CN109749994A - A kind of method of melanoma-associated antigen A3 killing human liver cancer cell - Google Patents

Abstract

A kind of method of melanoma-associated antigen A3 killing human liver cancer cell, using grain-macrophage colony stimulating factor (GMCSF) and IL-4 (interleukin4, IL4) the inducing dendritic shape cell from human peripheral, it is co-cultured after MAGEA3 antigen sensibilization with T lymphocyte, it is effector cell with lymphocyte, respectively to express the hepatoma cell strain H4M of MAGEA3 antigen, the normal liver cell for not expressing MAGEA3 antigen as target cell, collects T lymphocyte and carry out tumor cytotoxicity test.

Description

A kind of method of melanoma-associated antigen A3 killing human liver cancer cell

Technical field

The present invention relates to a kind of methods of melanoma-associated antigen A3 killing human liver cancer cell, specifically with a kind of black The method of plain tumor antigen A3 killing human liver cancer cell.

Background technique

Currently known human melanoma antigen A3(melanomaantigengene, MAGEA3) in kinds of tumors tissue Middle expression, and also have in hepatocellular carcinoma (hepatocellularcarcinoma, HCC) expression of upper frequency, but It is not expressed in addition to placenta and testis in normal tissue, therefore is ideal target antigen in anti-tumor immunotherapy.Dendron shape Cell (dendriticcells, DCs) is the professional antigen presenting cells that can uniquely activate primary tape T cell in vivo, and DCs is being lured Leading body and generating in specificity antineoplastic immunity reaction has especially important effect.

Summary of the invention

Materials and methods；The recombinant plasmid pGEX4T1MAGEA3 of plasmid and cell strain containing MAGEA3cDNA be this room voluntarily Building.Hepatoma cell strain H4M and normal liver cell strain L02 builds strain by pathology department, the first affiliated hospital, Zhongshan University originally culture. Main agents RPMI1640 culture medium is purchased from Gibcobrl company；Fetal calf serum is purchased from Hyclone company；Fluorescein isothiocynate (FITC) mouse anti human CD83, the mankind of mouse anti human CD80, CD86 monoclonal antibody and phycoerythrin (PE) label marked Leukocyte antigen DR monoclonal antibody is purchased from Pharmingen company；Rabbit-anti people's MAGE A3 polyclonal antibody is purchased from NeoMarkers company.The 1 MAGE A3 of isolation and purification recombinant plasmid pGEX 4T of MAGE A3 albumen converts Escherichia coli BL21 vulcanizes β D galactoside (IPTG) inducing expression through isopropyl.Take the bacterium solution of 1000mL inducing expression, 10000g, 4 DEG C centrifugation 5min；Bacterial sediment is resuspended with 50mL phosphate buffer (phosphatebufferedsaline, PBS), ice bath, Ultrasound cracking bacterium, it is 1% that TritonX100, which is added, to final volume score, is mixed, ice bath places 30min；12000g, 4 DEG C of centrifugations 20min；Supernatant is taken, using 5 mL glutathione sulfurtransferases of lauryl sodium sulfate-polyacrylamide gel electrophoresis (glutathin S transferase, GST) chromatographs post separation fusion protein, through SDS PAGE and gel thin-layer scanning identification The purity of purifying protein；PBS dialyses for 24 hours, filtration sterilization, -70 DEG C of preservations.The in vitro culture and antigen sensibilization of DCs uses Ficoll routinely separates 5 donors with normal peripheral bloods and obtains mononuclearcell, and adjusting its cell density is 2 × 106/mL, adds 1mL after cultivating 3h in 37 DEG C, the CO2 incubator that volume fraction is 5%, sucks suspension cell, DCs training is added in 24 orifice plates It supports base (the RPMI 1640 of containing rhGM CSF100ng/mL, rhIL 450ng/mL), in 37 DEG C, the CO2 item that volume fraction is 5% It is cultivated under part, every 3d is changed liquid 1 time.DCs dendron shape shape is observed by inverted microscope, while using flow cytomery The expression of the surface DCs HLA DR, CD80, CD83, CD86.The 7th day DCs is collected, adjustment cell concentration is 3 × 106/mL, It is inoculated in 24 orifice plates, is separately added into 50,100,200,300 μ L, GST albumen (20 μ g/ of GSTMAGEA3 albumen (20 μ g/mL) ML) 100 μ L and PBS100 μ L are cultivated for 24 hours；It is added TNF α (50U/mL), continues to cultivate 2d.Training is passed in the culture of cell strain respectively Support normal liver cell strain L02 and hepatoma cell strain H4M；Creep plate, immune group is made in cell suspension (concentration is 1 × 105/mL) Change the expression of dyeing detection MAGE A3 albumen.The preparation of T lymphocyte and the external evoked of CTL take with 1 peripheral body, Ficoll routinely separates mononuclearcell, and adjusting its cell density with culture solution is 1 × 106/mL；According to lymphocyte and DCs After the ratio mixed culture that cell quantity ratio is 5: 1, divide following groups: A group is (not empty with the lymphocyte of DCs mixed culture White control group)；B group is the unsupported fusion protein of lymphocyte+DCs(, is substituted with PBS)；C group is lymphocyte+DCs(load 20 μ g/mL fusion protein, 50 μ L)；D group is that lymphocyte+DCs(loads 20 μ g/mL fusion protein, 100 μ L)；E group is that lymph is thin Born of the same parents+DCs(loads 20 μ g/mL fusion protein, 200 μ L)；F group is that lymphocyte+DCs(loads 20 μ g/mL fusion protein, 300 μ L)； G group is that lymphocyte+DCs(loads 20 μ g/mLGST protein 10,0 μ L).Every 3d half, which is measured, changes liquid, supplements PHA and rhIL 2, to promote Into the maturation of DCs, enhance the HLA-II antigen of DCs；It is cultivated in 37 DEG C, volume fraction 5%CO2, saturated humidity incubator 7d, 4h before experiment terminates, every hole are added the MTT20 μ L of the 5mg/mL of Fresh, observe under inverted microscope；1000r/min It is centrifuged 5min, removes supernatant, DMSO150 μ L is added in every hole, 5min is vibrated, in reading absorbance (D) at 570nm in microplate reader Value, is calculated according to the following formula stimulus index: Is=D experimental group/D blank control group.Then, adjustment lymphocyte and DCs cell number Amount is than being respectively 10: 1,20: 1,50: 1,100: 1, and the grouping also according to above-mentioned A to G repeats the above experiment step, to compare Difference group lymphocytes proliferative capacities, every group counting 3 times, be averaged.Using not with DCs mixed culture lymphocyte as Blank control.The lethal effect of CTL is to cultivate the 7th day lymphocyte for effector cell, with the H4M liver cancer of the MAGE A3 positive Cell is target cell, is 10: 1 by effect target ratio (cell quantity ratio), lymphocyte and target cell is added simultaneously in following each group Killing experiments are carried out, wherein target cell is 2 × 104/hole, and effector cell adjusts accordingly；Every group separately sets 3 multiple holes, with PBS It is that 5%CO2 incubator is incubated for 48h in 37 DEG C, volume fraction for blank control.Be grouped as follows: I group is liver cancer cells+through loading The lymphocyte of the DCs stimulation of GST MAGE A3 fusion protein；II group stimulates for liver cancer cells+DCs through load GST albumen Lymphocyte；III group of lymphocyte for liver cancer cells+unsupported GST MAGE A3 fusion protein DCs stimulation；IV group is Liver cancer cells+the lymphocyte without DCs stimulation；V group is lymphocyte (effector cell)；VI group for liver cancer cells, (target is thin Born of the same parents)；Using the normal liver cell L02 of MAGE A3 feminine gender as target cell, above-mentioned experimental group is repeated.Then, adjustment effect target is than difference It is 20: 1,50: 1,100: 1, the grouping also according to above-mentioned I to VI repeats the above experiment step, compares feelings with more different effect targets The lethal effect of CTL under condition.The killing activity that mtt assay detects lymphocyte cultivates preceding 4h suction in end and abandons supernatant, and addition is new The MTT(5mg/mL of fresh configuration) 20 holes μ L/, be placed in 37 DEG C, volume fraction be 5%CO2 incubator be incubated for 4h；Supernatant is abandoned, is added The hole DMSO150 μ L/ vibrates at room temperature to crystal and dissolves；On enzyme mark detector, each hole is surveyed after being returned to zero with blank control wells D570 value calculates D < according to 0 value, and calculate killing rate: [ (it is thin that D imitates target cell-D effect to D target cell-for p killing/%= Born of the same parents) ]/D target cell × 100%.Statistical procedures are completed using statistic software SPSS 100.

As a result: the cytotoxic T lymphocyte (CTL) of the DCs activation of load MAGEA3 antigen has obviously liver cancer cells Lethal effect, normal liver cell control group (P < 0.05) is higher than to the killing rate conspicuousnesses of liver cancer cells.

Goal of the invention: human melanoma antigen A3(melanomaantigengene, MAGEA3) in kinds of tumors tissue Middle expression, and also have in hepatocellular carcinoma (hepatocellularcarcinoma, HCC) expression of upper frequency, but It is not expressed in addition to placenta and testis in normal tissue, therefore is ideal target antigen in anti-tumor immunotherapy.Dendron shape Cell (dendritic cells, DCs) is the professional antigen presenting cells that can uniquely activate primary tape T cell in vivo, and DCs exists Inducing body to generate in specificity antineoplastic immunity reaction has especially important effect.In the present invention, with the MAGE A3 of purifying Albumen is that antigen induction DCs excites specificity cell toxicity T lymphocyte (cytotoxicTlymphocyte, CTL) thin to HCC Born of the same parents carry out killing experiment in vitro, provide experimental basis for the immunization therapy of HCC for clinical application MAGEA3 development.

Technical solution: using grain-macrophage colony stimulating factor (GMCSF) and IL-4 (interleukin4, IL4) the inducing dendritic shape cell from human peripheral co-cultures after MAGEA3 antigen sensibilization with T lymphocyte, with lymphocyte For effector cell, respectively to express the hepatoma cell strain H4M of MAGE A3 antigen, not express the normal liver cell of MAGEA3 antigen For target cell, collects T lymphocyte and carry out tumor cytotoxicity test.

Invention is the utility model has the advantages that the dendritic cell vaccine of MAGEA3 proteantigen sensitization can activate the CTL of specificity, in body The stronger cytotoxicity to human hepatoma cell strain is induced outside.

Preferred forms: drug therapy is used.

Claims (3)

1. a kind of method of melanoma-associated antigen A3 killing human liver cancer cell, using grain-macrophage colony stimulating factor (GMCSF) and IL-4 (interleukin4, IL4) inducing dendritic shape cell from human peripheral, through MAGEA3 antigen sensibilization Afterwards with T lymphocyte co-culture, be effector cell with lymphocyte, respectively with express MAGEA3 antigen hepatoma cell strain H4M, The normal liver cell for not expressing MAGEA3 antigen is target cell, collects T lymphocyte and carries out tumor cytotoxicity test.

2. according to the method described in claim 1, it is characterized in that: after MAGEA3 antigen sensibilization with T lymphocyte co-culture, with Lymphocyte is effector cell, respectively to express the hepatoma cell strain H4M of MAGEA3 antigen, not express the normal of MAGEA3 antigen Liver cell is target cell, collects T lymphocyte and carries out tumor cytotoxicity test.

3. according to the method described in claim 1, it is characterized in that: respectively to express the hepatoma cell strain H4M, no of MAGEA3 antigen The normal liver cell for expressing MAGEA3 antigen is target cell, collects T lymphocyte and carries out tumor cytotoxicity test.