CN106282109A - A kind of DC CTL test kit for hepatocarcinoma and preparation method thereof - Google Patents
A kind of DC CTL test kit for hepatocarcinoma and preparation method thereof Download PDFInfo
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Abstract
The present invention provides a kind of DC CTL cell culture kit for liver cancer treatment and preparation and application thereof.This test kit uses multiple hepatoma cell line surface antigen to provide present antigen for DC cell, uses the CTL of this DC cell induction that the hepatoma carcinoma cell expressing not synantigen is had stronger killing activity.Meanwhile, other reagent needed for DC CTL cell is cultivated by this test kit are optimized and rename, to adapt to industrialized requirement.
Description
Technical field
The present invention relates to the cellular immunotherapy field of tumor.It is specifically related to a kind of DC-CTL cell for liver cancer treatment
Cultivate the method for preparation and use of test kit.
Background technology
Hepatocarcinoma onset is hidden, and grade of malignancy is high, mostly has been middle and advanced stage, and the natural immunity in the patient during morbidity, especially special
Specific immunological hypofunction, the weak curative effect of Radiotherapy chemotherapy, send out and metastasis in easily there is liver, only some patients can accept
Operative treatment, radical excision rate is relatively low, and Postoperative recurrent rate is high.Since the 90's of 20th century, the sickness rate of China's hepatocarcinoma
Having been raised to the 2nd of malignant tumor, there are about 130,000 people every year and die from hepatocarcinoma, death toll accounts for the whole world total people of PLC mortality
The 44% of number.Therefore, strategy and the Therapeutic Method of finding treatment and prevention liver cancer recurrence transfer are significant.
Adoptive immunity cell therapy refer to autologous or alloimmune effector lymphocyte's infusion that In vitro culture is activated amplification to
Patient, kills tumor cell in the patient or the Therapeutic Method of excitating organism anti tumor immune response.Adoptive immunotherapy
Advantage be mainly manifested in the following aspects.First, immunocyte processes in vitro, can walk around tumor patient vivo immunization
All mechanism of obstacle.Second, the activation of immunocyte and cytokine mediated by some, the present biotechnology of effect process
Cytokine profiles, tumor antigen etc. can be produced on a large scale so that external a large amount of amplification tumor vaccine cells are possibly realized.The
Three, the secondary work of serious poison that the Activated in Vitro amplification of immunocyte can be avoided some biological preparation to widely apply in vivo and bring
With.In recent years, many clinical researches use immunocytes are as a kind of auxiliary treatment means for the treatment of cancer, and achieve good
Effect.These researchs show that single cell therapy is little to the therapeutic effect of solid tumor, but for operation or chemicotherapy future trouble
The recurrence of person and the rate of transform have good inhibition.
At present, after relatively effective adoptive immunity cell therapy predominantly uses dendritic cell (DC) co-culturing, inducing
CTL cell therapy, i.e. DC-CTL treats.DC cell is to have now been found that the professional antigen presenting cell (APC) that function is the most powerful, its
There is abundant antigen capture molecule, antigen presentation molecule, co-stimulators in surface, has the angtigen presentation of uniqueness and swash
Send out function.Related immunological basic research and tumor research show, DC is the center ring starting, regulate and control and maintain immunne response
Joint.Bridge and pivotal role is played in the interaction of tumor cell and T lymphocyte.The DC and initial T of ripe activation drench
Bar cell interaction can be with inducing antigen-specific cytotoxic T lymphocyte (CTL), and cytotoxic T lymphocyte
It it is a kind of specific T-cells that antigenic substance is had lethal effect.Therefore, it can using DC as carrier, through tumor antigen body
Outer impact makes its sensitization, it is ensured that tumor antigen is effectively absorbed, offers, and then and by co-culturing of DC with CTL cell makes
CTL cell obtains the ability of specific killing tumor cell.
The CTL cell therapeutic approach major defect of tumor burden DC induction is: the tumor associated antigen (TAA) being currently known
Less, clinical practice is restricted；Have to be understood that the MHC background of patient, specify the HLA type of tumor antigen peptide, decorrelation
Epitope sequences；The heterogeneity of tumor cell and genetic instability, may not necessarily induce optimal exempting from known antigen polypeptide
Epidemic disease is reacted, and needs prophylaxis of tumours antigen immune to escape the probability of variation.Additionally, due to the technical side that each research unit uses
Case is different, causes occurring Railway Project in DC-CTL incubation:DC cell concentration is few, is not enough to stimulate CTL to produce
Specificity；The non-full maturity of DC cell, it is impossible to effectively offer tumor antigen；CD3 in the cell of results+CD8+CD28+'s
CTL cell proportion is relatively low, can not produce expected effect after feedback.Therefore it provides one that DC-CTL cell can be made both to have had is special
Property can reach again the cell culture protocol of effective quantity and be particularly important.
Summary of the invention
The present invention provides a kind of DC-CTL test kit for liver cancer treatment, and described test kit comprises LSA-A reagent, LSA-
B reagent, LSA-C reagent, DC-A reagent, DC-B reagent, CTL-A reagent, CTL-B reagent.
Described LSA-A reagent is that Ficoll separates liquid, uses it as the separation agent of PERIPHERAL BLOOD MONONUCLEAR CELL.
Described LSA-B reagent is the phosphate buffer of PH=7.3 ± 0.1, use it as be coated plate clean, blood dilute
Release and cell washing reagent.
Described LSA-C reagent is (α-Galcer) containing 200-400ng/ml alpha-galactosylceramide (preferably 200ng/ml)
And the phosphate buffer of multiple hepatoma cell line surface antigen.Specifically, the multiple hepatoma cell line surface that this reagent comprises
Antigen refers to 400-600ug/ml HepG2 hepatoma cell line surface antigen (preferably 400ug/ml), 400-600ug/ml QGY-
7703 hepatoma cell line surface antigens (preferably 400ug/ml), 400-600ug/ml SMMC-7721 hepatoma cell line surface antigen
One in (preferably 400ug/ml), 400-600ug/ml EGHC-9901 hepatoma cell line surface antigen (preferably 400ug/ml)
Or it is several.More specifically, the multiple hepatoma cell line surface antigen that this reagent comprises is by the method using liquid nitrogen multigelation
Obtain.
Described DC-A reagent is the anti-CD14 monoclonal antibody containing 50-500ng/ml (preferably 100ng/ml), 50-500ng/
The phosphate buffer of ml anti-CD11c monoclonal antibody (preferably 100ng/ml), uses its coated Tissue Culture Flask (ware) can
Act on DC cell sorting.
Described DC-B reagent is the preferred 1200U/ml of the GM-CSF(containing 1000-1500U/ml), 1000-1500U/ml TGF-
β (preferably 1200U/ml), the preferred 800U/ml of 600-1000U/ml IL-4() and 600-1000U/ml IFN-γ is (preferably
1640 culture medium 800U/ml), use it as induction DC cell differentiation and the culture medium expanded.
Described CTL-A reagent is containing 50-500ng/ml CD 3-resisting monoclonal antibody (preferably 100ng/ml) and 50-500ng/
The phosphate buffer of ml anti-CD28 monoclonal antibody (preferably 100ng/ml), uses its coated Tissue Culture Flask (ware) can
Act on the stimulation activation of T cell.
Described CTL-B reagent is containing 80-100ng/ml CD 3-resisting monoclonal antibody (preferably 80 ng/ml), 80-100 ng/
Ml anti-CD28 monoclonal antibody (preferably 80 ng/ml), 80-120ng/ml phytohaemagglutinin (PHA) (preferably 80 ng/ml),
The preferred 200U/ml of 200-400U/ml IL-2(), 1640 culture medium of 50-100U/ml IL-1 α (preferably 80 U/ml), use
It makees the culture medium that induction CTL occurs and expands, and meanwhile, this reagent also serves as the training of the DC-CTL cell after DC, CTL merge
Support.
Jointly, the described all reagent comprised for the DC-CTL test kit of liver cancer treatment all carry out antibacterial, fungus
And endotoxin content detection, and antibacterial, fungal detection be feminine gender, endotoxin content ＜ 2EU/ml.
The invention has the beneficial effects as follows: the present invention be extracted hepatoma cell line HepG2, QGY-7703, SMMC-7721,
The surface antigen of EGHC-9901, uses it to provide present antigen for DC cell so that DC cell obtains the anti-of multiple hepatoma carcinoma cell
Prime information, uses the CTL of this DC cell induction that the hepatoma carcinoma cell expressing not synantigen is had stronger killing activity, can be used for
There is hepatocarcinoma in prevention hepatitis B and liver cirrhosis patient, it is possible to as operation of liver cancer with treatment and the one of chemicotherapy due to disease progression
Plant auxiliary treatment means, can play and improve therapeutic effect and prevent the effect of liver cancer recurrence.Further, the present invention is by external training
Reagent, antigen needed for supporting DC-CTL are named and form test kit, formulate the test kit preparation method of standard simultaneously and make
The Standard Operating Procedure cultivated as external DC-CTL cell by method, to adapt to the requirement of industrialization.
Accompanying drawing explanation
Fig. 1 is the canonical plotting using BCA protein quantification kit measurement tumor cell line surface antigen protein amount.
Protein concentration (mg/ml)=1.32* sample mean light absorption value-0.1821 as seen from the figure, usually, tumor used in the present invention
The amount of antigen that the extracting method of cell line surface antigen is extracted is 11.3mg/1*108Individual cell.
Fig. 2 is the Phenotypic examination result of the DC cell using flow cytometry to cultivate test kit of the present invention.
Fig. 3 is the Phenotypic examination result of the DC-CTL cell using flow cytometry to cultivate test kit of the present invention.
The DC-CTL cell of Fig. 4 test kit of the present invention cultivation and the DC-CTL cell of cellar culture are to hepatoma cell line
The Mortaility results cartogram of HepG2.
Detailed description of the invention
Below in conjunction with the accompanying drawings and the present invention is elaborated further by detailed description of the invention.In embodiment, test kit is concrete
The experimental procedure using operation to recommend by businessman is carried out, and agents useful for same or instrument unreceipted production firm person are and can pass through city
Available from customary commercial
The extraction of embodiment 1 hepatoma cell line surface antigen
The cultivation of 1.1 hepatoma cell line.Hepatoma cell line used by the present invention include HepG2, QGY-7703, SMMC-7721,
EGHC-9901, all cells system is adherent growth cell and is the recovery cultivation of our company's tumor cell storehouse.HepG2、QGY-
7703, EGHC-9901 cell line uses the DMEM culture medium containing 10% hyclone (Gibco company, article No.: 10099141)
(Gibco company, article No.: 31966047) is cultivated, and SMMC-7721 cell line uses containing 10% hyclone
1640 culture medium (Gibco company, article No.: 61870036).
Passing on of 1.2 hepatoma cell line.Cell passes on when cultivating to degree of converging about 95%.Discard archeocyte to cultivate
Base, rinses cell twice with PBS.(Beijing relies on Hua Mao bio tech ltd to add 0.25% trypsinization liquid.
Article No.: PY0018A) so that it is uniformly it is paved with culture dish.Examine under a microscope cellular morphology, when most cells is from fusiformis
When attached cell is converted into the suspension cell of circle, pats Tissue Culture Flask with hands and make cell slide from culture bottle inwall, then
Blow wall with fresh culture fluid, make cell be evenly distributed in culture fluid, blow even rear cell be divided into two parts make amplification culture or
Centrifugal collection carries out Antigen extraction.
1.3 freeze-thaw methods extract tumor cell line antigen.The tumor cell line that digestion is collected is concentrated into 1ml and proceeds to freeze
Depositing pipe, cell concentration is 1*108Individual/ml, puts it in preprepared liquid nitrogen, is taken out by tumor cell and put after 10min
Enter in 37 DEG C of water-baths, proceed to rapidly in liquid nitrogen after all dissolving.After repeating above-mentioned frozen process experiment step 5-10 time, suspension is turned
Entering EP pipe, 10000 leave the heart 15 minutes, discard precipitation, collect supernatant and are tumor cell line antigen protein.BCA protein quantification
Test kit (Wei Ao bio tech ltd, Shanghai, article No.: WB0124) detection antigen protein content (Fig. 1), and use
The filter of 0.22um carries out aseptic filtration.
Embodiment 2 is prepared for the DC-CTL test kit of hepatocarcinoma
2.1 LSA-A preparation of reagents.Described LSA-A reagent is that Ficoll separates liquid (General Electric's Medical Group, article No. 17-
5442-02/03).
2.2 LSA-B preparation of reagents.Described LSA-B reagent is the phosphate buffer of PH=7.3 ± 0.1, its compound method
For well known in the art: NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO44.3mmol/L, KH2PO41.4mmol/L,
Aseptic redistilled water is used to mix and be settled to 1L, with concentrated hydrochloric acid regulation pH value to 7.2.
2.3 LSA-C preparation of reagents.Described LSA-C reagent is containing 200ng/ml alpha-galactosylceramide (α-Galcer)
Tumor cells of hepatocellular carcinoma system antigen described in (Guangzhou, Guangzhou Bo Bai trade Co., Ltd, article No.: KRN7000) and embodiment 1.Mainly
Including 400ug/ml HepG2 hepatoma cell line surface antigen, 400ug/ml QGY-7703 hepatoma cell line surface antigen,
400ug/ml SMMC-7721 hepatoma cell line surface antigen, 400ug/ml EGHC-9901 hepatoma cell line surface antigen.
2.4 DC-A preparation of reagents.Described DC-A reagent is containing 100ng/ml anti-CD14 monoclonal antibody (sub-novacine skill
Company, article No.: PAB4880), 100ng/ml anti-CD11c monoclonal antibody (Asia novacine skill company, article No.: PAB4887)
Phosphate buffer.
2.5 DC-B preparation of reagents.Described DC-B reagent is containing 1200U/ml GM-CSF(Beijing hundred Pu Saisi biotechnology
Company limited, article No.: GMF-H4214), 1000-1500U/ml TGF-β (Wei Huan bio tech ltd, Shanghai, article No.:
3711S) 800U/ml IL-4(Invitrogen company, article No.: RRIL410) and (the fluffy tail feather in the Shanghai life of 800U/ml IFN-γ
Thing Science and Technology Ltd., article No.: orb201503) 1640 culture medium.
2.6 CTL-A preparation of reagents.Described CTL-A reagent is containing 100ng/ml CD 3-resisting monoclonal antibody (sub-novacine
Skill company, article No.: MAB4959) and 100ng/ml anti-CD28 monoclonal antibody (Asia novacine skill company, article No.: MAB4730)
Phosphate buffer.
2.7 CTL-B preparation of reagents.Described CTL-B reagent is to resist containing 80ng/ml CD 3-resisting monoclonal antibody, 80ng/ml
CD28 monoclonal antibody, 80ng/ml phytohaemagglutinin (PHA) (it is Bioisystech Co., Ltd that Beijing reaches section, article No.:
CL76003K), 200U/ml IL-2(Invitrogen company, article No.: PA5-46923), (life is public for 50-100U/ml IL-1 α
Department, article No.: LHC0811M) 1640 culture medium.
Embodiment 3 is for the DC-CTL test kit using method of hepatocarcinoma
3.1 culture bottles are coated.DC-A reagent and each 10ml of CTL-A reagent described in embodiment 2 is used to be separately added into two T75 thin
Born of the same parents' culture bottle, is placed in 4 DEG C and is coated overnight.
The separation of 3.2 PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC).Use the 50-that anticoagulant heparin blood sampling tube venae vasorum gathers
80ml peripheral blood, 2000r/mim is centrifuged 10min, draws upper serum with pipettor, 56 degrees Celsius of inactivation 30min.Cell precipitates
Resuspended with LSA-B reagent described in equivalent embodiment 2, it is slowly added to the LSA-A described in cell precipitation equal-volume embodiment 2 try
In agent, arranging centrifuge nature rising or falling speed, 2000r/min is centrifuged 20min, draws middle tunica albuginea confluent monolayer cells, use after being centrifuged
LSA-B reagent cleans and i.e. obtains PBMC above-mentioned twice.
3.3 DC-CTL cultivate.Discard be coated in bottle be coated liquid, with LSA-B reagent clean be coated bottle once, will separate
PBMC 10ml CTL-B reagent resuspended and access DC-A reagent and be coated bottle, be placed in 37 DEG C of 5% CO2Cultivating in incubator, 2 is little
Time after, DC cell can be adherent, and now, careful upper strata suspension cell of drawing, access CTL-A is coated bottle and cultivates, and DC-A is coated bottle
Interior attached cell continues to cultivate after adding 10ml DC-B reagent.According to cell growth status supplemented medium in follow-up cultivation,
DC cell is cultivated and is added LSA-C reagent 1ml to cultivating system when the 5th day, and cell is cultivated to DC cell when the 7th day and CTL
Cell merges, and uses CTL-B reagent to cultivate, and cell carries out antibacterial, fungus, endotoxin detection when cultivating to fortnight
And carry out cell counting, detect qualified rear harvesting.
Embodiment 4 cell phenotype detects
4.1 DC cells are cultivated and are sampled to when the 7th day, use flow cytometer detection antibody PE Mouse Anti-Human CD86 (beautiful
BD company of state, article No.: 555658), APC Mouse Anti-Human HLA-DR (U.S. company BD, article No.: 559866) enters
Row dyeing, the expression of each cell mass of flow cytometry analysis.The cell of experimental result Explicit Expression CD86 is 81.4%, table
The cell reaching HLA-DR is 92.7%(Fig. 2).Illustrate that method therefor of the present invention and agents useful for same stimulate the DC cellular maturity cultivated
Well.
4.2 DC-CTL cells are cultivated and are sampled to during fortnight, use flow cytometer detection antibody Percp-CyTM5.5 Mouse
Anti-Human CD3(U.S. company BD, article No.: 560835), APC Mouse Anti-Human CD8(U.S. company BD, goods
Number: 555369), PE Mouse Anti-Human CD28(U.S. company BD, article No.: 553297) dye, fluidic cell
The expression of each cell mass analyzed by instrument.Experimental result display CD3+ cell proportion reaches 97.0%, CD3+CD8+CD28+ cell proportion
Reach 81.3%(Fig. 3).Illustrate that method therefor of the present invention and agents useful for same stimulate CTL cell proportion in the cell cultivated bigger.
The killing activity of hepatocellular carcinoma H22 is detected by embodiment 5 DC-CTL
5.1 target cell inoculations.The trypsinization liquid using 0.25% will be in the hepatoma cell line HepG2 cell of exponential phase
Digesting from culture bottle and proceed in 50ml centrifuge tube, 800 leave heart 3min, abandon supernatant.With the DMEM containing 10% hyclone
Culture medium re-suspended cell also counts, and adjusting target cell concentration is 5*105Individual/ml, accesses 24 orifice plates, every hole 500ul, connects cell
Culture plate be placed in CO2 gas incubator 5% CO2Hatch 24 hours for 37 DEG C.
5.2 effector lymphocytes act on target cell.Test kit of the present invention is used to cultivate the DC-CTL to fortnight and routine
The DC-CTL cell that method is cultivated sets three multiple holes as target cell, every kind of cell.1500 leave heart 5min, abandon supernatant, use
CellTracker Fluorescent Probes(life company article No.: C34565) working solution re-suspended cell, incubated at room
20min.It is centrifuged and discards CellTracker Fluorescent Probes working solution, wash cell 3 times with PBS, add effect
The resuspended counting of culture medium needed for answering cell to cultivate, adjusts cell concentration by corresponding effect target ratio (1:1,5:1,10:1,20:1),
Access 24 orifice plates containing target cell, every hole 500ul.The culture plate connecting cell is placed in 5% CO237 DEG C of incubators 24 hours.
5.3 target cell apoptosis rate detections.By cell in culture plate to centrifuge tube, and wash twice with 4 DEG C of PBS, use 200ul
1X Binding Buffer(U.S. company BD, article No.: 556454) re-suspended cell.Take 100ul cell to 5ml round bottom pipe, addition
5ul PE Annexin V(U.S. company BD, article No.: 556422) and 5ul 7-AAD(U.S. company BD, article No.: 559925),
After lucifuge dyeing 20min, wash away excess stain agent with 1X Binding Buffer, with the 1X Binding of 400 μ l
Buffer re-suspended cell, carries out flow cytometry, uses CellTracker Deep Red dye to enclose door during analysis, point
In analysis CellTracker Deep Red dye negative cells group, the expression of 7AAD+Annexin V+ cell is target cell
Apoptosis rate, does apoptosis curve according to testing result.The display of the present embodiment experimental result uses of the present invention thin for hepatocarcinoma
The fragmentation effect to hepatoma cell line HepG2 of the DC-CTL cell that the DC-CTL test kit of born of the same parents is cultivated to be substantially better than conventional training
The DC-CTL cell supported.(Fig. 4).
Claims (9)
1. the DC-CTL cell culture kit for liver cancer treatment, it is characterised in that include following several reagent: LSA-A
Reagent, LSA-B reagent, LSA-C reagent, DC-A reagent, DC-B reagent, CTL-A reagent, CTL-B reagent.
DC-CTL test kit the most according to claim 1, it is characterised in that described LSA-C reagent is containing 200-400 ng/
Ml alpha-galactosylceramide and the phosphate buffer of multiple hepatoma cell line surface antigen.
DC-CTL test kit the most according to claim 1, it is characterised in that described DC-A reagent is containing 50-500ng/ml
Anti-CD14 monoclonal antibody and the phosphate buffer of 50-500ng/ml anti-CD11c monoclonal antibody.
DC-CTL test kit the most according to claim 1 and 2, it is characterised in that described multiple hepatoma cell line surface antigen
Specifically include that 400-600ug/ml hepatoma cell line HepG2 surface antigen, 400-600ug/ml hepatoma cell line QGY-7703 table
Face antigen, 400-600ug/ml hepatoma cell line SMMC-7721 surface antigen, 400-600ug/ml hepatoma cell line EGHC-
One or more in 9901 surface antigens.
5. the DC-CTL cell culture processes for hepatocarcinoma, it is characterised in that employ the DC-described in claim 1-5
CTL cell culture kit.
DC-CTL cultural method the most according to claim 6, it is characterised in that make in described DC-CTL cell cultivation process
Present antigen is provided for DC cell with LSA-C reagent.
DC-CTL cultural method the most according to claim 6, it is characterised in that make in described DC-CTL cell cultivation process
Sorting and the activation of DC cell is carried out with DC-A reagent.
8. the purposes in preparing DC-CTL cell of any one reagent described in claim 1-4.
9. the application in preparing DC-CTL cell of the DC-CTL cell culture processes described in claim 5-7.
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