【摘要】 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。更多还原

【Abstract】 Background and Objective:Our previous studies have shown that dendritic cell(DC)-tumor cell fusion vaccine can induce specific antitumor response against esophageal carcinoma cells.This study was to investigate the inhibitory effect of intratumor injection of the antigen-specific cytotoxic T lymphocytes(CTLs) induced by DC-tumor cell fusion vaccine against subcutaneously transplanted esophageal carcinoma cells in nude mice,and to analyze the influence of DC/tumor cell fusion vaccine on proliferation and apoptosis of esophageal carcinoma cells.Methods:Fusion cell vaccine of mature DCs with EC109 cells were generated by the polyethylene glycol(PEG) protocol and the antigen-specific CTLs were induced.The models of transplanted human esophageal carcinoma in nude mouse were established using EC-109 cell line.Thirty-three nude mice with subcutaneous tumors were randomly divided into three groups.Subcutaneous tumors of group A(n=11),group B(n=11) and group C(n=11) were intratumorally injected with the CTLs induced by DC/tumor fusion vaccine,T lymphocytes and RPMI 1640 medium respectively once a week.After four weeks of intratumor injection,the nude mice were killed and the nodules were anatomized.The mean volume and weight of tumors of each group were measured,and the tumor inhibitory rates of the Group A and the Group B were calculated and compared.The expression of proliferating cell nuclear antigen(PCNA) was detected by immunohistochemistry(S-P method).The mean PCNA-label index(LI) of three groups was compared.The cell cycle and cell apoptosis of the xenograft tumor cells were analyzed by flow cytometry.The mean S-phase fraction(SPF) and the mean rate of cell apoptosis of three groups was compared respectively.Results:Both the mean volume and the mean weight of xenograft tumors in group A(881.45±31.14 mm3 and 0.88±0.04 g) were significantly smaller than those of group B(1493.37±51.67 mm3 and 1.38±0.07 g) and group C(2065.77±87.55 mm3 and 2.04±0.11 g).The tumor inhibitory rates of Group A was significantly higher than that of group B(56.86% vs.32.35%,F=1218.08,P=0.001).The mean PCNA-LI of xenograft tumors was less in the group A(26.83±0.95)% than in the group B(51.82±1.51)% and group C(68.93±2.40)%(F=1528.39,P=0.000).The mean SPF of xenograft tumors was less in the group A(12.46±0.36)% than in the group B(29.39±0.96)% and the group C(42.25±1.43)%(P<0.05).The mean apoptotic rate of xenograft tumors was less in the group A(38.03±1.21)% than in the group B(17.75±0.56)% and the group C(6.59±0.22)%(P<0.05).Conclusion:The model of subcutaneous xenograft tumors in nude mice using human esophageal carcinoma cell line EC-109 has been successfully established.CTLs induced by DC/tumor fusion vaccine has specific antitumor immunity efficacy in vivo.CTLs can inhibit the proliferation of tumor cells and induce apoptosis of tumor cells in local tumors.更多还原