【摘要】 目的:探讨体内抑制胰腺癌细胞表面的FasL表达,能否抑制胰腺癌细胞对免疫系统的反击作用。方法:反义核酸技术扩增C57BL/6小鼠FasL mRNA 35~371位点核苷酸序列互补的cDNA片断,构建pBlast质粒后,转染人Panc-1胰腺癌细胞株,蛋白质印迹观察细胞株FasL蛋白表达的变化,ELISA法检测培养上清可溶性FasL水平。将细胞注射到C57BL/6小鼠胰腺包膜下,观测瘤体的直径变化,免疫组化观测肿瘤组织内浸润淋巴细胞的数量。结果:在稳定转染编码反义FasL cDNA的质粒后,胰腺癌细胞表面的FasL表达明显下调;其FasL蛋白表达下降了80%;FasL表达下调对体外生长的胰腺癌细胞无明显影响,但能减少同系免疫活性鼠体内肿瘤的发展。转染了质检DNA的胰腺癌细胞接种组瘤体的平均直径为1.53 mm,而未转染质检DNA的胰腺癌细胞接种组为2.98 mm,两组比较差异有统计学意义,P<0.01。与未来转染组相比,转染组CD45+TIL细胞数量明显增多,是未转染组的3.23倍,P<0.01。结论:胰腺癌细胞表面FasL表达的下调,能在体内增加免疫系统的抗肿瘤能力,从而为"肿瘤反击"促进肿瘤细胞浸润的学说提供了有力的证据。更多还原

【Abstract】 OBJECTIVE: To investigate if the supression of FasL expression in pancreatic cancer cells can inhibite tumor cell immune counterattack in vivo.METHODS: A cDNA fragment complementary,when transcribed,from nucleotides 35 to 371 of the mouse FasL mRNA sequence was amplified by PCR.The fragment was cloned into the StuI site of pBlast expression vector.Panc-1 cells were transfected with plasmid DNA.The suppression of FasL expression in stably transfected clones was determined by Western blot for FasL protein.Soluble FasL was detected in the culture supernatant of Panc-1 cells by ELISA.C57BL/6 mice were injected with tumor cells suspended.Tumor growth was monitored by measuring the width and length of the tumors.The level of lymphocyte infiltration in the excised tumors was assessed.RESULTS: FasL expression in hepatic cancer cells was down-regulated following stable transfection with a plasmid encoding antisense FasL cDNA,and with 80% decrease of FasL expression in plasmidtrans-trans pected cells.Down-regulation of FasL expression on pancreatic cancer cells had no effect on tumor growth in vitro,but significantly reduced tumor development in syngeneic immunocompetent mice in vivo.Mean tumor diameters was 1.53 mm in the group transfected with plasmid DNA,versus 2.98 mm in the group not transfected with plasmid DNA,and the difference was significant between the two groups,P<0.01.Compared with the group not transfected with plasmid DNA,the absolute number of CD45+ TIL was increased by 3.23 times in the group transfected with plasmid DNA,P<0.01.CONCLUSION: Down-regulation of FasL expression by pancreatic cancer cells results in an improved anti-tumor immune challenge in vivo,providing experimental evidence for "counterattack" as a mechanism of tumor immune evasion.更多还原