摘要目的: 增强人肝癌细胞的免疫原性以激活宿主免疫细胞识别杀伤相应的肝癌细胞。方法: 以脂质体介导小鼠H-2K^b基因转染肝癌细胞株，并检测H-2K^b抗原表达情况，然后用H-2K^b基因转染后的肝癌细胞体外激活效应细胞杀伤活性，再进行裸鼠动物实验。结果: H-2K^b基因转染人肝癌细胞株HepG₂后，Southern印迹杂交结果显示，H-2K^b基因整合于肿瘤细胞染色体中，RNA点杂交结果显示，H-2K^bDNA已转录成mRNA。免疫组化及流式细胞仪检测显示，H-2K^b抗原已在肝癌细胞胞膜上表达。转染后肝癌细胞在体外能强烈诱发效应细胞毒性，这种细胞毒性现象在裸鼠实验中得到进一步验证。结论: 异源MHC-Ⅰ类基因H-2K^b转染肝癌细胞后可增强其免疫原性并激活免疫效应细胞杀伤活性。

《中国图书资料分类法》分类号R735.7

The Immological Rejection Activated by Human Heptocarcinoma Transferred with Murine H-2K^b Gene

Zhang ZhixiangYuan AiliWang XiaoningZhang LiZhou Dianyuan

(The PLA Hospital in Hong Kong, Shenzhen 518048)

AbstractObjective: To strengthen the immunogenicity of hepatocarcinoma cells and activate immnological cells recognizing and killing the tumor cells. Methods： The murine MHC-Ⅰ gene H-2K^b which can express immunologial rejection antigens was transfected into human hepatocarcinoma cells HepG₂ by liposome DNA mediated gene transfer. The transfection and transcription of H-2K^b gene were detected by molecular hybridization techniques. The exogeous antigens expressed on the membrane of transfected tumor cells were detected with ABC immunohistochemical method and flow cytometer. ［³H］ release assays were used to detect the recognizing and killing effects of lymphocytes to HepG₂ cells transferred with murine H-2K^b gene. The nude mice experiment was used to further verify CTL cells killing active. Results: Southern blot hybridization showed that the H-2K^b gene was integrated into the chromosome of HepG₂ cells. The RNA dot blot hybridization showed that there was transcription of H-2K^b DNA in the transfected tumor cells. ABC immunohistochemical method and flow cytometer detection showed that the murine H-2K^b antigens were expressed on the membrane of HepG₂ cells. ［³H］ release assays showed that the cytotoxicyty to HepG₂ cells transfected with H-2K^b gene was obviously higher than that to control cells. The results demonstrated that the growth of hepatocarcinoma cells which were transferred with H-2K^b gene was obviously inhibited. Conclusion： The murine MHC-Ⅰ gene H-2K^b could be transferred into the human hepatocarcinoma cells and expressed on the membrane of transferred cells. The HepG₂ cells transferred with H-2K^b gene could induce human effective lymphocytes to recognize and kill these transferred tumor cells.

Key words

Key wordsH-2K^b gene； immunogenicity； hepatocarcinoma； gene transfer

肿瘤免疫原性降低是造成肿瘤细胞免疫逃逸的主要原因之一，增强肿瘤细胞的免疫原性以激活宿主免疫系统识别和杀伤相应的肿瘤细胞可能成为一种治疗肿瘤的有效手段。本实验通过基因转染方法将小鼠MHC-Ⅰ类基因H-2K^b转染人肝癌细胞株HepG₂，并使此基因抗原在肝癌细胞膜上表达，从而人为地将肝癌细胞转变成带异源免疫性状的细胞，提高肝癌细胞的免疫原性，激发宿主免疫效应细胞杀伤活性。

1材料与方法

1.1脂质体介导H-2K^b基因质粒与PSV₂质粒共转染肝癌细胞

HepG₂细胞培养18 h，将H-2K^b基因与脂质体混合液20 μl加入培养细胞液中培养48 h（方法参照GIBCO公司产品说明书）。细胞传代后加入400 μg/mlG-418，实验同时设不加H-2K^b基因对照及单独PSV₂转染对照。细胞克隆经H-2K^b斑点杂交筛选。H-2K^b基因重组表达质粒由E*Weiss教授惠赠^［1］。

1.2转染细胞H-2K^b基因存在及表达情况检测

1.2.1Southern印迹杂交

制备Dig-H-2K^b DNA探针，按常规方法杂交（方法参照Mannheim公司产品说明书）。

1.2.2RNA点杂交

按常规方法进行。

1.2.3ABC免疫组化实验

操作按试剂盒（Vector公司产品）说明书进行。抗H-2K^b抗原单克隆抗体系ATCC产品。

1.2.4流式细胞仪检测

制备细胞悬液，加入抗H-2K^b抗原单克隆抗体，4℃过夜，洗涤后加入荧光标记羊抗鼠IgG抗体，37℃，40 min。

1.3H-2K^b基因转染后体外诱发效应细胞杀伤活性实验

1.3.1效应细胞制备

按常规方法进行。

1.3.2效应细胞杀伤活性检测

收获处于对数生长期的H-2K^b转染阳性、PSV₂转染阳性及未转染外源基因的HepG₂肝癌细胞，各加［³H］-TdR(中国原子能研究所产品)20 μCi。37℃，2 h。同时设最大释放组和自然释放组。测定CPM值，计算特异杀伤率。

1.4裸鼠实验

2～4周龄裸鼠（购自上海医科大学）随机分3组，每组10只，分别为接种H-2K^b转染HepG₂肝癌细胞组、PSV₂质粒转染HepG₂肝癌细胞组和未转染外源基因HepG₂肝癌细胞组，待肿瘤生长面积约为300 mm²后，将已制备好的人胎儿脾效应细胞每隔4 d分4次注射到3组裸鼠皮下肿瘤内，观察肿瘤生长情况。实验在第一军医大学无菌动物饲养室进行。实验数据用χ±SD表示，用SAS：软件包均数间比较，SNK法。

2结果

2.1脂质体介导H-2K^b基因表达质粒与PSV₂质粒共转染HepG₂肝癌细胞（图1）

H-2K^b与PSV₂质粒共转染HepG₂肝癌细胞后经G-418筛选，第14天出现25个细胞克隆，而未转染PSV₂者在第5天细胞全部死亡。上述25个细胞克隆DNA经H-2K^b DNA点杂交筛选出4个H-2K^b阳性克隆，显示此4个克隆细胞H-2K^b基因转染成功。

图1图中显示共转染小鼠H-2K^b基因与PSV₂质粒后，

用G418筛选出来的人肝癌细胞HepG₂克隆

Fig. 1Human hepatocarcinoma cell clone HepG₂

cotransfected with H-2K^b gene and PSV₂ plasmid

was selected with G418

2.2H-2K^b基因转染后在HepG₂肝癌细胞中表达

2.2.1H-2K^b基因转染后在HepG₂肝癌细胞中转录

H-2K^b基因转染HepG₂肝癌细胞后，筛选的4个细胞克隆RNA与Dig-H-2K^b DNA探针杂交结果阳性，显示H-2K^b基因转染成功后，能在细胞内转录成H-2K^b mRNA。

2.2.2H-2K^b基因转染后在HepG₂肝癌细胞中表达

ABC免疫组化检测显示H-2K^b基因转染后HepG₂肝癌细胞膜部位可见棕色显色。对照组无此现象（图2）。流式细胞仪检测显示，在相同细胞浓度下，H-2K^b基因转染组HepG₂肝癌细胞显示荧光信号，对照组无此现象（图3）。上述结果说明，H-2K^b基因外显子能在HepG₂肝癌细胞膜上表达H-2K^b抗原。

图2ABC免疫组化结果显示小鼠H-2K^b