靶向表达的逆转录病毒载体LN.ALBTRS.TK的构建

目的:构建出在肝细胞中具靶向表达潜能的逆转录 TK 基因载体。方法:用 p2335A-1中的一段2.0Kb的人白蛋白组织特异性转录调节序列(ALBTRS)取代 pSTK 中的 SV40启动子,所构建的载体命名为 LN.ALB-TRS.TK。结果:载体 LN.ALBTRS.TK 的结构中,含有 ALBTRS 启动子,具有在肝细胞中特异表达白蛋白的潜能,载体经酶切鉴定表明结构符合要求。结论:成功地构建出具靶向表达潜能的逆转录载体 LN.ALBTRS.TK。     

靶向表达的逆转录病毒载体LN．ALBTRS．TK的构建

目的 构建出在肝细胞中具靶向表达潜艇的逆转录TK基因载体。方法 用p2335A-1中的一段2.0kb的人白蛋白组织特异性转录调节序列（ALBTRS）取代pSTK中的SV40启动子，所构建的载体命名为LN.ALBTRS.TK。结果 载体LN.ALBTRS.TK的结构中，含有ALBTRS启动子，具有在表达白蛋白的肝细胞中特异表达的潜能，载全经酶切鉴定表明结构符合要求。结论 成功地构建出具靶向表达潜能的逆转录载体LN.ALBTRS.TK。     

靶向表达的逆转录病毒载体LN．ALBTRS．TK的构建

目的：构建出在肝细胞中具靶向表达潜能的逆转录TK基因载体。方法：用p2335A-l中的一段2．0Kb的人白蛋白组织特异性转录谓节序列(ALBTRS)取代pSTK中的SV40启动子，所构建的载体命名为LN．ALB-TRS．TK。结果：载体LN．ALBTRS．TK的结构中。含有ALBTRS启动子，具有在肝细胞中特异表达白蛋白的潜能，载体经酶切鉴定表明结构符合要求。结论；成功地构建出具靶向表达潜能的逆转录载体LN．ALBTRS．TK。     

逆转录病毒载体LN．ALBTRS．TK体外靶向表达研究

逆转录HSV—TK载体用于肝癌的转基因治疗已在动物实验证明有良好效果．但目前尚无满意的靶向表达载体可供使用,本研究采用PCR、Southern blot及Northern blot等方法．对本室构建的逆转录载体LN．ALBTRS．TK在体外对多种靶细胞进行转染和表达的检测．结果表明：LN．ALBTRS．TK能转染多种肿瘤细胞．包括肝癌细胞及非肝癌细胞．并在表达白蛋白的肝癌细胞中可检测到mRNA的转录和HSV—TK的表达产物,而在不表达白蛋白的非肝癌细胞中无HSV—TK的mRNA转录和HSV—TK的蛋白产物表达。这一结果表明：LN．ALBTRS．TK逆转录载体能正确的在期望的细胞中靶向表达．具备一定的组织特异性。     

体外垂体生长激素腺瘤转录靶向性基因治疗的实验研究

目的评价人生长激素启动子调控的基因治疗系统对垂体生长激素腺瘤的靶向性治疗作用。方法构建生长激素启动子调控的基因系统，体外转染垂体生长激素腺瘤GH3细胞；观察治疗基因HSV—TK在细胞中的表达靶向性，以及该系统杀伤细胞的能力和杀伤靶向性。结果HSV—TK蛋白能够在GH3细胞中靶向性表达，予以更昔洛韦后，GH3细胞增殖速度明显减慢，而对照细胞无明显杀伤作用（P〈0．01）。结论生长激素启动子调控的基因治疗系统能有效地、靶向性地抑制GH3细胞增殖。     

去唾液酸糖蛋白-人端粒酶逆转录酶-胸苷激酶/丙氧鸟苷的靶向促HepG2细胞凋亡作用及旁观者效应

目的 观察去唾液酸糖蛋白(AF)-pGL3-人端粒酶逆转录酶(hTERT)-胸苷激酶(TK)对肝癌细胞株HepG2细胞生长及凋亡的影响.方法 培养细胞并构建pGL3-hTERT-TK质粒、AF脂质体复合物后,转染HepG2细胞和L02细胞,通过单光子液闪计数仪,脱氧核糖核苷酸末端转移酶介导的缺口末端标记法和流式细胞仪观察自杀基因对肝癌细胞生长和凋亡的影响,以及其对自杀基因的旁观者效应.结果 在肝癌细胞HepG2中,TK基因可以被hTERT启动子驱动高效的表达,而不影响正常肝细胞L02的生长,AF通过识别去唾液酸糖蛋白受体结合到HepG2细胞表面,其携带的TK基因更易进入肝癌细胞,同时增强自杀基因TK的高效表达,在旁观者效应机制的参与下,肝癌细胞总的凋亡率达85%±3%,而正常肝细胞则仅为16%±2%.结论 AF-pGL3-hTERT-TK可以靶向攻击肝癌细胞,对正常肝细胞几乎无影响,其基因传递系统具备靶向治疗肝癌的潜力.     

多药耐药基因启动子高表达载体的构建与鉴定

目的克隆多药耐药基因(MDR1)启动子,构建并鉴定真核表达载体pcDNA3-MDR1启动子.方法采用PCR方法从白血病多药耐药K562-AO2细胞中扩增MDR1 启动子,克隆入T载体,酶切后与pcDNA3连接,电泳及DNA测序进行鉴定.结果 PCR克隆出 MDR1启动子,成功克隆入T载体,DNA测序证实序列正确,通过连接成功构建含正确目的基因的表达载体pcDNA3 -MDR1启动子.结论成功构建了MDR1启动子高表达载体,为靶向治疗耐药肿瘤构建载体提供了实验基础.     

不同启动子驱动HDV核酶的逆转录病毒载体构建

目的分别构建CMV、H1、tRNA、U2、U3和U6启动子驱动反式丁型肝炎病毒核酶（HDV核酶）的逆转录病毒表达载体。在这些载体中,HDV核酶设计为靶向HBV基因序列PreS2和C区。方法用PCR技术分别扩增CMV、U2和U3启动子,并连入pMD18-T载体。合成靶向PreS2和C区HDV核酶并利用SalⅠ和H indⅢ的酶切位点分别连入逆转录病毒表达载体pLEGFP（pLEGFP-R z）。然后利用BamHⅠ和SalⅠ的酶切位点分别把CMV、H1、tRNA、U2、U3和U6启动子连入重组载体pLEGFP-R z。所有的重组载体经PCR和酶切的方法验证。结果成功地构建了分别含有6种启动子驱动HDV核酶的逆转录病毒载体。结论这些重组载体的构建为筛选高效表达核酶的启动子奠定基础。同时这些重组载体也可用于进一步研究HDV核酶抑制HBV复制的效率。     

肺癌的自杀基因放射导向治疗实验研究

目的：利用放射敏感性调控序列诱导单纯疱疹病毒胸苷激酶（HSV－TK）基因的肿瘤靶向表达，以提高肺癌自杀基因治疗的选择性和有效性。方法：利用基因重组构建放射可调控的HSV－TK表达载体；转染肺癌A549细胞系，观察γ线照射后细胞HSV－TK表达，以及丙氧鸟苷（GCV）作用下细胞相对存活率的变化。利用裸鼠肺癌模型观察不同处理后的抑瘤效应。结果：γ线可诱导HSV－TK在被转染肺癌细胞表达显增强，呈剂量依赖性；经放射诱导后，转染细胞对GCV的敏感性明显升高（P＜0．05），细胞生工活性显受抑；放射联合GCV可明显抑制感染tgEgr-HyTK的裸鼠移植瘤，并可使50％的肿瘤完全消退。结论：由辐射敏感性启动子诱导的自杀基因肿瘤靶向性表达，是一种高产、安全的肺癌基因治疗新策略。     

肝细胞特异性表达Cre重组酶转基因小鼠的建立

目的 构建在小鼠肝细胞中特异表达Cre重组酶的转基因小鼠。方法 利用聚合酶链反应(PCR)获得小鼠肝细胞特异性启动子——白蛋白启动子，指导Cre重组酶在肝细胞中特异表达。通过受精卵显微注射的方法，将转基因载体导入小鼠受精卯中，获得转基因小鼠。利用逆转录聚合酶链反应(RT～PCR)检测转基因小鼠中Cre重组酶转录的组织特异性，并将转基因小鼠与Smad4条件基因打靶小鼠进行杂交，利用PCR和Southern Blot进一步检测Cre重组酶在小鼠体内表达的组织特异性及其介导lox P位点之间发生重组的活性。结果 获得了白蛋白启动子，构建了转基因载体pAlb-Cre。将转基因载体进行显微注射，共注射了837枚小鼠受精卵。PCR检测显示53只子代小鼠中有7只整合了Cre重组酶基因，Southern杂交结果证实共获得6只基因组上整合Cre重组酶基因的首建者小鼠。RT—PCR结果表明其中3只阳性小鼠的子代鼠在肝脏和睾丸中正确转录了外源基因。将在肝脏和睾丸中正确转录了外源基因的两只阳性鼠与Smad4条件基因打靶的小鼠杂交，通过PCR检测发现Cre转基因与Smad4条件基因打靶双阳性的子代鼠在肝脏中特异表达Cre重组酶，并能介导基因组中loxP序列间的基因重组，此结果通过Southern杂交得到了进一步证实。结论 成功构建了在小鼠肝细胞中特异表达Cre重组酶的转基因小鼠，为利用条件基因打靶技术研究基因在肝脏发育及相关疾病中的功能及机制奠定了基础。     

Plasmoviruses: nonviral/viral vectors for gene therapy.

We have generated a chimeric gene transfer vector that combines the simplicity of plasmids with the infectivity and long-term expression of retroviruses. We replaced the env gene of a Moloney murine leukemia virus-derived provirus by a foreign gene, generating a plasmid that upon transfer to tumor cells generates noninfectious retroviral particles carrying the transgene. We added to this plasmid an independent expression cassette comprising a cytomegalovirus promoter, an amphotropic retroviral envelope, and a polyadenylylation signal from simian virus 40. These constructs were designed to minimize the risk of recombination generating replication-competent retroviruses. Their only region of homology is a 157-bp sequence with 53% identity. We show that the sole transfection of this plasmid in various cell lines generates infectious but defective retroviral particles capable of efficiently infecting and expressing the transgene. The formation of infectious particles allows the transgene propagation in vitro. Eight days after transfection in vitro, the proportion of cells expressing the transgene is increased by 10-60 times. There was no evidence of replication-competent retrovirus generation in these experiments. The intratumoral injection of this plasmid, but not of the control vector lacking the env gene, led to foci of transgene-expressing cells, suggesting that the transgene had propagated in situ. Altogether, these "plasmoviruses" combine advantages of viral and non-viral vectors. They should be easy to produce in large quantity as clinical grade materials and should allow efficient and safe in situ targeting of tumor cells.     

Midkine启动子调控的HSV-TK基因重组腺病毒的构建

目的构建以人midkine（MK）启动子调控的TK基因的重组复制缺陷型腺病毒。方法以Adeno-XTM表达试剂盒为基础，应用分子克隆技术，将穿梭质粒pShuttle的CMV启动子替换为MK启动子，并将由pHSV-106获取HSV-TK基因的编码序列亚克隆至其下游，酶切鉴定阳性重组穿梭质粒pShuttle-MK-TK。通过I-CeuⅠ和PI-SceⅠ两个稀有酶切位点．将目的基因与腺病毒质粒DNA（pAdeno-X）进行体外连接，获得含目的基因的重组腺病毒质粒DNA，后者经限制性内切酶PacⅠ切割后，两端露出反向末端重复序列（ITR），利用脂质体转染293细胞，获得含有目的基因重组腺病毒上清，PCR检测。结果酶切结果显示．MK启动子与TK基因均正向插入pShuttle中．TK位于MK启动子的下游。PCR检测显示重组腺病毒中含有MK启动子及TK基因片段。结论体外连接法可成功构建以人MK启动子调控的TK基因的重组腺病毒。     

逆转录病毒介导HSV-TK基因治疗胰腺癌——实验研究

目的:探讨应用逆转录病毒载体介导HSV-TK基因治疗实验性人胰腺癌细胞系8988的价值。方法:HSV-TK被定向克隆入逆转录病毒载体pMNSM的SV_(40) 下游。重组逆转录病毒载体pMNS-TK-M转染至逆转录病毒包装细胞PA317细胞,产生的重组病毒将HSV-TK转入人胰腺癌细胞系8988细胞内。结果:Southern-blot试验及药敏试验均证实HSV-TK基因已整合至细胞DNA中并完全表达。体外试验证实,HSV-TK阳性8988细胞对ACV的敏感性较母细胞明显为高;裸鼠移植瘤试验证实,腹腔注射ACV有明显阻止移植瘤的形成以及对移植瘤的治疗作用。结论:HSV-TK/ACV有体内治疗胰腺癌的作用,可作为胰腺癌基因治疗的潜在方法之一。     

Gene therapy for the treatment of hepatoma by retroviral-mediated gene transfer of the herpes simplex virus thymidine kinase

We previously demonstrated that a foreign gene transferred by means of a retroviral vector can be expressed selectively in hepatoma cells when a liver-specific promoter was used to direct its expression. We now describe an approach for the treatment of hepatoma by the introduction of herpes simplex virus thymidine kinase (HSV-TK) gene. Expression of HSV TK gene in hepatoma cells was evaluated as an elimination system for a potential use in therapies. A murine retroviral vector was constructed in which the HSV-TK gene was expressed under control of the murine albumin enhancer and promoter elements. Replication-defective vector viral particles were obtained by transfer of the vector DNA into the ecotropic packaging cell line psi2 and were used to infect murine hepatoma cells. The introduction of the HSV-TK gene into hepatoma cells by infection of the recombinant retrovirus did not affect their proliferation at all. The sensitivity of those infected cells to the toxic effects of the nucleoside analog ganciclovir was found to be significantly increased by transfer of the HSV-TK gene. The difference in sensitivity between infected and uninfected cells to ganciclovir concentrations should give the utility for a clinical application indicating the feasibility of gene therapy toward hepatoma by the retroviral-mediated HSV-TK gene transfer.     

Liver-directed gene therapy: quantitative evaluation of promoter elements by using in vivo retroviral transduction.

Liver-directed gene therapy will be applicable tomany inherited diseases. Although various protocols have been devised for invivo delivery of retrovirus, comparison of hepatocyte transduction frequencieshas been difficult due to variations in retroviral titer and a paucity of DNAdata. We have previously reported an in vivo rat hepatocyte transductiontechnique which involves 70% hepatectomy followed 24 hr later by portal veininjection of retrovirus during hepatic in-flow occlusion. In this study, weemployed this method and concentrated retroviral preparations to achievetransduction of up to 15% of hepatocytes as determined by a quantitative PCRassay. As an initial step toward identifying promoters which lead to high-levellong-term expression of retroviral transduced genes, we used our in vivodelivery system to compare the Moloney murine leukemia virus long terminalrepeat (LTR) promoter with the promoter for the large subunit of murine RNApolymerase II (Pol-II). Human alpha 1-antitrypsin (hAAT) was used as thereporter gene to facilitate long-term analysis of expression. Serum hAAT levelswere higher for the Pol-II promoter (143 ng/ml) than for the LTR promoter (50ng/ml). This difference was consistent with the higher transduction frequencyobserved for the Pol-II-hAAT vector. Although serum hAAT expression wassustained for up to 1 year in six of eight Pol-II-hAAT-transduced rats and threeof five LTR-hAAT-transduced rats and was proportional to hAAT mRNA level andproviral DNA frequency, in vivo expression was significantly lower than intransduced tissue culture cells. We conclude that a high frequency of in vivotransduction can be achieved by using retroviral vectors and our rapidtransduction protocol, but transduced gene expression remains a serious problem.The quantitative assays described herein will facilitate in vivo comparisons ofgene regulatory elements.     

Brain-specific expression of an exogenous gene after i.v. administration

The treatment of brain diseases with gene therapy requires the gene to be expressed throughout the central nervous system, and this is possible by using gene targeting technology that delivers the gene across the blood-brain barrier after i.v. administration of a nonviral formulation of the gene. The plasmid DNA is targeted to brain with pegylated immunoliposomes (PILs) using a targeting ligand such as a peptidomimetic mAb, which binds to a transporting receptor on the blood-brain barrier. The present studies adapt the PIL gene targeting technology to the mouse by using the rat 8D3 mAb to the mouse transferrin receptor. Tissue-specific expression in brain and peripheral organs of different exogenous genes (beta-galactosidase, luciferase) is examined at 1-3 days after i.v. injection in adult mice of the exogenous gene packaged in the interior of 8D3-PIL. The expression plasmid is driven either by a broadly expressed promoter, simian virus 40, or by a brain-specific promoter taken from the 5' flanking sequence of the human glial fibrillary acidic protein (GFAP) gene. The transgene is expressed in both brain and peripheral tissues when the simian virus 40 promoter is used, but the expression of the exogenous gene is confined to the brain when the transgene is under the influence of the brain-specific GFAP promoter. Confocal microscopy colocalizes immunoreactive bacterial beta-galactosidase with immunoreactive GFAP in brain astrocytes. These studies indicate that tissue-specific gene expression in brain is possible after the i.v. administration of a nonviral vector with the combined use of gene targeting technology and tissue-specific gene promoters.     

TK gene combined with mIL-2 and mGM-CSF genes in treatment of gastric cancer

AIM: Cancer gene therapy has received more and more attentions in the recent decade. Various systems of gene therapy for cancer have been developed. One of the most promising choices is the suicide gene. The product of thymidine kinase (TK) gene can convert ganciclovir (GCV) to phosphorylated GCV, which inhibits the synthesis of cell DNA, and then induces the cells to death. Cytokines play an important role in anti-tumor immunity. This experiment was designed to combine the TK gene and mIL-2/mGM-CSF genes to treat gastric cancer, and was expected to produce a marked anti-tumor effect. METHODS: TK gene was constructed into the retroviral vector pLxSN, and the mIL-2 and mGM-CSF genes were inserted into the eukaryotic expressing vector pIRES. The gastric cancer cells were transfected by retroviral serum that was harvested from the package cells. In vitro study, the transfected gastric cancer cells were maintained in the GCV- contained medium, to assay the cell killing effect and bystander effect. In vivo experiment, retroviral serum and cytokines plasmid were transfected into tumor-bearing mice, to observe the changes of tumor volumes and survival of the mice. RESULTS: In vitro experiment, 20 % TK gene transduced cells could cause 70-80 % of total cells to death. In vivo results showed that there was no treatment effect in control group and TK/GCV could inhibit the tumor growth. The strongest anti-tumor effect was shown in TK+mIL-2+mGM-CSF group. The pathologic examination showed necrosis of the cancer in the treated groups. CONCLUSION: TK/GCV can kill tumor cells and inhibit the tumor growth in vivo. IL-2 and GM-CSF strongly enhance the anti-tumor effect. Through the retrovirus and liposome methods, the suicide gene and cytokine genes are all expressed in the tissues.     

载体法筛选乙型肝炎病毒S基因小干扰RNA靶位体系的建立

目的 构建4个针对乙型肝炎病毒(HBV)S基因序列的小发央RNA(shRNA)表达载体，作用于增强型绿色荧光蛋白(EGFP)和HBV S基因融合表达载体，观察shRNA对融合蛋白的抑制作用，筛选出有效靶位。方法 利用pAVU6 27质粒，设计并掏建4个shRNA表达载体，与融合表达载体共转染AD293细胞，荧光显做镜下观察融合蛋白的荧光表达情况并通过流式细胞仪分析shRNA对融合蛋白的抑制作用。同时，逆转录聚合酶链反应法验证其筛选的有效性。结果 成功掏建shRNA表达载体和HBs-EGFP融合表达载体；4组shRNA表达载体均可不同程度抑制融合基因的表达，其中以579位点最有效，荧光表达抑制率为69．8％，RNA水平抑制率为74．6％。结论 筛选出有效抑制HBV S基因的siRNA靶位。