A kind of cultural method of the autologous peripheral blood lymphocyte using the stimulation of TLR7 agonistTechnical field
The invention belongs to field of immune cell culture in vitro, in particular to it is a kind of using TLR7 agonist stimulationThe cultural method of autologous peripheral blood lymphocyte.
Background technique
Peripheral blood lymphocytes (Peripheral blood lymphocyte) abbreviation PBL, mainly in blood circulationLymphocyte, be made of T cell and B cell；Lymphocyte in lymphoid tissue has following key property: 1. specific:Lymphocytic cell surface has antigen receptor, to identify antigen；The antigenic variant of different lymphocytes is different, each receptorIt can only be combined with corresponding antigen, here it is specificity；For antigen receptor type there are about 1,000,000 kinds, the lymphocyte of whole body is totalVarious antigens can be identified altogether；2. convertibility: most of lymphocytes are in stationary state in regular；Only whenCertain lymphocyte is just activated after being stimulated by corresponding antigens, this process is known as convertibility, need to generally pass through 40 hours,Significant change has occurred in lymphocyte form, the small lymphocyte by one 5~7 μm of diameter is changed into one 20~30 μmLarge lymphocyte；The metabolism of cell enhances, form significant change, and core increases, and chromatin attenuates, and kernel is obvious, in cell spaceRibosomes increases, and cytoplasm dyeing is in stronger basophilla；After lymphocyte transformation, division and proliferation is carried out, wherein most can shapeAt the effector cell for participating in immune response, as can destroying the T effector cell of target cell, or the thick liquid cell of energy secretory antibody；TheseCell metabolism is active, can generate immunological effect, but the service life is short (about a couple of days or several weeks)；3. memory: lymphocyte is through antigenic activationAfter conversion, in the cell that division growth is formed, some is converted into the lymphocyte of quiescent condition, referred to as Memorability once again(T or B) lymphocyte, the service life is long, and up to several years or lifelong presence, they can turn rapidly after encountering corresponding antigens stimulationEffector cell is turned to, removes antigen in time, makes body from morbidity；These characteristics of lymphocyte, it is normal to maintain lymphocyteForm and physiological dynamic moving.
Existing publication: CN102816735B provides a kind of autologous peripheral blood strangury stimulated using TLR7 agonistThe cultural method of bar cell is to keep effector cell group living by adding Multiple Antibodies and cell factor into serum free mediumChange；But its operating process is complicated, and the period is long, accuracy is poor, and Lymphocyte expansion efficiency and activity be not high.CN104357394AThe cultural method of autologous peripheral blood lymphocyte DC-CIK a kind of is provided, external CTLA4 antibody, the PD-1 antibody of loading is to coverCTLA4, PD-1 molecule for covering all CIK cell surfaces prevent it from combining with receptor corresponding on DC, reduce T and adjust cellContent, but the specificity of tumour cell is ignored, external source serum free medium and antibody dosage are more, and it is at high cost, and may go outThe existing factors such as antagonistic effect and miscellaneous bacteria influence, and can reduce Lymphocyte expansion efficiency and activity, and then influence tumour cellFragmentation effect.
Summary of the invention
The purpose of the present invention is to provide a kind of cultures of autologous peripheral blood lymphocyte using the stimulation of TLR7 agonistMethod, to solve the problems mentioned in the above background technology.
To achieve the above object, the invention provides the following technical scheme:
Basic terms:
GM-CSF: macrophage colony stimulating factor
IL-2:: interleukin 2
IL-1a: interleukin 1 a
IL-4: interleukin-4
IL-1 β: interleukin-1 beta
PGE2: prostaglandin E2
TNF-α: tumor necrosis factor-alpha
CIK cell: the killing cell of cytokine profiles induction
A kind of DC cell: culture side of the autologous peripheral blood lymphocyte using the stimulation of TLR7 agonist of Dendritic CellsMethod includes the following steps:
(1) with mononuclearcell separating pipe and Percoll layering liquid isolated from adult peripheral blood mononuclearcell andMononuclearcell is carried out in vitro culture by autoserum on peripheral blood lymphocytes culture medium；
(2) it carries out cytokine activation to the mononuclearcell of in vitro culture to irritate: by the mononuclearcell of step (1)Sub-bottle carries out differential stimulus activation after culture 2 hours.After continuing culture 24 hours, OKT3 antibody final concentration is added in culture bottle 2For 90~100ng/ml, OKT28 antibody final concentration of 90~100ng/ml, IL-2 final concentration of 900~1000IU/ml, GM-Final concentration of 950~the 100IU/mL of CSF final concentration of 450~500U/ml, IL-1a；It is dense eventually that GM-CSF is added in culture bottle 1Degree is 950~1000U/ml, IL-4 final concentration of 950~1000U/ml, 37 DEG C, continue to cultivate under the conditions of 5%~7%CO 2；30~40ug/ml of autologous tumor antigens is added after continuing culture 48 hours, TLR7 agonist is added after continuing culture 24 hoursIL-1 β, final concentration of 8~10ng/ml is added after continuing culture 24 hours in final concentration of 8~12ng/ml；PGE2 is final concentration of450~500ng/ml；TNF-α, final concentration of 8~10ng/ml, induced cellular activation is added after continuing culture 24 hours；ContinueCulture closed bottle after 24 hours；
(3) ratio that concussion mixes is replenished in time with peristaltic pump in each culture bottle liquidometer real-time detection liquid levelFor the autoserum and peripheral blood lymphocytes culture medium of 1:1, every 48 hours replacement half culture mediums keep cell densityIt is 1.2 × 106~1.8 × 106/ ml, after closing bottle culture 7 days, the lymph for obtaining the specific High Fragmentation tumour cell ability of tool is thinBorn of the same parents' subgroup；
Peripheral blood lymphocytes culture medium is used in the step (1), every milliliter contains following component: RPMI-1460 trainingIt is solidifying to support base 0.6-0.8ml, fetal calf serum 0.2-0.3ml, gentamicin 200-500ng, L-Glutamine 300-500ng, plantCollect element (PHA) 600-800ng.
Further: the concrete operations of the step (1) are as follows:
1. Percoll is added and is layered liquid after the dust-free paper wiping that 50ml mononuclearcell separating pipe is infiltrated through alcohol is clean15ml；
2. extracting the adult peripheral blood in blood taking bag, it is dispensed into 50ml mononuclearcell separating pipe, every pipe blood 30ml；In gnotobasis, 3000rpm is centrifuged 20 minutes, is layered as plasma layer, mononuclearcell layer, red blood cell layer from top to bottom；
3. the upper plasma of step 2. is stood 20min under 42 DEG C of environment, then stood under -18 DEG C of environment10min；4000rpm is centrifuged 5min, collects serum, adds IL-12 to final concentration of 1000pg/ml to get autoserum；
4. the mononuclearcell layer of slow aspiration step 2. is added 10ml physiological saline and is resuspended, adds phenylalanine firstEster 1-2ml, then plus physiological saline is to 40ml, mixes, sampling is for cell quantity detection, cell subsets content detection, 300gLower centrifugation 5min；
5. step abandons supernatant after being 4. centrifuged, peripheral blood lymphocytes culture medium is added, cell density is adjusted to 1.2 ×106~1.8 × 106/ ml is added to concussion in culture bottle (No. 1) and mixes.37 DEG C of constant temperature, the training of 5%~7% CO2 percent by volumeIt supports and is cultivated 2 hours in case；
6. by celliferous media transfer in culture bottle (No. 1) into another culture bottle (No. 2), No. 1 culture bottle bottomRetain attached cell.Peripheral blood lymphocytes culture medium, adjustment cell concentration to 1 × 10 are added into No. 1 culture bottle6/Ml, willNo. 1 and No. 2 culture bottles are all placed in 37 DEG C, cultivate 3 days in 5%~7%CO, 2 concentration incubator；
7. carrying out cytokine activation stimulation to the mononuclearcell in No. 2 culture bottles: being added eventually into No. 2 culture bottlesConcentration is the INF- γ of 1000IU/ml；Add the autoserum of culture total volume 5% again, 37 DEG C, under the conditions of 5.0%CO 2Culture；After 24 hours, OKT3 antibody, OKT28 antibody, IL-2, GM-CSF and IL-1a is added, OKT3 antibody is final concentration ofThe final concentration of 500U/ of 100ng/ml, OKT28 antibody final concentration of 100ng/ml, IL-2 final concentration of 1000IU/ml, GM-CSFMl, IL-1a final concentration of 100IU/mL, 37 DEG C, continue culture 120 hours under the conditions of 6.0%CO 2, according to the growth of cellSituation carries out fluid infusion and replacement culture medium operation, cell density is made to be maintained at 1.2 × 106~1.8 × 106/ml；
8. step 6. after, in No. 1 culture bottle cell carry out cytokine activation stimulation: every 48 hours to No. 1 cultivateGM-CSF 1000U/ml, IL-41000U/ml are added in bottle, and replaces half culture medium, 37 DEG C, under the conditions of 6.0%CO 2 afterAfter continuous culture 24 hours, the final concentration of 30~40ug/ml of autologous tumor antigens is added, TLR7 is added after continuing culture 24 hoursIL-1 β, final concentration of 10ng/ml is added after continuing culture 24 hours in the final concentration of 8~12ng/ml of agonist；PGE2 is dense eventuallyDegree is 500ng/ml；TNF-α is added after continuing culture 24 hours, final concentration of 10ng/ml induces DC cell maturation, continues to trainIt supports 48 hours；
Cultivated 9. the culture in two culture bottles is mixed, by cell growth status carry out fluid infusion andCulture medium is replaced, cell density is made to be maintained at 1.2 × 106~1.8 × 106/ml.After continuing culture 7 days, it is special to obtain toolProperty High Fragmentation tumour cell ability lymphocyte subgroup, every culture harvests cell for 21 days in batches, and samples and be used for cell quantityDetection, cell subsets content detection.
Further, the peripheral blood lymphocytes culture medium of the use in the step (1), using preceding sterilizing, and water-bath37 DEG C are preheated to, concussion is uniform and removes bubble removing.
Further, the condition of culture in step (1)~(3) is temperature is 37 DEG C, CO2 percent by volume isIt is cultivated in the CO2 incubator that content is 6%, relative saturation humidity is 100%.
Further, step (2) gnotobasis is Laminar Flow Room or superclean bench.
It is preferred according to the present invention, contain such as the following group for every milliliter of peripheral blood lymphocytes culture medium described in step (1)Point, RPMI-1460 culture medium 0.65ml, fetal calf serum 0.25ml, gentamicin 320ng, L-Glutamine 450ng, plant are solidifyingCollect element (PHA) 650ng.
Preferred according to the present invention, the step (3) uses ultrasonic level gage real-time measurement liquid level.
Preferred according to the present invention, 8. the final concentration of 35ug/ml of autologous tumor antigens is added in the step, continue to cultivateThe final concentration of 10ng/ml of TLR7 agonist is added after 24 hours,
Further, specific step is as follows for the preparation of step (2) autologous tumor antigens:
1. aseptic operation acquires the tumor tissues of patient's body, it is placed in RPMI1640 culture medium and transports to sterile realityRoom is tested, is cleaned twice with PBS, and tumor tissue is cut into 1.0mm3 cell with sterile scissors；
2. the small tissue blocks shredded are put into 0.25%Trypsin/0.05%EDTA, in 37 DEG C of isothermal vibration instrumentIsothermal vibration 30min；
3. concussion after the completion of, be added PBS terminate digestion, and use the screen to filtrate of 100um, by filtrate centrifugation abandoning supernatant, bottomLayer is tumour cell；
4. obtained tumour cell is laid on culture bottle with RPMI1640 culture medium, it is placed in 37 DEG C, 5.0% CO, 2 concentrationIt is cultivated in incubator, this is the 0th generation cell；
5. reach 80%~90% to the cell fusion degree in culture bottle, 0.25% Trypsin/ is used0.05%EDTA digests adherent tumour cell, carries out secondary culture, and culture is arrived thin with harvest when the third generationBorn of the same parents；
6. harvesting cell: when reaching 80%~90% to the cell fusion degree in culture bottle, using 0.25%Trypsin/0.05%EDTA digests adherent tumour cell, is attached in 50ml centrifuge tube, 500g, and 5min centrifugation is abandonedSupernatant is removed, cell is resuspended with physiological saline, 500g, 5min centrifugation discard supernatant；
7. the cell after centrifugation is resuspended with physiological saline, being tuned into density is 2 × 106/ ml is sub-packed in 5ml cryopreservation tube,It is placed directly in liquid nitrogen, takes out cell after 20min and melt naturally, 3 times repeatedly；
8. the liquid after multigelation is placed in 50ml centrifuge tube, 400g is centrifuged 10min, takes supernatant, and as tumour is anti-It is former.
In CIK cultivating system, CIK cell is differentiated by the T cell of induced activation, and CD3 molecule can transduceActivation signals caused by T cell antigen Receptor recognition antigen, therefore CD3mAb is added, T cell activation efficiency can be improved；CD28Developed by molecule and T cell surface, it provides costimulatory molecules signal, this is the essential a part of T cell activation, thereforeCD28mAb, which is added, can be improved T cell activation efficiency；And IFN-γ is by activating T cell and natural killer cells (NK cell)The type cytokines with antiviral, immunological regulation and antitumor properties generated.Can be integrated to interferon gamma receptor(IFNGR), activation antigen presenting cells are to promote the differentiation of I type T helper cell (Th1 cell)；PHA can promote cellMitosis, to improve the amplification efficiency of CIK cell.IL-1 α can promote cell secretion IL-2, costimulation CD8+/IL-1α+cell and can be with fragmentation unit tumour cell.
Compared with prior art, the beneficial effects of the present invention are:
1, with mononuclearcell separating pipe and Percoll layering liquid isolated from adult peripheral blood mononuclearcell andAutoserum, the method than traditional secondary centrifuging separation mononuclearcell is easy to operate, high-efficient；
2, the present invention passes through the mononuclearcell sub-bottle in vitro culture, No. 1 culture bottle OKT3 antibody, OKT28 antibodyAnd cytokine activation irritates, after autologous tumor antigens, cell factor and TLR7 agonist culture is added in No. 2 culture bottles, soBottle culture is closed afterwards, can effectively improve specific effector cell mass activation efficiency and amplification efficiency.It is detected, activates and expandEfficiency significantly improves than existing methods.
3, nutriment needed for the culture medium that uses of the present invention has Lymphocyte expansion, with liquid level sensor, in real timeLiquid level is detected, the autoserum and peripheral blood lymphocytes culture medium of mixing, standard easy to operate are replenished in time with peristaltic pumpTrue property is high.
4, it is cultivated under constant temperature gnotobasis, autologous tumor antigens and process product autoserum is sufficiently used, to selfThe killing of tumour cell is more targeted, and creates optimal conditions for specific lymphocyte amplification, and it is thin to significantly improve lymphBorn of the same parents' culture efficiency.
Detailed description of the invention
The amplification curve diagram of Fig. 1, embodiment 1 and comparative example difference incubation time cell；
Fig. 2, embodiment 1 compare the killing activity experimental result histogram of breast cancer cell line with comparative example difference effect target,It is comparative example that a left side, which is 1 right side of embodiment, in figure；
Fig. 3, embodiment 1 and comparative example cell phenotype are analyzed, and it is comparative example that a left side, which is 1 right side of embodiment, in figure；
The amplification curve diagram of Fig. 4, embodiment 1 and the different incubation time autologous peripheral blood lymphocytes of embodiment 2；
Fig. 5, embodiment 1 and the different effect targets of embodiment 2 compare the killing activity experimental result column of breast cancer cell lineFigure；
Fig. 6, embodiment 1 and 2 cell phenotype of embodiment are analyzed, and it is embodiment 2 that a left side, which is 1 right side of embodiment, in figure
The amplification curve of Fig. 7, embodiment 1, embodiment 3 and comparative example difference incubation time autologous peripheral blood lymphocyteFigure；
The killing activity that Fig. 8, embodiment 1, embodiment 3 compare breast cancer cell line with comparative example difference effect target tests knotFruit histogram, it be 3 right side of embodiment is comparative example that a left side, which is in embodiment 1, in figure；
Fig. 9, embodiment 1, embodiment 3 and comparative example cell phenotype are analyzed, and it is that embodiment 3 is right that a left side, which is in embodiment 1, in figureFor comparative example
Specific embodiment
Below in conjunction in the embodiment of the present invention, technical solution of the present invention is clearly and completely described, it is clear thatDescribed embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the implementation in the present inventionExample, every other embodiment obtained by those of ordinary skill in the art without making creative efforts belong toThe scope of protection of the invention.
Technical solution of the present invention is further elaborated below with reference to embodiment, but institute's protection scope of the present invention is unlimitedIn this.
Raw material explanation
PBMC picks up from blood of cancer patients, obtains after separation.
IL-2, IL-4 are purchased from Beijing Qi Weiyi at Science and Technology Ltd..
IL-1 α, IL-1 β is purchased from R&D Systems company, 10 μ g/ branch.
TNF-α is purchased from Beijing Ke Xin Biotechnology Co., Ltd.
GM-CSF is purchased from the source Beijing Tong Lihai Biotechnology Co., Ltd.
PGE2 is purchased from Wuhan Sino-American Biotechnology Company.
Phenyalanine methyl ester is purchased from the Shanghai chemical Science and Technology Ltd. of special uncle.
Percoll is layered liquid, and phytolectin (PHA) is purchased from Sigma company.
RPMI-1460 culture medium is purchased from Jiangsu Kai Ji Biotechnology Ltd..
Fetal calf serum is purchased from Beijing Quan Shijin biotechnology (TransGen Biotech) Co., Ltd.
Gentamicin is purchased from the joyful beauty Biotechnology Co., Ltd in Wuhan.
L-Glutamine is purchased from Hubei Hong Xinrui space Fine Chemical Co., Ltd.
TLR7 agonist-R848 is purchased from Shenzhen Xin Bosheng Biotechnology Co., Ltd.
Mononuclearcell separating pipe is purchased from 31 hematopoiesis Technology Co., Ltd. of Fujian.
The purpose of the present embodiment is to keep those skilled in the art abundant by the comparison with prior art embodimentTechnical solution of the present invention and corresponding technical effect are solved, various reagents used in the present embodiment can be bought on the marketIt arrives, after the reagent of different manufacturers production is according to this field conventional treatment, not will cause the difference of the present embodiment experimental result.
Embodiment 1
A kind of cultural method of the autologous peripheral blood lymphocyte using the stimulation of TLR7 agonist, includes the following steps:
1. Percoll is added and is layered liquid after the dust-free paper wiping that 50ml mononuclearcell separating pipe is infiltrated through alcohol is clean15ml；
2. extracting the adult peripheral blood in blood taking bag, it is dispensed into 50ml mononuclearcell separating pipe, every pipe blood 30ml；In Laminar Flow Room, 3000rpm is centrifuged 20 minutes, is layered as plasma layer, mononuclearcell layer, red blood cell layer from top to bottom；
3. the upper plasma of step 2. is stood 20min under 42 DEG C of environment, then stood under -18 DEG C of environment10min；4000rpm is centrifuged 5min, collects serum, adds IL-12 to final concentration of 1000pg/ml to get autoserum；
4. the mononuclearcell layer of slow aspiration step 2. is added 10ml physiological saline and is resuspended, adds phenylalanine firstEster 1-2ml, then plus physiological saline is to 40ml, mixes, sampling is for cell quantity detection, cell subsets content detection, 300gLower centrifugation 5min；
5. step abandons supernatant after being 4. centrifuged, peripheral blood lymphocytes culture medium is added, cell density is adjusted to 1.2 ×10~1.8 × 106/ ml is added to concussion in culture bottle (No. 1) and mixes.37 DEG C of constant temperature, 6%CO2 percent by volume incubatorMiddle culture 2 hours；
6. by celliferous media transfer in culture bottle (No. 1) into another culture bottle (No. 2), No. 1 culture bottle bottomRetain attached cell.Peripheral blood lymphocytes culture medium, adjustment cell concentration to 1 × 10 are added into No. 1 culture bottle6/ ml,No. 1 and No. 2 culture bottles are all placed in 37 DEG C, cultivated 3 days in 2 concentration incubator of 6.0%CO；
7. carrying out cytokine activation stimulation to the mononuclearcell in No. 2 culture bottles: being added eventually into No. 2 culture bottlesConcentration is the INF- γ of 1000IU/ml；Add the autoserum of culture total volume 5% again, 37 DEG C, under the conditions of 5.0%CO 2Culture；After 24 hours, OKT3 antibody, OKT28 antibody, IL-2, GM-CSF and IL-1a is added, OKT3 antibody is final concentration ofThe final concentration of 500U/ of 100ng/ml, OKT28 antibody final concentration of 100ng/ml, IL-2 final concentration of 1000IU/ml, GM-CSFMl, IL-1a final concentration of 100IU/mL, 37 DEG C, continue culture 120 hours under the conditions of 6.0%CO 2, according to the growth of cellSituation carries out fluid infusion and replacement culture medium operation, cell density is made to be maintained at 1.2 × 106~1.8 × 106/ml；
8. step 6. after, in No. 1 culture bottle cell carry out cytokine activation stimulation: every 48 hours to No. 1 cultivateGM-CSF 1000U/ml, IL-41000U/ml are added in bottle, and replaces half culture medium, 37 DEG C, under the conditions of 6.0%CO 2 afterAfter continuous culture 24 hours, the final concentration of 3ng/ml of autologous tumor antigens is added, TLR7 agonist is added after continuing culture 24 hoursIL-1 β, final concentration of 10ng/ml is added after continuing culture 24 hours in final concentration of 8ng/ml；The final concentration of 500ng/ of PGE2ml；TNF-α is added after continuing culture 24 hours, final concentration of 10ng/ml induces DC cell maturation, continues culture 48 hours；
It cultivates 9. the culture in two culture bottles is mixed, is pressed by cell growth status with peristaltic pumpThe autoserum and peripheral blood lymphocytes culture medium that 1:1 ratio supplement mixes, every 48 hours replacement half culture mediums make thinIntracellular growth density is maintained at 1.2 × 106~1.8 × 106/ml.After continuing culture 7 days, it is thin to obtain the specific High Fragmentation tumour of toolThe lymphocyte subgroup of born of the same parents' ability, every culture harvest cell for 21 days in batches, and sample for cell quantity detection, cell subsetsContent detection.
Step 5. in every milliliter of peripheral blood lymphocytes culture medium contain following component, RPMI-1460 culture medium 0.6ml,Fetal calf serum 0.2ml, gentamicin 200ng, L-Glutamine 300ng, phytolectin (PHA) 600ng.
Specific step is as follows for autologous tumor antigens preparation:
1. aseptic operation acquires the tumor tissues of patient's body, it is placed in RPMI1640 culture medium and transports to sterile realityRoom is tested, is cleaned twice with PBS, and tumor tissue is cut into 1.0mm3 cell with sterile scissors；
2. the small tissue blocks shredded are put into 0.25%Trypsin/0.05%EDTA, in 37 DEG C of isothermal vibration instrumentIsothermal vibration 30min；
3. concussion after the completion of, be added PBS terminate digestion, and use the screen to filtrate of 100um, by filtrate centrifugation abandoning supernatant, bottomLayer is tumour cell；
4. obtained tumour cell is laid on culture bottle with RPMI1640 culture medium, it is placed in 37 DEG C, 6.0% CO, 2 concentrationIt is cultivated in incubator, this is the 0th generation cell；
5. reach 80%~90% to the cell fusion degree in culture bottle, 0.25% Trypsin/ is used0.05%EDTA digests adherent tumour cell, carries out secondary culture, and culture is arrived thin with harvest when the third generationBorn of the same parents；
6. harvesting cell: when reaching 80%~90% to the cell fusion degree in culture bottle, using 0.25%Trypsin/0.05%EDTA digests adherent tumour cell, is attached in 50ml centrifuge tube, 500g, and 5min centrifugation is abandonedSupernatant is removed, cell is resuspended with physiological saline, 500g, 5min centrifugation discard supernatant；
7. the cell after centrifugation is resuspended with physiological saline, being tuned into density is 2 × 106/ ml is sub-packed in 5ml cryopreservation tube,It is placed directly in liquid nitrogen, takes out cell after 20min and melt naturally, 3 times repeatedly；
8. the liquid after multigelation is placed in 50ml centrifuge tube, 400g is centrifuged 10min, takes supernatant, and as tumour is anti-It is former.
The comparative example of embodiment 1 is a kind of cultural method of autologous peripheral blood lymphocyte DC-CIK of CN104357394A,The main distinction is autologous tumor antigens, TLR7 agonist whether are added in medium component and incubation, and additionTime, concentration.
Experimental result
The autologous peripheral blood lymphocyte made from the 1st, 7,14,18,21 day collection the present embodiment after closing bottle culture respectivelyIt is tested for the following terms.
1, the measurement of cell Proliferation multiple
It will be counted again with blood counting chamber after the cell of acquirement Trypan Blue, and record count is as a result, with trainingSupport number of days be abscissa, using amplification times as ordinate (amplification times=by current total viable cell ÷ starting in the 1st day cultureMononuclearcell number) mapping, as a result as shown in table 1 and Fig. 1.
The number of amplification of 1 embodiment 1 of table and comparative example difference incubation time cell
Embodiment 1 and comparative example culture are in slowly to grow in the 1-7 days cells substantially known to above-mentioned table 1 and Fig. 1Gesture starts to reach logarithmic growth phase after the 7th day, reaches greatest measure by the 21st day.The cells expanded of embodiment 1 is highIn comparative example, so the method for embodiment 1 can be improved cell amplification efficiency.
2, cell killing activity is tested
The CIK cell for taking culture to the 14th day, with breast carcinoma cell strain (being purchased from Shanghai Mei Yan Biotechnology Co., Ltd)In the ratio of 40:1,20:1 and 10:1 be laid in 96 orifice plates 37 DEG C, percent by volume be 6%CO2 under conditions of carry out total trainingIt supports, every hole is added 10 μ L of CCK-8 reagent and is dyed after culture for 24 hours, in the incubator (37 DEG C, percent by volume 7%CO2 the OD value for) detecting each hole after incubation 2h under the wavelength of 450nm, is calculated as follows the killing rate of CIK cell, tiesFruit is as shown in table 2 and figure 2.
Cell survival rate (%)=(experimental group A450Average value-zeroing group A450Average value)/(comparative example A450Average value-Zeroing group A450Average value) × 100%
Kill tumor activity (%)=1- cell survival rate
2 embodiment 1 of table compares the killing activity (%, X ± SD) of breast cancer cell line with comparative example difference effect target
*P<0.05
It can be seen that 10-40:1 effect target than in range, 1 cell of embodiment and comparative example CN104357394A cellStronger lethal effect is all had to breast cancer cell, and lethal effect increases with the increase of effect target ratio；In same effect target ratioIn the case of, 1 cell of embodiment is significantly higher than comparative example CN104357394A cell (P < 0.05) to the killing activity of breast cancer.
2, cell phenotype is analyzed
(1) 1 cell of embodiment and comparative example CN104357394A cell of culture to the 14th day are taken respectively, and transfer becomes a mandarinFormula detection pipe, adds 3mLPBS to mix well, centrifuge washing cell (centrifugal condition: 1600rpm, 5min).Incline centrifugation supernatant,According to cell sample concentration and volume, with appropriate PBS suspension cell, adjustment cell concentration is 1x107/mL；
(2) fluorescence labeled cell: monoclonal antibody to be detected, monoclonal antibody to be detected is added in label flow cytometer detection pipeIncluding CD3+CD56+, CD3+CD8+, CD4+CD25+, every kind of amount of antibody is 8 μ l.It is outstanding that 100ul cell sample to be measured is added in every pipeLiquid；
(3) gently rotation mixes in vortex oscillator, and room temperature, which is protected from light, is incubated for 15min；
(4) plus 3mLPBS is washed, and centrifugation, 1600rpm, 5min, incline supernatant, 500ulPBS suspension precipitating is added, sufficientlyIt mixes；
(5) machine testing on, as a result as shown in table 3 and figure 3.
3 embodiment 1 of table and comparative example cell phenotype analysis (%)
| Embodiment 1 | Comparative example CN104357394A |
| CD3+CD56+ | 42.8 | 34.5 |
| CD3+CD8+ | 80.2 | 60.6 |
| CD4+CD25+ | 12.1 | 1.2 |
The results show that the ratio of the CD3+CD56+ cell on its surface of the CIK cell of embodiment 1 of culture 14 days is42.8%, CD3+CD8+ is 80.2%, is above comparative example.CD3+CD56+ and CD3+CD8+ is that the main effects of CIK is thinBorn of the same parents group.Because CD3+CD56+ is the effector cell group of non-specific killing tumour, therefore the raising of its cell proportion is to antitumorThe expansion of spectrum has very positive meaning.The ratio of CD3+CD56+ cell and CD3+CD8+ are higher, and it is stronger that cell kills tumor activity,Thus having confirmed has preferable cancer cell fragmentation effect by using the CIK cell of method culture of the invention.
Embodiment 2
Method as described in Example 1, the difference is that, every milliliter of peripheral blood lymphocytes culture medium containing as followsComponent: RPMI-1460 culture medium 0.65ml, fetal calf serum 0.25ml, gentamicin 320ng, L-Glutamine 450ng, plantAgglutinin (PHA) 650ng；The final concentration of 40ug/ml of autologous tumor antigens be added, the final concentration of 10ng/ of TLR7 agonistml。
Experimental result
Detection process is as described in Example 1.Through detecting, as a result as shown in table 4 and figure 4.
1, the measurement of cell Proliferation multiple
The number of amplification of 4 embodiment 1 of table and the different incubation time autologous peripheral blood lymphocytes of embodiment 2
The results show that culture, at 1~21 day, the amplification times of the CIK cell of embodiment 1 and embodiment 2 are at any timeElapse and be gradually increasing, 1~14 day when, remains basically stable, 14~18 days due to cell radix is larger and passage, embodiment2 amplification times are higher than 1,18~21 days amplification efficiencies of embodiment and increase, and reach maximum value when culture was by the 21st day, with realityIt applies example 1 to compare, 2 amplification times of embodiment are higher.Nutrient media components, autologous tumor antigens, TLR7 agonist is further changed to addDosage compares test, as a result, it has been found that under conditions of embodiment 2, cell number of amplification highest.
2, cytotoxicity experiment, as a result as shown in table 5, Fig. 5
5 embodiment 1 of table and the different effect targets of embodiment 2 compare the killing activity (%, X ± SD) of breast cancer cell line
*P<0.05
The results show that the CIK cell of culture to the 14th day is above 70% to the High Fragmentation power of breast cancer cell.Imitate targetThan for 20:1, CIK cell kills tumor activity highest.The CIK cell killing rate of embodiment 2 is when effect target is than for 40:192.01%, when effect target ratio is 20:1, reach 98.35%, compared with Example 1, averagely killing rate increases.Further changeBecome nutrient media components, autologous tumor antigens, TLR7 agonist additive amount and compare test, as a result, it has been found that the condition of embodiment 2Under, to the killing activity highest of breast cancer cell line.
3, cell phenotype is analyzed, as a result as shown in table 5, Fig. 5
6 embodiment 1 of table and 2 cell phenotype of embodiment analysis (%)
| Embodiment 1 | Embodiment 2 |
| CD3+CD56+ | 42.8 | 46.7 |
| CD3+CD8+ | 80.2 | 85.4 |
| CD4+CD25+ | 12.1 | 14.6 |
The results show that the ratio of the CD3+CD56+ cell on its surface of the CIK cell of embodiment 2 of culture 14 days is46.7%, 85.4%, the CD3+CD56+ and CD3+CD8+ of CD3+CD8+ is the main effects cell group of AIL.With 1 phase of embodimentThan being significantly improved by the content that embodiment 2 cultivates obtained CIK cell its CD3+CD56+ cell.
Embodiment 3
Method as described in Example 1, the difference is that autologous tumor antigens are added without, TLR7 agonist.With comparisonThe main distinction of example is the time of medium component and cell factor addition, concentration.
Experimental result
Detection process is as described in Example 1.Through detecting, as a result as shown in table 7, Fig. 7
1, the measurement of cell Proliferation multiple
The number of amplification of 7 embodiment 1 of table, embodiment 3 and comparative example difference incubation time autologous peripheral blood lymphocyte
The results show that culture is at 1~21 day, the passage at any time of the amplification times of CIK cell and be gradually increasing, 1~It is substantially in slow ascendant trend at 7 days, making amplification times substantially due to cell radix is larger and passage within 7~18 days is inIt ramps, 18~21 days amplification efficiencies increase, and reach maximum value, 3 cells expanded of embodiment when culture was by the 21st dayHigher than comparative example, it is seen that method provided by the present invention, medium component and the time of cell factor addition, concentration are playedEffect.3 cells expanded of embodiment is lower than embodiment 1, it is seen that method provided by the invention addition autologous tumor antigens,The effect that TLR7 agonist is played.
2, cytotoxicity experiment, as a result as shown in table 8, Fig. 8
8 embodiment 1 of table, embodiment 3 and comparative example difference effect target compare breast cancer cell line killing activity (%, X ±SD)
*P<0.05
The results show that the CIK cell of culture to the 14th day is above 60% to the High Fragmentation power of breast cancer cell, implementIt is 70.09% that example 3, which imitates CIK cell killing rate of the target than being 10:1 when, imitates CIK cell killing rate of the target than for 20:1 when and is87.65, CIK cell killing rate when effect target is than for 20:1 is.Compared with comparative example, effect target ratio is 10:1, embodiment when 20:13 averagely killing rates are higher by 5% or more, it is seen that method provided by the present invention, medium component and cell factor addition whenBetween, the effect that is played of concentration.3 cell killing rate of embodiment is compared lower than embodiment 1, it is seen that method provided by the invention addsThe effect for adding autologous tumor antigens, TLR7 agonist to be played.
3, cell phenotype is analyzed, as a result such as table 9, shown in Fig. 9
9 embodiment 1 of table, embodiment 3 and comparative example cell phenotype analysis (%)
| Embodiment 1 | Embodiment 3 | Comparative example CN104357394A |
| CD3+CD56+ | 42.8 | 40.1 | 34.5 |
| CD3+CD8+ | 80.2 | 71.9 | 60.6 |
| CD4+CD25+ | 12.1 | 6.34 | 1.2 |
The results show that the ratio of the CD3+CD56+ cell on its surface of the CIK cell of embodiment 3 of culture 14 days is40.1%, 71.9%, the CD3+CD56+ and CD3+CD8+ of CD3+CD8+ is the main effects cell group of AIL.With comparative example phaseThan being improved largely by the content that embodiment 3 cultivates obtained CIK cell its CD3+CD56+, CD3+CD8+ cell, it is seen thatThe effect that method provided by the present invention, medium component and the time of cell factor addition, concentration are played.CD3+CD56+, the content of CD3+CD8+ cell be lower than embodiment 1, it is seen that method addition autologous tumor antigens provided by the invention, TLR7 swashThe effect that dynamic agent is played.
It should be noted that, in this document, relational terms such as first and second and the like are used merely to a realityBody or operation are distinguished with another entity or operation, without necessarily requiring or implying between these entities or operationThere are any actual relationship or orders.Moreover, the terms "include", "comprise" or its any other variant are intended toCover non-exclusive inclusion, so that the process, method, article or equipment for including a series of elements not only includes thatA little elements, but also including other elements that are not explicitly listed, or further include for this process, method, article orThe intrinsic element of equipment.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be withA variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understandingAnd modification, the scope of the present invention is defined by the appended.