背景与目的:我们前期的研究表明树突细胞/肿瘤细胞融合疫苗能有效诱导体外抗食管癌细胞的特异性免疫应答,在此基础上,本研究进一步探讨树突细胞/肿瘤细胞融合疫苗诱导抗原特异性细胞毒性T淋巴细胞(cytotoxic T lymphocytes,CTLs)对食管...背景与目的:我们前期的研究表明树突细胞/肿瘤细胞融合疫苗能有效诱导体外抗食管癌细胞的特异性免疫应答,在此基础上,本研究进一步探讨树突细胞/肿瘤细胞融合疫苗诱导抗原特异性细胞毒性T淋巴细胞(cytotoxic T lymphocytes,CTLs)对食管癌细胞株EC-109裸鼠皮下移植瘤的体内抑瘤作用及对肿瘤细胞增殖和凋亡的影响。方法:①采用聚乙二醇法制备树突细胞/肿瘤细胞融合疫苗并诱导抗原特异性CTLs产生。②建立食管癌EC-109细胞裸鼠皮下移植瘤模型,将成功荷瘤的裸鼠随机分为3组,分别以融合细胞激活的CTLs(A组,n=11)、未激活的T淋巴细胞(B组,n=11)以及RMPI-1640培养液(C组,n=11)作为效应细胞进行瘤内注射,每周一次。③4周后处死动物剥离肿瘤组织,测量并比较各组肿瘤组织的平均体积及平均湿重,计算抑瘤率。④免疫组化SP法检测3组裸鼠移植瘤增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达。⑤流式细胞仪检测3组肿瘤细胞周期及细胞凋亡率,比较各组肿瘤细胞的S期细胞平均百分比(S-phase fraction,SPF)以及细胞平均凋亡率。结果:①A组肿瘤组织的平均体积显著小于B组和C组,差异有统计学意义[(881.45±31.14)mm3vs.(1493.37±51.67)mm3,(2065.77±87.55)mm3,F=950.67,P=0.00],其平均湿重亦显著小于B组和C组,差异有统计学意义[(0.88±0.04)gvs.(1.38±0.07)g,(2.04±0.11)g,F=1335.90,P=0.00];A组抑瘤率明显高于B组,差异有统计学意义(56.86%vs.32.35%,F=1218.08,P=0.001)。②A组肿瘤的平均PCNA-LI明显低于B组和C组,差异有统计学意义[(26.83±0.95)%vs.(51.82±1.51)%,(68.93±2.40)%,F=1528.39,P=0.000]。③A组肿瘤细胞平均SPF值明显低于B、C两组,差异有统计学意义[(12.46±0.36)%vs.(29.39±0.96)%,(42.25±1.43)%,F=2188.05,P=0.001]。④A组肿瘤细胞平均凋亡率明显高于B、C两组,差异有统计学意义[(38.03±1.21)%vs.(17.75±0.56)%,(6.59±0.22)%,F=4565.51,P=0.001]。结论:本研究成功建立人食管癌EC-109细胞裸鼠皮下移植瘤模型,证实DC/EC-109细胞融合疫苗体外诱导的抗原特异性CTLs瘤内直接注射具有抑制食管癌裸鼠移植瘤生长的作用,其机制可能是通过抑制肿瘤细胞增殖及诱导肿瘤细胞凋亡发挥其抗肿瘤作用。展开更多
背景与目的:细胞因子信号抑制因子(suppressor of cytokine signaling1,SOCS1)在肿瘤免疫负反馈调节中起重要作用,下调其表达可以有效增强机体的抗肿瘤免疫反应。本研究应用RNA干扰(RNA interference,RNAi)技术下调SOCS1基因表达联合OK-...背景与目的:细胞因子信号抑制因子(suppressor of cytokine signaling1,SOCS1)在肿瘤免疫负反馈调节中起重要作用,下调其表达可以有效增强机体的抗肿瘤免疫反应。本研究应用RNA干扰(RNA interference,RNAi)技术下调SOCS1基因表达联合OK-432刺激成熟的树突细胞(dendritic cell,DC)体外诱导特异性抗肿瘤作用,初步探讨其作用机制。方法:利用特异性小干扰RNA(siRNA)下调DC中SOCS1的表达,同时用肝癌HepG2细胞裂解物负载DC,OK-432刺激DC成熟,观察DC的形态特征,流式细胞仪检测刺激前后DC的表型变化,奥马蓝(AlamarBlue)法检测成熟DC对自身淋巴细胞的激活和增殖作用,LDH法检测其对HepG2、EC109细胞和K562细胞的杀伤作用。结果:DC体外诱导培养成功,设计的siRNA片段能有效下调iDC中SOCS1的表达,OK-432刺激DC成熟,CD80、CD83、CD86、HLA-DR等DC表面抗原明显表达上调,而负载肝癌抗原对DC表型无明显影响;SOCS1的下调可以促进DC成熟,负载肝癌抗原的DC能有效刺激自体淋巴细胞增殖,T细胞增殖率为(110.7±22.2)%,同时产生针对HepG2细胞的特异性杀伤作用,特异性细胞毒T淋巴细胞活性为(54.0±13.2)%。而针对EC109细胞和K562细胞的杀伤率仅为(10.0±30.7)%和(14.5±15.5)%。结论:RNAi下调SOCS1表达,OK432刺激负载肝癌全细胞抗原的成熟DC可以产生高效而特异性的抗肝癌的免疫应答。展开更多
<Abstract>Background and Objective:Cytokine-induced killer(CIK) cells and autologous dendritic cells-CIK(DCCIK) cells co-cultured with autologous dendritic cells(DCs) and CIK cells are commonly used for immunoth...<Abstract>Background and Objective:Cytokine-induced killer(CIK) cells and autologous dendritic cells-CIK(DCCIK) cells co-cultured with autologous dendritic cells(DCs) and CIK cells are commonly used for immunotherapy recently.We compared the anti-tumor immune response of CIK cells,autologous DC-CIK cells,and semi-allogeneic DC-CIK cells to explore a more effective anti-tumor adoptive immunotherapy approach.Met hods:Peripheral monocytes were isolated from patients with renal carcinoma,lung cancer,or maxillary squamous cell carcinoma and their healthy adult children.Isolated cells were cultured and induced as DCs and CIK cells in vitro.CIK cells from patients were co-cultured with autologous DCs and DCs from their children respectively,generating DC-CIK cells and semi-allogeneic DC-CIK cells.The anti-tumor activities of autologous CIK cells,autologous DC-CIK cells,and semi-allogeneic DC-CIK cells were measured by LDH assay.Intracellular staining was used to test the secretion of cytokines.Flow cytometry was applied for detecting the phonotype changes of these three types of cells.Cell proliferation and cell apoptosis were detected by 5,6carboxyfluorescein diacetate succinimidyl ester(CFSE) and Annexin V/PI respectively.Result s:Compared with autologous CIK cells and DC-CIK cells,semi-allogeneic DC-CIK cells significantly enhanced the anti-tumor activity and IFN-γ secretion,reduced IL-4 secretion,increased the ratio of CD3+CD56+ cells and CD3+CD8+ cells,decreased the number of CD4+CD25+ cells,promoted cell proliferation,and lessened cell apoptosis.Conclusions:Semi-allogeneic DC-CIK cells had a stronger anti-tumor effect than did autologous CIK cells and DC-CIK cells.Our results provided experimental evidence for clinical application of DC-CIK cells.展开更多
基金supported by Poten Biomedical Technology Development Co.,Ltd.
文摘<Abstract>Background and Objective:Cytokine-induced killer(CIK) cells and autologous dendritic cells-CIK(DCCIK) cells co-cultured with autologous dendritic cells(DCs) and CIK cells are commonly used for immunotherapy recently.We compared the anti-tumor immune response of CIK cells,autologous DC-CIK cells,and semi-allogeneic DC-CIK cells to explore a more effective anti-tumor adoptive immunotherapy approach.Met hods:Peripheral monocytes were isolated from patients with renal carcinoma,lung cancer,or maxillary squamous cell carcinoma and their healthy adult children.Isolated cells were cultured and induced as DCs and CIK cells in vitro.CIK cells from patients were co-cultured with autologous DCs and DCs from their children respectively,generating DC-CIK cells and semi-allogeneic DC-CIK cells.The anti-tumor activities of autologous CIK cells,autologous DC-CIK cells,and semi-allogeneic DC-CIK cells were measured by LDH assay.Intracellular staining was used to test the secretion of cytokines.Flow cytometry was applied for detecting the phonotype changes of these three types of cells.Cell proliferation and cell apoptosis were detected by 5,6carboxyfluorescein diacetate succinimidyl ester(CFSE) and Annexin V/PI respectively.Result s:Compared with autologous CIK cells and DC-CIK cells,semi-allogeneic DC-CIK cells significantly enhanced the anti-tumor activity and IFN-γ secretion,reduced IL-4 secretion,increased the ratio of CD3+CD56+ cells and CD3+CD8+ cells,decreased the number of CD4+CD25+ cells,promoted cell proliferation,and lessened cell apoptosis.Conclusions:Semi-allogeneic DC-CIK cells had a stronger anti-tumor effect than did autologous CIK cells and DC-CIK cells.Our results provided experimental evidence for clinical application of DC-CIK cells.
