CN104560883A - Cancer cell/DC fusion tumor vaccine for expressing Endoglin and preparation method for cancer cell/DC fusion tumor vaccine - Google Patents
Cancer cell/DC fusion tumor vaccine for expressing Endoglin and preparation method for cancer cell/DC fusion tumor vaccine Download PDFInfo
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Abstract
The invention discloses a cancer cell/DC (Dendritic Cell) fusion tumor vaccine for expressing Endoglin and a preparation method for the cancer cell/DC fusion tumor vaccine. According to the cancer cell/dendritic cell fusion tumor vaccine for expressing Endoglin and a preparation method for the cancer cell/dendritic cell fusion tumor vaccine, the method for preparing fusion cells comprises the step of fusing tumor cells and dendritic cells to obtain the fusion cells; the tumor cells are recombinant cells for expressing the Endoglin or the recombinant cells which contain the gene for encoding the Endoglin; the Endoglin is the protein of A1) or A2) below: the A1) is the protein of which the amino acid sequence is SEQ ID No.1; the A2) is the protein which has the same function as that of the protein of the A1) and is derived from the protein of the A1) performing substituting and/or deleting and/or adding on one or more amino acid residues of the amino acid sequence which is shown as the SEQ ID No.1.
Description
Technical field
Cancerous cell/the DC that the present invention relates to expressing tumor vascular marker molecule in biomedical sector merges tumor vaccine and preparation method thereof.
Background technology
The immunization therapy of malignant tumor has become a kind of new Therapeutic Method outside operation, radiation and chemotherapy.Wherein tumor-specific cytotoxicity T lymphocyte (cytotoxic T lymphocyte, CTL) therapy of adopting is the research emphasis of immunotherapy of tumors always.The effective ways that tumor cell and dendritic cell (dendritic cells, DC) merge as a kind of CTL of induction are widely accepted.But the therapeutic effect of fusion bacterin is still undesirable, new method is needed to strengthen effectiveness and the targeting of fused cell induction CTL.
Endoglin is (also known as CD105, differentiation group 105) be a kind of tumor vascular labelled molecule, it is homodimer transmembrane glycoprotein, molecular weight is 180kDa, transforming growth factor-β (transforming growth factor, TGF-β) part of receptor, controllable cell, to the reaction of TGF-β, promotes endothelial cell proliferation by suppressing the biological effect of TGF-β.
Summary of the invention
Technical problem to be solved by this invention how to treat tumor.
For solving the problems of the technologies described above, the present invention provide firstly the method preparing fused cell.
The method preparing fused cell provided by the present invention, comprises and tumor cell and dendritic cell is carried out merging the step obtaining fused cell;
Described tumor cell is M or N:
M, described tumor cell are the reconstitution cell of expressing Endoglin;
N, described tumor cell are the reconstitution cell of the gene containing the described Endoglin of coding;
Described Endoglin is following A 1) or protein A2):
A1) aminoacid sequence is the protein of SEQ ID No.1;
A2) in the aminoacid sequence shown in SEQ ID No.1 through replacement and/or disappearance and/or add that one or several amino acid residue obtains have identical function by A1) derivative protein.
Above-mentioned A2) in protein can synthetic, also can first synthesize its encoding gene, then carry out biological expression and obtain.Above-mentioned A2) in the encoding gene of protein by the codon by lacking one or several amino acid residue in the DNA sequence shown in the 1st of SEQ ID No.2 the-1683 nucleotide, and/or carry out the missense mutation of one or several base pair, and/or the coded sequence of the fluorescin shown in the 1705th-2424 nucleotide connecting SEQ ID No.2 at its 5 ' end and/or 3 ' end obtains.
Wherein, the 1st-1683 nucleotide coding aminoacid sequences of SEQ ID No.2 are the Endoglin of SEQ ID No.1, the 1705th-2424 nucleotide coding EGFP of SEQ ID No.2.
Above-mentionedly prepare in the method for fused cell, the C-terminal of described Endoglin or N-terminal can merge His, Flag, GST, MBP, His-MBP, HA, eGFP, eCFP, eYFP, Myc, His-Myc, His-AviTag, Sumo, His-Sumo, SNAP-Tag or Halo Tag label.
Above-mentionedly prepare in the method for fused cell, the tumor cell of described expression Endoglin is that the encoding gene of described Endoglin is imported the reconstitution cell obtained in receptor tumor cells.
Above-mentionedly prepare in the method for fused cell, the encoding gene of described Endoglin is following A11) or A21) or A31) shown in nucleic acid molecules:
A11) DNA molecular shown in the 1st-1683 nucleotide of SEQ ID No.2 in sequence table;
A21) nucleotide sequence and A11) limited has more than 75% or 75% homogeneity, and the cDNA molecule of the described Endoglin that encodes or genomic DNA molecule;
A31) under strict conditions with A11) nucleotide sequence hybridization that limits, and the cDNA molecule of the described Endoglin that encodes or genomic DNA molecule.
Term used herein " homogeneity " refers to the sequence similarity with native sequence nucleic acid." homogeneity " comprises and has 75% or higher with the 1st-1683 nucleotide sequences of SEQ ID No.2 of the present invention, or 85% or higher, or 90% or higher, or the nucleotide sequence of 95% or higher homogeneity.Homogeneity can with the naked eye or computer software evaluate.Use computer software, the homogeneity between two or more sequence can represent with percentage ratio (%), and it can be used for evaluating the homogeneity between correlated series.
Above-mentionedly prepare in the method for fused cell, described stringent condition is in the solution of 2 × SSC, 0.1%SDS, hybridizes and wash film 2 times, each 5min at 68 DEG C, again in the solution of 0.5 × SSC, 0.1%SDS, hybridizes and wash film 2 times, each 15min at 68 DEG C.
More than above-mentioned 75% or 75% homogeneity, can be the homogeneity of more than 80%, 85%, 90% or 95%.
Above-mentionedly prepare in the method for fused cell, described reconstitution cell expresses described Endoglin.
Above-mentionedly prepare in the method for fused cell, described receptor tumor cells is hepatoma carcinoma cell, specifically can be HepG2.
Above-mentionedly prepare in the method for fused cell, described dendritic cell is the dendritic cell prepared from peripheral blood.
For solving the problems of the technologies described above, present invention also offers fused cell.
Fused cell provided by the present invention is the fused cell utilizing the above-mentioned method preparing fused cell to obtain.
For solving the problems of the technologies described above, present invention also offers and treat and/or prevent tumour medicine.
The active component treating and/or preventing tumour medicine provided by the present invention is following H1 and/or H2:
H1, described fused cell;
The T lymphocyte of the secretion of gamma-IFN that H2, described fused cell inducer T lymphocyte obtain.
Above-mentionedly treat and/or prevent in tumour medicine, described in treat and/or prevent tumour medicine and can be and treat and/or prevent liver-cancer medicine.
For solving the problems of the technologies described above, present invention also offers fused cell and preparing the application that treats and/or prevents in tumour medicine or the application in the T lymphocyte of preparation secretion of gamma-IFN.
In above-mentioned application, described fused cell can be the fused cell utilizing the above-mentioned method preparing fused cell to obtain.
In above-mentioned application, described in treat and/or prevent tumour medicine and can be and treat and/or prevent liver-cancer medicine.
For solving the problems of the technologies described above, present invention also offers Endoglin and preparing the application treated and/or prevented in tumour medicine.
In above-mentioned application, described in treat and/or prevent tumour medicine and can be and treat and/or prevent liver-cancer medicine.
For solving the problems of the technologies described above, present invention also offers the biomaterial relevant to Endoglin and preparing the application treated and/or prevented in tumour medicine.
The biomaterial relevant to Endoglin provided by the present invention is in preparation treats and/or prevents in tumour medicine application, and described biomaterial is following E1) to E20) in any one:
E1) to encode the nucleic acid molecules of described Endoglin;
E2) containing E1) expression cassette of described nucleic acid molecules;
E3) containing E1) recombinant vector of described nucleic acid molecules;
E4) containing E2) recombinant vector of described expression cassette;
E5) containing E1) recombinant microorganism of described nucleic acid molecules;
E6) containing E2) recombinant microorganism of described expression cassette;
E7) containing E3) recombinant microorganism of described recombinant vector;
E8) containing E4) recombinant microorganism of described recombinant vector;
E9) containing E1) the transgenetic animal cell system of described nucleic acid molecules;
E10) containing E2) the transgenetic animal cell system of described expression cassette;
E11) containing E3) the transgenetic animal cell system of described recombinant vector;
E12) containing E4) the transgenetic animal cell system of described recombinant vector;
E13) containing E1) the transgenic animal tissue of described nucleic acid molecules;
E14) containing E2) the transgenic animal tissue of described expression cassette;
E15) containing E3) the transgenic animal tissue of described recombinant vector;
E16) containing E4) the transgenic animal tissue of described recombinant vector;
E17) containing E1) transgenic animal organ of described nucleic acid molecules;
E18) containing E2) transgenic animal organ of described expression cassette;
E19) containing E3) transgenic animal organ of described recombinant vector;
E20) containing E4) transgenic animal organ of described recombinant vector.
In above-mentioned application, the expression cassette of the nucleic acid molecules containing coding Endoglin E2), refer to the DNA that can express Endoglin in host cell, the promoter that the encoding gene that this DNA not only can comprise startup Endoglin is transcribed, also can comprise the terminator stopping Endoglin encoding gene and transcribe.Further, described expression cassette also can comprise enhancer sequence.
Available existing expression vector establishment contains the recombinant vector of described Endoglin encoding gene expression cassette.
In above-mentioned application, described carrier can be plasmid, glutinous grain, phage or viral vector.
In above-mentioned application, E5)-E8) described in microorganism can be yeast, antibacterial, algae or fungus.
In above-mentioned application, described transgenetic animal cell system, described transgenic animal tissue and described transgenic animal organ all do not comprise propagating materials.
In an embodiment of the invention, the encoding gene of described Endoglin is imported in hepatocellular carcinoma H22 by the recombinant vector of the expression cassette of the encoding gene containing described Endoglin.The recombinant vector that described recombinant vector obtains for replacing the fragment on pLVX-Puro carrier between BamH I and Xba I recognition site with the DNA molecular in sequence table shown in SEQ ID No.2.
In above-mentioned application, described in treat and/or prevent tumour medicine and can be and treat and/or prevent liver-cancer medicine.
For solving the problems of the technologies described above, the T lymphocyte that present invention also offers the secretion of gamma-IFN that described fused cell inducer T lymphocyte obtains is preparing the application treated and/or prevented in tumour medicine.
In above-mentioned application, described in treat and/or prevent tumour medicine and can be and treat and/or prevent liver-cancer medicine.
For solving the problems of the technologies described above, present invention also offers reconstitution cell and preparing the application treated and/or prevented in tumour medicine.
Above-mentioned reconstitution cell in preparation treats and/or prevents in tumour medicine application, described in treat and/or prevent tumour medicine and can be and treat and/or prevent liver-cancer medicine.
For solving the problems of the technologies described above, present invention also offers following P1)-P5) in any one treats and/or prevents tumour medicine:
What P1), utilize described Endoglin to prepare treats and/or prevents tumour medicine;
What P2), utilize the described biomaterial relevant to Endoglin to prepare treats and/or prevents tumour medicine;
What P3), utilize described fused cell to prepare treats and/or prevents tumour medicine;
What P4), utilize the tumor cell of described expression Endoglin to prepare treats and/or prevents tumour medicine;
Prepared by the T lymphocyte of the secretion of gamma-IFN P5), utilizing described fused cell inducer T lymphocyte to obtain treats and/or prevents tumour medicine.
Above-mentionedly treat and/or prevent in tumour medicine, described in treat and/or prevent tumour medicine and can be and treat and/or prevent liver-cancer medicine.
In the present invention, T lymphocyte can be the T lymphocyte deriving from human peripheral.
Experiment proves, the T lymphocyte of the secretion of gamma-IFN that fused cell DC/HepG2 (Eng+) of the present invention is induced significantly can delay tumor growth: the volume of the tumor after the T lymphocyte treatment of the secretion of gamma-IFN of inducing through DC/HepG2 (Eng+) be respectively DC, HepG2, HepG2 (Eng+), HepG2 (pLVX-Puro), DC/HepG2, DC/HepG2 (pLVX-Puro) and PBS 0.429 times, 0.295 times, 0.300 times, 0.328 times, 0.479 times, 0.487 times, 0.319 times.The T lymphocyte of the secretion of gamma-IFN that fused cell DC/HepG2 (Eng+) of the present invention is induced obviously can extend lotus people Liver Cancer Bearing Nude Mice life span: compare with PBS, DC, HepG2, HepG2 (Eng+), HepG2 (pLVX-Puro), DC/HepG2 with DC/HepG2 (pLVX-Puro), when the survival rate of lotus people Liver Cancer Bearing Nude Mice is respectively 80%, 60%, 40%, 20% and 0, the lotus people Liver Cancer Bearing Nude Mice of the T lymphocyte treatment of the secretion of gamma-IFN that DC/HepG2 (Eng+) induces all extended apart from the time that the 1st time is treated.Experiment proves, the T lymphocyte of the secretion of gamma-IFN that the DC/HepG2 (Eng+) of the application induces can obviously Tumor suppression growth, prolongation lotus people Liver Cancer Bearing Nude Mice life span.
Accompanying drawing explanation
Fig. 1 is the expression that Western blot method detects Endoglin albumen in HepG2 (Eng+).
Fig. 2 is Double fluorescence staining method qualification fused cell.Wherein, red arrow indication is DC/HepG2 (Eng+), and green arrow indication is HepG2 (Eng+), and blue arrow indication is DC.
Fig. 3 is that DC/HepG2 (Eng+) treats tumor presence.Wherein, A is the situation of change of gross tumor volume after the T lymphocyte treatment tumor of the secretion of gamma-IFN that DC/HepG2 (Eng+) induces, and B is the survival condition of nude mice after the T lymphocyte treatment tumor of the secretion of gamma-IFN that DC/HepG2 (Eng+) induces.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.
Experimental technique in following embodiment, if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
PLVX-Puro carrier in following embodiment is Nanjing Jin Sirui Products, and article No. is RP20513.
HepG2 cell in following embodiment is bought from ATCC cell bank, and article No. is HB-8065
tM.
Inbred line Female nude mice (BALB/c Nude Mice) in following embodiment is bought from Shanghai Bang Yao Bioisystech Co., Ltd.
RhGM-CSF (Recombinant human Granulocyte/Macrophagecolony-stimulating factor in following embodiment, recombined human granulocyte macrophage colony simulating factor) be R & D Systems Products, article No. is 215-GM-010; RhIL-4 (Recombinant human interleukin-4, recombinant human interleukin--4) is R & D Systems Products, and article No. is 204-IL-010; RhIL-2 (Recombinant humaninterleukin-2, recombinant human interleukin--2) is R & D Systems Products, and article No. is 202-IL-010; RhTNF-α (Recombinant human tumor necrosis factor-α, recombination human tumor necrosis factor-alpha) is R & D Systems Products, and article No. is 210-TA-005.
T lymphocyte in following embodiment is the T lymphocyte deriving from human peripheral.
The preparation of embodiment 1, fused cell
The preparation method of fused cell, comprises S1) and S2):
S1) reconstitution cell is obtained by the encoding gene of Endoglin importing tumor cell;
S2) described reconstitution cell and dendritic cell are merged obtain described fused cell.
Concrete grammar is as follows:
1, the preparation of reconstitution cell
BamH I on pLVX-Puro carrier and the DNA fragmentation between Xba I recognition site are replaced with the DNA molecular shown in SEQ ID No.2 in sequence table, keep other sequences of pLVX-Puro constant, obtain recombinant vector pLVX-Puro/Eng, this recombinant vector pLVX-Puro/Eng expresses the Endogl in shown in SEQ ID No.1.This recombinant vector pLVX-Puro/Eng is imported in HepG2 cell, obtains the reconstitution cell containing Endogl in coded sequence and EGFP coded sequence, by this reconstitution cell called after HepG2 (Eng+).
Wherein, the Endoglin shown in the 1st-1683 nucleotide coding SEQ ID No.1 of SEQ ID No.2, the 1705th-2424 nucleotide coding EGFP of SEQ ID No.2.
Western blot method is utilized to detect the expression of Endogl in HepG2 (Eng+), primary antibodie is the Endogl in antibody (product of Abcam, article No. is ab169545), two resist for horseradish peroxidase-labeled goat anti-rabbit igg, and DAB develops the color result as shown in Figure 1.Result shows to express Endogl in albumen in restructuring cancerous cell HepG2 (Eng+).
By in pLVX-Puro vector introduction HepG2 cell, obtain the reconstitution cell of pLVX-Puro carrier, by this reconstitution cell called after HepG2 (pLVX-Puro).
Cultivate HepG2, HepG2 (Eng+) and HepG2 (pLVX-Puro), obtain being in the HepG2 of logarithmic (log) phase respectively, be in the HepG2 (Eng+) of logarithmic (log) phase and be in the HepG2 (pLVX-Puro) of logarithmic (log) phase.
2, the preparation of dendritic cell
S1, the blood PBS buffer getting people dilute, blood after dilution is added slowly and is placed with (volume of the blood after dilution is the twice of the volume of lymphocyte separation medium) in the centrifuge tube of lymphocyte separation medium, make the blood after dilution slowly run down into lymphocyte separation medium skin lamination along tube wall, make the blood suspension after dilution on lymphocyte separation medium;
S2,2300rpm, centrifugal 30min, centrifuge raising speed and reduction of speed are 1 grade;
S3, liquid in pipe layering are plasma layer from top to bottom successively, PBMC (PERIPHERAL BLOOD MONONUCLEAR CELL) layer, separating medium, GCL, red blood cell layer.Handle with care, the cloud tunica albuginea confluent monolayer cells in the middle of drawing with 1mL syringe, collects in new 50mL centrifuge tube, obtains PBMC liquid;
S4, add 4 times of 37 DEG C of incomplete RIPM 1640 (namely not containing the RIPM 1640 of serum) washings to PBMC liquid volume, 1500rpm, centrifugal 10min; Use 4 times again to 37 DEG C of infull RIPM 1640 (namely not containing the RIPM 1640 of serum) washing of PBMC liquid volume, 1100rpm, 10min; Use 4 times again to 37 DEG C of infull RIPM 1640 (namely not containing the RIPM 1640 of serum) washing of PBMC liquid volume, 1000rpm, centrifugal 10min;
S5, with complete RIPM 1640 (namely containing the RIPM 1640 of serum) re-suspended cell, to be put in culture bottle.At 37 DEG C, 5%CO
2cultivate 2 hours (attached cell is the precursor (being designated as the DC that DC cultivates the 0th day) of dendritic cell (DC)) in incubator, add 1000U/mL rhGM-CSF and 500U/mL rhIL-4, be placed in 37 DEG C, 5%CO
2in incubator.Within in DC incubation every three days, change liquid by complete RIPM 1,640 half amount containing 1000U/mL rhGM-CSF, 500U/mL rhIL-4;
S6, to cultivate the 5th day DC at DC be suspended state substantially, adds the rhTNF-α of 25ng/mL, continue to be placed in 37%, 5%CO
2cultivate in incubator, cultivate at DC and obtain ripe DC on the 7th day.The cellular morphology of this ripe DC is that volume becomes large, and have root hair shape burr shape projection in colony growth, form is irregular.
The level of CD83, CD86, HL Α-DR and HL Α-ABC is expressed through the above-mentioned mature dendritic cell of cells were tested by flow cytometry (DC).First anti-HLA-DR antibody (the ebioscience product with FITC-respectively by above-mentioned mature dendritic cell (DC), article No. is 11-9956-42), anti-HLA-ABC antibody (the ebioscience product of FITC-, article No. is 11-9983-41), anti-CD83 antibody (the ebioscience product of FITC-, article No. is 11-0839-42) and anti-CD86 antibody (the ebioscience product of PE-, article No. is 25-0869-42) react 45 minutes in PBS, three times are washed with PBS, upper machine analysis after reaction terminates.Result shows ripe DC high expressed CD83, CD86, HLA-DR and HLA-ABC, reach 85.7 ± 4.79% respectively, 93.8 ± 2.75%, 97.06 ± 1.30%, 99.37 ± 0.35%, compared with the DC (precursor of DC) cultivating the 0th day with DC, the expression of CD83, CD86, HLA-DR has had significantly increases, and shows that the dendritic cell (DC) obtained in above-mentioned steps is ripe DC.
3, the preparation of fused cell
The ripe DC of step 2 is washed one time with the RIPM 1640 not containing serum by 3.1.Abandon supernatant, add 500 μ L Diluent C (NEB, B8003S) resuspended one-tenth individual cells suspension.Separately add 1 μ L PKH26 stock solution (Sigma) mixing at 500 μ L Diluent C, then add immediately in cell, incubated at room 4min.Add 1mL calf serum stop 1min, with 6mL complete RIPM 1640 culture medium wash three times for subsequent use, obtain dye after DC, above step needs lucifuge to carry out.
HepG2 (Eng+) the tumor cell staining procedure being in logarithmic (log) phase of 3.2 steps 1: the RIPM 1640 of tumor cell not containing serum washes one time.Abandon supernatant, add the PBS resuspended one-tenth individual cells suspension of 1mL containing 0.1% (mass percent concentration) BSA.Separately add 3 μ L FITC stock solutions (Sigma company) mixings at 1mL containing the PBS of 0.1 (mass percent concentration) %BSA, then add immediately in cell, hatch 8min for 37 DEG C, every 2min concussion once.Add 1mL calf serum and stop 1min, wash three times by 6mL complete RIPM 1640 culture medium, obtain the tumor cell after dyeing, above step needs lucifuge to carry out.
3.3, by the tumor cell mixing after the dyeing of the DC after the dyeing of step 3.1 and step 3.2, topple over supernatant after the centrifugal 10min of 1500rpm, do not add culture fluid and hatch 4min in 38 DEG C.The PEG2000250 μ L of preheating at 38 DEG C is slowly added along centrifugal tube wall, will rotating centrifugal pipe slowly in dropping process, cell is fully contacted with PEG2000.Then 38 DEG C, 4min is hatched.Slowly add 40mL PBS along tube wall to stop merging, 1200rpm, centrifugal 10min washs one time, adds culture fluid and is placed in 37 DEG C, 5%CO containing the complete DMEM of 10% hyclone
2suspension cell is collected after cultivating 24h in incubator, Double fluorescence staining method as follows selects fused cell: detect the cell in above-mentioned cell suspension to be measured, the HepG2 (Eng+) being in logarithmic (log) phase of step 1 only sends green fluorescence and does not send red fluorescence, the ripe DC of step 2 only sends red fluorescence and does not send green fluorescence, the fused cell of the two can send red fluorescence and send green fluorescence again, select the fused cell (Fig. 2) that can send red fluorescence and send again green fluorescence, by this fused cell called after DC/HepG2 (Eng+).Result shows, the percentage rate that fused cell DC/HepG2 (Eng+) occurs is 65%.
According to the method described above, the HepG2 (Eng+) being in logarithmic (log) phase is replaced with the HepG2 cell being in logarithmic (log) phase that step 1 obtains, other steps are constant, obtain the fused cell DC/HepG2 of HepG2 and DC.
According to the method described above, the HepG2 (Eng+) being in logarithmic (log) phase is replaced with the HepG2 (pLVX-Puro) being in logarithmic (log) phase that step 1 obtains, other steps are constant, obtain the fused cell DC/HepG2 (pLVX-Puro) of HepG2 (pLVX-Puro) and DC.
The lymphocytic generation of T of embodiment 2, DC/HepG2 (Eng+) external evoked secretion of gamma-IFN
In triplicate, the concrete steps at every turn repeating to test are as follows in experiment:
Obtain the T lymphocyte of the secretion of gamma-IFN that DC/HepG2 (Eng+) induces by the following method, and with enzyme-linked immunospot assay (enzyme l inked immunospot assay, ELISPOT) the IFN-γ detecting T lymphocytic emiocytosis is carried out, 1 × Washing buffer used, biotin labeled antibody, enzyme mark Avidin is HumanIFN-gamma precoated ELISPOT kit, and (Human IFN-gamma precoated ELISPOT kit is Bioisystech Co., Ltd's product for reaching section, article No. is DKW22-1000-048) in reagent, concrete steps are as follows:
1) take out the orifice plate in test kit, in every hole, add infull RIPM 1640 culture medium of 200 μ L, room temperature leaves standstill 5-10 minute, is outwelled by liquid.
2) DC/HepG2 (Eng+) 3 × 10 of the embodiment 1 suspended by infull RIPM 1640 culture medium is added to every Kong Jun
4individual and T lymphocyte 3 × 10
5individual.Every hole 100 μ L amount of liquid, often organizes 4 multiple holes.
3) hatch: build plate lid, put into incubator and cultivate 5 days, obtain the T lymphocyte of the secretion of gamma-IFN that DC/HepG2 (Eng+) induces.
4) cleavage step 3) DC/HepG2 (Eng+) the T lymphocyte of secretion of gamma-IFN of inducing: pouring aperture inner cell and culture medium.In every hole, add the deionized water of 200 μ L, 4 DEG C of pre-coolings, 15 minutes hypotonic lysis cells placed by 4 DEG C of refrigerators.
5) wash plate: topple over the liquid in culture plate, each every hole 200 μ L 1 × Washing buffer wash, and wash 5 times altogether, stop 60-80 second at every turn.For the last time, after pouring out the liquid in culture plate, culture plate is tipped upside down in absorbent paper to no liquid in culture plate.
6) detect antibody incubation: by with sterilized water according to biotin labeled antibody: the biotin labeled antibody that the dilution proportion of sterilized water=1:1000 obtains adds in every hole of culture plate, every hole 100 μ L.Hatch 1 hour for 37 DEG C.
7) wash plate: topple over the liquid in culture plate, add 1 × Washing buffer, every hole 200 μ L, washs 5-6 time.Each stop 60-80 second.For the last time, in absorbent paper, button is dry.
8) enzyme connection Avidin hatch: by with sterilized water according to enzyme mark Avidin: the enzyme mark Avidin that the dilution proportion of sterilized water=1:100 obtains adds in every hole of culture plate, every hole 100 μ L.Hatch 1 hour for 37 DEG C.
9) wash plate: topple over the liquid in culture plate, add 1 × Washing buffer, every hole 200 μ L, washs 5-6 time.Each stop 60-80 second.For the last time, in absorbent paper, button is dry.
10) develop the color: freshly prepared AEC nitrite ion working solution is added each experimental port, every hole 100 μ L.Room temperature lucifuge leaves standstill 15-50 minute.
11) color development stopping: topple over the liquid in culture plate, opens plate base, spends tap water culture plate positive and negative and base 5 times, color development stopping.
12) plate dries rear CTL instrument designing analysis of threshold, and records speckle parameter, does statistical analysis.
According to the method described above, DC/HepG2 (Eng+) is replaced with DC/HepG2 respectively, DC/HepG2 (pLVX-Puro), DC, HepG2 (Eng+), HepG2 and HepG2 (pLVX-Puro), obtain the T lymphocyte of DC/HepG2 induction respectively, the T lymphocyte that DC/HepG2 (pLVX-Puro) induces, the T lymphocyte of DC induction, the T lymphocyte that HepG2 (Eng+) induces, the T lymphocyte that the T lymphocyte of HepG2 induction and HepG2 (pLVX-Puro) induce, and the ELISpot testing result of the amount of the IFN-γ of these T lymphocytic emiocytosis.
Result shows, the T lymphocyte secretion of gamma-IFN hardly that HepG2 (Eng+), HepG2 and HepG2 (pLVX-Puro) are external evoked; The speckle number of the IFN-γ of the T lymphocytic emiocytosis that DC/HepG2 (Eng+) induces is all higher than the speckle number of the IFN-γ of DC/HepG2, DC/HepG2 (pLVX-Puro) and the secretion of DC inducer T lymphocyte.Show, DC/HepG2 (Eng+) can the lymphocytic generation of T of efficient secretion inducing IFN-γ.
The T lymphocyte of the secretion of gamma-IFN that embodiment 3, fused cell DC/HepG2 (Eng+) induce is to the therapeutical effect of tumor
In triplicate, the concrete steps at every turn repeating to test are as follows in experiment:
The T lymphocyte that the T lymphocyte that the T lymphocyte of secretion of gamma-IFN that the T lymphocyte of the secretion of gamma-IFN of being induced by the DC/HepG2 (Eng+) of embodiment 2, the T lymphocyte of the secretion of gamma-IFN of DC/HepG2 induction, DC/HepG2 (pLVX-Puro) induce, the T lymphocyte of the secretion of gamma-IFN of DC induction, HepG2 (Eng+) induce, HepG2 induce and the T lymphocyte that HepG2 (pLVX-Puro) induces are suspended in PBS respectively, obtain cell content respectively and are 10
5the T Lymphocyte suspension of the secretion of gamma-IFN that the DC/HepG2 (Eng+) of individual/μ L induces, the T Lymphocyte suspension of the secretion of gamma-IFN of DC/HepG2 induction, the T Lymphocyte suspension of the secretion of gamma-IFN that DC/HepG2 (pLVX-Puro) induces, the T Lymphocyte suspension of the secretion of gamma-IFN of DC induction, the T Lymphocyte suspension that HepG2 (Eng+) induces, the T Lymphocyte suspension that the T Lymphocyte suspension of HepG2 induction and HepG2 (pLVX-Puro) induce.
Get 20 4-5 inbred line Female nude mice in age in week (BALB/c Nude Mice), every only in oxter, right side subcutaneous vaccination 5 × 10
6individual HepG2 hepatoma carcinoma cell.Measure tumor major diameter and minor axis, according to formula TV=1/2 × a × b twice weekly
2calculate gross tumor volume.Treat that tumor average volume grows to about 100mm
3time, (in the T Lymphocyte suspension of the secretion of gamma-IFN that DC/HepG2 (Eng+) induces, the lymphocytic content of T of secretion of gamma-IFN is 10 to inject the T cell suspension of the secretion of gamma-IFN that 100 μ L DC/HepG2 (Eng+) induce in the vein to every lotus tumor BalB/c nude mice
7individual/100 μ L) treat, first time injection is designated as treatment the 0th day, the T Lymphocyte suspension of the secretion of gamma-IFN that the above-mentioned DC/HepG2 of 100 μ L (Eng+) induces within 36th day, is all injected respectively in treatment the 6th day, treatment the 12nd day, treatment the 18th day, treatment the 24th, treatment the 30th day and treatment, the volume (Fig. 3 and table 1) of lotus tumor BalB/c nude mouse tumor is measured respectively before the injection on the per injection same day, the time-to-live of every mice is recorded 1st day, the survival rate (Fig. 3 and table 2) of statistics mice from treatment.
According to the method described above, the T Lymphocyte suspension of the secretion of gamma-IFN of being induced by DC/HepG2 (Eng+) respectively replaces with PBS, the T Lymphocyte suspension of the secretion of gamma-IFN of DC/HepG2 induction, the T Lymphocyte suspension of the secretion of gamma-IFN that DC/HepG2 (pLVX-Puro) induces, the T Lymphocyte suspension of the secretion of gamma-IFN of DC induction, the T Lymphocyte suspension that HepG2 (Eng+) induces, the T Lymphocyte suspension that the T Lymphocyte suspension of HepG2 induction and HepG2 (pLVX-Puro) induce, other steps are all constant, obtain PBS respectively, the T lymphocyte of the secretion of gamma-IFN of DC/HepG2 induction, the T lymphocyte of the secretion of gamma-IFN that DC/HepG2 (pLVX-Puro) induces, the T lymphocyte of the secretion of gamma-IFN of DC induction, the T lymphocyte that HepG2 (Eng+) induces, the T lymphocyte that the T lymphocyte of HepG2 induction and HepG2 (pLVX-Puro) induce is to therapeutic effect (Fig. 3 of tumor, table 1 and table 2).
Volume (the mm of tumor after the T lymphocyte treatment tumor of table 1, different cell induction
3)
When treatment the 36th day, the T lymphocyte that volume is respectively DC, HepG2, HepG2 (Eng+), HepG2 (pLVX-Puro), DC/HepG2 and DC/HepG2 (pLVX-Puro) induce of tumor after the T lymphocyte treatment of the secretion of gamma-IFN of inducing through DC/HepG2 (Eng+) and 0.429 times, 0.295 times, 0.300 times, 0.328 times, 0.479 times, 0.487 times, 0.319 times of PBS.
Result shows, the T lymphocyte of the secretion of gamma-IFN that DC/HepG2 (Eng+) induces significantly can delay tumor growth, and its to the therapeutic effect of tumor significantly better than DC, HepG2, HepG2 (Eng+), HepG2 (pLVX-Puro), DC/HepG2 (pLVX-Puro) and DC/HepG2 induction T lymphocyte to the therapeutic effect of tumor.
Table 2, the lotus people Liver Cancer Bearing Nude Mice time that distance is treated for the 1st time when different survival rates (my god)
| Survival rate | 80％ | 60％ | 40％ | 20％ | 0 |
| PBS | 85 | 94 | 106 | 119 | 133 |
| DC/HepG2(pLVX-Puro) | 144 | 166 | 173 | 185 | 194 |
| DC/HepG2 | 112 | 145 | 169 | 175 | 183 |
| DC/HepG2(Eng+) | 196 | 203 | 203 | 208 | 231 |
| DC | 134 | 168 | 182 | 188 | 190 |
| HepG2 | 76 | 89 | 98 | 110 | 112 |
| HepG2(Eng+) | 88 | 102 | 104 | 108 | 119 |
| HepG2(pLVX-Puro) | 77 | 98 | 99 | 106 | 110 |
Result shows, the T lymphocyte of the secretion of gamma-IFN that DC/HepG2 (Eng+) induces obviously can extend lotus people Liver Cancer Bearing Nude Mice life span: the T lymphocyte of inducing with PBS and DC, HepG2, HepG2 (Eng+), HepG2 (pLVX-Puro), DC/HepG2 with DC/HepG2 (pLVX-Puro) is compared, when the survival rate of lotus people Liver Cancer Bearing Nude Mice is respectively 80%, 60%, 40%, 20% and 0, the lotus people Liver Cancer Bearing Nude Mice of the T lymphocyte treatment of the secretion of gamma-IFN that DC/HepG2 (Eng+) induces all extended apart from the time that the 1st time is treated; When the survival rate 0% of lotus people Liver Cancer Bearing Nude Mice, the T lymphocytic 1.22 times, 2.06 times, 1.94 times, 2.1 times, 1.26 times, 1.19 times that the time is respectively DC, HepG2, HepG2 (Eng+), HepG2 (pLVX-Puro), DC/HepG2 and DC/HepG2 (pLVX-Puro) induce of lotus people Liver Cancer Bearing Nude Mice distance the 1st treatment of the T lymphocyte treatment of the secretion of gamma-IFN that DC/HepG2 (Eng+) induces.At treatment and viewing duration, the phenomenons such as mice is all without piloerection, and appetite declines, and behavioral activity is abnormal.
The T lymphocyte of the secretion of gamma-IFN that the DC/HepG2 (Eng+) of the application induces obviously can delay the growth of tumor, extends lotus people Liver Cancer Bearing Nude Mice life span.
Claims (10)
1. prepare the method for fused cell, it is characterized in that: described method comprises is undertaken merging the step obtaining fused cell by tumor cell and dendritic cell;
Described tumor cell is M or N:
M, described tumor cell are the reconstitution cell of expressing Endoglin;
N, described tumor cell are the reconstitution cell of the gene containing the described Endoglin of coding;
Described Endoglin is following A 1) or protein A2):
A1) aminoacid sequence is the protein of SEQ ID No.1;
A2) in the aminoacid sequence shown in SEQ ID No.1 through replacement and/or disappearance and/or add that one or several amino acid residue obtains have identical function by A1) derivative protein.
2. method according to claim 1, is characterized in that: the tumor cell of described expression Endoglin is that the encoding gene of described Endoglin is imported the reconstitution cell obtained in receptor tumor cells.
3. method according to claim 1 and 2, is characterized in that: the encoding gene of described Endoglin is following A11) or A21) or A31) shown in nucleic acid molecules:
A11) DNA molecular shown in the 1st-1683 nucleotide of SEQ ID No.2 in sequence table;
A21) nucleotide sequence and A11) limited has more than 75% or 75% homogeneity, and the cDNA molecule of the described Endoglin that encodes or genomic DNA molecule;
A31) under strict conditions with A11) nucleotide sequence hybridization that limits, and the cDNA molecule of the described Endoglin that encodes or genomic DNA molecule.
4. method according to claim 1 and 2, is characterized in that: described receptor tumor cells is hepatoma carcinoma cell.
5. method according to claim 1, is characterized in that: described dendritic cell is the dendritic cell prepared from peripheral blood.
6. the fused cell that in claim 1-5, arbitrary described method obtains.
7. treat and/or prevent tumour medicine, it is characterized in that: described in treat and/or prevent tumour medicine active component be following H1 and/or H2:
H1, fused cell according to claim 6;
The T lymphocyte of the secretion of gamma-IFN that described in H2, claim 6, fused cell inducer T lymphocyte obtains.
8. fused cell according to claim 6 is preparing the application that treats and/or prevents in tumour medicine or the application in the T lymphocyte of preparation secretion of gamma-IFN.
9. the application of following M1 or M2 or M3:
M1, claim 1 or the Endoglin described in 2 or 3 are preparing the application treated and/or prevented in tumour medicine;
The application treated and/or prevented in tumour medicine prepared by M2, the biomaterial relevant to the Endoglin described in claim 1 or 2 or 3; Described biomaterial is following E1) to E20) in any one:
E1) nucleic acid molecules of coding claim 1 or the Endoglin described in 2 or 3;
E2) containing E1) expression cassette of described nucleic acid molecules;
E3) containing E1) recombinant vector of described nucleic acid molecules;
E4) containing E2) recombinant vector of described expression cassette;
E5) containing E1) recombinant microorganism of described nucleic acid molecules;
E6) containing E2) recombinant microorganism of described expression cassette;
E7) containing E3) recombinant microorganism of described recombinant vector;
E8) containing E4) recombinant microorganism of described recombinant vector;
E9) containing E1) the transgenetic animal cell system of described nucleic acid molecules;
E10) containing E2) the transgenetic animal cell system of described expression cassette;
E11) containing E3) the transgenetic animal cell system of described recombinant vector;
E12) containing E4) the transgenetic animal cell system of described recombinant vector;
E13) containing E1) the transgenic animal tissue of described nucleic acid molecules;
E14) containing E2) the transgenic animal tissue of described expression cassette;
E15) containing E3) the transgenic animal tissue of described recombinant vector;
E16) containing E4) the transgenic animal tissue of described recombinant vector;
E17) containing E1) transgenic animal organ of described nucleic acid molecules;
E18) containing E2) transgenic animal organ of described expression cassette;
E19) containing E3) transgenic animal organ of described recombinant vector;
E20) containing E4) transgenic animal organ of described recombinant vector;
The T lymphocyte of the secretion of gamma-IFN that described in M3, claim 6, fused cell inducer T lymphocyte obtains is preparing the application treated and/or prevented in tumour medicine.
10. following P1)-P5) in any one treats and/or prevents tumour medicine:
What P1), utilize claim 1 or the Endoglin described in 2 or 3 to prepare treats and/or prevents tumour medicine;
What P2), utilize the biomaterial described in claim 9 to prepare treats and/or prevents tumour medicine;
What P3), utilize the fused cell described in claim 6 to prepare treats and/or prevents tumour medicine;
What P4), utilize the tumor cell of the expression Endoglin described in claim 1 or 2 to prepare treats and/or prevents tumour medicine;
What P5), utilize the T lymphocyte of the secretion of gamma-IFN that described in claim 6, fused cell inducer T lymphocyte obtains to prepare treats and/or prevents tumour medicine.
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| XING GU，ET AL: "Transforming Growth Factor beta1 Gene Variation Leu10Pro Affects Secretion and Function in Hepatic Cells", 《DIG DIS SCI》 * |
| 张利旺: "全细胞性肿瘤抗原以不同方式负载树突状细胞诱导肝癌特异性T细胞反应的实验研究", 《中国博士学位论文全文数据库医药卫生科技辑》 * |
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| CN109251961A (en) * | 2018-06-28 | 2019-01-22 | 广西医科大学 | A kind of method of unicellular sequencing detection activating T cell |
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