间充质干细胞基因治疗肿瘤新进展

骨髓间充质干细胞(MSC)是骨髓内除造血干细胞和多功能前祖细胞外另一种成体干细胞,具有很强的自我更新能力、多向分化潜能.该细胞具有明显的趋向肿瘤移动并与肿瘤组织楔合的生长特性.虽然骨髓间充质干细胞有向肿瘤干细胞转化的风险,但其作为一种克隆载体可以转导治疗基因在体内获得长期稳定表达,对肿瘤的靶向治疗有着广阔的前景.     

Vectors for cancer gene therapy

Many viral and non-viral vector systems have now been developed for gene therapy applications. In this article, the pros and cons of these vector systems are discussed in relation to the different cancer gene therapy strategies. The protocols used in cancer gene therapy can be broadly divided into six categories including gene transfer to explanted cells for use as cell-based cancer vaccines; gene transfer to a small number of tumour cells in situ to achieve a vaccine effect; gene transfer to vascular endothelial cells (VECs) lining the blood vessels of the tumour to interfere with tumour angiogenesis; gene transfer to T lymphocytes to enhance their antitumour effector capability; gene transfer to haemopoietic stem cells (HSCs) to enhance their resistance to cytotoxic drugs and gene transfer to a large number of tumour cells in situ to achieve nonimmune tumour reduction with or without bystander effect. Each of the six strategies makes unique demands on the vector system and these are discussed with reference to currently available vectors. Aspects of vector biology that are in need of further development are discussed in some detail. The final section points to the potential use of replicating viruses as delivery vehicles for efficient in vivo gene transfer to disseminated cancers.     

针对核干细胞因子腺病毒干扰载体构建与干扰作用观察

目的构建针对核干细胞因子(Nucleostemin)基因寂寞的RNA干扰腺病毒载体,观察其干扰作用。方法根据克隆的全长Nucleostemin基因(1 650bp,GenBank接受序号:AY825265)设计RNA干扰反向重复序列,采用分子生物学组装腺病毒载体,制备高效价RNAi重组腺病毒。实验分为Backbone-P1P2重组腺病毒感染组(P1P2干扰组)、Backbone-P3P4重组腺病毒感染组(P3P4干扰组)、Backbone空质粒病毒感染对照组(B组)和无病毒感染对照组。不同剂量腺病毒液RNAi(分别为60、30、15和7.5μL)转染F6细胞及U937细胞,采用蛋白质印迹法检测F6细胞及U937细胞中核干细胞因子的蛋白表达水平,采用实时荧光定量PCR(QPCR)检测U937细胞中核干细胞因子的mRNA表达水平。计数法绘制细胞生长曲线,判定细胞活力;流式细胞术检测细胞周期变化。结果靶向核干细胞因子基因的腺病毒RNA干扰载体(Backbone-P1P2,Backbone-P3P4)被成功构建。蛋白质印迹法检测结果显示,F6细胞内不同梯度浓度腺病毒RNA干扰后,核干细胞因子表达量分别为1.23、4.48、5.03和5.75,随P3P4片段RNA干扰浓度梯度下降而核干细胞因子表达量呈上升趋势。P1P2干扰组蛋白水平为0.219±0.051,与空质粒转染对照组的2.174±0.196比较明显降低,F=279.4,P<0.001;P3P4干扰组蛋白水平为0.0164±0.003,与空质粒转染对照组比较明显降低,F=363.4,P<0.001。P1P2干扰组基因表达水平为1.585±0.20,与空质粒转染对照组的3.932±0.271比较明显降低,F=145.9,P<0.001;P3P4干扰组基因表达水平为1.328±0.251,与空质粒转染对照组比较明显降低,F=149.9,P<0.001。细胞周期实验发现,核干细胞因子干扰后,P1P2腺病毒RNA干扰组为44.89±4.04,与空质粒转染对照组的54.74±3.28对比减低,F=10.75,P=0.031;P3P4干扰组为45.00±3.82,与空质粒转染对照组对比减低,F=11.23,P=0.029;S期+G2M期细胞P1P2干扰组为55.11±2.81,与空质粒转染对照组的45.26±1.72对比升高,F=26.82,P=0.007;P3P4干扰组为55.00±2.64,与空质粒转染对照组相比明显升高,F=28.67,P=0.006。表明核干细胞因子在细胞周期中阻碍G2/M检验点通过。结论成功构建针对核干细胞因子基因沉默的RNA干扰腺病毒载体,该病毒载体可特异有效的沉默下调核干细胞因子基因的表达,为今后研究核干细胞因子功能奠定了基础。     

靶向尿激酶受体uPAR基因的shRNA表达载体的构建及鉴定

背景与目的：尿激酶受体（uPAR）与肿瘤的侵袭和转移密切相关,抑制uPAR的表达,可以抑制肿瘤的转移。本研究构建uPAR基因短发夹RNA（shRNA）的表达质粒,为舌鳞状细胞癌RNA干涉（RNAi）介导的基因治疗打下基础。方法：根据GenBank数据库提供的uPAR基因序列,选择设计合成能转录shRNA的DNA序列,将它们插入真核表达载体pWH1构建重组质粒,用酶切的方法进行鉴定;最后将构建成功的特异性表达载体（pWH1-uPAR）转染至舌鳞状细胞癌,通过RT-PCR、免疫细胞化学和Western blot观察其对舌鳞状细胞癌uPAR mRNA和蛋白水平表达的影响。利用MTT实验检测细胞增殖能力的变化。结果：成功构建了uPAR shRNA表达载体;与Ts细胞和转染空载体pWH1的Ts细胞相比,转染pWH1-uPAR表达载体的Ts细胞uPAR mRNA和蛋白的表达明显降低。MTT结果显示uPAR shRNA对Ts细胞增殖的抑制率为32.9%。结论：设计构建的真核表达载体可以特异性干扰uPAR基因的表达,为进一步研究uPAR基因的功能和舌鳞状细胞癌治疗提供有效的方法和手段。     

肝癌细胞中RhoA小干扰RNA载体的构建与鉴定

 目的 构建靶向RhoA基因的RNA干扰（RNAi）载体。方法 根据人RhoA基因序列设计两条短双链，人工合成后，连接到真核表达载体Pgenesil-1中，构成Pgenesil-1-siRhoA1、Pgene-sil-1-siRhoA2两个重组质粒，转入人肝癌细胞（HEPG2）中，Western blot方法检测RhoA-siRNA对RhoA表达的抑制效果，确定一个重组质粒进行下一步研究。结果 两质粒都有沉默RhoA基因的作用，Pgen-esil-1-siRhoA1重组质粒作用更强烈一些，使用Pgenesil-1-siRhoA1质粒进行下一步研究。结论 Pgen-esil-1-siRhoA重组质粒的成功构建，为研究RhoA与肝细胞癌侵袭转移的关系提供实验模型。     

肺腺癌耐药相关基因BC006151原核表达载体的构建及蛋白表达

背景与目的肺癌细胞对化疗药物的耐受是肺癌患者生存率低的重要原因之一。我们在前期研究中发现了一条肺腺癌耐药相关的基因BC006151,本研究拟构建肺腺癌耐药相关基因的原核表达载体PGEX-4T-1-BC006151,诱导其表达及鉴定目的蛋白,从而揭示该基因与肺腺癌耐药的关系。方法以RNA为模板RT-PCR扩增,产物与质粒PGEX-4T-1用BamHI和EcoRI双酶切,用T4DNA连接酶将二者连接,连接物转化DH5α菌,重组克隆菌经测序鉴定确认。将带有目的片段的重组克隆载体转化BL21表达菌,SDS-PAGE电泳检测表达产物,Western blot检测GST融合蛋白表达。结果经酶切鉴定及DNA测序,重组质粒中已插入了目的基因片段,与GenBank中公布的BC006151基因序列一致。重组克隆载体经BL21表达菌表达,获得40KD的条带(其中目的蛋白13KD,GST标签26KD)。结论成功构建了肺腺癌耐药相关基因BC006151原核表达载体,并成功诱导了目的蛋白表达,GST亲和纯化,为进一步制备该基因的多克隆、单克隆抗体奠定了基础。     

骨肉瘤基因治疗的现状分析

目前,医学界普遍承认治疗骨肉瘤的最佳方法是基因治疗,常用的有抑癌基因治疗、反义基因治疗、自杀基因治疗、免疫基因治疗、联合基因治疗等.但是无论哪种基因治疗方法,都必须具备安全的基因载体.骨肉瘤的基因治疗方法近几年有所突破,在广泛应用的基础上,还应进一步强调基因载体的重要性.     

脑胶质瘤基因治疗载体

基因载体是将治疗基因导入肿瘤细胞的载体,在基因治疗中起关键作用。目前常用的基因治疗载体分为生物载体和非生物载体两大类。生物载体包括病毒载体、细菌载体和干细胞载体。研究显示生物载体虽然靶向性好,转导效率高但存在遗传安全隐患;非生物载体包括脂质体和纳米材料类载体,这类载体制备简单,安全可控但转导效率低。建立安全有效的脑胶质...     

Prodrug cancer gene therapy

There is no effective treatment for late stage and metastatic cancers of colorectal, prostate, pancreatic, breast, glioblastoma and melanoma cancers. Novel treatment modalities are needed for these late stage patients because cytotoxic chemotherapy offers only palliation, usually accompanied with systemic toxicities and poor quality of life. Gene directed enzyme prodrug therapy (GDEPT), which concentrates the cytotoxic effect in the tumor site may be one alternative. This review provides an explanation of the GDEPT principle, focusing on the development, application and potential of various GDEPTs. Current gene therapy limitations are in efficient expression of the therapeutic gene and in tumor-specific targeting. Therefore, the current status of research related to the enhancement of in situ GDEPT delivery and tumor-specific targeting of vectors is assessed. Finally, GDEPT versions of stem cell based gene therapy as another potential treatment modality for progressed tumors and metastases are discussed. Combinations of traditional, targeted, and stem cell directed gene therapy could significantly advance the treatment of cancer.     

Current Status of Retroviral Vector Mediated Gene Transfer into Human Hematopoietic Stem Cells

Genetic modification of hematopoietic stem cells (HSCs) has been proposed as a treatment strategy for a variety of hematologic diseases, tracking marked cells or conferring resistance to chemotherapeutic agents. Despite early enthusiasm, the results of clinical studies involving gene transfer into HSCs have not resulted in therapeutic benefits for the vast majority of treated patients. This review describes the limitations and advances that have been made in the areas of gene transfer vectors, identification of the appropriate HSCs to target for genetic modifications and the methods used to perform gene transfer.     

Current Status of Retroviral Vector Mediated Gene Transfer into Human Hematopoietic Stem Cells

Genetic modification of hematopoietic stem cells (HSCs) has been proposed as a treatment strategy for a variety of hematologic diseases, tracking marked cells or conferring resistance to chemotherapeutic agents. Despite early enthusiasm, the results of clinical studies involving gene transfer into HSCs has not resulted in therapeutic benefits for the vast majority of treated patients. This review describes the limitations and advances that have been made in the areas of gene transfer vectors, identification of the appropriate HSCs to target for genetic modifications and the methods used to perform gene transfer.     

可调控腺病毒载体Ad-RUDsRed构建及其在结肠癌SW620细胞中表达的实验研究

目的:构建带有RU486可调控红色荧光蛋白(DsRed)的复制缺陷型腺病毒Ad-RUDsRed,用于肿瘤基因治疗.方法:构建带有RU486调控系统和DsRed基因的腺病毒穿梭质粒pDC-RUDsRed,筛选阳性克隆,酶切、测序鉴定.利用AdMaxTM系统重组得到腺病毒Ad-RUDsRed,进行扩增、纯化和滴度测定;腺病毒Ad-RUDsRed感染SW620细胞细胞株后分别加入0、1×10-10、1×10-9、1×10-8、1×10-7和1×10-6mol/L的RU486诱导,利用荧光显微镜和流式细胞仪检测Ad-RUDsRed调控功能.结果:PCR结果证实,成功构建可调控腺病毒载体Ad-RUDsRed,病毒滴度3.46×1010pfu/mL.DsRed的表达与RU486剂量相关,无RU486时,报告基因DsRed几乎没有表达.给予诱导剂RU486,腺病毒栽体可以诱导表达红色荧光蛋白,RU486剂量达1×10-6mol/L时DsRed的表达量达到最大值.结论:成功构建携带RU486可调控DsRed的复制缺陷型腺病毒Ad-RUDsRed,通过RU486可以诱导调控DsRed的表达.为肿瘤的基因治疗奠定了基础.     

Functional characterization of hepatoma-specific stem cell antigen-2

Identification of tumor-specific antigens and genetic pathways may lead to potential diagnostic and therapeutic applications in cancer treatment. cDNA microarray has been used in cancer gene profiling, but the broad spectrum of data accruing and narrow signal-to-noise range of this technology have limited its use in rapid identification of highly differentially expressed tumor genes. Here, we used a modified suppression subtractive hybridization (SSH) method to isolate a small number of highly differentially expressed genes from murine hepatoma cells. For functional analysis of these hepatoma-specific genes, we employed the small interference RNA (siRNA)-mediated gene silencing method with lentiviral vectors, which have the advantages of high delivery efficiency and long lasting effect. Stem cell antigen-2 (Sca-2) was identified as one of the highest differentially expressed tumor antigens. Lentiviral siRNA successfully suppressed >90% of Sca-2 expression and the suppression lasted longer than 3 mo. Interestingly, inhibition of Sca-2 induced rapid hepatoma cell apoptosis, and the survival Sca-2-negative hepatoma cells exhibited high sensitivity to extrinsic tumor necrosis factor alpha (TNF-alpha) apoptosis signal but not intrinsic apoptosis signal. Analysis of TNF receptor 1 (TNFR1) by flow cytometry and Western blotting indicated that Sca-2 expression downregulated cell surface but not de novo synthesis of TNFR1 in the hepatoma cells. Together, our results suggested that Sca-2 was a signal transducer situated at the nexus of surface molecules regulating death receptor-mediated apoptosis. The technology illustrated that this method can deduce a small number of highly differentially expressed tumor genes that may have diagnostic and therapeutic potential.     

Adenovirus-mediated transfer of tris-shRNAs induced apoptosis of nasopharyngeal carcinoma cell in vitro and in vivo

RNA interference (RNAi) is an evolutionary conserved mechanism for specific gene silencing. There are currently numerous cancer therapy clinical trials based on RNAi technology. Using an adenoviral system as a delivery mediator of RNAi, we investigated the therapeutic effects of targeting three genes simultaneously in vitro and in vivo. In this study, we constructed an recombinant adenoviral shRNA expression system as Adv-pEGFP-shVEGF-shTERT-shBcl-xl for multi-genes silencing. Our results showed that the adenoviral vector can achieve above 90% of transfection efficiency and induced obvious apoptosis in CNE-2 cell both in vitro and in vivo compared with targeting the TERT alone or controlled group.     

Immunotherapy for neuroblastoma using syngeneic fibroblasts transfected with IL-2 and IL-12

Cytokine-modified tumour cells have been used in clinical trials for immunotherapy of neuroblastoma, but primary tumour cells from surgical biopsies are difficult to culture. Autologous fibroblasts, however, are straightforward to manipulate in culture and easy to transfect using nonviral or viral vectors. Here we have compared the antitumour effect of fibroblasts and tumour cells transfected ex vivo to coexpress interleukin-2 (IL-2) and IL-12 in a syngeneic mouse model of neuroblastoma. Coinjection of cytokine-modified fibroblasts with Neuro-2A tumour cells abolished their in vivo tumorigenicity. Treatment of established tumours with three intratumoral doses of transfected fibroblasts showed a significant therapeutic effect with reduced growth or complete eradication of tumours in 90% of mice, associated with extensive leukocyte infiltration. Splenocytes recovered from vaccinated mice showed enhanced IL-2 production following Neuro-2A coculture, and increased cytotoxicity against Neuro-2A targets compared with controls. Furthermore, 100% of the tumour-free mice exhibited immune memory against tumour cells when rechallenged three months later. The potency of transfected fibroblasts was equivalent to that of tumour cells in all experiments. We conclude that syngeneic fibroblasts cotransfected with IL-2 and IL-12 mediate therapeutic effects against established disease, and are capable of generating immunological memory. Furthermore, as they are easier to recover and manipulate than autologous tumour cells, fibroblasts provide an attractive alternative immunotherapeutic strategy for the treatment of neuroblastoma.