重组腺病毒介导自杀基因HSV1-tk转染内皮祖细胞的实验研究

[目的]构建含有自杀基因单纯疱疹病毒胸腺激酶基因( HSV1-tk)的重组腺病毒载体,并感染大鼠骨髓来源的内皮祖细胞(EPCs),以期用于抗肿瘤血管生成的基因治疗及核素报告基因显像.[方法]从大鼠骨髓中分离、培养EPCs,利用流式细胞术进行鉴定.构建重组腺病毒载体Ad5-HSV1-tk-EGFP,并感染EPCs,以腺病毒Ad5-EGFP为对照.利用RT-PCR法、Westernblot免疫印迹法检测HSV1-tk的表达,利用MTT法检测更昔洛韦(GCV)对病毒感染后EPCs的杀伤作用.[结果]流式细胞术结果显示从大鼠骨髓中分离出的EPCs阳性表达CD34(80.09％)和CD 133(81.75％),RT-PCR及Western blot免疫印迹结果显示HSV1-tk基因在转录及蛋白水平可以正确表达,MTT结果显示HSV-tk/GCV自杀基因系统对EPCs细胞具有明显的杀伤作用.[结论]重组腺病毒Ad5-HSV1-tk-EGFP感染EPCs后可以在细胞中成功表达,并且感染后自杀基因具有生物学活性,从而为利用EPCs作为载体进行肿瘤靶向基因治疗、报告基因显像提供实验依据.     

HSV1-tk报告基因真核表达载体的构建及其在人肺腺癌AGZY细胞中的表达

目的：构建含有HSV1-tk（ Herpes simplex virus type 1 thymidine kinase, HSV1-tk）基因的真核表达载体,并检测其在人肺腺癌AGZY细胞系中的表达。方法应用PCR反应从质粒pHSV106质粒中扩增HSV1-tk基因后与pMD18-T载体连接,构建重组质粒pHSV1-tk/18T。将测序正确的重组质粒插入plRES2-EGFP 载体内,通过LipofectamineTM 2000将表达载体转染人肺腺癌AGZY细胞。结果酶切鉴定结果表明扩增的HSV1-tk基因序列正确;用荧光显微镜观察HSV1-tk基因的转入和表达;RT-PCR和Western blot结果显示在AGZY细胞中HSV1-tk基因在转录水平和蛋白水平均可以正确表达。MTT结果显示转染后AGZY细胞与未转染细胞在细胞增殖能力方面无明显差别。结论成功构建HSV1-tk报告基因的真核表达载体,在人肺腺癌AGZY细胞中能有效表达。     

动脉灌注GE7系统介导Ⅰ型单纯疱疹病毒胸腺嘧啶核苷激酶基因导入大鼠卵巢癌细胞

目的 采用大鼠诱发性卵巢癌模型,观察经肿瘤动脉灌注结合GE7导入系统对Ⅰ型单纯疱疹病毒胸腺嘧啶核苷激酶基因(HSV1-tk)转导的效率和靶向性.方法 构建GE7四元复合物,将诱发的荷瘤大鼠9只随机分成A组、B组和C组,A组经卵巢动脉注入四元复合物(8μg/只),B组注入同体积生理盐水,C组经尾静脉注入四元复合物(8μg/只).给药72 h后,分别以逆转录聚合酶链反应(RT-PCR)和Western blot检测大鼠的肿瘤、心、肝、脾、肺及肾脏组织的HSV1-tk mRNA及蛋白的表达.结果 A组肿瘤组织中HSV1-tk mRNA和蛋白均高表达,而其他组织中则无表达或表达极少;B组未见HSV1-tk mRNA及蛋白的表达;C组肿瘤组织中未见HSV1-tkmRNA和蛋白的表达,肝脏、脾脏、肺脏及肾脏组织中则有少量的表达.RT-PCR半定量检测结果显示,A组卵巢肿瘤组织中的HSV1-tk mRNA含量(1.692±0.221)明显高于C组卵巢肿瘤组织(0.012±0.002;P<0.01).结论 动脉灌注结合GE7导入系统对HSV1-tk有较高的转导率和靶向性,为HSV1-tk/GCV治疗系统在卵巢癌基因治疗的应用提供了新的策略.

Abstract：

Objective To observe the gene and protein expression of herpes simplex virus type Ⅰ thymidine kinase (HSV1-tk) in the ovarian tumor tissues and other organs after arterial infusion of HSV1-tk gene mediated by GE7 delivery system.Methods GE7-polylysine/pCMV-HSV1-tk/polylysine-HA20complexes were constructed.Nine rats with induced ovarian tumor were divided into 3 groups, injecting the 4-element complexes or saline buffer through the ovarian artery and complexes through the tail vein,respectively.The ovarian tumors, hearts, livers, spleens, lungs and kidneys were obtained at 72 hours after injection.RT-PCR and Western Blot were preceeded to determine the expression of HSV1-tk gene and protein in the tumor tissues and other organs.Results In the group of arterial injection with 4-element complexes, the HSV1-tk gene and protein were expressed strongly in the tumor tissues, while little or none was detected in other organs.In the group of arterial injection with saline buffer, no HSV1-tk gene and protein was detected in both tumor tissues and other organs.In the group of tail vein injection, none was detected in tumor tissues and only little was found in the livers, spleens, lungs and kidneys.Conclusion High target and gene transfer rates can be obtained when HSV1-tk gene is transferred via the artery route mediated by GE7 delivery system.HSV1-tk protein can be expressed after the gene transfer.The results may provide a new strategy for target killing of HSV1-tk/GCV system in ovarian tumors.     

肿瘤自杀基因靶向治疗的研究进展

自杀基因治疗是目前肿瘤基因治疗中的研究热点.本文重点从自杀基因的种类、载体、作用机制和发展应用前景等几方面进行综述.其中HSV1-tk/GCV自杀基因系统是研究最多、临床价值最为明确的自杀基因系统之一.治疗机制上除通过直接作用和旁观者效应发挥作用外,还从自杀基因联合使用、趋化因子等细胞因子作用、自杀基因与放射治疗联合作用、自杀基因与免疫治疗联合使用等多方面进行了探索和研究.众多研究结果认为自杀基因治疗是一种具有良好应用前景的肿瘤治疗方法,但在临床应用上还有很多问题需要解决,如将自杀基因安全、高效地转染到人体肿瘤组织和细胞中的方法,如何能使自杀基因在靶组织中稳定表达并发挥作用,各种治疗方法的协同作用等,均有待于我们在今后的研究中进一步解决.     

核医学基因显像在肿瘤研究中的应用进展

基因显像对指导肿瘤的基因治疗有着重要的意义.核医学基因显像是目前基因显像的主要手段,能够对靶基因分布的部位、数量及活性程度进行可视化的定性和定量检测,并在治疗前预测疗效、治疗中监测基因表达、治疗后评价疗效等方面发挥着重要的作用.现综述近年来有关核医学基因显像在肿瘤研究中的相关进展.     

核医学基因显像在肿瘤研究中的应用进展

基因显像对指导肿瘤的基因治疗有着重要的意义。核医学基因显像是目前基因显像的主要手段，能够对靶基因分布的部位、数量及活性程度进行可视化的定性和定量检测，并在治疗前预测疗效、治疗中监测基因表达、治疗后评价疗效等方面发挥着重要的作用。现综述近年来有关核医学基因显像在肿瘤研究中的相关进展。     

HSV1-tk/GCV系统联合Topotecan治疗人卵巢癌的动物实验研究

目的探讨Topotecan能否增强HSV1-tk/GCV自杀基因系统对卵巢癌的体内治疗作用.方法 先用携带tk基因的重组逆转录病毒上清转染人卵巢癌细胞系SKOV-3,用含G418的培养液筛选抗性克隆(命名为SKOV-3/TK).PCR方法检测tk基因整合情况.用SKOV-3细胞建立荷瘤鼠模型作为对照组和Topotecan组.用SKOV-3与SKOV-3/TK细胞按82比例混合细胞建立者为HSV1-tk/GCV组和HSV1-tk/GCV联合Topotecan组;从用药第1天开始每5天测量肿瘤体积一次,至用药结束后一周,绘制肿瘤生长曲线,计算抑瘤率,并取瘤组织做病理学检查.结果与对照组比较,HSV1-TK/GCV组和Topotecan组、联合用药组抑瘤率分别为38.8%、25.3%和89.7%,差异均有显著性,P＜0.01.组间两两比较差异亦有显著性,P＜0.01.病理显示实验组出现不同程度点、片状坏死,以联合用药组为重.结论HSV1-tk/GCV自杀基因系统具有强大的杀伤肿瘤效应及旁观者效应,联合Topotecan化疗将起到协同作用.     

ATRA联合HSV1-TK/GCV系统治疗卵巢癌的实验研究

目的 探讨全反式维甲酸（all-trans retinoic acid,ATRA）能否提高Ⅰ型单纯疱疹病毒胸苷激酶（herpes simplex virus thymidine kinase,HSV1-TK）/丙氧鸟苷（ganciclovir,GCV）系统对卵巢癌的体内治疗作用。方法 用携带tk基因的重组逆转录病毒上清转染人卵巢癌细胞系SKOV-3,用含G418的培养液筛选获得抗性克隆（命名为SKOV-3/TK）。采用PCR方法 检测HSV1-tk基因整合情况。分别用SKOV-3细胞建立皮下移植瘤裸鼠模型作为对照组和ATRA组,用SKOV-3细胞与SKOV-3/TK细胞按8∶2比例混合细胞建立的动物模型作为HSV1-tk/GCV组和HSV1-tk/GCV联合AT-RA组。于接种细胞后15天开始实验用药,从用药第1天开始每5天测量肿瘤体积1次,至用药结束后2周,绘制肿瘤生长曲线,计算抑瘤率,并取瘤组织做病理学检查。结果 与对照组比较,ATRA组、HSV1-tk/GCV组抑瘤率分别为25.5%、38.8%,联合用药组抑瘤率高达67.8%,有统计学意义,P〈0.05。病理结果 显示实验组出现不同程度点、片状坏死,以联合用药组为重。结论 HSV1-TK/GCV系统对卵巢癌有体内杀伤作用;ARTA可以提高HSV1-TK/GCV系统对卵巢癌的体内杀伤作用。     

纳米脂质体在肿瘤治疗及影像监测中的应用

纳米脂质体作为抗癌药物、治疗基因载体,用于肿瘤靶向治疗和基因治疗,较传统治疗方法能获得更好的疗效.同时包载显像剂对肿瘤细胞和肿瘤血管生成等进行靶向显像,可为了解肿瘤对治疗的反应提供依据.     

肿瘤基因治疗中HSV1-TK的靶向性研究

自杀基因HSV1-TK可选择性杀伤表达HSV1-TK的细胞,在肿瘤的基因治疗中尤为重要.HSV1-TK 除了可以引起细胞自杀作用外,还有独特的旁观者效应,已经被美国FDA批准进入Ⅲ期临床试验研究.近几年人们把HSV1-TK的研究重点放在了肿瘤靶向性治疗上,力求提高HSV1-TK的转移效率以及HSV1-TK基因表达的特异性,使抗肿瘤治疗更有效,更安全.     

Imaging transgene expression with radionuclide imaging technologies

A variety of imaging technologies are being investigated as tools for studying gene expression in living subjects. Noninvasive, repetitive and quantitative imaging of gene expression will help both to facilitate human gene therapy trials and to allow for the study of animal models of molecular and cellular therapy. Radionuclide approaches using single photon emission computed tomography (SPECT) and positron emission tomography (PET) are the most mature of the current imaging technologies and offer many advantages for imaging gene expression compared to optical and magnetic resonance imaging (MRI)-based approaches. These advantages include relatively high sensitivity, full quantitative capability (for PET), and the ability to extend small animal assays directly into clinical human applications. We describe a PET scanner (microPET) designed specifically for studies of small animals. We review "marker/reporter gene" imaging approaches using the herpes simplex type 1 virus thymidine kinase (HSV1-tk) and the dopamine type 2 receptor (D2R) genes. We describe and contrast several radiolabeled probes that can be used with the HSV1-tk reporter gene both for SPECT and for PET imaging. We also describe the advantages/disadvantages of each of the assays developed and discuss future animal and human applications.     

Cytoplasmically retargeted HSV1-tk/GFP reporter gene mutants for optimization of noninvasive molecular-genetic imaging

To optimize the sensitivity of imaging HSV1-tk/GFP reporter gene expression, a series of HSV1-tk/GFP mutants was developed with altered nuclear localization and better cellular enzymatic activity, compared to that of the native HSV1-tk/GFP fusion protein (HSV1-tk/GFP). Several modifications of HSV1-tk/GFP reporter gene were performed, including targeted inactivating mutations in the nuclear localization signal (NLS), the addition of a nuclear export signal (NES), a combination of both mutation types, and a truncation of the first 135 bp of the native hsv1-tk coding sequence containing a "cryptic" testicular promoter and the NLS. A recombinant HSV1-tk/GFP protein and a highly sensitive sandwich enzyme-linked immunosorbent assay for HSV1-tk/GFP were developed to quantitate the amount of reporter gene product in different assays to allow normalization of the data. These different mutations resulted in various degrees of nuclear clearance, predominant cytoplasmic distribution, and increased total cellular enzymatic activity of the HSV1-tk/GFP mutants, compared to native HSV1-tk/GFP when expressed at the same levels. This appears to be the result of improved metabolic bioavailability of cytoplasmically retargeted mutant HSV1-tk/GFP enzymes for reaction with the radiolabeled probe (e.g., FIAU). The analysis of enzymatic properties of different HSV1-tk/GFP mutants using FIAU as a substrate revealed no significant differences from that of the native HSV1-tk/GFP. Improved total cellular enzymatic activity of cytoplasmically retargeted HSV1-tk/GFP mutants observed in vitro was confirmed by noninvasive imaging of transduced subcutaneous tumor xenografts bearing these reporters using [(131)I]FIAU and a gamma-camera.     

Enhanced efficiency and specificity of ovarian cancer gene therapy in rats with a novel nonviral gene delivery system (GE7) via intraovarian artery perfusion approach

Transfer of the herpes simplex virus type I-thymidine kinase gene, followed by the administration of ganciclovir (HSV1-tk/GCV) into ovarian cancer-derived cell line either in vitro or transplanted into nude mice has been shown to provide a potential strategy for the gene therapy of ovarian cancer. We investigated the antitumor effects of HSV1-tk/GCV strategy with a chemically induced rat ovarian cancer model and a tumor-selective gene delivery by a novel nonviral gene delivery system (GE7) through the ovarian artery and tail vein. We demonstrated the expression of a reporter gene, beta-gal gene, as well as HSV1-tk gene in tumors and other organs, evaluated the overall antitumor effects after the GCV treatment and analyzed the tumor cell cycle phase distribution. Via the ovarian artery route, the expressions of beta-gal and HSV1-tk in tumors were significantly stronger than those expressed in such organs as the hearts, livers, spleens, lungs and kidneys. However, no beta-gal and HSV1-tk were detected in the tumor tissues when administrated via the tail vein, and little was found in other organs. The cell cycle analysis showed that the total S-phase of tumor cells in the test intra-arterial treatment group was considerably higher than that of the controls. The weight of the tumor tissues in the group treated by the intra-arterial route (4.06+/-2.12 g) was much less than the group treated intravenously (18.25+/-8.34 g) (P<.01). These findings indicated that the administration of GE7/HSV1-tk complex via the ovarian artery route could be a promising avenue of future human ovarian cancer treatment.     

Focused ultrasound enhanced molecular imaging and gene therapy for multifusion reporter gene in glioma-bearing rat model

The ability to monitor the responses of and inhibit the growth of brain tumors during gene therapy has been severely limited due to the blood-brain barrier (BBB). A previous study has demonstrated the feasibility of noninvasive in vivo imaging with ¹²³I-2′-fluoro-2′-deoxy-5-iodo-1-β-D-arabinofuranosyluracil (¹²³I-FIAU) for monitoring herpes simplex virus type 1 thymidine kinase (HSV1-tk) cancer gene expression in an experimental animal model. Here, we tested the enhancement of SPECT with ¹²³I-FIAU and ganciclovir (GCV) treatment in brain tumors after BBB disruption induced by focused ultrasound (FUS) in the presence of microbubbles. We established an orthotopic F98 glioma-bearing rat model with trifusion reporter genes. The results of this study showed that the rat model of HSV1-tk-expressing glioma cells could be successfully detected by SPECT imaging after FUS-induced BBB disruption on day 10 after implantation. Compared to the control group, animals receiving the GCV with or without sonication exhibited a significant antitumor activity (P < 0.05) of glioma cells on day 16 after implantation. Moreover, combining sonication with GCV significantly inhibited tumor growth compared with GCV alone. This study demonstrated that FUS may be used to deliver a wide variety of theranostic agents to the brain for molecular imaging and gene therapy in brain diseases.     

Application of bioluminescence imaging to therapeutic intervention of herpes simplex virus type I – Thymidine kinase/ganciclovir in glioma

Lentiviral vector containing the HSV1-tk and firefly luciferase (fLuc) gene was infected into C6 and C6-TL expressing HSV1-tk and fLuc gene was generated. C6-TL showed higher [¹²⁵I]IVDU uptake than C6. The survival rate of C6-TL decreased more rapidly with increasing GCV dose and was well correlated with fLuc activity. The images of microPET clearly demonstrated higher uptake of [¹⁸F]FHBG into the C6-TL tumor. Inhibition of tumor growth was observed in C6-TL tumor-bearing mice treated with GCV through tumor size measurement and bioluminescence imaging. The therapeutic effect of HSV1-tk/GCV system can be monitored using bioluminescent imaging and tumor size measurement.     

靶向非病毒载体一次及持续介导Ⅰ型单纯疱疹病毒胸腺嘧啶核苷激酶基因转导卵巢癌细胞的比较

Objective:To compare the transferring efficiency and killing effects of one time and continuous mediation with GE7,a non-viral targeted delivery system,in transfection of thymidine kinase gene of herpes simplex virus(HSV-tk)into ovarian cancer cells.Methods:GE7 was used to prepare recombinants with β-galactosidase(β-gal)and HSV1-tk;the recombinants were then used to transfect human ovarian cancer line CaOV3 once and continuously.β-gal staining was used to compare the efficiencies of one time and continuous mediation with GE7 system.Ganciclovior(GCV)was introduced into HSV1-tk transfected ovarian cells.Through drawing the cell growth curve and flow cytometry,the killing effects of GCV on once and continuously GE7/HSV1-tk transfected cells were observed.Results:We found that the one time and continuous exogenous gene transfer efficiencies were about 80% and 85%,respectively When 1 μg/mL GCV was used to treat ovarian cell transfected with HSV1-tk gene,growth inhibiting rates of ovarian cells of one time and continuous transferring were 82% and 90%,respectively;their apoptosis indices were 15 and 30,respectively.Under same GCV concentration.continuous mediation of GE7/pCMV-tk transfection into ovarian cancer cells had more significant inhibitory effect than one time mediation(P＜0.05).Conclusion:Compared with one time mediation,continuous mediation of transfection with GE7 gene delivery system has higher efficiency.Continuous mediation of GE7/HSV1-tk/GCV therapeutic gene system has more powerful killing effect.     

Noninvasive molecular imaging of hypoxia in human xenografts: comparing hypoxia-induced gene expression with endogenous and exogenous hypoxia markers

Tumor hypoxia is important in the development and treatment of human cancers. We have developed a novel xenograft model for studying and imaging of hypoxia-induced gene expression. A hypoxia-inducible dual reporter herpes simplex virus type 1 thymidine kinase and enhanced green fluorescence protein (HSV1-TKeGFP), under the control of hypoxia response element (9HRE), was stably transfected into human colorectal HT29 cancer cells. Selected clones were further enriched by repeated live cell sorting gated for hypoxia-induced eGFP expression. Fluorescent microscopy, fluorescence-activated cell sorting, and radioactive substrate trapping assays showed strong hypoxia-induced expression of eGFP and HSV1-tk enzyme in the HT29-9HRE cells in vitro. Sequential micropositron emission tomography (PET) imaging of tumor-bearing animals, using the hypoxic cell tracer (18)F-FMISO and the reporter substrate (124)I-FIAU, yielded similar tumor hypoxia images for the HT29-9HRE xenograft but not in the parental HT29 tumor. Using autoradiography and IHC, detailed spatial distributions in tumor sections were obtained and compared for the following hypoxia-associated biomarkers in the HT29-9HRE xenograft: (124)I-FIAU, (18)F-FMISO, Hoechst (perfusion), lectin-TRITC (functional blood vessels), eGFP, pimonidazole, EF5, and CA9. Intratumoral distributions of (124)I-FIAU and (18)F-FMISO were similar, and eGFP, pimonidazole, EF5, and CA9 colocalized in the same areas but not in well-perfused regions that were positive for Hoechst and lectin-TRITC. In enabling the detection of hypoxia-induced molecular events and mapping their distribution in vivo with serial noninvasive positron emission tomography imaging, and multiple variable analysis with immunohistochemistry and fluorescence microscopy, this human xenograft model provides a valuable tool for studying tumor hypoxia and in validating existing and future exogenous markers for tumor hypoxia.     

Functional coexpression of HSV-1 thymidine kinase and green fluorescent protein: implications for noninvasive imaging of transgene expression

Current gene therapy technology is limited by the paucity of methodology for determining the location and magnitude of therapeutic transgene expression in vivo. We describe and validate a paradigm for monitoring therapeutic transgene expression by noninvasive imaging of the herpes simplex virus type 1 thymidine kinase (HSV-1-tk) marker gene expression. To test proportional coexpression of therapeutic and marker genes, a model fusion gene comprising green fluorescent protein (gfp) and HSV-1-tk genes was generated (tkgfp gene) and assessed for the functional coexpression of the gene product, TKGFP fusion protein, in rat 9L gliosarcoma, RG2 glioma, and W256 carcinoma cells. Analysis of the TKGFP protein demonstrated that it can serve as a therapeutic gene by rendering tkgfp transduced cells sensitive to ganciclovir or as a screening marker useful for identifying transduced cells by fluorescence microscopy or fluorescence-activated cell sorting (FACS). TK and GFP activities in the TKGFP fusion protein were similar to corresponding wild-type proteins and accumulation of the HSV-1-tk-specific radiolabeled substrate, 2'-fluoro-2'-deoxy-1beta-D-arabinofuranosyl-5-iodo-uracil (FIAU), in stability transduced clones correlated with gfp-fluorescence intensity over a wide range of expression levels. The tkgfp fusion gene itself may be useful in developing novel cancer gene therapy approaches. Valuable information about the efficiency of gene transfer and expression could be obtained by non-invasive imaging of tkgfp expression with FIAU and clinical imaging devices (gamma camera, positron-emission tomography [PET], single photon emission computed tomography [SPECT]), and/or direct visualization of gfp expression in situ by fluorescence microscopy or endoscopy.     

Non-viral targeted delivery system mediates transfection of thymidine kinase gene of herpes simplex virus into ovarian cancer cells： a comparison between one time and continuous mediation

Objective  To compare the transferring efficiency and killing effects of one time and continuous mediation with GE7, a non-viral targeted delivery system, in transfection of thymidine kinase gene of herpes simplex virus (HSV-tk) into ovarian cancer cells. Methods  GE7 was used to prepare recombinants with β-galactosidase (β-gal) and HSV1-tk; the recombinants were then used to transfect human ovarian cancer line CaOV3 once and continuously. β-gal staining was used to compare the efficiencies of one time and continuous mediation with GE7 system. Ganciclovior (GCV) was introduced into HSV1-tk transfected ovarian cells. Through drawing the cell growth curve and flow cytometry, the killing effects of GCV on once and continuously GE7/HSV1-tk transfected cells were observed. Results  We found that the one time and continuous exogenous gene transfer efficiencies were about 80% and 85%, respectively. When 1 μg/mL GCV was used to treat ovarian cell transfected with HSV1-tk gene, growth inhibiting rates of ovarian cells of one time and continuous transferring were 82% and 90%, respectively; their apoptosis indices were 15 and 30, respectively. Under same GCV concentration, continuous mediation of GE7/pCMV-tk transfection into ovarian cancer cells had more significant inhibitory effect than one time mediation (P < 0.05). Conclusion  Compared with one time mediation, continuous mediation of transfection with GE7 gene delivery system has higher efficiency. Continuous mediation of GE7/HSV1-tk/GCV therapeutic gene system has more powerful killing effect. Supported by a grant from the National Natural Sciences Foundation of China (No. 39800144).