小鼠H-2Kb基因转染人肝癌细胞后激活免疫排斥反应的研究
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作者：张智翔 袁爱力 王小宁 张力 周殿元
单位：张智翔 袁爱力 王小宁 张力 周殿元(驻香港部队医院 深圳 518048)
关键词：H-2Kb基因；免疫原性；肝癌细胞；基因转染
中国肿瘤生物治疗杂志000216
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-2Kb基因转染肝癌细胞株，并检测H-2Kb抗原表达情况，然后用H-2Kb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-2Kb基因转染人肝癌细胞株HepG2后，Southern印迹杂交结果显示，H-2Kb基因整合于肿瘤细胞染色体中，RNA点杂交结果显示，H-2KbDNA已转录成mRNA。免疫组化及流式细胞仪检测显示，H-2Kb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-2Kb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。
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《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-2Kb Gene
Zhang Zhixiang Yuan Aili Wang Xiaoning Zhang Li Zhou Dianyuan
(The PLA Hospital in Hong Kong, Shenzhen 518048)
Abstract Objective: To strengthen the immunogenicity of hepatocarcinoma cells and activate immnological cells recognizing and killing the tumor cells. Methods： The murine MHC-Ⅰ gene H-2Kb 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-2Kb 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-2Kb gene. The nude mice experiment was used to further verify CTL cells killing active. Results: Southern blot hybridization showed that the H-2Kb gene was integrated into the chromosome of HepG2 cells. The RNA dot blot hybridization showed that there was transcription of H-2Kb DNA in the transfected tumor cells. ABC immunohistochemical method and flow cytometer detection showed that the murine H-2Kb antigens were expressed on the membrane of HepG2 cells. [3H] release assays showed that the cytotoxicyty to HepG2 cells transfected with H-2Kb gene was obviously higher than that to control cells. The results demonstrated that the growth of hepatocarcinoma cells which were transferred with H-2Kb gene was obviously inhibited. Conclusion： The murine MHC-Ⅰ gene H-2Kb could be transferred into the human hepatocarcinoma cells and expressed on the membrane of transferred cells. The HepG2 cells transferred with H-2Kb gene could induce human effective lymphocytes to recognize and kill these transferred tumor cells.
, 百拇医药
Key words
Key words H-2Kb 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-2Kb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-2Kb基因质粒与PSV2质粒共转染肝癌细胞
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HepG2细胞培养18 h，将H-2Kb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-2Kb基因对照及单独PSV2转染对照。细胞克隆经H-2Kb斑点杂交筛选。H-2Kb基因重组表达质粒由E*Weiss教授惠赠[1]。
1.2 转染细胞H-2Kb基因存在及表达情况检测
1.2.1 Southern印迹杂交
制备Dig-H-2Kb DNA探针，按常规方法杂交(方法参照Mannheim公司产品说明书)。
1.2.2 RNA点杂交
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按常规方法进行。
1.2.3 ABC免疫组化实验
操作按试剂盒(Vector公司产品)说明书进行。抗H-2Kb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-2Kb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。
1.3 H-2Kb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。
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1.3.2 效应细胞杀伤活性检测
收获处于对数生长期的H-2Kb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-2Kb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。
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2 结 果
2.1 脂质体介导H-2Kb基因表达质粒与PSV2质粒共转染HepG2肝癌细胞(图1)
H-2Kb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-2Kb DNA点杂交筛选出4个H-2Kb阳性克隆，显示此4个克隆细胞H-2Kb基因转染成功。
图1 图中显示共转染小鼠H-2Kb基因与PSV2质粒后，用G418筛选出来的人肝癌细胞HepG2克隆
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Fig. 1 Human hepatocarcinoma cell clone HepG2
cotransfected with H-2Kb gene and PSV2 plasmid
was selected with G418
2.2 H-2Kb基因转染后在HepG2肝癌细胞中表达
2.2.1 H-2Kb基因转染后在HepG2肝癌细胞中转录
H-2Kb基因转染HepG2肝癌细胞后，筛选的4个细胞克隆RNA与Dig-H-2Kb DNA探针杂交结果阳性，显示H-2Kb基因转染成功后，能在细胞内转录成H-2Kb mRNA。
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2.2.2 H-2Kb基因转染后在HepG2肝癌细胞中表达
ABC免疫组化检测显示H-2Kb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-2Kb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-2Kb基因外显子能在HepG2肝癌细胞膜上表达H-2Kb抗原。
图2 ABC免疫组化结果显示小鼠H-2Kb抗原在
人HepG2肝癌细胞膜上表达
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Fig. 2 The results of ABC immunohistochemitry assays showed
that the H-2Kb antigens could be expressed on the membrane
of human hepatocarcinoma cells HepG2
图3 流式细胞仪结果显示人肝癌细胞小鼠
H-2Kb抗原阳性的免疫荧光信号
Fig. 3 The results of flow cytometry showed that there
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were H-2Kb antigens positive immunologic fluorescence signals
A: Smaple； B: Contrast
2.3 H-2Kb基因转染后HepG2肝癌细胞激活人胎儿脾效应细胞杀伤活性
结果见表1。表中显示H-2Kb基因转染后的HepG2肝癌细胞激活胎儿脾效应细胞杀伤活性明显高于对照组(P<0.01)，提示H-2Kb抗原作为异源免疫激活物能体外激发效应细胞识别杀伤功能。
表1 H-2Kb基因转染后HepG2细胞体外激活
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人胎儿脾效应细胞杀伤活性
Tab. 1 The killing effects of human lymphocytes on HepG2 cells Cells
N
The percent specific lysis
(%)
HepG2 transferred with H-2Kb
12
82.73±8.91△
HepG2 transferred with PSV2
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12
27.43±5.70△
HepG2 no transferred
12
24.20±7.07△
△ P<0.01
2.4 裸鼠实验
结果见表2。表中显示H-2Kb基因转染阳性的HepG2肝癌细胞接种于裸鼠后，人胎儿脾效应细胞对裸鼠肿瘤有明显杀伤效应，肿瘤平均面积缩小，成活天数延长，与PSV2转染组及未转染外源基因组差别有统计学意义(P<0.01)。表2 人胎儿脾效应细胞对裸鼠内HepG2细胞杀伤作用
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Tab. 2 The killing activity of human lymphocytes to HepG2 cells in nude mice Tumor node
N
The average area before
injected with lymphocytes
(mm2)
The average area after
injected with lymphocytes
(mm2)
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The average survival
days of nude mice
H-2Kb positive
10
310.3±17.7
130.7±39.6
93.2±6.5△
PSV2 positive
10
308.4±14.7
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30.0±5.0△
No transferred with foreign gene
10
307.6±19.1
298.7±16.6△
28.1±4.1△
△ P<0.01
3 讨 论
本实验将与宿主种属差异极大的小鼠MHC-Ⅰ基因H-2Kb转染到人肝癌细胞HepG2中，通过免疫组化及流式细胞仪检测显示H-2Kb基因在细胞膜上表达H-2Kb抗原。为了验证这种异源表达产物能否提高肝癌细胞的免疫原性，本实验将人胎儿脾分离出来的淋巴细胞作为效应细胞，进行细胞杀伤试验，结果显示，人胎儿脾效应细胞杀伤H-2Kb基因转染的HepG2细胞效率明显高于单纯转染PSV2质粒及未转染外源基因的细胞，此结果提示，转染H-2Kb基因后，人肝癌细胞株HepG2细胞免疫原性增强，在体外激发效应细胞识别杀伤靶细胞功能。为了进一步在体内验证这种细胞毒性现象，本实验将HepG2细胞株接种到免疫缺陷的裸鼠皮下，待肿瘤生长到一定程度后再直接注射人胎儿脾效应细胞于肿瘤体内，结果显示转染了H-2Kb基因的肿瘤其生长受到明显抑制，裸鼠存活时间也明显延长。近年来，从提高肿瘤自身免疫原性着手的基因治疗受到重视，研究多集中于将同源MHC-Ⅰ类和MHC-Ⅱ类基因转染肿瘤细胞，刺激肿瘤抗原和MHC免疫应答。1984年，Hui等[2]将H-2Kb基因导入胸腺细胞癌AKR小鼠肿瘤中，可使其在纯系小鼠中形成肿瘤的能力下降，此后其它有关的研究也支持这一结论[3～7]。1992年，美国密执安州立大学Nabel领导的小组，将HLA-B7表达质粒用脂质体包裹，直接注射到黑色素瘤结节中，在肿瘤细胞膜上表达HLA-B7抗原，诱导肿瘤患者特异性CTL免疫因素形成[8]。
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上述研究给我们很大启发，我们试图找出一种能直接表达强免疫原性抗原的基因，使其在肿瘤细胞膜上表达，刺激机体产生强烈的免疫排斥反应。移植免疫理论认为，移植物与宿主种属差异越大，即MHC抗原系统之间差异越大，抗移植物的免疫排斥反应越强烈。因此，我们选择小鼠MHC-Ⅰ类基因H-2Kb，将其导入到人类肝癌细胞株中，并在人肝癌细胞膜上表达鼠H-2Kb抗原，从而将人肝癌细胞变成带有异源免疫性状的细胞，增强其特异性，以激发人淋巴细胞产生类似于抗移植物的免疫排斥效应，识别杀伤癌细胞。
张智翔,男,37岁,博士,主要从事肝病方面的研究
参 考 文 献
1，Mellor AL, Golden L, Weiss E, et al. Expression of murine H-2Kb histocompatibility antigen in cells transformed with cloned H-2 genes. Nature, 1982, 298: 529
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2，Hui K, Grosveld F, Festenstein H. Rejection of transplantable AKR leukemia cells following MHC DNA mediated cell transformation. Nature, 1984, 311: 750
3，Chia KY, Lim SP, Oei AA, et al. Acquisition of immunogenicity by AKR leukemic cells following DNA mediated gene transfer is assocated with the reduction of constitutive reactive superoxide radicals. Int J Cancer, 1994, 57: 216
4，Hui K, Chia TF. Eradication of tumor growth via biolistic transformtion with allogeneic MHC genes. Gene Ther, 1997, 4: 762
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5，Tanaka K, Issebacher KJ, Khoury G, et al. Reversal of oncogenesis by the expression of a major histocompatibility complex class 1 gene. Science, 1985, 228: 26
6，Grelik E, Jay G, Kwiatkowski B, et al. Increasitivity to MHC-nonrestricted lysis of B16 melanoma cells by transfection with class 1 H-2Kb gene. J Immunol, 1990, 145: 1621
7，Ramakrishna V, Eisenthal A, Shornick Y, et al. Increased projection of MHC and tumor antigens in murine B16-BL6 melanoma induced by hydrostatic pressure and chemical crosslinking. Cancer Immunol Immunother, 1993, 36: 293
8，Nabel GJ, Nabel GE, Tang ZY, et al. Direct gene transfer with DNA-liposome complexes in melanoma: Expression, biologic activity, and lack of toxicity in humans. Proc Natl Acad Sci USA, 1993, 90: 11307
(1999-07-19收稿； 2000-04-17修回), 百拇医药
单位：张智翔 袁爱力 王小宁 张力 周殿元(驻香港部队医院 深圳 518048)
关键词：H-2Kb基因；免疫原性；肝癌细胞；基因转染
中国肿瘤生物治疗杂志000216
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-2Kb基因转染肝癌细胞株，并检测H-2Kb抗原表达情况，然后用H-2Kb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-2Kb基因转染人肝癌细胞株HepG2后，Southern印迹杂交结果显示，H-2Kb基因整合于肿瘤细胞染色体中，RNA点杂交结果显示，H-2KbDNA已转录成mRNA。免疫组化及流式细胞仪检测显示，H-2Kb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-2Kb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。
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《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-2Kb Gene
Zhang Zhixiang Yuan Aili Wang Xiaoning Zhang Li Zhou Dianyuan
(The PLA Hospital in Hong Kong, Shenzhen 518048)
Abstract Objective: To strengthen the immunogenicity of hepatocarcinoma cells and activate immnological cells recognizing and killing the tumor cells. Methods： The murine MHC-Ⅰ gene H-2Kb 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-2Kb 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-2Kb gene. The nude mice experiment was used to further verify CTL cells killing active. Results: Southern blot hybridization showed that the H-2Kb gene was integrated into the chromosome of HepG2 cells. The RNA dot blot hybridization showed that there was transcription of H-2Kb DNA in the transfected tumor cells. ABC immunohistochemical method and flow cytometer detection showed that the murine H-2Kb antigens were expressed on the membrane of HepG2 cells. [3H] release assays showed that the cytotoxicyty to HepG2 cells transfected with H-2Kb gene was obviously higher than that to control cells. The results demonstrated that the growth of hepatocarcinoma cells which were transferred with H-2Kb gene was obviously inhibited. Conclusion： The murine MHC-Ⅰ gene H-2Kb could be transferred into the human hepatocarcinoma cells and expressed on the membrane of transferred cells. The HepG2 cells transferred with H-2Kb gene could induce human effective lymphocytes to recognize and kill these transferred tumor cells.
, 百拇医药
Key words
Key words H-2Kb 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-2Kb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-2Kb基因质粒与PSV2质粒共转染肝癌细胞
, http://www.100md.com
HepG2细胞培养18 h，将H-2Kb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-2Kb基因对照及单独PSV2转染对照。细胞克隆经H-2Kb斑点杂交筛选。H-2Kb基因重组表达质粒由E*Weiss教授惠赠[1]。
1.2 转染细胞H-2Kb基因存在及表达情况检测
1.2.1 Southern印迹杂交
制备Dig-H-2Kb DNA探针，按常规方法杂交(方法参照Mannheim公司产品说明书)。
1.2.2 RNA点杂交
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按常规方法进行。
1.2.3 ABC免疫组化实验
操作按试剂盒(Vector公司产品)说明书进行。抗H-2Kb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-2Kb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。
1.3 H-2Kb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。
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1.3.2 效应细胞杀伤活性检测
收获处于对数生长期的H-2Kb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-2Kb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。
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2 结 果
2.1 脂质体介导H-2Kb基因表达质粒与PSV2质粒共转染HepG2肝癌细胞(图1)
H-2Kb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-2Kb DNA点杂交筛选出4个H-2Kb阳性克隆，显示此4个克隆细胞H-2Kb基因转染成功。
图1 图中显示共转染小鼠H-2Kb基因与PSV2质粒后，用G418筛选出来的人肝癌细胞HepG2克隆
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Fig. 1 Human hepatocarcinoma cell clone HepG2
cotransfected with H-2Kb gene and PSV2 plasmid
was selected with G418
2.2 H-2Kb基因转染后在HepG2肝癌细胞中表达
2.2.1 H-2Kb基因转染后在HepG2肝癌细胞中转录
H-2Kb基因转染HepG2肝癌细胞后，筛选的4个细胞克隆RNA与Dig-H-2Kb DNA探针杂交结果阳性，显示H-2Kb基因转染成功后，能在细胞内转录成H-2Kb mRNA。
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2.2.2 H-2Kb基因转染后在HepG2肝癌细胞中表达
ABC免疫组化检测显示H-2Kb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-2Kb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-2Kb基因外显子能在HepG2肝癌细胞膜上表达H-2Kb抗原。
图2 ABC免疫组化结果显示小鼠H-2Kb抗原在
人HepG2肝癌细胞膜上表达
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Fig. 2 The results of ABC immunohistochemitry assays showed
that the H-2Kb antigens could be expressed on the membrane
of human hepatocarcinoma cells HepG2
图3 流式细胞仪结果显示人肝癌细胞小鼠
H-2Kb抗原阳性的免疫荧光信号
Fig. 3 The results of flow cytometry showed that there
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were H-2Kb antigens positive immunologic fluorescence signals
A: Smaple； B: Contrast
2.3 H-2Kb基因转染后HepG2肝癌细胞激活人胎儿脾效应细胞杀伤活性
结果见表1。表中显示H-2Kb基因转染后的HepG2肝癌细胞激活胎儿脾效应细胞杀伤活性明显高于对照组(P<0.01)，提示H-2Kb抗原作为异源免疫激活物能体外激发效应细胞识别杀伤功能。
表1 H-2Kb基因转染后HepG2细胞体外激活
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人胎儿脾效应细胞杀伤活性
Tab. 1 The killing effects of human lymphocytes on HepG2 cells Cells
N
The percent specific lysis
(%)
HepG2 transferred with H-2Kb
12
82.73±8.91△
HepG2 transferred with PSV2
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12
27.43±5.70△
HepG2 no transferred
12
24.20±7.07△
△ P<0.01
2.4 裸鼠实验
结果见表2。表中显示H-2Kb基因转染阳性的HepG2肝癌细胞接种于裸鼠后，人胎儿脾效应细胞对裸鼠肿瘤有明显杀伤效应，肿瘤平均面积缩小，成活天数延长，与PSV2转染组及未转染外源基因组差别有统计学意义(P<0.01)。表2 人胎儿脾效应细胞对裸鼠内HepG2细胞杀伤作用
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Tab. 2 The killing activity of human lymphocytes to HepG2 cells in nude mice Tumor node
N
The average area before
injected with lymphocytes
(mm2)
The average area after
injected with lymphocytes
(mm2)
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The average survival
days of nude mice
H-2Kb positive
10
310.3±17.7
130.7±39.6
93.2±6.5△
PSV2 positive
10
308.4±14.7
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30.0±5.0△
No transferred with foreign gene
10
307.6±19.1
298.7±16.6△
28.1±4.1△
△ P<0.01
3 讨 论
本实验将与宿主种属差异极大的小鼠MHC-Ⅰ基因H-2Kb转染到人肝癌细胞HepG2中，通过免疫组化及流式细胞仪检测显示H-2Kb基因在细胞膜上表达H-2Kb抗原。为了验证这种异源表达产物能否提高肝癌细胞的免疫原性，本实验将人胎儿脾分离出来的淋巴细胞作为效应细胞，进行细胞杀伤试验，结果显示，人胎儿脾效应细胞杀伤H-2Kb基因转染的HepG2细胞效率明显高于单纯转染PSV2质粒及未转染外源基因的细胞，此结果提示，转染H-2Kb基因后，人肝癌细胞株HepG2细胞免疫原性增强，在体外激发效应细胞识别杀伤靶细胞功能。为了进一步在体内验证这种细胞毒性现象，本实验将HepG2细胞株接种到免疫缺陷的裸鼠皮下，待肿瘤生长到一定程度后再直接注射人胎儿脾效应细胞于肿瘤体内，结果显示转染了H-2Kb基因的肿瘤其生长受到明显抑制，裸鼠存活时间也明显延长。近年来，从提高肿瘤自身免疫原性着手的基因治疗受到重视，研究多集中于将同源MHC-Ⅰ类和MHC-Ⅱ类基因转染肿瘤细胞，刺激肿瘤抗原和MHC免疫应答。1984年，Hui等[2]将H-2Kb基因导入胸腺细胞癌AKR小鼠肿瘤中，可使其在纯系小鼠中形成肿瘤的能力下降，此后其它有关的研究也支持这一结论[3～7]。1992年，美国密执安州立大学Nabel领导的小组，将HLA-B7表达质粒用脂质体包裹，直接注射到黑色素瘤结节中，在肿瘤细胞膜上表达HLA-B7抗原，诱导肿瘤患者特异性CTL免疫因素形成[8]。
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上述研究给我们很大启发，我们试图找出一种能直接表达强免疫原性抗原的基因，使其在肿瘤细胞膜上表达，刺激机体产生强烈的免疫排斥反应。移植免疫理论认为，移植物与宿主种属差异越大，即MHC抗原系统之间差异越大，抗移植物的免疫排斥反应越强烈。因此，我们选择小鼠MHC-Ⅰ类基因H-2Kb，将其导入到人类肝癌细胞株中，并在人肝癌细胞膜上表达鼠H-2Kb抗原，从而将人肝癌细胞变成带有异源免疫性状的细胞，增强其特异性，以激发人淋巴细胞产生类似于抗移植物的免疫排斥效应，识别杀伤癌细胞。
张智翔,男,37岁,博士,主要从事肝病方面的研究
参 考 文 献
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(1999-07-19收稿； 2000-04-17修回), 百拇医药