CN103981146A - Group of antitumor T lymphocytes and preparation method thereof - Google Patents
Group of antitumor T lymphocytes and preparation method thereof Download PDFInfo
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Abstract
The invention provides a group of antitumor T lymphocytes. The group of antitumor T lymphocytes are used for representing the following five types of lymphocyte membrane molecules to a certain extent: human leukocyte differentiation antigens CD3, human leukocyte differentiation antigens CD4, human leukocyte differentiation antigens CD8, human Alpha Beta T cell receptors and human Gamma Delta T cell receptors, thereby inhibiting the proliferation of breast cancers and hepatoma cell lines, inducing the apoptosis of cancer cells as well as inhibiting the growth of the in-vivo tumors of tumor-bearing mice suffering from the hepatoma and the breast cancers. If the antitumor T lymphocytes provided by the invention are matched with the traditional surgery, chemotherapy and radiation therapy, a small quantity of residues and diffused tumor cells are removed and killed in a biological therapy mode after a large quantity of tumor cells are removed by the conventional therapy method, thereby improving and consolidating the tumor therapy and reducing the tumor relapse.
Description
Technical field
The present invention relates to one group of antitumor T lymphocyte populations and preparation method thereof.
Background technology
The immunotherapy of tumour in recent years more and more comes into one's own.The immunotherapy of tumour mainly comprises tumor vaccine, monoclonal antibody technique, cytokine therapy, adoptive cellular immunotherapy etc. at present.Wherein adoptive cellular immunotherapy is to point to the immunocyte that tumour patient transmission has anti-tumor activity, direct killing tumour or excitating organism antineoplastic immune effect, thus reach the object for the treatment of tumour.The main sexual cell of adopting comprises the killer cell (anti-CD3MeAbactivatedkiller cells) that killer cell (LAK), tumor infiltrating lymphocyte (TIL), cytokine induced kill cell (CIK), the CD3 monoclonal antibody of Lymphokine activate etc.It should be noted that the latent effect of gamma delta T cells in adoptive immunotherapy.Gamma delta T cells is the specifc immunity cell type between acquired immunity and the natural immunity, has antigen-specific recognition function and without MHC restriction, can amplification under IL-2 stimulates, and present in vitro the function of killing and wounding kinds of tumors.The gamma delta T cells of take has demonstrated good result as basic immunotherapy in the I phase clinical studyes such as lung cancer, kidney, malignant melanoma, and especially metastatic malignant melanoma, points out it may become the new way for the treatment of tumour.CD8+T cell (TC or CTL cell) can kill and wound the target cell of antigen expressed, and it is important effector cell at anti-virus infection, acute allotype transplant rejection and killing tumor cell on.In normal body, CD8+T cell exists with the form of the tranquillization T cell that do not activate.It must, through antigen activates and under auxiliary (TH) cell synergy of T, could differentiation and development be effect killer T cell (TC).Clinical case report CD8+T has different effects to dissimilar tumour.At present, there is no the lymphocyte populations that report has the mixing of antitumor action.
Summary of the invention
The invention provides the one group of antitumor T lymphocyte populations that suppresses propagation, the cancer cell specific induction of apoptosis of mammary cancer and hepatoma cell strain and suppress the growth of liver cancer mammary cancer tumor-bearing mice in-vivo tumour, the present invention also provides the preparation method of described one group of antitumor T lymphocyte populations.
The invention provides one group of antitumor T lymphocyte populations, it comprises that the cell of expressing human leukocyte differentiation antigen CD3 accounts for 71.63%～86.32%, the cell of expressing human leukocyte differentiation antigen CD4 accounts for 16.43%～27.37%, the cell of expressing human leukocyte differentiation antigen CD8 accounts for 65.58%～76.86%, the cell of expressing people α β φt cell receptor accounts for 11.90%～17.51%, expresses the cell 69.95%～83.74% of human gamma delta t cells acceptor.
The present invention also provides the preparation method of above-mentioned one group of antitumor T lymphocyte populations, comprises the steps:
Step 1: remove leukocyte depletion filter, after filtering whole blood, white corpuscle remains in filter;
Step 2: use phosphoric acid buffer to rinse filter for removing white blood cell, obtain the mixed solution of PBS, white corpuscle and part blood;
Step 3: by the centrifugal blood plasma that discards of the mixed solution of phosphoric acid buffer, white corpuscle and part blood;
Step 4: add phosphoric acid buffer to abandoning in plasmapheretic cell mixture, carefully mix formation mixed solution;
Step 5: mixed solution is carefully superimposed on cellular segregation liquid centrifugal, is now divided into from top to bottom four layers in centrifuge tube: the first layer is plasma layer; The second layer is ring-type oyster white mononuclearcell layer; The 3rd layer is transparent separated liquid layer; The 4th layer is red corpuscle layer; Collect second layer cell and put into the test tube of phosphoric acid damping fluid, after fully mixing, centrifugal, supernatant discarded is stayed precipitation, and then repeated washing, finally precipitates and obtain separated peripheral blood mononuclear cell;
Step 6: separated peripheral blood mononuclear cell is incubated to 37 ℃, 5%CO
2incubator, with after antitumor T lymphocyte populations special culture media incubated overnight, collects the lymphocyte suspending;
Step 7: go down to posterity once in this antitumor T lymphocyte populations special culture media, make cell concn maintain 5 * 10 for every 2-3 days
5～10 * 10
5individual cell/mL, the condition of cultivating that at every turn goes down to posterity is 37 ℃, 5%CO
2, more than passing for 5 generations, the culture condition in per generation is identical, obtains antitumor T lymphocyte populations;
Wherein, described antitumor T lymphocyte populations special culture media is to be in serum-free lymphocytes culture medium, to add the substratum that isopentenylpyrophosphate and Human Inter Leukin-2 obtain.
Described phosphoric acid buffer is for containing 135mM NaCl, 4.7mM KCl, 10mM Na
2hPO
4, 2mM NaH
2pO
4and the aqueous solution that pH value is 7.4.
In described step 1, the specification of filter for removing white blood cell is 2 400ml of unit, and whole blood is 400ml, and the phosphoric acid buffer in described step 2 is 40-80ml.
Centrifugation rate in described step 3 is 1500-2500 rev/min, and centrifugation time is 15-20 minute.
The phosphoric acid buffer adding in described step 4 is 25-40ml.
Cellular segregation liquid in described step 5 is purchased from Tianjin Hao Yang biological products Science and Technology Ltd., production code member is LTS1077, the volume ratio of enchylema and cellular segregation liquid is 1.5:1-2:1, and after mixing, centrifugal speed is 2000-2500 rev/min, and centrifugation time is 15-20 minute.
In described step 5, collecting second layer cell is the test tube of putting into containing the phosphoric acid buffer of 20-45ml, and mixing centrifugation rate with phosphoric acid buffer is 2000-2500 rev/min, and centrifugation time is 10-15 minute, then with twice of same rotating speed and time repeated washing.
The pH of described antitumor T lymphocyte populations special culture media is 7.2-7.4, consumption is 30 milliliters, wherein, the concentration of isopentenylpyrophosphate in this antitumor T lymphocyte populations special culture media is 200 μ g/L, and the concentration of Human Inter Leukin-2 in this antitumor T lymphocyte populations special culture media is 1,000,000 U/L.
The present invention also provides a kind of antitumor drug, and it take one group of above-mentioned antitumor T lymphocyte populations is activeconstituents.
Antitumor T lymphocyte populations provided by the present invention, expression to a certain degree following five kinds of T lymphocyte membrane molecules: human leukocyte differentiation antigen CD3, human leukocyte differentiation antigen CD4, human leukocyte differentiation antigen CD8, people
α βφt cell receptor, people
γ δφt cell receptor, thus the propagation of mammary cancer and hepatoma cell strain can be suppressed, and cancer cell specific induction of apoptosis, can also suppress the growth of liver cancer mammary cancer tumor-bearing mice in-vivo tumour; The antitumor T lymphocyte populations of the present invention coordinates traditional operation, chemotherapy and radiation treatment, can reach in routine treatment and remove after massive tumor cell, re-use biology therapeutic modality and remove, kill and wound the tumour cell of a small amount of residual or diffusion, can improve and consolidate oncotherapy, and reduce the recurrence of tumour.
Accompanying drawing explanation
Fig. 1 is that the isolated peripheral blood mononuclear cell PBMC of each embodiment (before stimulation) passes through the comparing result of flow cytometer phenotypic evaluation with antitumor T lymphocyte populations (after stimulating).
Fig. 2 is breast cancer cell MCF-7, MDA-MB-231, MDA-MB-453, MDA-MB-468 and antitumor T lymphocyte populations compare 1:1 by three different effect targets, 5:1, after 10:1 effect, the detected result at enzyme-linked immunosorbent assay instrument OD595nm place, wherein X-coordinate is T lymphocyte and the effect target ratio of tumour cell, and ordinate zou is cell survival rate.
Fig. 3 is breast cancer cell MCF-7, MDA-MB-231, MDA-MB-453, MDA-MB-468 and antitumor T lymphocyte populations acted on respectively after 24,48,72,96 hours, detected result at enzyme-linked immunosorbent assay instrument OD595nm place, wherein X-coordinate is the action time of T lymphocyte and tumour cell, and ordinate zou is cell survival rate.
Fig. 4 is that T lymphocyte treatment group, T lymphocyte pretreated group and control group are along with the increase of injection T lymphocyte number of days, the impact on the tumor growth of MDA-MB-231 tumor-bearing mice.Wherein left figure X-coordinate is injection T lymphocyte number of days, and ordinate zou is mouse interior tumor volume size; Right figure X-coordinate is injection T lymphocyte number of days, and ordinate zou is Mouse Weight.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method; Material used, reagent etc., if no special instructions, all can obtain from commercial channels.
1 * PBS is for containing 135mM NaCl below, 4.7mM KCl, 10mM Na
2hPO
4, 2mMNaH
2pO
4and the aqueous solution that pH value is 7.4.
Embodiment 1
The present embodiment provides the method for separated antitumor T lymphocyte populations from filter for removing white blood cell, comprises the steps:
1) from Shenzhen Blood Center, obtain Yi Ge 2 units (400ml) filter for removing white blood cell, blood station is used filter for removing white blood cell to filter after 400 milliliters of whole bloods (normal people's voluntary blood donation), and 99% white corpuscle remains in filter.
2) use 60 milliliters of 1 * PBS to rinse filter for removing white blood cell, obtain the mixed solution of PBS, white corpuscle and part blood.
3) mixed solution of PBS, white corpuscle and part blood is discarded to blood plasma for centrifugal 20 minutes with 1500 revs/min.
4) to abandoning in plasmapheretic cell mixture, add 30 milliliters of 1 * PBS, carefully mix formation mixed solution.
5) mixed solution is carefully superimposed on the liquid level of cellular segregation liquid (purchased from Tianjin Hao Yang biological products Science and Technology Ltd., production code member is LTS1077) to mixed solution: cellular segregation liquid=2:1 (volume ratio).
6) centrifugal 20 minutes with 2000 revs/min.Now in centrifuge tube, be divided into from top to bottom four layers: the first layer is plasma layer; The second layer is ring-type oyster white mononuclearcell layer; The 3rd layer is transparent separated liquid layer; The 4th layer is red corpuscle layer.Collect second layer cell and put into the test tube containing 1 * PBS of 20ml, after fully mixing, with 2000 revs/min centrifugal 10 minutes, supernatant discarded is stayed precipitation, then with 1 * PBS of 20ml with same rotating speed and time repeated washing twice, finally precipitation obtains separated peripheral blood mononuclear cell PBMC.
7) separated PBMC is incubated to 37 ℃, 5%CO
2incubator, nutrient solution is 30 milliliters of antitumor T lymphocyte populations special culture medias.This antitumor T lymphocyte populations special culture media is that (Tianjin Hao Yang biological products Science and Technology Ltd. produces at serum-free lymphocytes culture medium, production code member is CIK2013) middle isopentenylpyrophosphate (the Sigma company that adds, production code member is I0503) and Human Inter Leukin-2 (IL-2) (the accurate word S10970016 of traditional Chinese medicines, Beijing Sihuan Biopharmaceutical Co., Ltd., specification is 500,000 U/ bottles) substratum that obtains, the concentration of isopentenylpyrophosphate in this antitumor T lymphocyte populations special culture media is 200 μ g/L, the concentration of Human Inter Leukin-2 in this antitumor T lymphocyte populations special culture media is 1,000,000 U/L.The pH of this antitumor T lymphocyte populations special culture media is 7.2-7.4.After incubated overnight, attached cell is monocyte, suspension be lymphocyte.The lymphocyte of suspension culture is transferred in new culturing bottle and continues to cultivate.
8) within every 2-3 days, in this antitumor T lymphocyte populations special culture media, go down to posterity once, make cell concn maintain (5～10) * 10
5individual cell/mL.The condition of cultivating that at every turn goes down to posterity is 37 ℃, 5%CO
2, biography 5 generations (culture condition in per generation is identical) obtains antitumor T lymphocyte populations, abandons nutrient solution supernatant after cell centrifugation, with 1 * PBS, is made into cell suspension 5 * 10
6/ ml, gets 200 microlitres and carries out flow cytometer detection.Use respectively anti-human CD3, CD4, CD8, TCR α β, TCR γ δ monoclonal antibody (is bought the company in biolegend, article No. is respectively 317307, 317407, 301005, 306707, 331207) by flow cytometer, detect human leukocyte differentiation antigen CD3, CD4, CD8 and people α β, (detection method is referring to Biggs MJ for the expression of gamma delta T cells acceptor, et al.2011.J.R.Soc.Interface.8:1462.), the cell that flow cytometer result shows to express in this antitumor T lymphocyte populations human leukocyte differentiation antigen CD3 accounts for 86.32% and (accounts for the per-cent of the total cell of antitumor T lymphocyte populations, lower same), the cell of expressing human leukocyte differentiation antigen CD4 accounts for 16.43%, the cell of expressing human leukocyte differentiation antigen CD8 accounts for 76.86%, the cell of expressing people α β φt cell receptor account for 13.68% and the cell of human gamma delta t cells acceptor account for 73.33%.Meanwhile, under similarity condition, detect 6) in peripheral blood mononuclear cell PBMC, its result is as shown in Figure 1.
Embodiment 2
The present embodiment provides the method for separated antitumor T lymphocyte populations from filter for removing white blood cell, comprises the steps:
1) from Shenzhen Blood Center, obtain Yi Ge 2 units (400ml) filter for removing white blood cell, blood station is used filter for removing white blood cell to filter after 400 milliliters of whole bloods (normal people's voluntary blood donation), and 99% white corpuscle remains in filter.
2) use 50 milliliters of 1 * PBS to rinse filter for removing white blood cell, obtain the mixed solution of PBS, white corpuscle and part blood.
3) mixed solution of PBS, white corpuscle and part blood is discarded to blood plasma for centrifugal 15 minutes with 2000 revs/min.
4) to abandoning in plasmapheretic cell mixture, add 30 milliliters of 1 * PBS, carefully mix formation mixed solution.
5) mixed solution is carefully superimposed on the liquid level of cellular segregation liquid (purchased from Tianjin Hao Yang biological products Science and Technology Ltd., production code member is LTS1077) to mixed solution: cellular segregation liquid=1.5:1 (volume ratio).
6) centrifugal 15 minutes with 2500 revs/min.Now in centrifuge tube, be divided into from top to bottom four layers: the first layer is plasma layer; The second layer is ring-type oyster white mononuclearcell layer; The 3rd layer is transparent separated liquid layer; The 4th layer is red corpuscle layer.Collect second layer cell and put into the test tube containing 1 * PBS of 25ml, after fully mixing, centrifugal 15 minutes with 2500 revs/min, supernatant discarded is stayed precipitation, with 1 * PBS of 20ml, with same rotating speed and time repeated washing twice, finally precipitation obtains separated peripheral blood mononuclear cell (PBMC).
7) separated PBMC is incubated to 37 ℃, 5%CO
2incubator, nutrient solution is 30 milliliters of antitumor T lymphocyte populations special culture medias.This antitumor T lymphocyte populations special culture media is that (Tianjin Hao Yang biological products Science and Technology Ltd. produces at serum-free lymphocytes culture medium, production code member is CIK2013) middle isopentenylpyrophosphate (the Sigma company that adds, production code member is I0503) and Human Inter Leukin-2 (IL-2) (the accurate word S10970016 of traditional Chinese medicines, Beijing Sihuan Biopharmaceutical Co., Ltd., specification is 500,000 U/ bottles) substratum that obtains, the concentration of isopentenylpyrophosphate in this antitumor T lymphocyte populations special culture media is 200 μ g/L, the concentration of Human Inter Leukin-2 in this antitumor T lymphocyte populations special culture media is 1,000,000 U/L.The pH of this antitumor T lymphocyte populations special culture media is 7.2-7.4.After incubated overnight, attached cell is monocyte, suspension be lymphocyte.The lymphocyte of suspension culture is transferred in new culturing bottle and continues to cultivate.
8) within every 2-3 days, in this antitumor T lymphocyte populations special culture media, go down to posterity once, make cell concn maintain (5～10) * 10
5individual cell/mL.The condition of cultivating that at every turn goes down to posterity is 37 ℃, 5%CO
2, biography 5 generations (culture condition in per generation is identical) obtains antitumor T lymphocyte populations, abandons nutrient solution supernatant after cell centrifugation, with 1 * PBS, is made into cell suspension 5 * 10
6/ ml, gets 200 microlitres and carries out flow cytometer detection.Use respectively anti-human CD3, CD4, CD8, TCR α β, TCR γ δ monoclonal antibody (is bought the company in biolegend, article No. is respectively 317307, 317407, 301005, 306707, 331207) by flow cytometer, detect human leukocyte differentiation antigen CD3, CD4, CD8 and people α β, (detection method is referring to Biggs MJ for the expression of gamma delta T cells acceptor, et al.2011.J.R.Soc.Interface.8:1462.), shown in result, show that the cell of expressing human leukocyte differentiation antigen CD3 in this antitumor T lymphocyte populations accounts for 71.63%, the cell of expressing human leukocyte differentiation antigen CD4 accounts for 17.78%, the cell of expressing human leukocyte differentiation antigen CD8 accounts for 79.39%, the cell of expressing people α β φt cell receptor account for 11.90% and the cell of human gamma delta t cells acceptor account for 83.74%.Meanwhile, under similarity condition, detect 6) in peripheral blood mononuclear cell PBMC, its result is as shown in Figure 1.
Embodiment 3
The present embodiment provides the method for separated antitumor T lymphocyte populations from filter for removing white blood cell, comprises the steps:
1) from Shenzhen Blood Center, obtain Yi Ge 2 units (400ml) filter for removing white blood cell, blood station is used filter for removing white blood cell to filter after 400 milliliters of whole bloods (normal people's voluntary blood donation), and 99% white corpuscle remains in filter.
2) use 80 milliliters of 1 * PBS to rinse filter for removing white blood cell, obtain the mixed solution of PBS, white corpuscle and part blood.
3) mixed solution of PBS, white corpuscle and part blood is discarded to blood plasma for centrifugal 20 minutes with 2000 revs/min.
4) to abandoning in plasmapheretic cell mixture, add 40 milliliters of 1 * PBS, carefully mix formation mixed solution.
5) mixed solution is carefully superimposed on the liquid level of cellular segregation liquid (purchased from Tianjin Hao Yang biological products Science and Technology Ltd., production code member is LTS1077) to mixed solution: cellular segregation liquid=2:1 (volume ratio).
6) centrifugal 15 minutes with 2000 revs/min.Now in centrifuge tube, be divided into from top to bottom four layers: the first layer is plasma layer; The second layer is ring-type oyster white mononuclearcell layer; The 3rd layer is transparent separated liquid layer; The 4th layer is red corpuscle layer.Collect second layer cell and put into the test tube containing 1 * PBS of 40ml, after fully mixing, centrifugal 15 minutes with 2000 revs/min, supernatant discarded is stayed precipitation, with 1 * PBS of 40ml, with same rotating speed and time repeated washing twice, finally precipitation obtains separated peripheral blood mononuclear cell (PBMC).
7) separated PBMC is incubated to 37 ℃, 5%CO
2incubator, nutrient solution is 30 milliliters of antitumor T lymphocyte populations special culture medias.This antitumor T lymphocyte populations special culture media is that (Tianjin Hao Yang biological products Science and Technology Ltd. produces at serum-free lymphocytes culture medium, production code member is CIK2013) middle isopentenylpyrophosphate (the Sigma company that adds, production code member is I0503) and Human Inter Leukin-2 (IL-2) (the accurate word S10970016 of traditional Chinese medicines, Beijing Sihuan Biopharmaceutical Co., Ltd., specification is 500,000 U/ bottles) substratum that obtains, the concentration of isopentenylpyrophosphate in this antitumor T lymphocyte populations special culture media is 200 μ g/L, the concentration of Human Inter Leukin-2 in this antitumor T lymphocyte populations special culture media is 1,000,000 U/L.The pH of this antitumor T lymphocyte populations special culture media is 7.2-7.4.After incubated overnight, attached cell is monocyte, suspension be lymphocyte.The lymphocyte of suspension culture is transferred in new culturing bottle and continues to cultivate.
8) within every 2-3 days, in this antitumor T lymphocyte populations special culture media, go down to posterity once, make cell concn maintain (5～10) * 10
5individual cell/mL.The condition of cultivating that at every turn goes down to posterity is 37 ℃, 5%CO
2, biography 5 generations (culture condition in per generation is identical) obtains antitumor T lymphocyte populations, abandons nutrient solution supernatant after cell centrifugation, with 1 * PBS, is made into cell suspension 5 * 10
6/ ml, gets 200 microlitres and carries out flow cytometer detection.Use respectively anti-human CD3, CD4, CD8, TCR α β, TCR γ δ monoclonal antibody (is bought the company in biolegend, article No. is respectively 317307, 317407, 301005, 306707, 331207) by flow cytometer, detect human leukocyte differentiation antigen CD3, CD4, CD8 and people α β, (detection method is referring to Biggs MJ for the expression of gamma delta T cells acceptor, et al.2011.J.R.Soc.Interface.8:1462.), shown in result, show that the cell of expressing human leukocyte differentiation antigen CD3 in this antitumor T lymphocyte populations accounts for 80.76%, the cell of expressing human leukocyte differentiation antigen CD4 accounts for 25.44%, the cell of expressing human leukocyte differentiation antigen CD8 accounts for 67.71%, the cell of expressing people α β φt cell receptor account for 15.39% and the cell of human gamma delta t cells acceptor account for 69.95%.Meanwhile, under similarity condition, detect 6) in peripheral blood mononuclear cell PBMC, its result is as shown in Figure 1.
Embodiment 4
The present embodiment provides the method for separated antitumor T lymphocyte populations from filter for removing white blood cell, comprises the steps:
1) from Shenzhen Blood Center, obtain Yi Ge 2 units (400ml) filter for removing white blood cell, blood station is used filter for removing white blood cell to filter after 400 milliliters of whole bloods (normal people's voluntary blood donation), and 99% white corpuscle remains in filter.
2) use 40 milliliters of 1 * PBS to rinse filter for removing white blood cell, obtain the mixed solution of PBS, white corpuscle and part blood.
3) mixed solution of PBS, white corpuscle and part blood is discarded to blood plasma for centrifugal 15 minutes with 2500 revs/min.
4) to abandoning in plasmapheretic cell mixture, add 25 milliliters of 1 * PBS, carefully mix formation mixed solution.
5) mixed solution is carefully superimposed on the liquid level of cellular segregation liquid (purchased from Tianjin Hao Yang biological products Science and Technology Ltd., production code member is LTS1077) to mixed solution: cellular segregation liquid=2:1 (volume ratio).
6) centrifugal 20 minutes with 2500 revs/min.Now in centrifuge tube, be divided into from top to bottom four layers: the first layer is plasma layer; The second layer is ring-type oyster white mononuclearcell layer; The 3rd layer is transparent separated liquid layer; The 4th layer is red corpuscle layer.Collect second layer cell and put into the test tube containing 1 * PBS of 45ml, after fully mixing, centrifugal 10 minutes with 2500 revs/min, supernatant discarded is stayed precipitation, with 1 * PBS of 45ml, with same rotating speed and time repeated washing twice, finally precipitation obtains separated peripheral blood mononuclear cell (PBMC).
7) separated PBMC is incubated to 37 ℃, 5%CO
2incubator, nutrient solution is 30 milliliters of antitumor T lymphocyte populations special culture medias.This antitumor T lymphocyte populations special culture media is that (Tianjin Hao Yang biological products Science and Technology Ltd. produces at serum-free lymphocytes culture medium, production code member is CIK2013) middle isopentenylpyrophosphate (the Sigma company that adds, production code member is I0503) and Human Inter Leukin-2 (IL-2) (the accurate word S10970016 of traditional Chinese medicines, Beijing Sihuan Biopharmaceutical Co., Ltd., specification is 500,000 U/ bottles) substratum that obtains, the concentration of isopentenylpyrophosphate in this antitumor T lymphocyte populations special culture media is 200 μ g/L, the concentration of Human Inter Leukin-2 in this antitumor T lymphocyte populations special culture media is 1,000,000 U/L.The pH of this antitumor T lymphocyte populations special culture media is 7.2-7.4.After incubated overnight, attached cell is monocyte, suspension be lymphocyte.The lymphocyte of suspension culture is transferred in new culturing bottle and continues to cultivate.
8) within every 2-3 days, in this antitumor T lymphocyte populations special culture media, go down to posterity once, make cell concn maintain (5～10) * 10
5individual cell/mL.The condition of cultivating that at every turn goes down to posterity is 37 ℃, 5%CO
2, biography 5 generations (culture condition in per generation is identical) obtains antitumor T lymphocyte populations, abandons nutrient solution supernatant after cell centrifugation, with 1 * PBS, is made into cell suspension 5 * 10
6/ ml, gets 200 microlitres and carries out flow cytometer detection.Use respectively anti-human CD3, CD4, CD8, TCR α β, TCR γ δ monoclonal antibody (is bought the company in biolegend, article No. is respectively 317307, 317407, 301005, 306707, 331207) by flow cytometer, detect human leukocyte differentiation antigen CD3, CD4, CD8 and people α β, (detection method is referring to Biggs MJ for the expression of gamma delta T cells acceptor, et al.2011.J.R.Soc.Interface.8:1462.), shown in result, show that the cell of expressing human leukocyte differentiation antigen CD3 in this antitumor T lymphocyte populations accounts for 75.94%, the cell of expressing human leukocyte differentiation antigen CD4 accounts for 27.37%, the cell of expressing human leukocyte differentiation antigen CD8 accounts for 65.58%, the cell of expressing people α β φt cell receptor account for 17.51% and the cell of human gamma delta t cells acceptor account for 80.08%.Meanwhile, under similarity condition, detect 6) in peripheral blood mononuclear cell PBMC, its result is as shown in Figure 1.
Embodiment 5, antitumor T lymphocyte populations anti-tumor activity are identified.
1. antitumor T lymphocyte populations suppresses multiple Cells Proliferation of Human Breast Cancer
1.1 antitumor T lymphocyte populations and breast cancer cell effect target ratio
1) by the breast cancer cell MCF-7 having inoculated, MDA-MB-231, MDA-MB-453, MDA-MB-468, (buying the Shanghai cell bank in the Chinese Academy of Sciences) put into incubator and cultivated, at the bottom of being paved with hole to cell monolayer (96 hole flat underside), every hole 200 microlitres.Respectively by effect target than 1:1,5:1,10:1 adds the antitumor T lymphocyte populations of embodiment 1, and (concentration is 5 * 10
6/ ml), each experimental group arranges five parallel multiple holes.
2) 5%CO
2, to hatch 12 hours for 37 ℃, every hole adds 20ul tetrazolium bromide (MTT) solution (5mg/ml, i.e. 0.5%MTT), continues to cultivate 4 hours.
3) stop cultivating, abandon supernatant, every hole adds 200ul dimethyl sulfoxide (DMSO), puts low-speed oscillation 10min on vibrator, and crystallisate is fully dissolved.After the light absorption value in each hole of measurement, enzyme-linked immunosorbent assay instrument OD595nm place, calculate cell survival rate, as shown in Figure 2, along with antitumor T lymphocyte populations and breast cancer cell effect target ratio increase, the survival rate of breast cancer cell obviously declines.
1.2 antitumor T lymphocyte populations and breast cancer cell action time
1) by the breast cancer cell MCF-7 having inoculated, MDA-MB-231, MDA-MB-453, MDA-MB-468, (buying the Shanghai cell bank in the Chinese Academy of Sciences) put into incubator and cultivated, at the bottom of being paved with hole to cell monolayer (96 hole flat underside), every hole 200 microlitres.(concentration is 5 * 10 by effect target, than 1:1, to add the antitumor T lymphocyte populations of embodiment 1
6/ ml), each experimental group arranges five parallel multiple holes.
2) 5%CO
2, to hatch 24,48,72,96 hours for 37 ℃, every hole adds 20ul tetrazolium bromide (MTT) solution (5mg/ml, i.e. 0.5%MTT), continues to cultivate 4 hours.
3) stop cultivating, abandon supernatant, every hole adds 200ul dimethyl sulfoxide (DMSO), puts low-speed oscillation 10min on vibrator, and crystallisate is fully dissolved.Measure the light absorption value in each hole at enzyme-linked immunosorbent assay instrument OD595nm place after, calculate cell survival rate, as shown in Figure 3, along with the prolongation of antitumor T lymphocyte populations and breast cancer cell action time, the survival rate of breast cancer cell obviously declines.
2. antitumor T lymphocyte populations suppresses the growth that mammary cancer is closed knurl mouse interior tumor.
Get surrounding female Bal/c nude mice in age, be divided at random three groups, 10 every group.The curative effect difference of more antitumor T lymphocyte populations treatment group, antitumor T lymphocyte populations pretreated group and control group.It is 2 * 10 that breast cancer cell MDA-MB-231 cell (buying in Shanghai cell bank) is equipped to cell concn with 1 * PBS
6the MDA-MB-231 cell suspension of individual cells/ml, nude mice left side armpit subcutaneous vaccination MDA-MB-231 cell suspension, 100 microlitres/only, the experimental group that is wherein grouped into antitumor T lymphocyte populations pretreated group is first to inoculate 1 * 10 before inoculation breast cancer cell
8the antitumor T lymphocyte populations of individual/milliliter (from embodiment 1 pass logarithmic phase propagation in the 5th generation process antitumor T lymphocyte populations), be all to treat that tumour grows to 100mm
3time begin treatment.T cell therapy group: antitumor T lymphocyte populations concentration is 1 * 10
8individual/milliliter (from embodiment 1 pass logarithmic phase propagation in the 5th generation process antitumor T lymphocyte populations), nude mice intravenous injection, 100 microlitres/only.T lymphocyte pretreated group and control group: intravenous injection 1 * PBS, 100 microlitres/only.Inject weekly twice, inject continuously after surrounding the relatively difference in size of the gross tumor volume of T lymphocyte treatment group, T lymphocyte pretreated group and control group, as shown in Figure 4, T lymphocyte pretreated group is compared with control group, and obviously diminishing does not appear in gross tumor volume; T lymphocyte treatment group is compared with control group, and obviously diminishing appears in mouse tumor volume.As can be seen here, T lymphocyte pretreated group does not make immune function of mice rebuild, and continues to use antitumor T lymphocyte populations to have obvious restraining effect to the tumor growth of MDA-MB-231 tumor-bearing mice, and tumour inhibiting rate surpasses 70%.
Claims (10)
1. one group of antitumor T lymphocyte populations, it is characterized in that, it comprises that the cell of expressing human leukocyte differentiation antigen CD3 accounts for 71.63%～86.32%, the cell of expressing human leukocyte differentiation antigen CD4 accounts for 16.43%～27.37%, the cell of expressing human leukocyte differentiation antigen CD8 accounts for 65.58%～76.86%, the cell of expressing people α β φt cell receptor accounts for 11.90%～17.51%, and the cell of expressing human gamma delta t cells acceptor accounts for 69.95%～83.74%.
2. the preparation method of one group of antitumor T lymphocyte populations according to claim 1, is characterized in that, comprises the steps:
Step 1: remove leukocyte depletion filter, after filtering whole blood, white corpuscle remains in filter;
Step 2: use phosphoric acid buffer to rinse filter for removing white blood cell, obtain the mixed solution of PBS, white corpuscle and part blood;
Step 3: by the centrifugal blood plasma that discards of the mixed solution of phosphoric acid buffer, white corpuscle and part blood;
Step 4: add phosphoric acid buffer to abandoning in plasmapheretic cell mixture, carefully mix formation mixed solution;
Step 5: mixed solution is carefully superimposed on cellular segregation liquid centrifugal, is now divided into from top to bottom four layers in centrifuge tube: the first layer is plasma layer; The second layer is ring-type oyster white mononuclearcell layer; The 3rd layer is transparent separated liquid layer; The 4th layer is red corpuscle layer; Collect second layer cell and put into the test tube of phosphoric acid damping fluid, after fully mixing, centrifugal, supernatant discarded is stayed precipitation, and then repeated washing, finally precipitates and obtain separated peripheral blood mononuclear cell;
Step 6: separated peripheral blood mononuclear cell is incubated to 37 ℃, 5%CO
2incubator, with after antitumor T lymphocyte populations special culture media incubated overnight, collects the lymphocyte suspending;
Step 7: go down to posterity once in this antitumor T lymphocyte populations special culture media, make cell concn maintain 5 * 10 for every 2-3 days
5～10 * 10
5individual cell/mL, the condition of cultivating that at every turn goes down to posterity is 37 ℃, 5%CO
2, more than passing for 5 generations, the culture condition in per generation is identical, obtains antitumor T lymphocyte populations;
Wherein, described antitumor T lymphocyte populations special culture media is to be in serum-free lymphocytes culture medium, to add the substratum that isopentenylpyrophosphate and Human Inter Leukin-2 obtain.
3. the preparation method of one group of antitumor T lymphocyte populations according to claim 2, is characterized in that, described phosphoric acid buffer is for containing 135mM NaCl, 4.7mM KCl, 10mMNa
2hPO
4, 2mM NaH
2pO
4and the aqueous solution that pH value is 7.4.
4. the preparation method of one group of antitumor T lymphocyte populations according to claim 3, is characterized in that, in described step 1, the specification of filter for removing white blood cell is 2 400ml of unit, and whole blood is 400ml, and the phosphoric acid buffer in described step 2 is 40-80ml.
5. the preparation method of one group of antitumor T lymphocyte populations according to claim 4, is characterized in that, the centrifugation rate in described step 3 is 1500-2500 rev/min, and centrifugation time is 15-20 minute.
6. the preparation method of one group of antitumor T lymphocyte populations according to claim 5, is characterized in that, the phosphoric acid buffer adding in described step 4 is 25-40ml.
7. the preparation method of one group of antitumor T lymphocyte populations according to claim 6, it is characterized in that, cellular segregation liquid in described step 5 is purchased from Tianjin Hao Yang biological products Science and Technology Ltd., production code member is LTS1077, the volume ratio of enchylema and cellular segregation liquid is 1.5:1-2:1, after mixing, centrifugal speed is 2000-2500 rev/min, and centrifugation time is 15-20 minute.
8. the preparation method of one group of antitumor T lymphocyte populations according to claim 7, it is characterized in that, in described step 5, collecting second layer cell is the test tube of putting into containing the phosphoric acid buffer of 20-45ml, mixing centrifugation rate with phosphoric acid buffer is 2000-2500 rev/min, centrifugation time is 10-15 minute, then with twice of same rotating speed and time repeated washing.
9. the preparation method of one group of antitumor T lymphocyte populations according to claim 8, it is characterized in that, the pH of described antitumor T lymphocyte populations special culture media is 7.2-7.4, consumption is 30 milliliters, wherein, the concentration of isopentenylpyrophosphate in this antitumor T lymphocyte populations special culture media is 200 μ g/L, and the concentration of Human Inter Leukin-2 in this antitumor T lymphocyte populations special culture media is 1,000,000 U/L.
10. an antitumor drug, is characterized in that, it take one group of antitumor T lymphocyte populations claimed in claim 1 is activeconstituents.
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