CN104288179A - Dendritic cell, preparation method thereof and application - Google Patents
Dendritic cell, preparation method thereof and application Download PDFInfo
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- CN104288179A CN104288179A CN201410356051.8A CN201410356051A CN104288179A CN 104288179 A CN104288179 A CN 104288179A CN 201410356051 A CN201410356051 A CN 201410356051A CN 104288179 A CN104288179 A CN 104288179A
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Abstract
The invention provides a dendritic cell, a preparation method thereof and application. Concretely, the invention provides application of combination of the dendritic cell and cytokine-induced killer cell to prepare a medicine for treating or inhibiting liver cancer, wherein the dendritic cell is capable of specifically recognizing liver cancer nidus. According to the embodiment in the invention, the combination of the dendritic cell and the cytokine-induced killer cell is capable of effectively treating or inhibiting liver cancer.
Description
Technical field
The present invention relates to biomedicine field, concrete, the present invention relates to dendritic cell and its production and use, more specifically, what the present invention relates to dendritic cell and cytokine-induced killer cell sexual cell is combined in the purposes prepared in medicine, the method preparing dendritic cell, medicine and dendritic cell.
Background technology
Hepatocarcinoma is Chinese second tumor killer, and annual death toll more than 30 ten thousand, accounts for 55% of whole world PLC mortality sum.The Main Means of current Hepatoma therapy is excision, liver transplantation, local ablation therapy, hepatic artery interventional therapy, radiotherapy, chemotherapy and other treatment.
In the world, any area finds all equally, and the chronic hepatopathy that any reason causes all may occur in hepatocarcinoma and play an important role in evolution.What epidemiology and experimentation all showed viral hepatitis and primary hepatocarcinoma has specific relation, at present clearer and more definite have B-mode, the third type and 3 kinds, fourth type with the related viral hepatitis of hepatocarcinoma, wherein close with hepatitis B and Relations with Liver Cancer, main manifestations be following some: hepatocarcinoma parallels with the incidence rate of HBsAg carrier.
But the treatment means of existing hepatocarcinoma still haves much room for improvement.
Summary of the invention
The present invention is intended to solve one of technical problem in correlation technique at least to a certain extent.For this reason, one object of the present invention is to propose that a kind of have can the means of effective Hepatoma therapy.
In a first aspect of the present invention, the present invention proposes a kind of dendritic cell (dendritic cell, and cytokine-induced killer cell sexual cell (cytokine-induced killer DC), CIK) be combined in the purposes prepared in medicine, described medicine is used for the treatment of or suppresses hepatocarcinoma, wherein, described specific for dendritic cells identification liver lesion.
Dendritic cell is the professional antigen presenting cell that body function is the strongest, and ripe dendritic cell can be absorbed efficiently, processed and present antigen, and can effectively activate primary tape T cell, is in startup, regulates and controls and maintains the important step of immunne response.
Cytokine-induced killer cell sexual cell is the T lymphocyte obtained after cytokine profiles co-cultivation a period of time in vitro by the peripheral blood PBMC of people (Peripheral Blood Monoclonal Cells), because this cell expresses CD3 and CD56 two kinds of membrane protein molecules simultaneously, therefore be also called NK cell (natural killer cell) sample T lymphocyte.Cytokine-induced killer cell aggressive cellular proliferation ability is strong, cultivates in vitro and within about two weeks, can make main effects cell (CD3
+, CD56
+) increment more than 1000 times; Cytokine-induced killer cell sexual cell toxic action is strong, is much better than traditional LAK cell and cytokine IFN-γ, interleukin II; Cytokine-induced killer cell sexual cell has cytotoxicity widely, kills tumor cell not injuring under Normocellular condition by number of ways targeting; Cytokine-induced killer cell sexual cell also has immunoregulation effect.DC and CIK cell use in conjunction will complete the overall process of tumor cell, killing off tumor cells.Research shows that DC cell can make suppressor T cell (CD4
+cD25
+) quantity reduce, reduced activity, so co-cultivation DC, CIK cell with cultivate merely compared with CIK cell at ability of cell proliferation, effector lymphocyte (CD3
+, CD8
+, CD3
+cD56
+) quantity and the aspects such as the cytotoxicity of tumor cell are significantly strengthened.
The DC-CIK immune cell therapy of tumor is the 4th kind of antineoplaston pattern after operation, radiotherapy, chemotherapy, autologous to tumor patient input, to increase and the immunocyte activated in vitro, direct killing tumor cell or excitating organism anti tumor immune response, thus the object reaching treatment tumor.For solid tumor patients after surgery, this treatment can residual tumor cells in purged body, obviously reduce the transfer after tumor operation and recurrence probability; For chemicotherapy patient, this treatment can be removed residual tumor cell, strengthens the sensitivity of chemicotherapy, be alleviated the toxic and side effects of chemicotherapy; For performing the operation and the Advanced cancers patient of chemicotherapy, this treatment can growth, the enhancing immunity of Tumor suppression, quality of making the life better, the extending life cycle.Tumor vaccine cells treatment exceedes tens thousand of example in the accumulative treatment case in the whole world, and efficacy and saferry is all more satisfactory.
Therefore, Combined with Dendritic cytokine-induced killer cell sexual cell immunotherapy and DC-CIK immunotherapy are incorporated in the treatment of anti-hepatocarcinoma, improve the lethal effect of anti-liver cancer immunity effector lymphocyte in prior art, by the interpolation of cytokine, find the lethal cell culture processes of new dendritic cell and the induction of cytokine Combined culture, significant at biological technical field.
According to embodiments of the invention, described dendritic cell can obtain through the following steps: (1) obtains peripheral blood, and described peripheral blood comes from the individuality carrying described focus; (2) from described peripheral blood, mononuclearcell is separated; (3) stimulate described mononuclearcell to differentiation of dendritic cells, to obtain described dendritic cell, wherein, comprise in step (3) and the specific antigen of described mononuclearcell with described focus contacted, described specific antigen be alpha-fetoprotein and Hepatitis B virus vaccine one of at least.
According to embodiments of the invention, described individuality suffers from chronic viral hepatitis B further.Inventor is surprised to find, and dendritic cell of the present invention (DC) can effectively treat chronic viral hepatitis B hepatocarcinoma with the combination of cytokine-induced killer cell sexual cell.
According to embodiments of the invention, step (3) comprises further: (3-1) utilizes M-CSF and recombinant human interleukin 4 to stimulate described mononuclearcell to differentiation of dendritic cells; (3-2) contacted with Hepatitis B virus vaccine with alpha-fetoprotein by the cell obtained in step (3-1), the weight concentration ratio of described alpha-fetoprotein and described Hepatitis B virus vaccine is 1:1; And (3-3) one of at least contacting the cell obtained in step (3-2) and IL-β, IL-6 and TNF-α, to obtain described dendritic cell.
In serum in patients with primary hepatic, alpha-fetoprotein (α-fetoprotein) AFP raises and can reach 250ug ~ 6mg/ml, (even reaching 9mg/ml), be equivalent to the decades of times of normal person and even tens thousand of times, therefore the mensuration of Serum AFP is the method for early diagnosis that current diagnosis primary hepatocarcinoma is the most frequently used, more responsive, specificity is stronger, has now been widely used in the generaI investigation of hepatocarcinoma, diagnosis, judgement therapeutic effect, prediction recurrence.AFP and tumor size have certain dependency, and namely tumor is less, and positive rate is lower, and AFP is also relevant to histological type, Carcinoma cell differentiation I level and II level, and AFP is relatively low, relatively high when III grade.Adopt AFP heteroplasmon and AFP Simultaneously test in recent years, diagnosing cancer of liver positive rate can be made to be increased to 92%, and false positive is lower than 15%.
Inventor is surprised to find, and obtains dendritic cell can significantly improve the specific recognition of obtained dendritic cell to liver lesion by adopting AFP (alpha-fetoprotein) to carry out stimulating.
According to embodiments of the invention, the concentration of described M-CSF is 1000U/mL, and the concentration of described recombinant human interleukin 4 is 500U/mL.
According to embodiments of the invention, the concentration of described alpha-fetoprotein and described Hepatitis B virus vaccine is separately 40 ~ 60 mcg/ml, most preferably 50 mcg/ml.
According to embodiments of the invention, the combination of described dendritic cell and cytokine-induced killer cell sexual cell is by carrying out Dual culture acquisition by described dendritic cell and described cytokine-induced killer cell sexual cell, wherein, when described Dual culture, the inoculative proportion of described dendritic cell and described cytokine-induced killer cell sexual cell is 1:8 ~ 12, preferred 1:10.
According to embodiments of the invention, described cytokine-induced killer cell sexual cell obtains through the following steps: (4-1) utilizes the mononuclearcell that obtains in IFN-γ stimulation step (2) 24 hours, wherein, the concentration of described IFN-γ is 1000U/ml; And the cell obtained in step (4-1) contacts with recombinated interleukin-2 with CD3 monoclonal antibody by (4-2), to obtain described cytokine-induced killer cell sexual cell, wherein, the concentration of described CD3 monoclonal antibody is the concentration 500U/ml of 50ng/ml, recombinated interleukin-2.
In a second aspect of the present invention, the present invention proposes a kind of method preparing dendritic cell, wherein, described specific for dendritic cells identification liver lesion.According to embodiments of the invention, described method comprises: (1) obtains peripheral blood, and described peripheral blood comes from the individuality carrying described focus; (2) from described peripheral blood, mononuclearcell is separated; (3) described mononuclearcell is stimulated to break up to dendritic cell direction, to obtain described dendritic cell, wherein, comprise in step (3) and the specific antigen of described mononuclearcell with described focus contacted, described specific antigen be alpha-fetoprotein and Hepatitis B virus vaccine one of at least.
According to embodiments of the invention, step (3) may further include: (3-1) utilizes M-CSF and recombinant human interleukin 4 to stimulate described mononuclearcell to differentiation of dendritic cells; (3-2) contacted with Hepatitis B virus vaccine with alpha-fetoprotein by the cell obtained in step (3-1), the weight concentration ratio of described alpha-fetoprotein and described Hepatitis B virus vaccine is 1:1; And (3-3) one of at least contacting the cell obtained in step (3-2) and IL-β, IL-6 and TNF-α, to obtain described dendritic cell.
According to embodiments of the invention, the concentration of described M-CSF is 1000U/mL, and the concentration of described recombinant human interleukin 4 is 500U/mL.
According to embodiments of the invention, the concentration of described alpha-fetoprotein and described Hepatitis B virus vaccine is separately 40 ~ 60 mcg/ml, most preferably 50 mcg/ml.
In a third aspect of the present invention, the present invention proposes a kind of medicine, described medicine is used for the treatment of or prevents liver cancer.According to embodiments of the invention, described medicine comprises: the combination of dendritic cell and cytokine-induced killer cell sexual cell, wherein, and described specific for dendritic cells identification liver lesion.
According to embodiments of the invention, described dendritic cell is prepared by foregoing method.
In a fourth aspect of the present invention, the present invention proposes a kind of dendritic cell, described specific for dendritic cells identification liver lesion.
According to embodiments of the invention, described dendritic cell is prepared by foregoing method.
Particularly, according to embodiments of the invention, the invention provides a kind of Combined with Dendritic cytokine-induced killer cell sexual cell cultural method for anti-hepatocarcinoma, it comprises the following steps:
Step a: the preparation of autologous plasma
In Biohazard Safety Equipment, the 40ml-60ml anticoagulated whole blood average mark in syringe is installed in 2 50mL sterile centrifugation tube, with centrifuge, centrifugal rotational speed is 2500rpm, centrifugal 10min, centrifugal complete after absorption upper plasma, 56 DEG C, 30min deactivation complement, is placed in 4 DEG C of water-baths and places 15 minutes, use centrifuge again, centrifugal speed is 2500rpm, centrifugal 20 minutes, and transfer supernatant is to new centrifuge tube.Labelling, is placed in-20 DEG C of refrigerators and preserves;
Step b: the separation of mononuclearcell
Buffer salt solution 25mL is not added, diluted blood cell, piping and druming limit, limit rotating centrifugal pipe, mixing by the hemocyte drawn after centrifugal described in step a.The diluted blood getting 30ml mixing is slowly added to and is equipped with in the 50mL centrifuge tube of lymphocyte separation medium.With centrifuge, centrifugal rotational speed is centrifugal 30 minutes of 2100rpm; Sucking-off tunica albuginea layer is in the centrifuge tube that 30ml buffer salt solution is housed, and with centrifuge, centrifugal speed is 2100rpm, centrifugal 15 minutes;
Step c: centrifuge washing, counting
By the cell suspension obtained in above-mentioned steps b, abandon supernatant, often pipe adds 5mL buffer salt solution, and re-suspended cell precipitates, and is transferred to a pipe, then draws after appropriate buffer salt solution rinsing is respectively managed and collect residual cell, and be settled to 30mL, keep sample counting.With centrifuge, centrifugal rotational speed 1500rpm, centrifugal 10 minutes;
Steps d: re-suspended cell, inoculation
By the cell suspension obtained in above-mentioned steps c, abandon supernatant, get 5mL culture fluid re-suspended cell precipitation, moving into surface area is in the culture bottle of 75 square centimeters, every bottle of 1*10
6the density of/mL, is placed in 37 DEG C, 5%CO
2in the cell culture incubator of saturated humidity, hatch 2 hours;
The cultivation of step e:DC cell and qualification
From the culture bottle described in above-mentioned steps d, sucking-off suspension cell is for subsequent use, adds rhGM-CSF1000U/ml, rhIL-4 500U/ml, and irritation cell, to DC cell differentiation, expands bottle; Alpha-fetoprotein and Hepatitis B virus vaccine 50 μ g/ml within 5th day, is added what cultivate; IL-β, IL-6, TNF α cytokine within 6th day, is added what cultivate; 7th day results DC cell, Trypan Blue detects cell viability, flow cytomery DC cell CD1a, CD11c and CD83 immunophenotypic marker;
The cultivation of step f:CIK cell and qualification
Suspension cell described in step e is re-seeded in culture bottle, adds IFN-γ 1000U/ml; CD350ng/ml monoclonal antibody and rhIL-2500U/ml is added after 24h; Cultivate; Sampling in 7th day, Trypan Blue detects cell viability, flow cytomery CIK cell CD3
+cD56
+immunophenotypic marker.
Step g: DC-CIK Coculture
The DC cell of collection is added CIK cell Combined culture, after last supplemented medium, antibacterial is carried out in sampling, fungus, endotoxin detect, confirm testing result feminine gender after the 4th day harvesting, Trypan Blue detects cell viability, flow cytomery CIK cell CD3
+cD56
+immunophenotypic marker;
Step h: cell harvesting
To be dispensed in 50ml centrifuge tube after the cell suspension mixing in cell culture bags, 45ml/ manages, trim.With centrifuge, centrifugal rotational speed is 1500rpm, 10min.Abandon supernatant, often pipe adds the normal saline that 5ml contains diluting plasma, and evenly, every 8 pipes close 1 pipe in piping and druming, then collect residual cell, trim with each centrifuge tube of a small amount of normal saline rinsing.With centrifuge, centrifugal rotational speed is 1500rpm, 10min.Abandon supernatant, add normal saline resuspended, be combined into 1 pipe, trim.With centrifuge, centrifugal rotational speed is 1500rpm, 10min.Abandon supernatant, flick at the bottom of centrifuge tube, add 10mL cell re-suspension liquid with pipet, blow and beat gently, re-suspended cell, first suct and run underneath to precipitation along tube wall clearly and scatter and blow and beat again, then add 10mL cell re-suspension liquid and the mixing of 5mL albumin.Prepare normal saline bag and put into super-clean bench, alcohol gauze wiping mouth of pipe place, open mouth of pipe vinyl cover and expose cork, open 50ml injector package, syringe is stretched into sucking-off cell suspension by inclination centrifuge tube.The syringe needle changing syringe is No. 9 syringe needles, and cell suspension upwards, injects, injected rear gas bleeding by bag of saline, and load packaging bag, pass-through box spreads out of.
In addition, cell culture medium according to the above embodiment of the present invention can also have following additional technical characteristic: the buffer salt solution described in described step b is PBS solution.
According to embodiments of the invention, the lymphocyte separation medium described in described step a is ficoll separating medium.
According to embodiments of the invention, the tunica albuginea layer in described step b is be divided into the intermediate layer of 3 layers after centrifugal completing.
According to embodiments of the invention, the culture fluid in described steps d is GT-T551 culture fluid.
According to embodiments of the invention, the expansion bottle described in described step e refers to 1 bottle of expansion in the 3rd day that cultivates 3 bottles, 3 bottles of expansions in the 6th day 9 bottles.
According to embodiments of the invention, the cultivation described in described step f is cultivated for expanding bottle.
According to embodiments of the invention, the cultivation described in described step f is cultivate in culture bag.
According to embodiments of the invention, the ratio of the DC cell described in described step g and CIK cell associating is 1:10.
According to embodiments of the invention, the content of the diluting plasma described in described step h is 4%.
Detailed description of the invention
Embodiments of the invention are described below in detail.Below by being exemplary with reference to the embodiment described, only for explaining the present invention, and limitation of the present invention can not be interpreted as.
Embodiment 1
Intrahepatic cholangiocellular carcinoma postoperative patient autologous blood DC-CIK immune cell therapy, particular content is as follows:
1, clinical data
Case 1, female, 69 years old, intrahepatic cholangiocellular carcinoma, for postoperative pathological examination checks confirmed cases.
2, main material
(a) consumptive material: lymphocyte separation medium (GE healthcare), GT-T551 culture medium (purchased from Bao Yi Bioisystech Co., Ltd), rhGM-CSF, rhIL-4, rhIL-2, IL-β, IL-6, IFN-γ, TNF α, alpha-fetoprotein, Hepatitis B virus vaccine, CD3 monoclonal antibody (Beijing plan Pharmaceutical far away responsibility company limited), 50ml centrifuge tube, 10ml centrifuge tube, 5ml pipet, 25ml pipet, Pasteur's tubule, Tissue Culture Flask, cell culture bags (Thermo), gentamycin (purchased from Yichang people's good fortune Pharmaceutical responsibility company limited).
(b) instrument: flow cytometer (beckman), Biohazard Safety Equipment (power health), cell culture incubator (power health), high speed centrifuge (Kubo field), inverted microscope (south of the River), ultra cold storage freezer (Haier), nitrogen storage tank (Thermo).
Concrete steps:
(1) Peripheral Blood of Patients with Hepatocellular Carcinoma 45mL is extracted with the syringe containing anticoagulant;
(2) in Biohazard Safety Equipment, 50mL anticoagulated whole blood is divided equally in the sterile centrifugation tube of two 50mL, the centrifugal 10min of 2500rpm;
(3) centrifugal complete after upper plasma (often pipe is about 10mL) is transferred in new centrifuge tube, 56 DEG C, 30min deactivation complement, 4 DEG C of water-bath 15min, 2500rpm*20min centrifugal segregation fibrin, is transferred to new centrifuge tube by supernatant, is placed in-20 DEG C of refrigerators for subsequent use;
(4) in remaining hemocyte, add PBS often pipe 25mL, at the bottom of piping and druming limit, limit rotating centrifugal pipe to mixing emphasis piping and druming tube wall and pipe, stay 1mL diluted blood to do five indexes of hepatitis b, syphilis, AIDS, hepatitis C detection.
(5) every for the diluted blood of mixing 30ml is slowly added to is equipped with in the 50ml centrifuge tube of 15mLficoll.The centrifugal 30min of 2100rpm, slow liter falls slowly;
(6) centrifugal complete after be divided into 3 layers, carefully draw middle tunica albuginea layer with pasteur pipet and go to and be equipped with in the centrifuge tube of 30mlPBS, often pipe 42.5mL, trim centrifugal 2100rpm, 10min;
(7) abandon supernatant, often pipe adds the PBS re-suspended cell of 5mL, and soft piping and druming is combined into a pipe, then is settled to 30ml with each pipe of PBS rinsing of 5mL, and keep sample counting, tightens lid centrifugal 2100rpm, 10min;
(8) abandon supernatant, use GT-T551 re-suspended cell, in step (7), cell counting is 7.3*10
7, by every bottle of 1*10
6the density of/ml is inoculated in 75cm
2culture bottle in, plant 2 bottles, being placed in temperature is 37 DEG C, CO
22h (being designated as the 0th day) is hatched in the cell culture incubator of % saturated humidity.
(9) sucking-off non-adherent cell is for subsequent use, rhGM-CSF1000U/ml (concentration is final concentration), rhIL-4500U/ml is added in attached cell, irritation cell is to DC cell differentiation, the 3rd day adherence rate cultivated is about 85%, culture fluid color becomes orange, and 1 bottle is expanded 3 bottles; Within 6th day, adherence rate is about 80%, and culture fluid color becomes orange 1 bottle and expands 3 bottles again; Within 5th day, add Hepatitis B virus vaccine 50 μ g/ml what cultivate, add alpha-fetoprotein according to the concentration of 1:1, the generation of stimulator antigen specific cell; Within 6th day, add IL-β, IL-6, TNF α cytokine what cultivate, the final concentration of often kind of cytokine is 10-100ng/ml, stimulates DC cell maturation; 7th day results DC cell; Sampling, Trypan Blue detects cell viability, flow cytomery DC cell CD1a, CD11c and CD83 immunophenotypic marker;
(10) suspension cell in step (9) is re-seeded in culture bottle, adds IFN-γ 1000U/ml;
A, the CD3 monoclonal antibody adding 50ng/ml after 1 day and rhIL-2500U/ml stimulate growth and the propagation of CIK cell;
Under b, the 4th clear water surface, observation of cell quantity is many, conglomeration is comparatively large, culture medium color becomes crocus, and transferred to by cell in culture bag, volume augmentation is to 200ml;
Under c, the 5th clear water surface, observation of cell volume increases, endochylema is few, core is large, and conglomeration is many, and culture medium becomes crocus fluid infusion to 600ml;
Under d, the 6th clear water surface, observation of cell growth conditions is good, and the golden yellow fluid infusion of culture medium color is to 1200ml, and mixing, samples coated plate and do antibacterial, fungal detection;
(11) DC cell is added in CIK cell culture bag in the ratio of 1:10 mix, continue to cultivate;
Within (12) the 9th days, expand bag.1200ml culture fluid is divided and is filled in two culture bag, wherein the culture fluid of 600ml is divided and be filled in a bag, and utilize supplementing culture medium to 1200ml, the culture fluid of other 600ml is divided and is filled in b bag, and utilize supplementing culture medium to 1500ml, two bags all will sample quality inspection;
(13) the 12nd days results a bag cells
A, subpackage: will be dispensed in a 50ml centrifuge tube after the cell suspension mixing in cell culture bags, 45ml/ manages, totally 27 pipes, trim.5ml cell suspension sample is stayed to do endotoxin and Gram’s staining detection and count;
B, centrifugal collecting cell: 1500rpm*10min;
C, for the first time centrifuge washing: abandon supernatant, often pipe adds 5ml normal saline (diluting plasma containing 4%), piping and druming is evenly, every 8 pipes close 1 pipe, totally 4 pipes, then collect residual cell with each centrifuge tube of a small amount of normal saline rinsing, trim, 1500rpm*10min;
D, second time centrifuge washing: abandon supernatant, add normal saline resuspended, be combined into 1 pipe, trim.With centrifuge, centrifugal rotational speed is 1500rpm, centrifugal 10min;
E, re-suspended cell: abandon supernatant, first flick at the bottom of centrifuge tube, makes cell precipitation loose.10ml cell re-suspension liquid is added again with 10ml pipet, blow and beat gently, re-suspended cell, start not direct sucking-off precipitation, to first suct clearly along tube wall run underneath to precipitation scatter blow and beat again (checked whether that granule precipitates, 70um metre filter cell precipitation will have been used if cannot not blow loosely) add again 5ml10 cell re-suspension liquid and 5ml albumin mixing.
F, by cell suspension inject by normal saline bag: prepare normal saline bag put into super-clean bench, alcohol gauze wiping mouth of pipe place, open mouth of pipe vinyl cover and expose cork, open 50ml injector package, do not encounter the body of below syringe graduation line, syringe is stretched into sucking-off cell suspension by inclination centrifuge tube, ensures sterile working.The syringe needle changing syringe is No. 9 syringe needles, and cell suspension upwards, injects, observes simultaneously and whether have precipitation, injected the gas of rear extraction respective volume by bag of saline.
G, inspection confirm: again checked whether precipitation, check whether injection pin hole place has liquid to ooze out and determines errorless rear loading packaging bag, and pass-through box spreads out of.
(14) the 13rd days results b bag cells, the same step of method (13)
(15) a, b cell counting is respectively 3.54*10
9, 4.57*10
9;
Embodiment 2:
Primary hepatocarcinoma II phase autologous patient blood DC-CIK immune cell therapy, particular content is as follows:
1, clinical data
Case 2, man, 66 years old, primary hepatocarcinoma II phase, for postoperative pathological examination checks confirmed cases.
2, main material
(a) consumptive material: lymphocyte separation medium (GE healthcare), GT-T551 culture medium (deriving from Bao Yi Bioisystech Co., Ltd), rhGM-CSF, rhIL-4, rhIL-2, IL-β, IL-6, IFN-γ, TNF α, Hepatitis B virus vaccine, CD3 monoclonal antibody (Beijing plan Pharmaceutical far away responsibility company limited), 50ml centrifuge tube, 10ml centrifuge tube, 5ml pipet, 25ml pipet, Pasteur's tubule, Tissue Culture Flask, cell culture bags (Thermo fisher), gentamycin (deriving from people from Yichang good fortune Pharmaceutical responsibility company limited).
(b) flow cytometer (bekman), Biohazard Safety Equipment (power health), cell culture incubator (power health), high speed centrifuge (Kubo field), inverted microscope (south of the River), ultra cold storage freezer (Haier), nitrogen storage tank (Thermo).
(1) Peripheral Blood of Patients with Hepatocellular Carcinoma 55ml is extracted with the syringe containing anticoagulant;
(2) in Biohazard Safety Equipment, 50ml anticoagulated whole blood is divided equally in the sterile centrifugation tube of two 50ml, the centrifugal 10min of 2500rpm;
(3) centrifugal complete after upper plasma (often pipe is about 12.5ml) is transferred in new centrifuge tube, 56 DEG C, 30min deactivation complement, 4 DEG C of water-bath 15min, 2500rpm*20min centrifugal segregation fibrin, is transferred to new centrifuge tube by supernatant, is placed in-20 DEG C of refrigerators for subsequent use;
(4) in remaining hemocyte, add PBS often pipe 25ml, at the bottom of piping and druming limit, limit rotating centrifugal pipe to mixing emphasis piping and druming tube wall and pipe, stay 1ml diluted blood to do five indexes of hepatitis b, syphilis, AIDS, hepatitis C detection.
(5) every for the diluted blood of mixing 30ml is slowly added to is equipped with in the 50ml centrifuge tube of 15ml ficoll.The centrifugal 30min of 2100rpm, slow liter falls slowly;
(6) centrifugal complete after be divided into 3 layers, carefully draw middle tunica albuginea layer with pasteur pipet and go to and be equipped with in the centrifuge tube of 30mlPBS, often pipe 42.5ml, trim centrifugal 2100rpm, 10min;
(7) abandon supernatant, often pipe adds 5mlPBS re-suspended cell, and soft piping and druming is combined into a pipe, then is settled to 30ml with each pipe of 5mlPBS rinsing, and keep sample counting, tightens lid centrifugal 2100rpm, 10min;
(8) abandon supernatant, use GT-T551 re-suspended cell, in step (7), cell counting is 8.1*10
7press according to every bottle of 1*10
6the density of/ml is inoculated in 75cm
2culture bottle in, plant 2 bottles, be placed in cell culture incubator, 37 DEG C, CO
2% hatches 2h (being designated as the 0th day).
(9) sucking-off non-adherent cell is for subsequent use, add rhGM-CSF1000U/ml (concentration is final concentration), rhIL-4500U/ml, irritation cell, to DC cell differentiation, is about 75% at the 3rd day adherence rate cultivated, culture fluid color becomes orange, and 1 bottle is expanded 3 bottles; ; Hepatitis B virus vaccine 50 μ g/ml within 5th day, is added, the generation of stimulator antigen specific cell what cultivate; Be about 80% at cultivation the 6th day adherence rate, culture fluid color becomes orange 1 bottle and expands 3 bottles again, adds IL-β, IL-6, TNF α cytokine, and the final concentration of often kind of cytokine is 10-100ng/ml, stimulates DC cell maturation; 7th day results DC cell; Sampling, Trypan Blue detects cell viability, flow cytomery DC cell CD1a, CD11c and CD83 immunophenotypic marker;
(10) suspension cell in step (9) is re-seeded in culture bottle, adds IFN-γ 1000U/ml;
A, the CD3 monoclonal antibody adding 50ng/ml after 1 day and rhIL-2500U/ml stimulate growth and the propagation of CIK cell
Under b, the 4th clear water surface, observation of cell quantity is many, and conglomeration is comparatively large, many, culture medium color becomes golden yellow, and transferred to by cell in culture bag, volume augmentation is to 300ml;
Under c, the 5th clear water surface, observation of cell volume increases, endochylema is few, core is large, and conglomeration is many, and culture medium becomes crocus fluid infusion to 600ml;
Under d, the 6th clear water surface, observation of cell growth conditions is good, and the golden yellow fluid infusion of culture medium color is to 1200ml, and mixing, samples coated plate and do antibacterial, fungal detection;
E, the 7th day results CIK cell; Trypan Blue detects cell viability, flow cytomery CIK cell CD3
+cD56
+immunophenotypic marker;
(11) DC cell is added in CIK cell culture bag in the ratio of 1:10 mix, continue to cultivate;
Within (12) the 9th days, expand bag.1200ml culture fluid is divided and is filled in two culture bag, wherein the culture fluid of 600ml is divided and be filled in a bag, and utilize supplementing culture medium to 1200ml, the culture fluid of other 600ml is divided and is filled in b bag, and utilize supplementing culture medium to 1500ml, two bags all will sample quality inspection;
(13) the 12nd days results a bag cells.
A, subpackage: will be dispensed in a 50ml centrifuge tube after the cell suspension mixing in cell culture bags, 45ml/ manages, totally 27 pipes, trim.5ml cell suspension sample is stayed to do endotoxin and Gram’s staining detection and count;
B, centrifugal collecting cell: 1500rpm*10min;
C, for the first time centrifuge washing: abandon supernatant, often pipe adds 5ml normal saline (diluting plasma containing 4%), evenly, every 8 pipes close 1 pipe in piping and druming, totally 4 manage, then collect residual cell, trim, 1500rpm*10min with each centrifuge tube of a small amount of normal saline rinsing;
D, second time centrifuge washing: abandon supernatant, add normal saline resuspended, be combined into 1 pipe, trim.1500rpm*10min；
E, re-suspended cell: abandon supernatant, first flick at the bottom of centrifuge tube, makes cell precipitation loose.10ml cell re-suspension liquid is added again with 10ml pipet, blow and beat gently, re-suspended cell, start not direct sucking-off precipitation, to first suct clearly along tube wall run underneath to precipitation scatter blow and beat again (checked whether that granule precipitates, 70um metre filter cell precipitation will have been used if cannot not blow loosely) add again 5ml10 cell re-suspension liquid and 5ml albumin mixing.
F, by cell suspension inject by normal saline bag: prepare normal saline bag put into super-clean bench, alcohol gauze wiping mouth of pipe place, open mouth of pipe vinyl cover and expose cork, open 50ml injector package, do not encounter the body of below syringe graduation line, syringe is stretched into sucking-off cell suspension by inclination centrifuge tube, ensures sterile working.The syringe needle changing syringe is No. 9 syringe needles, and cell suspension upwards, injects, observes simultaneously and whether have precipitation, injected the gas of rear extraction respective volume by bag of saline.
G, inspection confirm: again checked whether precipitation, check whether injection pin hole place has liquid to ooze out and determines errorless rear loading packaging bag, and pass-through box spreads out of.
(14) the 13rd days results b bag cells, the same step of method (13).
(15) cell counting is respectively 3.7*10
9, 4.72*10
9.
In the description of this description, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referred to the schematic representation of above-mentioned term.And the specific features of description, structure, material or feature can combine in an appropriate manner in any one or more embodiment or example.
Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.
Claims (10)
1. dendritic cell and cytokine-induced killer cell sexual cell be combined in the purposes prepared in medicine, described medicine is used for the treatment of or suppresses hepatocarcinoma, wherein, described specific for dendritic cells identification liver lesion,
Preferably, described dendritic cell obtains through the following steps:
(1) obtain peripheral blood, described peripheral blood comes from the individuality carrying described focus;
(2) from described peripheral blood, mononuclearcell is separated;
(3) stimulate described mononuclearcell to differentiation of dendritic cells, to obtain described dendritic cell,
Wherein,
Comprise in step (3) and the specific antigen of described mononuclearcell with described focus contacted, described specific antigen be alpha-fetoprotein and Hepatitis B virus vaccine one of at least,
Optionally, described individuality suffers from chronic viral hepatitis B further.
2. purposes according to claim 1, is characterized in that, step (3) comprises further:
(3-1) M-CSF and recombinant human interleukin 4 is utilized to stimulate described mononuclearcell to differentiation of dendritic cells;
(3-2) contacted with Hepatitis B virus vaccine with alpha-fetoprotein by the cell obtained in step (3-1), the weight concentration ratio of described alpha-fetoprotein and described Hepatitis B virus vaccine is 1:1; And
(3-3) one of at least contacting by the cell obtained in step (3-2) and IL-β, IL-6 and TNF-α, to obtain described dendritic cell,
Preferably, the concentration of described M-CSF is 1000U/mL, and the concentration of described recombinant human interleukin 4 is 500U/mL,
Preferably, the concentration of described alpha-fetoprotein and described Hepatitis B virus vaccine is separately 40 ~ 60 mcg/ml.
3. purposes according to claim 1, is characterized in that, the combination of described dendritic cell and cytokine-induced killer cell sexual cell by described dendritic cell and described cytokine-induced killer cell sexual cell are carried out Dual culture acquisition,
Wherein,
When described Dual culture, the inoculative proportion of described dendritic cell and described cytokine-induced killer cell sexual cell is 1:8 ~ 12, preferred 1:10.
4. purposes according to claim 3, is characterized in that, described cytokine-induced killer cell sexual cell obtains through the following steps:
(4-1) utilize the mononuclearcell that obtains in IFN-γ stimulation step (2) 24 hours, wherein, the concentration of described IFN-γ is 1000U/ml; And
(4-2) cell obtained in step (4-1) is contacted with recombinated interleukin-2 with CD3 monoclonal antibody, to obtain described cytokine-induced killer cell sexual cell, wherein, the concentration of described CD3 monoclonal antibody is the concentration 500U/ml of 50ng/ml, recombinated interleukin-2.
5. prepare a method for dendritic cell, wherein, described specific for dendritic cells identification liver lesion, described method comprises:
(1) obtain peripheral blood, described peripheral blood comes from the individuality carrying described focus;
(2) from described peripheral blood, mononuclearcell is separated;
(3) stimulate described mononuclearcell to the differentiation of dendritic cell direction, to obtain described dendritic cell,
Wherein,
Comprise in step (3) and the specific antigen of described mononuclearcell with described focus contacted, described specific antigen be alpha-fetoprotein and Hepatitis B virus vaccine one of at least,
Preferably, step (3) comprises further:
(3-1) M-CSF and recombinant human interleukin 4 is utilized to stimulate described mononuclearcell to differentiation of dendritic cells;
(3-2) contacted with Hepatitis B virus vaccine with alpha-fetoprotein by the cell obtained in step (3-1), the weight concentration ratio of described alpha-fetoprotein and described Hepatitis B virus vaccine is 1:1; And
(3-3) one of at least contacting by the cell obtained in step (3-2) and IL-β, IL-6 and TNF-α, to obtain ripe dendritic cell,
Preferably, the concentration of described M-CSF is 1000U/mL, and the concentration of described recombinant human interleukin 4 is 500U/mL.
6. method according to claim 5, is characterized in that, the concentration of described alpha-fetoprotein and described Hepatitis B virus vaccine is separately 40 ~ 60 mcg/ml.
7. a medicine, described medicine is used for the treatment of or prevents liver cancer, and it is characterized in that, described medicine comprises:
The combination of dendritic cell and cytokine-induced killer cell sexual cell,
Wherein, described specific for dendritic cells identification liver lesion.
8. medicine according to claim 7, is characterized in that, described dendritic cell is prepared by the method described in claim 5 or 6.
9. a dendritic cell, described specific for dendritic cells identification liver lesion.
10. dendritic cell according to claim 9, is characterized in that, described dendritic cell is prepared by the method described in claim 5 or 6.
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| CN105368777A (en) * | 2015-10-22 | 2016-03-02 | 黄月华 | Construction method of hepatitis vaccine sensitization dendritic cell inducing specific T cell |
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| CN105368777A (en) * | 2015-10-22 | 2016-03-02 | 黄月华 | Construction method of hepatitis vaccine sensitization dendritic cell inducing specific T cell |
| CN111690611A (en) * | 2020-06-08 | 2020-09-22 | 海南优尼科尔生物科技有限公司 | DC-CIK cell preparation and preparation method thereof |
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