共同培养的树突状细胞与CIK细胞对乳腺癌细胞杀伤活性的研究

目的:本研究将树突状细胞(dendritic cells,DCs)和细胞因子诱导的杀伤细胞(cytokine - induced killer cells,CIK)共同培养,获得DC- CIK细胞.观察DC - CIK细胞的体外增殖能力、表型变化及其对乳腺癌的细胞毒性作用.方法:从健康人外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)中诱导出DCs和CIK细胞,并将其按1∶10的比例共同培养4d获得DC - CIK细胞.检测DC - CIK细胞的增殖活性、表型变化及对乳腺癌细胞株SKBR-3的细胞毒活性.结果:DCs与CIK细胞共同培养后获得的DC-CIK细胞相对于单纯的CIK细胞具有更强增殖活性及成熟度,对乳腺癌细胞具有更强杀伤活性.结论:DCs与CIK细胞共同培养可使CIK细胞获得更高的增殖活性及更强的抑癌作用.     

抗原致敏的树突状细胞联合细胞因子诱导的杀伤细胞对肝癌细胞杀伤活性的研究

目的：观察肝癌细胞抗原致敏的树突状细胞（Dendritic cells,DC）与细胞因子诱导的杀伤细胞（Cytokine-induced killer cells,CIK）共培养后的细胞增殖活性、表型的变化及其对肝癌细胞HepG2的杀伤活性。方法：采集健康供者的外周血单个核细胞（Peripheral blood mononuclear cells,PBMNC）,常规诱导出DC、CIK,用肝癌细胞HepG2抗原冲击DC（Ag-DC）,并将其与CIK共培养（Ag-DC-CIK）,观察CIK和Ag-DC-CIK的细胞表型及增殖活性,并以肝癌细胞HepG2作为靶细胞,用MTT法检测CIK和Ag-DC-CIK的杀伤活性。结果：肝癌细胞抗原致敏的DC与CIK细胞共培养后,Ag-DC-CIK细胞群增殖活性明显增强,且高表达CD3+CD8+、CD3+CD56+双阳性细胞,其增殖活性和杀伤活性较单纯的CIK细胞更高（P<0.05）。结论：肿瘤抗原致敏的DC与CIK共培养获得的Ag-DC-CIK增殖活性和对肝癌细胞的杀伤活性显著高于CIK细胞。     

Effect of puerarin on the release of interleukin-8 in co-culture of human bronchial epithelial cells and neutrophils

Objective:To investigate the effect of puerarin on interleukin(IL)-8 mRNA expression and the protein release in the co-culture of human bronchial epithelial(BEAS-2B) cells and human neutrophils.Methods:BEAS2B cells and neutrophills were cultured separately and co-cultured with puerarin(50,100,and 200μg/mL) for a predetermined time.Cytokines in culture supematant were evaluated by protein array and IL-8 quantified by enzymelinked immunosorbent assay(ELISA).IL-8 mRNA expression was evaluated by real-time quantitative polymerase chain reaction(real-time qPCR).Results:The co-culture of BEAS-2B cells and neutrophils exhibited synergistic effects on IL-8 mRNA expression in BEAS-2B cells,but not in neutrophils after 12 h incubation(P<0.01),as compared with that in BEAS-2B cells or neutrophils alone.IL-8 protein release in the culture supematant was obviously elevated when BEAS-2B cells were co-cultured with human neutrophils as compared with that in the supematant of BEAS-2B cells or neutrophils alone after incubated for 2,6,12,and 18 h(P<0.01).Treatment with puerarin could significantly down-regulate the expression of IL-8 mRNA in BEAS-2B cells and IL-8 release in the supernatant of the co-culture of BEAS-2B cells and neutrophils(P<0.01).Conclusion:Puerarin could exhibit anti-inflammatory activity by suppressing IL-8 production from the co-culture of human bronchial epithelial cells and neutrophils.     

Ponatinib and gossypol act in synergy to suppress colorectal cancer cells by modulating apoptosis/autophagy crosstalk and inhibiting the FGF19/FGFR4 axis

Objective: To evaluate the efficacy of ponatinib plus gossypol against colorectal cancer HCT-116 and Caco-2 cells.Methods: Cells were treated with ponatinib and/or gossypol at increasing concentrations to evaluate synergistic drug interactions by combination index. Cell viability, FGF19/FGFR4, and apoptotic and autophagic cell death were studied.Results: Ponatinib(1.25-40 μM) and gossypol(2.5-80 μM)monotherapy inhibited HCT-116 and Caco-2 cell viability in a doseand time-dependent manner. The co...     

In Vitro Anti-tumor Immune Response Induced by Dendritic Cells Transfected with Recombinant Adenovirus Carrying Mutant K-ras Genes

Summary： The specific anti-tumor immune response induced by mouse bone marrow dendritic cells （DCs） lransfected with recombinant adenovirus carrying mutant k-ras genes was investighted. DCs were generated from mouse bone marrow in the presence of rmGM-CSF （3.3 ng/mL） and rmIL-4 （1.3 ng/mL） and detected by FACS, and then transfecled with the recombinant adenovirus encoding mutant k ras gene. The efficacy of transfection and T cell stimulating activity of DCs were detected. CTL activity of the mice vaccinated with DCs was observed. The resuhs showed thai DCs had dendritic veiled morphology. BmDCs highly expressed B7-1（80%）, B7-2（77%）, MHC Ⅱ （70%）, CDllc （65%）, CD40 （70%） and CD54 （96%） with FACS, and no significant difference in the expression was observed before and after the transfection （P〈0.05）. The DCs transfeeled by mutant k-ras gene could significantly stimulate lymphoeytes proliferation as compared with those transfeeted by Ad e or non-modified DCs （P〈0.05）. DC vaccine transfected by mutant k-ras gene could induce CTL activity against Lewis lung cancer, but not against B16. The specific eytotoxicity against Lewis lung cancer in Ad-k-ras/12-transdueed DC group was signifieantly higher than those in the control, vector and non transfeeted DCs groups （P〈0.05）. It was concluded that special antitumor response could be induced by DCs transfected with recombinant adenovirus carrying mutant k-ras genes.     

Potent antitumor effect elicited by gp96-peptide complexes pulsed by dendritic cell on mice of H22 liver cancer

Objective： To improve DC-based tumor vaccination, we studied whether dendritic ceils （DCs） which cocultured with H22 liver cancer cells-derived heat shock protein （HSP） glycoprotein 96 （gp96） affect the T cell-activating potential in vitro and the induction of tumor immunity in vivo. Methods： Maturation of murine bone marrow-derived DC was induced by GM-CSF plus IL-4, which mimiced the immunostimulatory effect of DC. Cocultured DC and gp96-peptide complexes were used to vaccine H22 liver cancer cells of mice. Using murine models we compared the immunogenecity of DC modified by gp96-peptides complexes derived from murine liver cancer cells alone or inactive tumor cells. To verify the specificity of the vaccine, in vitro assays were executed. Serum cytokine levels were quantified to explore the supposed pathway of DC modified by gp96 peptide complexes and its effect on antitumor immune response. Results： DC modified by gp96-peptide complexes can activate spleen lymphocyte and the latter can specifically kill H22 cells but not Ehrilich ascites carcinoma cells. Modified DC can induce potent tumor-antigenspecific immune response, augment the proliferation of Thl cells, and inhibit tumor growth. Conclusion： In this study, we have developed a novel DC-mediated tumor vaccine by combing the gp96 antigenic peptides complexes and inducing immune response against specific tumor cells, gp96 can be identified as a potent DC activator.     

Immunotherapeutic effects of dendiritic cells vaccine pulsed with tumor cell lysate in mice with pancreatic carcinoma

Objective To observe the immunotherapeutic effects of dendritic cells vaccine pulsed with tumor cell lystate on mice with pancreatic carcinoma. Methods Dendritic cells (MTSC4) were pulsed with tumor cells lysate. The immune preventative and immnotherapeutic effects of DC vaccines on mice with pancreatic carcinoma were assessed. Results After vaccination of the DC vaccines,mice remained tumor-free for at least 25 days in DCs vaccines group,but in other groups the subcutaneous implantation tumorigenesis were found beginning 3 to 9 days. CTL stimulated by DC vaccines effected cytolytic activity against pancreatic carcinoma cells. The survival period was obviously prolonged in DCs vaccines group (56 ±9)d than in other groups P<0.01) and tumors (1.4 ±0.8)g in DCs vaccines group were significantly smaller than that in other groups (P < 0. 05). Conclusion Tumor cell lysate-pulsed dendrtic cells vaccines can induce a specific and effective immune response against pancreatic carcinoma cell implanted in mice. 4     

Induction of T-cell immunity against leukemia by dendritic cells pulsed with total RNA isolated from leukemia cells

Objectives To assess the feasibility and efficacy of eliciting leukemia-specific T-cell responses in syngeneic mice in vitro and in vivo using dendritic cells (DCs) pulsed with total RNA from leukemia cells.Methods DCs generated from bone marrow culture in vitro in the presence of combined cytokines were pulsed with cellular total RNA isolated from cultured L615 cells by cationic lipid 1,2-dioleoyloxy-3-(trimethylammonium) propane (DOTAP). T-cell responses were evaluated by in vitro proliferation, and cytotoxicity assay. And in vivo immune protection and proghosis of mice with leukemia were studied.Results DCs pulsed with total RNA isolated from cultured L615 cells (DCs/RNA) were remarkably effective in stimulating L615-specific T-cell response in vitro, but did not cross-react with other leukemia cells from syngeneic mice. Vaccination of naive mice with viable DCs/RNA vaccine was able to partly protect from challenge with a lethal dose of live L615 cells, leading to low leukemia incidence and overall survival prolongation. Statistically significant survival was also observed in a low lethal dose of L615-bearing mice that received treatment using viable DCs∕RNA vaccine alone, suggesting that systemic administration of IL-2 could enhance the anti-tumor efficacy of leukemia RNA/DCs vaccine.Conclusions These data support the use of DCs/RNA vaccine as a feasible and effective route to elicit leukemia immunity against unidentified leukemia-associated antigens for treatment of leukemia-bearing animals.     

TNF-α gene-modified dendritic cells act as more potent adjuvants for peptide delivery to induce specific antitumor immunity in mice

Objective To investigate the antitumor immune efficiency of mouse dendritic cells (mDCs) by using adenovirus-mediated tumor necrosis factor-alpha (AdV-TNF-α) gene transfer.Methods MDCs infected with AdV-TNF-α and AdV-pLpA (no gene insert) at 100 multiplicity of infection (MOI) were analyzed by RNase protection assay for their cytokine secretion. Mixed lymphocyte reactions were also performed to analyze their capacity for alloantigen-presentation. C57BL/6 mice were challenged with R3LL tumor cells (Lewis lung carcinoma line) 10 days after vaccination with different engineered DCs and regular DCs as well.Results Compared to AdV-pLpA and mock-infected DCs, AdV-TNF-α-infected DCs displayed up-regulated expression of alpha tumor necrosis factor, interleukin-12 (IL-12), interleukin-18 (IL-18) and granulocyte macrophage colony stimulation factor (GM-CSF), and indicated stronger allogeneic T cell proliferative responses. Furthermore, vaccination of mice with dendritic cell tumor necrosis factor-alpha (DCTNF-α) pulsed with Mut1 peptide induced more efficient tumor-specific cytotoxic T lymphocyte (CTL) cytotoxicity against R3LL tumor cells in vitro and with efficient antitumor immunity in vivo. Conclusion This type of engineered DCs could be applied in clinical settings of DC-based cancer vaccines     

CIK细胞与DC-CIK细胞抗肿瘤效应的对照研究

目的比较细胞因子诱导的杀伤细胞（cytokine-induced killer cells,CIK）与树突状细胞（dendritic cells,DC）结合CIK细胞抗肿瘤效应。方法外周血单个核细胞诱导DC和CIK细胞,将DC与CIK共培养,以CIK细胞单独培养为对照。用MTT法测定杀伤活性,流式细胞术分析免疫表型,ELISA法测定干扰素-γ（IFN-γ）、白介素-12（IL-12）、肿瘤坏死因子-α（TNF-α）的水平。结果 DC-CIK细胞增殖能力明显高于CIK细胞（P〈0.05）,DC-CIK细胞共培养后,CD3＋CD8＋、CD3＋CD56＋细胞比率较相同条件下CIK细胞组显著增多（P〈0.05）,共培养3 d,DC-CIK细胞上清液中IL-12、IFN-γ、TNF-α水平均比CIK细胞单独培养的水平高（P〈0.01）,DC-CIK细胞对白血病细胞与淋巴瘤细胞的杀伤率显著高于CIK细胞（P〈0.01）。结论 DC-CIK细胞比CIK细胞有更强的抗肿瘤效应。