| BackgroundAlthough surgery,radiotherapy and chemotherapy have made some progress in reducing the mortality rate of patients and improving the quality of life in the treatment of malignant tumors,surgery is easy to relapse,radiotherapy and chemotherapy have more adverse reactions,patients need to bear great pain but still difficult to cure in the treatment process.In recent years,the application of immunotherapy with transformation lymphocytes has achieved great success.The design of immunotherapy is to give patients the activation of lymphocytes in vitro,and hope lymphocytes to kill and clear tumor cells.Immunotherapy also development and improvement step by step,from tumor infiltrating lymphocytes(TIL)which origin the tumor eluting cells,and then to lymphokine activated killer cells(LAK)which stimulated with IL-2,to cytokine induced killer cells(CIK)which stimulated with a variety of cytokines,and cytotoxic lymphocytes(CTL)which activated by tumor antigen peptide,and so on.The treatment effects of immunotherapy are limited.Until recently,chimeric antigen receptor T cells(CAR T)stand for the cellular immunotherapy in the clinical treatment of tumors.The reasons for the success of CAR T cells,which solve two key problems: the culture acquisition of activated lymphocytes growth massive in the body and continue to play the role of anti-tumor.CAR T cells include chimeric sequence CD8A-CD28-CD137-CD3 which induce T cell proliferative,long-term survival in vitro and vivo;another is to identify specific tumor antigen.We take CD19 CAR-T for example,which modify the receptor recognition of T cell antigen peptide fragment for anti-CD19 antibody,acute lymphocytic leukemia and chronic lymphocytic leukemia cells with CD19 antigen can be specific recognized and destructed.Despite this achievement,CAR-T cells also have defects including anti-tumor cytokine storm,drug resistance and off-target effects and other issues have gradually emerged in clinical,especially for CD19 CAR-T cells,killing CD19+ leukemia cells and normal B lymphocytes at the same time,it cause hypoimmunoglobulinemia,which cause the death of patients with infection of major hazards.The reason is that we do not find tumor-special marker of acute and chronic lymphocytic leukemia,just for CD19 antigen who often appear in leukemia cells and normal B cells.At present,there is an urgent need for the specific-biomarker of tumor cells.We found that High Mobility Group Chromosal Protein N2(HMGN2)is a protein which has ability of trend and specificity of the recognition for breast cancer,cervical cancer and other tumor cells,also improve anti-tumor effect of immune cells including cytotoxic T lymphocytes and natural killer cells.So we intend to construct the HMGN2-CD8A-CD28-CD137-CD3ζ fusion protein lentiviral vector,and recombinant T cell(referred to HMGN2-T cell)and observe its anti-tumor potential.ObjectiveExplore HMGN2-T cells whether express HMGN2-CD8A-CD28-CD137-CD3ζfusion protein,have function of proliferation and specific killing effects for tumor cells in vitro,can take HMGN2-T cells in clinical of tumor theatment.Methods1.Construction of HMGN2-CD8A-CD28-CD137-CD3 ζ fusion protein.Extract RNA of mononuclear cells from normal peripheral blood,then reverse transcription into c DNA,design PCR primers and amplify gene encoding area including specificity tumor recognition receptor HMGN2,transmembrane and hinge region CD8 A,intracellular stimulating signal area CD137 and CD28,signal transduction domain structure CD3ζ,take DNA with gel purification and HMGN2,CD8 A,CD28,CD137 and CD3ζ with TA cloning,Choose colony for gene sequencing.If the colony of sequencing is right,extract and purify the DNA plasmid.2.We connect HMGN2,CD8 A,CD28,CD137 and CD3ζ gene sequence together in sequence with molecular biology techniques,synthetic recombinant HMGN2-CD8A-CD28-CD137-CD3ζ(2880 bp)gene.Build recombinant HMGN2 fusion gene plasmid vector,detect gene sequence by PCR and gene sequencing technology identification,recombinant HMGN2 fusion gene is packaged with lentivirus,enrichment,purification and drops to measure.3.We take recombinant HMGN2 fusion gene to infect Jurkat cells with lentivirus and construct HMGN2-T cell.Detect recombinant HMGN2 fusion gene whether express in Jurkat cells by fluorescence microscope,flow cytometry and PCR.Detect the proliferation ability of recombinant HMGN2 fusion gene in Jurkat cells.Detect the proliferation ability of recombinant HMGN2-T cells by CCK-8 method,detect the levels of cytokines IL-2,IFN-γ,TNF-α in recombinant HMGN2-T cells with ELISA method,detect the apoptosis of recombinant HMGN2-T cells with flow cytometry in Annexin v-PE/7-AAD double staining;detect the cell cycle DNA contents of recombinant HMGN2-T cells with flow cytometry in PI single staining.4.Detect the killing ability of recombinant HMGN2-T cells.Lactate dehydrogenase(LDH)cytotoxicity experiment is used to test whether HMGN2-T cells can respectively kill lung cancer cell line LLC,cervical cancer cell line Hela,breast cancer cell line MCF7,liver cancer cell line Hep G2,and secretion levels of cytokine IL-2、IFN-γ、TNF-α.Results1 Exacted the RNA of HMGN2,CD8 A,CD137,CD28 and CD3ζ from normal human peripheral blood genomic and structured plasmid vectorExtracted total RNA from normal human peripheral blood mononuclear cells,reversed transcription into c DNA,respectively amplified specific tumor recognition receptor HMGN2(273 bp),transmembrane and hinge structure CD8A(708 bp),intracellular stimulating signal structures CD28(663 bp)and CD137(768 bp),signal conduction structure CD3ζ(492 bp)in PCR,DNA gel purification,then structured HMGN2-p GM-T,CD8A-p GM-T,CD28-p GM-T,CD137-p GM-T,CD3ζ-p GM-T plasmid vector,picked cloned gene sequencing.2 We restructured HMGN2 fusion protein GV358 plasmid vector,transfected to Jurkat cell and built recombinant HMGN2 fusion protein lentiviral.Application of PCR technology,we connectted five genes together and structured HMGN2-CD8A-CD28-CD137-CD3ζ(2880 bp)GV358 plasmid vector,picked cloned gene sequencing.The new recombinant of HMGN2-CD8A-CD28-CD137-CD3ζ plasmid vector named recombinant HMGN2 fusion protein.We transfected recombinant HMGN2 fusion protein to 293 T cells with GV358 plasmid,the Helper 1.0 plasmid,the Helper 2.0 plasmid.After 72 h,collected cells supernatant,took centrifugal concentration after removal of impurities,concentration and purification of lentivirus.Detected drops of recombinant HMGN2 fusion protein lentivirus was 1×108Tu/ml,under inverted fluorescence microscope photos after 72,293 T cells transfection with HMGN2 fusion protein lentivirus,saw large amounts of green fluorescent cells,and fluorescence rate was above 70%;PCR amplification HMGN2 fusion protein full length gene,it showed that lentivirus packaging successed.3 We used recombinant HMGN2 fusion protein lentiviral to transfect T cells,prepared HMGN2-T cells.We used recombinant HMGN2 fusion protein lentiviral to transfect T cell,successfully prepared HMGN2-CD8A-CD28-CD137-CD3ζ fusion protein,also called HMGN2-T cell.After transfection 72 h,cell refraction was strong,grow in good condition with ordinary microscope,saw a lot of green fluorescence Jurkat cells under the fluorescent microscope,the green fluorescence rate of HMGN2 fusion protein transfection Jurkat cells was 78.33% with flow cytometry.Extracted RNA from HMGN2-T cells,reversed transcription into c DNA,for PCR amplification of HMGN2 fusion protein length gene,gene sequencing was correct.Proliferation rate increased significantly in HMGN2 fusion protein transfection Jurkat cell by CCK-8 method.It showed that after transfection 24 h,48 h,72 h,compared with Jurkat cell and transfection GV358 empty plasmid Jurkat cells,HMGN2-T cell proliferation in 24 h(0.404±0.075)with the control group(0.363±0.103,0.371±0.097),had not significant difference(P = 0.499),but in the 48 h,72h(0.787±0.206,0.931±0.273)compared with control group had obvious enhancement,proliferation rate reached 48.66%,56.60% respectively,that HMGN2-T cell increased in proliferation effect,and as time extended HMGN2-T could significantly promote the Jurkat cell proliferation(P = 0.001).4 Secretion of cytokines IL-2 and TNF-α increased,TNF-γ levels without changed in HMGN2-T cellDetected cytokines IL-2,TNF-α,TNF-γ secretion of HMGN2 fusion protein transfection Jurkat cells after 24 h,48 h with ELISA.The experimental results showed that after 24 h,48h,cytokine IL-2 secretion of HMGN2-T cells(1054.205±94.815,1680.705±119.437)pg/ml was significantly higher than the Jurkat cells(795.25±16.263,882.735±36.790)pg/ml and GV358 Jurkat cells(540.46±8.188,801.735±25.434)pg/ml secretion levels at the same time points(P < 0.05),with time extended IL-2 of HMGN2-T cell secretion grew at a speed of 1.5 times(P = 0.009).Within 24 h,48h cytokine TNF-α secretion(305.373±16.598,662.534±48.485)pg/ml was significantly higher than the Jurkat cell(164.408±16.519,348.421±12.517)pg/ml and GV358 Jurkat cells(270.701±18.990,271.977±4.917)pg/ml at the same time of level(P < 0.05),in HMGN2-T cell,secretion of IL-2,TNF-α level had the trend of increasing over time.TNF-γ levels of HMGN2-T cell(1040.59±174.259,1013.652±84.402,1042.767-117.305)pg/ml between the three groups had no obvious change(P=0.991),and no difference changes at different time points(P = 0.764).5 HMGN2-T cells significantly increased in proliferation rate and apoptosis rate,metabolic rate increased fast.Detected HMGN2-T cells changes of apoptosis rate after 24 h,48h in flow cytometry Annexin v-PE/7-AAD double staining.Experimental results showed that,after 24 h,48h,HMGN2-T cells apoptosis rate compared with GV358 Jurkat cells(7.02% vs 3.57%,6.34% vs 7.02%),apoptosis rate increased by 3.45% and 5.5% respectively,had obvious difference(P=0.001),but HMGN2-T cells apoptosis in two time points(7.28%±0.99 vs 6.5%±0.72)had no significant change(P = 0.457).Detected HMGN2 fusion protein transfection Jurkat cells changes of cell DNA content after 24 h,48h in flow cytometry PI single staining.HMGN2-T cells compared to GV358 Jurkat cells,after transfection 24 h,G0/G1 phase cells decreased by 13.63%(38.926%±0.628 vs 5.556%±5.408),S phase cells increased 15.1%(55.94%±2.032 VS 40.846%±7.387),G2/M phase cells did not changed significantly(5.133%±1.532 VS 6.6%±1.992);HMGN2-T cells and GV358 Jurkat cells in 48 h,G0/G1 phase cells decreased by 8.72%(41.393%±3.425 vs 50.116%±8.575),S phase cells increased by 2.14%(47.3%±12.708 vs 45.163%±6.475),G2/M phase cells increased 1.92%(6.663%±3.133 vs 4.716%±2.511),it showed that transfection HMGN2-T cells compared with control group,the G0/G1 phase cells decreased,S phase and G2/M phase cells increased,cell proliferation rate significantly increased.6 In the process of anti-tumor cytokines of TNF-α,INF-γ secreted increased in HMGN2-T cells,could directly kill cervical cancer,lung cancer,liver cancer,breast cancer cell lines.Detected secretion of cytokine TNF-α,IFN-γ,IL-2 when HMGN2-T cells with Hela,LLC,Hep G2,MCF7 respectively in ELISA.IL-2 in Hela,LLC,Hep G2,MCF7 respectively,compared with the control group(2270.5±994.8 vs 1729±490.7,2997.5±1010.4 vs 2802.5±1115.1,3112.5±620.1 vs 2371±677.4,3221.5±615.89 vs 3596.5±453.2)% had no significant difference(P > 0.05);TNF-α levels in LLC(4332.5±887.4)%,MCF7(3611.5±1511.0)% and Hep G2(6256±660.4)% respectively,compared with the control group(2167.5±498.5,1116±237.5,3556.5±1004.7)% increased significantly,but in Hela cells(3002.5±306.1 vs 2282±395.9)% change level had no statistical difference(P > 0.05);secretion level of IFN-γ in LLC,MCF7 cells,Hela,Hep G2 respectively compared with the control group(18744.5±1545.0 vs 6781±1176.6,12852±3364.4 vs 4374±1347.7,15037.5± 4850.0 vs 3348±750.9,14282.5±760.1 vs 7332.5±1921.2)% increased significantly,the results showed that HMGN2-T cell on Hela,LLC,Hep G2 cells,MCF7 cells had obvious killing effect,and in the process of killing mainly secreted TNF-α,IFN-γ.Detected killing effect when HMGN2-T cells with LLC,Hela,Hep G2,MCF7 cells in LDH cytotoxic test.In LLC,effector-target ratio from 1:1,HMGN2-T cells killing rate(5.62±1.45 VS 8.92±3.47)% had no difference,to the 5:1,10:1,15:1,20:1,HMGN2-T cells for LLC cell specificity killing rate(22.33±4.75 VS 3.95±6.73,23.60±6.58 VS 3.81±1.07,43.81±5.73 VS 5.27±0.90,55.34±3.45 VS 3.81±1.89)% had obvious differences compared with control group,it showed that with the increase of effector-target ratio,killing rate increased;in MCF7 effector-target ratio from 5:1,the specificity killing rate(18.23±3.47,32.67± 7.44,58.78±16.51,66.74±8.38)%,the specificity killing rate of Hela(13.29±5.45,21.89±5.54,47.74±4.04,57.81±3.37)%,the specificity killing rate of Hep G2(19.58±5.22,29.57±10.17,56.22±10.26,64.18±25.30)% compared with the control group had obvious difference(P<0.05),with effector-target ratio increased,HMGN2-T cell had specificity killing effect respectively on Hela,LLC,Hep G2 cells,MCF7 reached 55.4%,66.4%,57.8%,64.1%.ConclusionWe successfully constructed recombinant HMGN2-CD8A-CD28-CD137-CD3ζ fusion gene lentiviral vector,infected to Jurkat cells and prepared HMGN2-T cells.HMGN2-T cells increased significantly in vitro,could directly kill cervical cancer,lung cancer,liver cancer,breast cancer cells.So HMGN2-T cells had the potential as a new type of cell immunotherapy in clinical application. |
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