人内皮抑素基因腺病毒表达载体的构建及其在乳腺癌细胞中的表达活性

引言近年来,阻止肿瘤血管生成,控制肿瘤的生长和转移,成为肿瘤治疗的一项新策略[1,2],其中采用转染血管生成抑制剂拮抗血管的生成,以内皮抑素最为理想[3]。本文通过构建人内皮抑素基因的腺病毒表达载体,研究内皮抑素在乳腺癌细胞中的表达,为进一步研究乳腺癌的抗血管基因治疗做好准备。1材料与方法1.1材料携带人内皮抑素基因(humanendostatin,hE)的质粒pBlast hEndostatin购于美国InvitroGen公司;腺病毒载体pCA13、pBGHE3及293细胞株购于MicrobixBiosystems公司;人乳腺癌细胞株MBD231和MCF7、人脐静脉内皮细胞株ECV304购于美国ATCC细胞库;LipofectAMINE2000转染试剂盒购自GibcoBRL公司;各种限制性内切酶和DNA连接酶购自NEB公司;QIAprepspin miniprepkit、QIAampDNABloodMiniKit购自QIAGEN公司;M PERMammalianProteinEx tractionReagent购自PIERCE公司;Western显色液LumiGLOchemiluminescentreag...     

重组人内皮抑素的纯化及抗肿瘤活性研究

[目的]从高效表达的基因工程菌中纯化重组人内皮抑素,对其抗肿瘤活性进行研究.[方法]经异丙基硫代-β-D-半乳糖苷(IPTG)诱导,重组人内皮抑素在大肠杆菌基因工程菌中以包涵体形式高效表达.通过凝胶层析纯化重组内皮抑素蛋白.应用鸡胚绒毛尿囊膜实验(CAM),肺癌细胞MTT试验及细胞迁移抑制实验,裸鼠皮下移植喉癌抑瘤实验、病理组织切片、免疫组化指标的测定等检测研究重组人内皮抑素的抗肿瘤活性.[结果]重组内皮抑素的复性率可达40%.重组内皮抑素在体外直接抑制肺癌细胞的增殖及迁移.用药21天,对裸鼠皮下移植喉癌的抑瘤率达到40.66%,HE染色、免疫组化及CAM实验表明其对肿瘤组织新生血管的生成有较强的抑制作用.[结论]重组人内皮抑素具有抑制肿瘤组织新生血管生成和直接抑制肿瘤细胞生长和迁移的双重抗肿瘤活性.     

腺病毒介导人血管抑素抑制乳腺癌生长的研究

目的：研究腺病毒介导的重组人血管抑制Plasminogen K5基因对乳腺癌的治疗作用。方法：通过PCR将人血纤维蛋白酶原的信号肽基因加至Plasminogen的K5结构域基因，克隆到质粒pCA13，并在293细胞中同源重组，得到腺病毒AdK5。将Ad-K5体外感染乳腺癌细胞B－Cap-37和血管内皮细胞ECV304，观察其对细胞生长的影响，同时在乳腺癌的裸鼠动物模型上，检测其对肿瘤生长的抑制作用。结果：在感染Ad-K5的B－Cap-37细胞中检测到K5基因mRNA的表达。Ad-K5对血管内皮细胞ECV304有抑制作用，但对癌细胞B-Cap-37没有直接抑制作用。动物实验表明Ad-K5能显著抑制肿瘤的生长。结论：K5基因能抑制乳腺癌实体瘤的生长。     

腺病毒介导人VEGF-siRNA对裸鼠移植骨肉瘤的抑制作用

目的:研究腺病毒介导人VEGF-siRNA对裸鼠移植骨肉瘤的治疗作用.方法:利用腺病毒重组技术将VEGF-siRNA基因克隆入增殖缺陷型腺病毒基因组中,构建重组腺病毒Ad-VEGF-siRNA,重组腺病毒体外感染骨肉瘤MG63细胞,RT-PCR检测MG63细胞VEGF的表达水平.建立荷人骨肉瘤MG63裸小鼠动物模型,以Ad-VEGF-siRNA瘤组织注射治疗,检测移植瘤组织中VEGF的表达情况及Ad-VEGF-siRNA对肿瘤生长和肺转移的抑制作用.结果:成功构建了表达VEGF-siRNA的重组腺病毒载体Ad-VEGF-siRNA;体外与体内实验检测Ad-VEGF-siRNA转染骨肉瘤细胞MG63后显著抑制VEGF表达.荷人骨肉瘤裸鼠经Ad-VEGF-siRNA治疗后,显示其对移植骨肉瘤生长有明显抑制作用(P<0.05),并且对骨肉瘤的肺转移有显著的抑制作用(P<0.05).结论:所构建的Ad-VEGF-siRNA可以有效抑制骨肉瘤中VEGF表达,使裸鼠移植骨肉瘤生长减慢,并且显著抑制荷瘤裸小鼠肺转移的发生.     

重组腺病毒介导小鼠内皮抑素对荷骨肉瘤MG-63细胞裸鼠肺转移的抑制

目的: 构建携小鼠内皮抑素(endostatin， ES)基因的重组腺病毒载体，观察其对荷骨肉瘤裸鼠肺转移的抑制，探讨内皮抑素表达水平与骨肉瘤肺转移的关系。方法:构建pDC315mEndo表达质粒，同源重组产生重组腺病毒AdmEndo。裸鼠右前肢皮下注射骨肉瘤MG63细胞建立移植瘤裸鼠模型；随机分为4组：小鼠内皮抑素腺病毒（AdmEndo）组，携带EGFP基因腺病毒（AdEGFP）组， PBS组，未接种肿瘤细胞裸鼠空白对照组。各组裸鼠每周分别注射相应药物200 μl，连续5次，观察各组动物移植瘤体积、瘤组织病理，ELISA法检测各组裸鼠血ES水平；7周后处死动物，观察有无肺转移及肺转移灶病理。结果：AdEGFP组肿瘤体积为（1.53±0.05） cm3,PBS组为（1.56±0.07） cm3, AdmEndo组为（0.91±0 .03） cm3，AdmEndo治疗的抑瘤率达40.7％。AdmEndo组裸鼠血内皮抑素表达水平明显高于AdEGFP组和PBS组（P<0.05）。AdmEndo组裸鼠肺部未发现肿瘤转移灶，其他两组肺部见大量散在转移灶，肺转移率分别为80%和90%。未发生肺转移裸鼠的ES水平显著高于发生肺转移的裸鼠（P<0.05）。结论：腺病毒介导的小鼠内皮抑素显著抑制了荷骨肉瘤裸鼠肺转移，内皮抑素表达水平与肺转移有着直接的关系。     

Ki67基因RNA干扰腺病毒表达载体的构建及其抗结肠肿瘤作用研究

目的:构建针对肿瘤增殖基因Ki67的小干扰RNA(Ki67-siRNA)腺病毒表达载体,体外研究其对人结肠癌细胞株SW620细胞生长影响,为结肠癌的基因治疗提供参考。方法:设计可形成小发夹结构的Ki67-siRNA对应模板DNA序列,克隆至缺陷型腺病毒质粒pCA13,构建Ki67-siRNA表达质粒pCA13-Ki67。鉴定正确后,将pCA13-Ki67与含有腺病毒右臂的质粒pBHGE3共转染至293细胞,包装成复制缺陷型腺病毒Ad-Ki67。大量扩增Ad-Ki67,氯化铯梯度离心纯化,测病毒滴度。Ad-Ki67感染人结肠癌细胞,结晶紫染色法检测细胞病理作用,MTT法检测细胞存活,Western印迹法检测Ki67表达。结果:酶切分析、测序鉴定表明pCA13-Ki67构建成功,PCR分析表明Ad-Ki67含有Ki67-siRNA序列。Ad-Ki67可显著抑制Ki67蛋白表达及结肠癌细胞生长。结论:含有Ki67-siRNA的重组腺病毒构建成功,其能抑制人结肠癌细胞Ki67基因表达和生长。     

携带p16基因增殖缺陷型腺病毒对胃癌细胞皮下移植瘤的治疗作用

目的:研究携带p16基因的增殖缺陷型腺病毒载体对裸鼠皮下胃癌细胞移植瘤的抗肿瘤活性。方法:PCR扩增p16cDNA,构建腺病毒载体pSuCMV-p16表达质粒,在293细胞内重组增殖缺陷型腺病毒AdCMV-p16;建立胃腺癌细胞SGC-7901皮下移植瘤裸鼠模型,分为3组(AdCMV-p16组、Ad-LacZ组、空白对照组),以2×108pfu/100μl的重组腺病毒Ad-CMV-p16直接瘤内多点注射,隔日1次,共5次,空白对照组以等量病毒保存液注射,定时测量瘤体生长情况,并以p16免疫组化、TUNEL凋亡检测等观察AdCMV-p16抗肿瘤的疗效。结果:携带p16基因的增殖缺陷型腺病毒AdCMV-p16对胃腺癌裸鼠移植瘤有明显的抑制作用,抑瘤率为58.12%,而对照病毒Ad-LacZ对胃癌移植瘤的抑瘤率仅为4.26%;病理学检查显示,病毒治疗的癌组织中细胞坏死以凋亡为主,癌细胞中检测到p16基因的表达。结论:携带p16基因的增殖缺陷型腺病毒能恢复胃癌细胞中p16基因的表达,对胃癌移植瘤生长有明显的抑制作用。     

腺病毒介导的内皮抑素基因治疗小鼠肺癌

目的 探讨内皮抑素对小鼠肺癌生长和转移的抑制作用及其对肿瘤内部新生血管的影响.方法 在C57BL/6小鼠背部皮下注射2×106 lewis肺癌(LLC)细胞,建立小鼠肺癌种植瘤模型,2周后,瘤内注射2×109 pfu内皮抑素腺病毒载体,观察内皮抑素对肿瘤生长、转移及生存率的影响,检测内皮抑素在肿瘤组织的原位表达和血液循环中的表达水平及持续时间.用免疫组化方法,检测肿瘤内部血管密度,观察治疗对肿瘤血管的影响.用透射电镜观察肿瘤细胞的凋亡情况.结果 免疫组化检测结果显示,内皮抑素蛋白在内皮抑素组的肿瘤组织中呈强阳性表达,而在空载体对照组和阴性对照组中呈阴性表达或很少量表达.用酶联免疫吸附实验(ELISA)法检测内皮抑素组血清内皮抑素浓度,第2周可达1540±560 ng/ml;1个月后,血清内皮抑素浓度降至对照水平.内皮抑素组的肿瘤体积和生存率,与空载体对照和阴性对照组比较,差异有统计学意义(P＜0.05).抗CD31抗体标记的肿瘤内血管密度(MVD)在内皮抑素组、空载体对照组和阴性对照组中,分别为37.5±4.6、65.2±5.8和68.5±4.5个/200倍视野,抗CD105抗体标记的肿瘤内MVD分别为10.5±3.2、39.7±5.6和42.4±4.8个/200倍视野,内皮抑素组与空载体对照组和阴性对照组比较,差异有统计学意义(P＜0.05).内皮抑素组的组织在电镜下呈凋亡相的肿瘤细胞多见.结论 腺病毒介导的内皮抑素基因可在体内高效、较长时间表达内皮抑素蛋白,对小鼠皮下种植瘤有一定的治疗作用,其作用的靶点是抑制新生血管的生成.     

重组腺病毒人内皮抑素的质量标准研究

目的: 建立重组腺病毒人内皮抑素的质量标准和检测方法. 方法: PCR法鉴定腺病毒载体的E2B区和插入基因,琼脂糖凝胶电泳检查重组病毒DNA酶切图谱.分光光度法测定病毒颗粒数,TCID50法测定病毒感染滴度.感染人肝癌细胞HepG2测定插入基因的表达量,感染人血管内皮细胞HM2测定表达产物的生物学活性.病毒的纯度分析采用A260/A280比值和HPLC法进行.采用A549细胞进行复制型腺病毒的检测.结果: 腺病毒载体E2B区和插入基因PCR扩增结果与理论相符,重组病毒DNA的酶切图谱与标准品一致.原液病毒颗粒数为2.4×1012 VP/ml,滴度为1.53×1011 IU/ml,比滴度为6.4% IU/VP;成品病毒颗粒数为1.0×1012 VP/ml,滴度为3.75×1010 IU/ml,比滴度为病3.8% IU/VP.以50 MOI重组腺病毒感染HepG2细胞48 h后培养上清人内皮抑素表达量为332 ng/ml.50 MOI重组腺病毒对血管内皮细胞生长的抑制率为55%.A260/A280为1.29,HPLC纯度为99.7%.复制型腺病毒为≤1RCA/3×1010 VP.其他各项检测指标均符合规定.结论: 建立了重组腺病毒人内皮抑素的质量标准及其检测方法,并用于该产品的质量控制.     

血管抑素基因转染抑制胆囊癌细胞的实验研究

目的:观察血管抑素基因转染对体外人胆囊癌细胞系GBC-SD细胞株增殖及血管抑素表达和裸鼠致瘤体积的影响,初步探讨血管抑素基因转染抑制胆囊癌细胞生长的可能性.方法:应用pcDNA3.1( )-angiostatin 基因转染GBC-SD胆囊癌细胞.观察细胞生长情况,制作细胞生长曲线;Western-blot法分析血管抑素的表达情况;裸鼠种植瘤模型观察肿瘤的体积和质量.结果:pcDNA3.1( )-angiostatin 基因转染的GBC-SD胆囊癌细胞生长明显受到抑制,血管抑素的蛋白表达也明显增加.转染的肿瘤细胞在裸鼠种植瘤明显小于阴性组和空白组.结论:血管抑素基因转染可能具有抑制胆囊癌细胞增殖及生长的作用.     

小鼠内皮抑素基因的克隆、表达及其抑瘤效应研究

克隆小鼠内皮抑素（ｅｎｄｏｓｔａｔｉｎ）基因,检测其表达蛋白的生物学活性,应用该蛋白治疗大鼠Ｃ６脑胶质瘤。方法：采用ＲＴ－ＰＣＲ法,从小鼠肝组织克隆ｅｎｄｏｓｔａｔｉｎ基因,重组入ｐＵＣ１９,测序后构建非融合表达载体ｐＢＶ２２０－ｅｎｄｏｓｔａｔｉｎ,使其在ＤＨ５α内经温度诱导表达,纯经ｅｎｄｏｓｔａｔｉｎ蛋白并用鸡胚绒毛膜尿囊膜实验和内皮细胞抑制实验检测其活性,经荷Ｃ６胶质瘤大鼠皮下注射该蛋白,     

Potent antitumor efficacy of an E1B 55kDa-deficient adenovirus carrying murine endostatin in hepatocellular carcinoma

Data from clinical trails have shown that the antitumoral effect of ONYX-015, an E1B 55kDa-deficient adenovirus, as monotherapy is insufficient. To enhance its efficiency, CNHK200-mE, another E1B 55kDa-deficient adenovirus armed with a mouse endostatin gene was constructed and its antitumoral activities against hepatocellular carcinoma (HCC) in vitro and in vivo were investigated. The selective replication and cytotoxicity of CNHK200-mE in Hep3B and HepGII cells independent of p53 status were confirmed via TCID50 and 3-(4,5dimetylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assays. Potent tumor growth suppression on SMMC-7721 xenografts in nude mice was observed and a synergistic effect of the carrier virus and the therapeutic gene was suggested. Moreover, in comparison with the nonreplicative adenovirus carrying the same therapeutic gene, amplified transgene expression of mouse endostatin in vitro and in vivo were confirmed by Western blotting and ELISA assay. The effective angiogenesis inhibition and replication of CNHK200-mE in nude mice xenografts were demonstrated by immunohistochemistry. In conclusion, the recombinant adenovirus CNHK200-mE is a replication-competent oncolytic virus mediating high expression of therapeutic gene. Because CNHK200-mE is capable of replicating in and lysing HCC cells selectively with effective tumor growth suppression and antiangiogenic activity on HCC xenografts in nude mice, it holds good potential for the treatment of HCC.     

siRNA靶向沉默hyrdC基因对胃癌细胞SGC-7901生长作用的研究

目的:观察RNA干扰靶向沉默hyrdC基因对胃癌SGC-7901细胞生长的影响.方法:将已成功构建的携带hyrdC基因以及沉默hyrdC基因的重组腺病毒Ad-hyrdC、Ad-shyrdC分别转染胃癌SGC-7901细胞,台盼蓝细胞计数和MTT法观察各组胃癌细胞生长情况,将胃癌细胞接种裸鼠皮下构建裸鼠荷瘤模型观察各组胃癌瘤体的生长情况.结果:Ad-shyrdC组细胞生长明显慢于Ad-hyrdC、Ad-null和正常对照组,差异有统计学意义,P=0.02;Ad-shyrdC组细胞的MTT值明显低于Ad-hyrdC、Ad-null和正常对照组,P=0.03;Ad-shyrdC组胃癌瘤体生长率较Ad-hyrdC、Ad-null组以及正常对照组明显减慢,P=0.02.结论:hyrdC基因具有促进胃癌细胞生长的功能,而沉默hyrdC基因可抑制胃癌细胞的生长,hyrdC基因有望成为胃癌基因治疗领域的新靶点.     

Inhibition of orthotopic human bladder tumor growth by lentiviral gene transfer of endostatin.

PURPOSE: Inhibitors of endothelial cell proliferation, such as endostatin, result in suppression of tumor-associated angiogenesis and can achieve growth-inhibitory effects depending on the type of tumor treated. The purpose of this study was to investigate whether local overexpression of endostatin could serve to diminish tumor growth of bladder cancer in vivo. EXPERIMENTAL DESIGN: We examined the capability of lentiviral-mediated gene transfer in vitro and therapeutic effects of lentivirus-based vectors expressing endostatin on tumor growth using an orthotopic human bladder tumor model. RESULTS: We found that self-inactivating lentivirus vectors containing green fluorescent protein, alone or in combination with endostatin, were capable of efficient and stable gene transfer to a variety of human bladder tumor cell lines. The production and secretion of endostatin from lentivirus-transduced KU-7 human bladder cancer cells was confirmed by Western blot and competitive enzyme immunoassay. Intravesical instillation of untransduced, green fluorescent protein control lentivirus-transduced, and endostatin-transduced KU-7 cells was performed in murine models to establish orthotopic tumors. Sustained long-term expression of endostatin was achieved in lentivirus-transduced orthotopic bladder tumors, and it was associated with decreased vascularization and inhibition of tumor growth. Lentivirus vector-mediated overexpression of endostatin did not affect the intrinsic production of basic fibroblast growth factor and vascular endothelial growth factor. CONCLUSIONS: These findings suggest that lentivirus-mediated gene transfer might represent an effective strategy for expression of angioinhibitory peptides to achieve inhibition of human bladder cancer proliferation and tumor progression.     

Adeno-associated virus-mediated delivery of a mutant endostatin in combination with carboplatin treatment inhibits orthotopic growth of ovarian cancer and improves long-term survival

A human ovarian cancer cell line, which migrates to mouse ovaries and establishes peritoneal carcinomatosis, was used to evaluate the cooperative effect of an antiangiogenic gene therapy combined with chemotherapy. The ovarian carcinoma cell line MA148 was genetically modified by "Sleeping Beauty" transposon-mediated delivery of DsRed2 fluorescent protein. Stable, high-level expression of DsRed protein enabled in vivo imaging of peritoneal dissemination of ovarian cancer. Both external and internal imaging, along with histopathology, showed migration of i.p. injected human ovarian cancer cell line to mouse ovaries. Using this model, we evaluated the effect of adeno-associated virus (AAV)-mediated expression of a mutant endostatin either alone or in combination with carboplatin treatment. A single i.m. injection of recombinant AAV (rAAV)-mutant human endostatin with P125A substitution (P125A-endostatin) showed sustained expression of mutant endostatin. Antiangiogenic gene therapy inhibited orthotopic growth of ovarian cancer and resulted in 33% long-term tumor-free survival. A single cycle of carboplatin treatment combined with mutant endostatin gene therapy resulted in 60% of the animals remaining tumor free for >200 days, which was significantly better than rAAV-LacZ and/or carboplatin. Combination treatment delayed tumor appearance in 40% of the animals, wherein the residual tumors were smaller in size with limited or no peritoneal metastasis. These studies suggest that AAV-mediated gene therapy of P125A-endostatin in combination with carboplatin is a useful method to inhibit peritoneal dissemination of ovarian carcinoma.     

Survivin promoter-regulated oncolytic adenovirus with Hsp70 gene exerts effective antitumor efficacy in gastric cancer immunotherapy

Gene therapy is a promising adjuvant therapeutic strategy for cancer treatment. To overcome the limitations of current gene therapy, such as poor transfection efficiency of vectors, low levels of transgene expression and lack of tumor targeting, the Survivin promoter was used to regulate the selective replication of oncolytic adenovirus in tumor cells, and the heat shock protein 70 (Hsp70) gene was loaded as the anticancer transgene to generate an AdSurp-Hsp70 viral therapy system. The efficacy of this targeted immunotherapy was examined in gastric cancer. The experiments showed that the oncolytic adenovirus can selectively replicate in and lyse the Survivin-positive gastric cancer cells, without significant toxicity to normal cells. AdSurp-Hsp70 reduced viability of cancer cells and inhibited tumor growth of gastric cancer xenografts in immuno-deficient and immuno-reconstruction mouse models. AdSurp-Hsp70 produced dual antitumor effects due to viral replication and high Hsp70 expression. This therapeutic system used the Survivin promoter-regulated oncolytic adenovirus vector to mediate targeted expression of the Hsp70 gene and ensure safety and efficacy for subsequent gene therapy programs against a variety of cancers.     

Gene therapy delivery of endostatin enhances the treatment efficacy of radiation.

BACKGROUND AND PURPOSE: To evaluate whether sustained expression of mouse endostatin by adeno-associated virus (AAV)-mediated gene transfer can enhance the treatment efficacy of ionizing radiation. MATERIALS AND METHODS: Mouse endostatin was cloned into recombinant AAV (rAAV) under the control of CMV beta-actin promoter. Recombinant mouse endostatin expressed via AAV gene transfer was tested for biological activity in endothelial cells. The impact of elevated serum levels of endostatin on tumor-induced angiogenesis was evaluated using an in vivo angiogenesis assay. The anti-tumor efficacy of combining rAAV-mediated endostatin delivery with radiation was evaluated in a human colorectal tumor model (HT29). RESULTS: Recombinant mouse endostatin expressed through an AAV vector (rAAV-mEndo) inhibited endothelial cell proliferation (by 40-45%) and migration (by 22-33%). Intramuscular injection of rAAV-mEndo (1x10(9) i.u.) led to a sustained serum endostatin level of approximately 500 ng/ml. Compared to control animals this endostatin level was sufficient to inhibit tumor cell-induced vessel formation (37 vs. 28.5, P<0.05) and delay the growth of HT29 xenografts (time from 200 to 1,000 mm(3), 21 vs. 34.5 days, P<0.05). When combined with ionizing radiation, elevated serum endostatin levels significantly enhanced the time for tumors to grow from 200 to 1,000 mm(3) (radiation, 34 days; endostatin plus radiation, 50 days, P<0.05). CONCLUSION: The delivery of endostatin via rAAV vectors may provide an effective means of enhancing the anti-tumor efficacy of radiation therapy.     

Ad-PTEN体外对SGC-7901胃癌细胞生长的抑制作用

目的：探讨腺病毒介导的PTEN基因表达体外对SGC-7901胃癌细胞生长抑制作用及其分子机制。方法：将携有PTEN基因的复制缺陷型腺病毒载体（Ad-PTEN）感染SGC-7901胃癌细胞,用RT-PCR法检测Ad-PTEN在细胞中的表达,光学显微镜及荧光显微镜下观察Ad-PTEN感染细胞前后形态的变化,MTT法检测Ad-PTEN对SGC-7901胃癌细胞生长的抑制作用,用流式细胞术（FCM）检测SGC-7901胃癌细胞凋亡率。RT-PCR分析Bax、Bcl-2、p53、Survivin细胞凋亡相关基因的表达。结果：Ad-PTEN基因组感染SGC-7901胃癌细胞后,RT-PCR结果显示PTEN目的基因能在SGC-7901胃癌细胞中转录,其表达可明显抑制该胃癌细胞的生长,并诱导细胞凋亡。其凋亡机制可能与Bax/Bcl-2比值、p53基因表达上调、Survivin下调有关。结论：重组腺病毒Ad-PTEN具有抑制SGC-7901胃癌细胞生长和诱导细胞凋亡的作用。     

人MUC4启动子驱动HSV-TK腺病毒靶向杀伤胃癌细胞的研究

目的：构建人MUC4启动子驱动下的HSV-TK重组腺病毒,研究其对胃癌细胞的靶向杀伤作用。方法：免疫荧光法检测MUC4在胃癌细胞系中的表达。克隆MUC4启动子区625bp活性序列,利用重组荧光素酶检测系统检测其在SGC-7901胃腺癌细胞及NUGC4胃印戒细胞癌细胞中的转录活性。以AdEasyTM腺病毒系统为载体,构建MUC4启动子驱动下的HSV-TK重组腺病毒,与前体药物GCV联合,检测其对上述两种胃癌细胞的细胞毒作用。结果：MUC4蛋白在两种胃癌细胞的胞浆和胞膜均有表达,而在成纤维细胞中无表达。克隆的MUC4启动子片段在SGC-7901和NUGC4细胞中具有强转录活性,转录活性高于强启动子SV40,而NIH3T3成纤维细胞系中几乎无转录活性。MUC4启动子驱动下的HSV-TK重组腺病毒与GCV联合能够诱导SGC-7901和NUGC4胃癌细胞凋亡,产生特异性靶向细胞毒作用。结论：人MUC4启动子驱动下的HSV-TK重组腺病毒联合GCV对SGC-7901和NUGC4胃癌细胞具有靶向杀伤作用,MUC4启动子可以作为胃癌靶向基因治疗的工具。