重组人内皮抑素腺病毒抑制肝癌裸鼠移植瘤生长

目的 观察重组人内皮抑素腺病毒(Ad/hEndo)对人肝癌裸鼠移植瘤生长的影响。方法 人脐静脉内皮细胞ECV-304经Ad/hEndo感染后,western印迹检测人内皮抑素的表达。人肝癌BEL-7402细胞移植到裸鼠背脊部后,检测Ad/hEndo对肝癌移植瘤生长的抑制作用。逆转录聚合酶链反应(RT-PCR)检测肿瘤组织中内皮抑素mRNA的表达。分析人内皮抑素在裸鼠体内的表达分布。结果 Western印迹检测到人内皮抑素基因在ECV-304细胞内高效表达。Ad/hEndo明显抑制人肝癌BEL-7402裸鼠移植瘤生长(F=4.061,P<0.05)。Ad/hEndo组血管密度计数为6.88±1.08,DMEM组为13.60±1.71(t=9.216,P<0.01)。瘤内注射Ad/hEndo后3d,RT-PCR在肿瘤组织检测到内皮抑素mRNA的表达,7d后表达不明显。人内皮抑素蛋白主要分布在肿瘤组织。结论 腺病毒介导的人内皮抑素基因在体内、体外获得高效表达,并明显抑制肝癌裸鼠移植瘤的生长与血管生成。     

树突状细胞诱导的抗肿瘤免疫诱导移植瘤细胞凋亡并抑制其增殖

目的研究树突状细胞(DC)体外诱导的细胞免疫能否抑制裸鼠移植瘤生长及其机制.方法联合应用粒/巨噬细胞集落刺激因子(GM-CSF)及白介素-4(IL-4)直接从肝癌患者外周血中培养出DC,以源于人肝癌细胞系HepG2肿瘤细胞的肿瘤抗原粗提物刺激DC,DC激活同源的T淋巴细胞产生细胞毒性T淋巴细胞(CTL),建立裸鼠人肝癌细胞系HepG2移植瘤模型.以CTL治疗裸鼠HepG2移植瘤并观察治疗效果,检测移植瘤标本肿瘤细胞凋亡情况.结果DC诱导的CTL通过诱导肿瘤细胞凋亡并抑制其增殖而抑制移植瘤生长.结论经肿瘤抗原激发的DC有可能在肿瘤的治疗中发挥重要作用.     

人舌癌移植瘤模型中血管内皮细胞来源的实验研究

目的 观察人舌癌裸鼠皮下移植瘤模型中肿瘤新生血管内皮细胞的来源.方法 通过皮下移植人舌癌Tca8113-Ml细胞建立人舌癌裸鼠移植肿瘤模型,并于肿瘤生长至直径1cm时切除肿瘤,对移植瘤进行病理组织学观察,利用免疫组织化学方法检测移植瘤内新生血管内皮细胞的来源,抗体为鼠抗人单克隆抗体CD34和大鼠抗小鼠单克隆抗体CD34;并进行微血管密度(microvessel density,MVD)计数及血管内皮生长因子(vascular endothelial growth factor,VEGF)表达检测.结果 右腋窝皮下接种人舌癌Tca8113-Ml细胞2周后,6只裸鼠的肿瘤直径都达到1 cm 以上,切片HE染色可见肿瘤组织病理形态为鳞状上皮细胞癌,MVD平均值为10.72±2.12,其中肿瘤新生血管内皮细胞大鼠抗小鼠CD34免疫组化结果是阳性,而鼠抗人CD34阴性.VEGF在6例移植瘤标本中均阳性表达, 平均阳性率为(67±5.6)%.结论 人舌癌移植瘤模型中血管内皮细胞主要来源于宿主裸鼠,并且可能与肿瘤细胞分泌的VFGE诱导有关.     

慢病毒重组ACE2基因表达载体的构建及其感染人脐静脉内皮细胞的研究

目的构建携带血管紧张素转换酶2（ACE2）基因的慢病毒表达载体,观察其感染人脐静脉内皮细胞后ACE2基因的转录及表达水平。方法采用PCR技术从含有ACE2基因的质粒克隆模板pc—DNA3．1-hygro（＋）一mACE2上钓取ACE2基因,并将ACE2基因重组到慢病毒载体表达质粒pGC—FU中,构建重组慢病毒载体表达质粒pGC—FU—ACE2,通过酶切、测序验证ACE2基因后,将pGC—FU—ACE2质粒和包装质粒pHelper1．0、pHelper2．0共同转染人胚胎肾上皮细胞系293T细胞,获得携带ACE2基因的重组慢病毒Lentiviral—ACE2;然后感染人脐静脉内皮细胞,通过RT—PCR、Western—blot检测ACE2基因的转录和蛋白表达水平。结果成功构建携带ACE2基因的慢病毒表达载体Lentiviral—ACE2,并获得大量高纯度的慢病毒浓缩液。目的基因ACE2能被重组慢病毒高效地导入人脐静脉血管内皮细胞,并能高水平表达。结论慢病毒表达载体Lentiviral—ACE2成功构建并能高效率感染人脐静脉内皮细胞,为研究ACE2对血管内皮细胞的影响和更深入地探索ACE2基因的功能奠定了基础。     

siRNA腺病毒体内抑制Survivin基因表达诱导裸鼠肿瘤细胞凋亡的研究

目的利用腺病毒siRNA载体介导的RNA干扰技术,在免疫缺陷鼠肝癌肿瘤动物模型中通过靶向沉默细胞凋亡因子Survivin的表达,探讨其在活体动物肿瘤组织中的作用。方法利用肝癌细胞株HepG2构建裸鼠肝癌肿瘤细胞动物模型,并按照病毒注射剂量分组,将已构建的Survivin-siRNA重组腺病毒注射入裸鼠肿瘤内,观察肿瘤生长状况并绘制肿瘤生长曲线,利用TUNEL反应测定肿瘤组织细胞内DNA的片段化观察细胞凋亡。结果注射AdsiR- NA-Survivin高、低剂量组肿瘤生长明显受到抑制,且抑制程度与注射的腺病毒浓度呈正相关;TUNEL实验表明注射AdsiRNA-Survivin高、低剂量组的肿瘤组织标本可见大量细胞核呈黄褐色、核质浓缩、细胞形态不规则的凋亡细胞。高剂量组、低剂量组、AdsiRNA-U6组、PBS组凋亡细胞数依次减少。结论腺病毒siRNA载体系统可应用于活体动物试验中;AdsiRNA-Survivin对裸鼠人肝癌移植瘤有明显抑制作用,与肿瘤感染数或浓度成正比,且能促进人肝癌肿瘤细胞的凋亡。     

重组腺病毒载体介导HOSM基因对肝癌细胞体外增殖的抑制作用

目的:构建含人抑瘤素M(human oncostatin M,HOSM)基因的复制缺陷型重组腺病毒载体AD-HOSM,观察其对人肝癌细胞株HepG2在体外生长抑制情况.方法:通过同源重组方法构建含HOSM基因的缺陷型重组腺病毒载体AD-HOSM,用报告基因AD-GFP检测腺病毒的对人肝癌细胞系的转染效率:人肝癌细胞株HepG2转染含HOSM基因的缺陷型腺病毒载体AD-HOSM后,用RT-PCR法检测外源HOSM基因在其中的表达;台盼蓝染色、细胞计数法检测AD-HOSM对HepG2的体外增殖抑制情况.结果:成功构建了重组腺病毒载体AD-HOSM,AD-HOSM对肝癌细胞HepG2有较高的转染效率,并且转染效率与病毒的剂量呈正相关.AD-HOSM感染HepG2细胞48h,HOSM基因在转染细胞能有效的表达.体外试验示,AD-HOSM转染的细胞的增殖却受到明显的抑制.结论:重组腺病毒介导HOSM表达能有效抑制HepG2在体外的增殖,提示重组腺病毒介导的HOSM基因治疗可能成为肝癌治疗的候选方案.     

抗端粒酶核酶抑制肝癌细胞裸鼠移植瘤生长的研究

设计针对端粒酶RNA组分的特异性核酶，研究该核酶对肝癌细胞裸鼠移植瘤生长的影响。构建含针对端粒酶RNA组分锤头状核酶基因的重组真核表达质粒pBBS212-RZ，以及建立人肝癌细胞株HepG2裸鼠移植瘤模型。将不同剂量的pBBS212-RZ用脂质体包裹后进行瘤体内多点注射，对照组注射生理盐水或空质粒载体pBBS212。连续注射2l天后，测量移植瘤体积和重量，检测瘤组织端粒酶活性。抗端粒酶特异性核酶使瘤组织端粒酶活性下降(抑制率为72％)，明显地抑制了肝癌细胞裸鼠移植瘤生长(抑制率为68％)，且存在剂量依赖性。抗端粒酶特异性核酶作为一种有效的端粒酶抑制剂，可抑制肝癌细胞的生长，有望在肝癌基因治疗中发挥作用。     

人肿瘤抑素Tumstatin基因慢病毒表达载体的构建及其蛋白表达

目的 构建Tumstatin基因慢病毒表达载体,进一步研究Tumstatin基因的功能及其在肿瘤治疗中的应用.方法 采用PCR技术从含有Tumstatin基因的质粒pSPORT1-Sfi扩增目的 基因Tumstatin,并将基因克隆到慢病毒载体表达质粒PGC-Fu[含增强型绿色荧光蛋白(EGFP)基因]中,构建慢病毒载体表达质粒pGC-FU-Tum,通过酶切、测序验证Tumstatin基因后,将pGC-FU-Tum质粒和包装质粒pHelper1.0、pHelpe2.0共同转染人胚胎肾上皮细胞系293T细胞,获得携带Tumstatin基因和EGFP基因的重组慢病毒GC-Fu-Tum,并转染人脐静脉血管内皮细胞HUVEC,Wstem blot检测目的 蛋白Tumstatin的表达.结果 pGC-FU-Tum中携有正确的Tumstatin基因;pGC-FUTurn共转染包装细胞293T能产生高浓度的重组慢病毒GC-FU-Tum;目的 基因Tumstatin能被重组慢病毒高效地转导入HUVEC,荧光显微镜下能直接观察到EGFP,Western blot能检测到Tumstatin蛋白在靶细胞中的表达.结论 成功构建了携带Tumstatin基因的重组慢病毒载体;其蛋白表达与EGFP-致..     

慢病毒介导的IGF1R基因干扰对A549细胞对紫杉醇敏感性的影响

目的 探讨慢病毒介导的RNA干扰技术沉默人肺腺癌A549细胞胰岛素样生长因子1受体(IGF1R)表达,并观察其对萦杉醇化疗敏感性的影响.方法 构建IGF1R重组慢病毒,感染A549细胞,建立稳定低表达IGF1R细胞株;应用RT-PCR及蛋白免疫印迹法检测IGF1R沉默效率;CCK8比色法检测IGF1R沉默后A549细胞对紫杉醇敏感性的影响.构建裸鼠移植瘤模型,检测紫杉醇对低表达IGF1R的A549细胞移植瘤生长的抑制作用.结果 成功构建IGF1R重组慢病毒,并建立了稳定低表达IGF1R的A549细胞株;紫杉醇对低表达IGF1R的A549细胞的半数抑制剂量由38.52 μg/L降至16.27 μg/L,敏感性提高2.37倍;低表达IGF1R的A549细胞移植瘤生长缓慢,与紫杉醇联合应用后,移植瘤生长显著抑制,体积抑瘤率达87.5％,重量抑瘤率达88.2％.结论 慢病毒介导的IGF1R基因沉默能抑制人肺腺癌细胞增殖,并显著提高腺癌细胞对紫杉醇的敏感性.     

索拉非尼联合顺铂对人肝癌裸鼠移植瘤生长的抑制作用

目的探讨多靶点酪氨酸激酶抑制剂索拉非尼联合顺铂（DDP）对人肝癌裸鼠皮下移植瘤生长的抑制作用。方法构建人肝癌细胞HepG2裸鼠皮下移植瘤模型,随机分为空白对照组、溶剂对照组、索拉非尼组、DDP组和联合用药组。观察用药前后肿瘤大小,测体质量,并计算瘤重,绘制肿瘤生长曲线;应用免疫组化检测肿瘤微血管密度（MVD）。结果索拉非尼和DDP单药均能抑制肿瘤生长,两药联合疗效明显增强（P〈0．05）。与对照组和顺铂组相比,索拉非尼组和联合用药组能明显抑制肿瘤血管生成,MVD值均明显降低,以联合用药组最为明显（P均〈0．05）。结论索拉非尼联合DDP能增强抑制人肝癌裸鼠移植瘤的生长及微血管生成。     

Berberine inhibits angiogenic potential of Hep G2 cell line through VEGF down-regulation in vitro

Background and Aim: Berberine, an herbal alkaloid, has been reported to have promotion potential of apoptosis and anticancer effect on a variety of human tumor cells. To obtain more specific understanding of those consequences of berberine on hepatocellular carcinoma (HCC) and the tumor microenvironment, we conducted in vitro experiments to investigate the inhibitory effect of berberine on tumor‐induced angiogenesis using HCC cells and human umbilical vein endothelial cells (HUVECs). Methods: Human umbilical vein endothelial cell growth was quantified with the CCK‐8 cell proliferation assay; cell migration was observed with a Boyden chamber (Transwell, Corning, Lowell, MA, USA), and angiogenesis was assessed by endothelial tube formation in Matrigel in vitro. In addition, VEGF level was determined by ELISA and VEGF mRNA expression by RT‐PCR. Results: Berberine inhibited the capacity of HCC to stimulate HUVEC's proliferation, migration and endothelial tube formation, suggesting that berberine could influence the cross‐talk between the HCC cell and vascular endothelial cells. These results demonstrate berberine's antiangiogenesis property and its clinical potential as an inhibitor of tumor angiogenesis. Subsequently analyses reveal that berberine prevents secretion of VEGF from HCC and down‐regulates VEGF mRNA expression. Conclusion: These findings strongly suggest that berberine is a potential antiangiogenic agent and a promising antitumor drug for HCC.     

Antiangiogenic property of pigment epithelium-derived factor in hepatocellular carcinoma

Pigment epithelium-derived factor (PEDF) is one of the most powerful endogenous antiangiogenic reagents discovered to date. Its antiangiogenic potential in neoplastic disease remains unclear. In this study, we investigated antiangiogenic property of PEDF in hepatocellular carcinoma (HCC), a typical hypervascular tumor. In HCC cell lines, constitutive messenger RNA and protein expression of PEDF varied. Genomic DNA encoding the PEDF gene was the same in the cell lines examined by Southern blotting. In chemically induced hypoxic conditions, secreted PEDF protein was suppressed in contrast to elevation of vascular endothelial growth factor protein. When PEDF was overexpressed by gene transfer, proliferation and migration of endothelial cells were inhibited in conditioned media derived from all HCC cell lines. However, the serum concentration of PEDF, as measured by enzyme-linked immunosorbent assay, was decreased in patients with cirrhosis or HCC complicated by cirrhosis compared to healthy volunteers and patients with chronic hepatitis. According to the endothelial cell proliferation assay, the serum PEDF of patients with HCC had antiangiogenic activity. Moreover, intratumoral injection of a PEDF-expressing plasmid in athymic mouse models caused significant inhibition of preestablished tumor growth. In conclusion, PEDF plays a role in the angiogenic properties of HCC. Reduction of serum PEDF concentration associated with the development of chronic liver diseases may contribute to the progression of HCC. In addition, gene therapy using PEDF may provide an efficient treatment for HCC.     

东亚钳蝎镇痛抗肿瘤肽体外靶向肝细胞癌的实验研究

背景：肝细胞癌（HCC）是全球最常见的恶性肿瘤之一,目前手术、介入、放化疗等常规治疗效果均不理想,开发有效、毒副作用相对较小的新药成为研究者关注的重点。目的：观察东亚钳蝎镇痛抗肿瘤肽（AGAP）基因真核表达载体对HCC细胞的靶向表达和毒性作用。方法：真核表达质粒pAFP．AGAP经酶切和测序鉴定后转染甲胎蛋白（AFP）阳性人HCC细胞株HepG2以及AFP阴性人宫颈癌细胞株HeLa和正常人肝细胞株LO2。逆转录聚合酶链反应（RT—PCR）检测AGAP mRNA表达,CCK-8方法检测细胞毒性作用。结果：各组细胞转染质粒pAFP-AGAP后．AGAP mRNA表达仅见于AFP阳性HepG2细胞．而AFP阴性HeLa细胞和LO2细胞均无AGAPmRNA表达。HepG2细胞转染质粒pAFP—AGAP48h后形态发生明显改变,细胞生长显著受抑（P〈0．01）．48h和72h时抑制率分别为44．4％和74．6％,而HeLa细胞和LO2细胞的形态和生长均未受明显影响。结论：AGAP基因真核表达载体可实现HCC基因治疗的靶向性和高效性,有望成为HCC靶向基因治疗的有力工具。     

siRNA干扰纤维蛋白原α链基因对人肝癌细胞增殖和凋亡的影响

背景：纤维蛋白原（FG）是由肝细胞合成、分泌的糖蛋白。除凝血和纤溶外,FG在细胞增殖、血管发生、肿瘤的发生、进展和转移中亦发挥重要作用。肝细胞癌患者血浆FG水平显著高于健康人。目的：观察以RNA干扰技术抑制FG仅链（FGA）基因对人肝癌细胞增殖和凋亡的影响。方法：构建FGAsiRNA干扰质粒,将重组质粒pU6．FGAsiRNA稳定转染入人肝癌细胞株HepG2,同时设置空质粒载体pU6转染组作为对照。以RT－PCR、蛋白质印迹法在mRNA和蛋白质水平验证干扰效果,以CCK－8实验和流式细胞术检测各组HepG2细胞的增殖和凋亡情况。结果：FGAsiRNA转染组HepG2细胞FGAmRNA和蛋白表达明显低于空载体转染组,细胞增殖显著受抑（P〈0．001）,细胞凋亡显著增加（P〈0．001）。结论：RNA干扰技术能有效下调人肝癌细胞的FGA表达,抑制肿瘤细胞生长增殖并促进其凋亡。FGA可能成为肝细胞癌治疗的潜在靶点之一。     

Inhibitory effect of endostatin expressed by human liver carcinoma SMMC7721 on endothelial cell proliferation in vitro

AIM: To construct a stable transfectant of human liver carcinoma cell line SMMC7721 that could secret human endostatin and to explore the effect of human endostatin expressed by the transfectant on endothelial cell proliferation. METHODS: Recombinant retroviral plasmid pLncx-Endo containing the cDNA for human endostatin gene together with rat albumin signal peptide was engineered and transferred into SMMC7721 cell by lipofectamine. After selection with G418, endostatin-transfected SMMC7721 cells were chosen and expanded. Immunohistochemical staining and Western blot were used to detect the expression of human endostatin in transfected SMMC7721 cells and its medium. The conditioned medium of endostatin-transfected and control SMMC7721 cells were collected to cultivate with human umbilical vein endothelial cells for 72 hours. The inhibitory effect of endostatin, expressed by transfected SMMC7721 cells, on endothelial proliferation in vitro was observed by using MTT assay. RESULTS: A 550 bp specific fragment of endostatin gene was detected from the PCR product of endostatin-transfected SMMC7721 cells. Immunohistochemistry and Western blot analysis confirmed the expression and secretion of foreign human endostatin protein by endostatin-transfected SMMC7721 cells. In vitro endothelial proliferation assay showed that 72 hours after cultivation with human umbilical vein endothelial cells, the optical density (OD) in group using the medium from endostatin-transfected SMMC7721 cells was 0.51 +/- 0.06, lower than that from RPMI 1640 group (0.98 +/- 0.09) or that from control plasmid pLncx-transfected SMMC7721 cells (0.88 +/- 0.11). The inhibitory rate for medium from endostatin-transfected SMMC7721 cells was 48%, significantly higher than that from empty plasmid pLncx-transfected SMMC7721 cells (10.2%, P<0.01). CONCLUSION: Human endostatin can be stably expressed by SMMC7721 cell transferred with human endostatin gene and its product can significantly inhibit the proliferation of human umbilical vein endothelial cell in vitro.     

人胰岛素样生长因子Ⅱ/P4启动子调控胸苷激酶载体的构建

目的 探讨人胰岛素样生长因子Ⅱ/(IGF-Ⅱ)P4启动子调控单纯疱疹病毒胸苷激酶(HSV-tk)/丙氧鸟苷(GCV)自杀基因系统体外对人肝癌细胞的选择性杀伤效应.方法用分子生物学方法构建IGF-Ⅱ P4启动子和巨细胞病毒启动子(CMV)调控下HSV-tk 与增强型绿色荧光蛋白(EGFP)融合基因穿梭质粒,并用脂质体转染法将其转入肝癌细胞系HepG2及宫颈癌细胞系HeLa细胞中,在荧光显微镜下观察荧光表达情况.加入GCV使总浓度分别为0、1.0、10.0和100.0μg/ml,用四甲基偶氮唑盐实验检测HSV-tk/GCV系统对各种细胞的杀伤作用,用RT-PCR法检测转染前后胸苷激酶(tk)和EGFP mRNA的表达情况.用方差分析进行统计学处理.结果酶切鉴定和测序分析证实重组质粒构建成功;转染此穿梭质粒的细胞中仅有HepG2细胞检测到绿色荧光;RT-PCR检测到tk和EGFP的mRNA仅在HepG2细胞中表达;pDC316-tkEGFP-CMV和pDC316-tkEGFP-P4转染的HepG2细胞均出现明显的细胞生长抑制现象,两者转染细胞的抑制率分别为6.95%±0.67%、24.99%±1.53%、49.68%±1.68%和71.85%±3.28%,4.83%±0.35%、17.34%±1.15%、30.17%±1.30%和40.39%±0.82%,转染后者对HepG2细胞的抑制率低(F=24.055,P<0.05),pDC316-tkEGFP-CMV转染的HeLa细胞也出现明显的细胞生长抑制现象,其抑制率分别是6.36%±0.83%、23.95%±1.72%、45.13%±1.64%和69.38%±3.17%.转染质粒pDC3 16-tkEGFP-P4的HeLa细胞未发现明显的细胞抑制现象.结论成功构建IGF-ⅡP4启动子调控携带tk与EGFP融合基因穿梭质粒载体,转染后对人肝癌细胞系HepG2有选择性杀伤作用,为进一步靶向性肝癌腺病毒基因治疗奠定基础.

Abstract：

Objective To investigate the selective killing effect of herpes simplex virus-thymidine kinase/ganciclovir(HSV-tk/GCV)suicide gene system controlled by human IGF-II P4 promoter on HCC cells in vitro.Methods Recombinant shuttle plasmid vectors driven by IGFⅡ P4 promoter and driven by CMV promoter were constructed by techniques of gene recombination.The recombinant shuttle plasmids were then transfected into HepG2 and HeLa cells by techniques of lipidosome transfection.EGFP expression was detected by fluoroscopy.Tk and EGFP mRNA expression were detected by RT-PCR.The selective killing effect after GCV application was determined with MTT method.Statistical analysis was performed with ANOVA analysis.Results Identification of pDC3 16-tkEGFP-P4 by enzyme digestion and sequencing analysis showed that the construction of the recombinant shuttle plasmid was correct.It was found that green fluorescence protein could only be seen in HepG2 cells.not in HeLa cells.The results of RT-PCR showed only two bands could be seen in the samples of pDC316-tkEGFP-P4 transfected HepG2 cells.The growth of HepG2 cells transfected with pDC316-tkEGFP-CMV and pDC316-tkEGFP-P4 were inhibited remarkably,the growth inhibition rates were 6.95% ± 0.67%,24.99% ± 1.53%,49.68% ±1.68%,71.85% ± 3.28% and 4.83% ± 0.35% vs 17.34% ± 1.15%,30.17% ± 1.30%,40.39% ± 0.82%(F = 24.055,P < 0.05),respectively. The growth of HeLa cells transfected with pDC316-tkEGFP-CMV were also inhibed,the growth inhibition rates were 6.36% ± 0.83%,23.95% ± 1.72%,45.13% ± 1.64%and 69.38 % ± 3.17%,respectively. The growth of HeLa cells transfected with pDC316-tkEGFP-P4 was not inhibed. The growth inhibition rates were 0.91 ± 0.04,1.18 ± 1.32,1.19 ± 0.10 and 1.32 ± 0.05(F = 26.469,P < 0.01),respectively. Conclusion The shuttle plasmid vector carrying the tkEGFP fusion protein gene driven by IGF-Ⅱ P4 promoter has been constructed successfully and its specific expression in HepG2 cells provided the basis for targeted gene therapy for HCC.     

Superantigen-SEA gene modified tumor vaccine for hepatocellular carcinoma： An in vitro study

AIM: To construct an eukaryotic superantigen gene expression vector containing the recombinant gene of SEA and CD80 molecule transmembrane region (CD80TM), and to express staphylococcus enterotoxin A (SEA) on the membrane of hepatocellular carcinoma (HCC) cell to form a superantigen gene modified tumor vaccine for HCC. METHODS: SEA and linker-CD80TM gene were amplified through PCR from plasmid containing cDNA of SEA and CD80. Gene fragments were then subcloned into the multiple cloning sites of retroviral vector pLXSN. Recombinant plasmid was transferred into HepG2 cells mediated with lipofectamine, positive clones were selected in culture medium containing G418. RT-PCR and indirect immunofluorescence studies confirmed that SEA was expressed specifically on HCC cell membrane. INFgamma-ELISPOT study demonstrated that SEA protein was expressed on the membrane of HCC cells. Cytotoxicity of HepG2-SEA primed CTLs (SEA-T) was analyzed by (51)Cr release assay. T cells cultured with rhIL-2 (IL-2-T) were used as control. RESULTS: Restriction digestion and sequence analyses confirmed the correctness of length, position and orientation of inserted fusion genes. SEA was expressed on the surface of HepG2 cells, HepG2-SEA had strong stimulating effect on production of HepG2 specific CTL (P<0.001). SEA-T had enhanced cytotoxicity to HepG2 cells (P<0.05). CONCLUSION: Tumor cell membrane expressed superantigen can be used to reinforce the immune effect of tumor cell vaccine for HCC, which provides a new method of the enhanced active immunotherapy for HCC.