immunophenotype detection method, is characterized in that:
First prepare a kind of human gamma delta t cells, comprising: (1) prepares tubercule bacillus thermotolerance antigen with the tubercule bacillus cultivated, and its concrete steps are:
Step 1: tubercule bacillus thalline seed weight in wet base 50 ~ 100 grams is inoculated in Soviet Union's general formula liquid nutrient medium of 200ml, static gas wave refrigerator 2 weeks in 37 DEG C of incubators, then be dispensed in Soviet Union's general formula liquid nutrient medium of the 250ml containing 10% ~ 30% new-born calf serum, every bottle of 50ml, be distributed into 4 bottles, then continue in 37 DEG C of incubators static gas wave refrigerator 4 weeks, collect the bacterial suspension cultivated, through 4000 revs/min, centrifugal 30 minutes to gather in the crops tubercule bacillus thalline;
Step 2: by the thalline collected through brine 2 times, then use the distilled water resuspension thalline of 2 times of volumes, 115 DEG C of autoclaved 20 minutes, collect supernatant liquor, are Mtb-Hag for centrifugal 30 minutes by 4000 revs/min;
(2) stimulate PBMCs with the Mtb-HAg of preparation, add cytokine rhIL-2 and rhIL-21, its concrete steps are simultaneously:
Detection in peripheral blood of patients underwent is gathered with blood cell separator or 50ml syringe under aseptic condition, 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 30 minutes, with the hemocyte of physiological saline two-fold dilution precipitation, human lymphocyte parting liquid and dilute blood add in centrifuge tube in the ratio of 1:2, 2000 revs/min, centrifugal 20 minutes, careful absorption tunica albuginea layer, with brine 2 times, rotating speed is respectively 1600 revs/min, 1300 revs/min, all centrifugal 7 minutes, namely peripheral blood mononuclear cell is obtained, being placed in by the PBMCs of above-mentioned separation containing stimulant and cytokine is in RPMI1640, AIM-V or GT-T551 substratum of Mtb-HAg, 50 ~ 500IU/ml rhIL-2 of 1 ~ 10 μ g/ml, 5 ~ 20ng/ml rhIL-21 and 1% ~ 10% autologous plasma, adjusts cell density to be (1 ~ 2) × 10
6/ ml, proceeds in Tissue Culture Plate, culturing bottle or culture bag, in 37 DEG C, 5%CO
2cultivate with in the incubator of saturated humidity,
(3) cell cultures is carried out half amount replacing nutrient solution and is added the rhIL-2 of equivalent after 72 hours, within every 2 ~ 3 days, carry out adding the nutrient solution containing equivalent rhIL-2 according to cell growth state, to maintain cell density for (0.5 ~ 2.0) × 10 later
6/ ml;
(4) according to cell growth state, 10th ~ 15 days harvested cells;
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, 48 hours colonies start to become large, cultivate and namely can see large colony and irregular mononuclearcell in 6 ~ 10 days, auspicious Ji's Albert'stain Albert is carried out to the cultivation cell of 10 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 l 0.4% Trypan Blue liquid, viable cell does not dye, and dead cell dyes blueness, counted under microscope;
Get the cell of the 0th, 7,14,21 and 28 day respectively, wash 2 times with the PBS received containing 5% new-born calf serum and 0.1% nitrine, adjustment cell density is 1 × 10
6/ ml, 50 μ l cell suspensions are added in each detector tube, add the fluorescent-labeled antibody dyeing of AntiCD3 McAb-FITC and anti-tcr γ δ-PE, 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.
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| Aurelie Thedrez,et al.IL-21-mediated potentiation of antitumor cytolytic and proinflammatory responses of human Vγ9Vδ2 T cells for adoptive immunotherapy.《The Journal of Immunology》.2009,第182卷第3423-3431页. * |
| 陈勇 等.刺激γδ+T细胞增殖的结核杆菌多肽抗原的纯化与活性鉴定.《细胞与分子免疫学杂志》.2003,第19卷(第1期),第86-89页. * |
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