融合基因GM-CSF-BZLF1重组腺病毒表达载体的构建

目的构建粒细胞-巨噬细胞集落刺激因子(GM-CSF)和EB病毒即刻早期基因(BZLF1)融合基因的重组腺病毒表达载体。方法采用逆转录-聚合酶链反应分别获得GM-CSF和BZLF1编码序列的cDNA,应用剪接式重叠延伸(SOE)技术将两段基因通过多肽接头(Gly4Ser)3的DNA序列进行连接,构建融合基因GM-CSF-BZLF1。将融合基因GM-CSF-BZLF1定向亚克隆至pAdTrack-CMV质粒,在原核细胞E.coliBJ5183中完成穿梭质粒与骨架质粒pAdEasy-1的同源重组,构建融合基因GM-CSF-BZLF1真核表达载体pAd-GM-CSF-BZLF1。将真核表达载体pAd-GM-CSF-BZLF1转染293细胞,获得复制缺陷型重组腺病毒vAd-GM-CSF-BZLF1。RT-PCR鉴定感染重组腺病毒的293细胞中GM-CSF-BZLF1基因的表达。结果 GM-CSF-BZLF1基因插入重组腺病毒表达载体的预期位置,且插入序列完全正确;感染重组腺病毒vAd-GM-CSF-BZLF1的293细胞中检测到融合基因GM-CSF-BZLF1的转录表达。结论成功地构建了融合基因GM-CSF-BZLF1重组腺病毒表达载体,为进一步探讨GM-CSF-BZLF1的功能提供了理论基础和实验依据。     

LHR真核表达载体构建及表达

目的构建黄体生成素受体（LHR）真核表达质粒载体和建立稳定高表达LHR的乳腺癌细胞株。方法KpnI／Hpa工双酶切LHR-cDNA质粒载体获得目的基因,定向克隆构建pcDNA3．1（4-）真核表达载体,酶切图谱和序列分析鉴定;重组质粒载体经脂质体转染MCF-7乳腺癌细胞,G418筛选,RT-PCR、细胞内cAMP测定,筛选鉴定高表达LHRMCF-7细胞。结果重组质粒pcDNA3．1（4-）-LHR酶切图谱分析结果、序列分析证明pcDNA3．1（4-）-LHR载体中LHR序列与GenBank的LHR基因序列完全相符。RT-PCR检测MCF-7细胞LHRmRNA,MCF-7细胞内cAMP浓度测定,证实MCF-7细胞高表达LHR。结论构建并转染pcDNA3．1（4-）-LHR真核表达载体,在MCF-7细胞中稳定表达,为探讨hCG对乳腺癌细胞的作用提供实验基础。     

Efficient and non-toxic gene transfer to cardiomyocytes using novel generation amplicon vectors derived from HSV-1

OBJECTIVE: The feasibility of gene transfer to myocardial tissue using viral vectors was investigated over the last few years. In this study we report gene transfer using a recently described improved of Herpes simplex virus (HSV-1)-derived amplicon vectors and demonstrate that these vectors are a powerful and potentially very interesting tool for gene transfer into neonatal primary as well as in adult cardiac myocytes. METHODS AND RESULTS: Non-pathogenic HSV-1 amplicon vectors simultaneously expressing GFP and LacZ were constructed using a novel helper system that yields essentially helper-free vector particles. These vectors were used to infect either cultured primary neonatal rat cardiomyocytes or adult cardiac tissue. Transgenic expression was quantified using a FACS (GFP) or X-gal staining (LacZ). Infection of primary cardiomyocytes showed efficient transduction even at very low multiplicity of infection (MOI), and expression increased with the infectious dose. By investigating release of lactate dehydrogenase (LDH) or spontaneous beating of the cells, we failed to detect cytotoxic effects in cardiomyocytes infected at high MOI. Thin slices of adult cardiac tissue placed in medium containing vectors also showed very good levels of transduction, without any evidence of toxic effects. CONCLUSIONS: Helper-free amplicon vectors very efficiently transduce genes into cardiomyocytes. Our results indicate similar or better transduction efficiencies than those reported using other vector systems. Furthermore, the very high transgenic capacity of amplicon vectors (up to 150 kbp) makes these vectors a unique and very suitable system to transduce large genomic sequences into cardiomyocytes.     

Treatment of acetaminophen-induced acute liver failure in the mouse with conditionally immortalized human hepatocytes

BACKGROUND/AIMS: Liver failure is a life threatening condition currently treated by palliative measures and, when applicable, organ transplantation. The use of a bioartificial organ capable of fulfilling the main functions of the liver would represent an attractive alternative. However, the shortage of suitable donor cells, and their limited growth ability have impeded the development of this strategy. We investigated whether lentiviral vectors allow for conditional immortalization of human hepatocytes and whether these immortalized hepatocytes could reverse lethal acute liver failure. METHODS: We exposed primary human hepatocytes to Cre-excisable lentiviral vectors coding for SV40T Antigen, telomerase, and/or Bmi-1 and tested the functionality of the resulting cell lines. Therapeutic potential of immortalized hepatocytes were tested in a murine model of acetaminophen-induced hepatic injury. RESULTS: The immortalized hepatocytes grew continuously yet were non-tumorigenic, stopped proliferating when exposed to Cre recombinase, and conserved defining properties of primary hepatocytes, including the ability to secrete liver-specific proteins and to detoxify drugs. The implantation of encapsulated immortalized human hepatocytes rescued mice from lethal doses of acetaminophen. CONCLUSIONS: Lentiviral vectors represent tools of choice for immortalization of non-dividing primary cells, and lentivirally immortalized human hepatocytes are promising reagents for cell-based therapy of acute liver failure.     

逆转录病毒载体介导IL 6基因转导成纤维细胞的表达

目的将ＩＬ６基因转导至成纤维细胞ＮＩＨ３Ｔ３，并使转染株有效地表达ＩＬ６，为ＩＬ６转基因治疗奠定基础．方法利用重组载体构建技术将质粒ｐＵＣＩＬ６ｃＤＮＡ的目的片段连接于逆转录病毒载体上，并以脂质体介导的方法将重组载体转染包装细胞ＰＡ３１７，以Ｇ４１８筛选克隆细胞，浓缩克隆细胞上清以制备重组病毒液，继之感染ＮＩＨ３Ｔ３细胞后，进行Ｓｏｕｔｈｅｒｎｂｌｏｔ和Ｎｏｒｔｈｅｒｎｂｌｏｔ分析，检测目的基因在靶细胞的整合与转录水平．结果成功地构建了重组载体ｐＺＩＰＩＬ６ｃＤＮＡ，筛选出抗生较强的克隆细胞，制备了高滴度的重组病毒液．杂交结果表明转导株３Ｔ３ＩＬ６具有ＩＬ６基因的整合和相应ｍＲＮＡ的高表达．结论ＩＬ６基因能稳定整合至靶细胞并进行有效的转录表达，为ＩＬ６基因治疗的应用奠定了可靠的基础     

Comparison of gene therapy strategies for treatment of liver diseases

Abstract   Liver-directed gene therapy can be aimed at replacing a missing gene product, overexpressing or ectopically expressing a gene product in the liver, generating proteins that are normally not produced in the liver (e.g. hormones, vaccines), down-regulating specific gene expression and targeted repair of genetic mutations. A common critical requirement for achieving these goals is the availability of efficient methods for transferring DNA and RNA into target organ in vivo . Both recombinant viruses and non-viral vectors are being explored for transferring nucleic acids to cells in vitro and in vivo . This review compares the characteristics of these vectors in the context of their potential application in liver-directed gene therapy for various inherited or acquired disorders.     

Mechanism of autoimmune hepatic fibrogenesis induced by an adenovirus encoding the human liver autoantigen cytochrome P450 2D6

Autoimmune hepatitis type 2 (AIH-2) is a severe autoimmune liver disease with unknown etiology. We recently developed the CYP2D6 mouse model for AIH-2, in which mice are challenged with an adenovirus (Ad-2D6) expressing human cytochrome P450 2D6 (hCYP2D6), the major autoantigen in AIH-2. Such mice develop chronic hepatitis with cellular infiltrations and generation of hCYP2D6-specific antibodies and T cells. Importantly, the CYP2D6 model represents the only model displaying chronic fibrosis allowing for a detailed investigation of the mechanisms of chronic autoimmune-mediated liver fibrogenesis. We found that hCYP2D6-dependent chronic activation of hepatic stellate cells (HSC) resulted in an increased extracellular matrix deposition and elevated expression of α-smooth muscle actin predominantly in and underneath the liver capsule. The route of Ad-2D6 infection dramatically influenced the activation and trafficking of inflammatory monocytes, NK cells and hCYP2D6-specific T cells. Intraperitoneal Ad-2D6 infection caused subcapsular fibrosis and persistent clustering of inflammatory monocytes. In contrast, intravenous infection caused an accumulation of hCYP2D6-specific CD4 T cells throughout the liver parenchyma and induced a strong NK cell response preventing chronic HSC activation and fibrosis. In summary, we found that the location of the initial site of inflammation and autoantigen expression caused a differential cellular trafficking and activation and thereby determined the outcome of AIH-2-like hepatic damage and fibrosis.     

非病毒类载体靶向治疗前列腺癌研究进展

在治疗前列腺癌的各种传统方法中,普遍存在着杀伤癌细胞的同时对正常组织器官造成损伤的现象。近年来兴起的靶向治疗在一定程度上能很好地解决这一问题。但作为靶向治疗的载体,病毒类载体存在免疫原性大、毒性大等缺点,而非病毒类载体靶向治疗前列腺癌往往能克服这些缺点。本文综述了以阳离子聚合物、脂质体和壳聚糖聚合物为主的非病毒类载体包裹基因药物靶向治疗前列腺癌的研究进展。     

非病毒靶向载体递送小干扰RNA的研究进展

近年来由小干扰RNA(siRNA)所介导的RNA干扰(RNAi)技术发展迅速,如何高效、安全地将siRNA转运至靶组织是决定这一技术应用前景的关键问题。本文重点围绕构建siRNA的非病毒靶向载体递送系统的研究进展进行综述。     

Preventing HIV-1 Tat-induced neuronal apoptosis using antioxidant enzymes: mechanistic and therapeutic implications

HIV-1 proteins, especially gp120 and Tat, elicit reactive oxygen species (ROS) and cause neuron apoptosis. We used antioxidant enzymes, Cu/Zn superoxide dismutase (SOD1) and glutathione peroxidase (GPx1) to study signaling and neuroprotection from Tat-induced apoptosis. SOD1 converts superoxide to peroxide; GPx1 converts peroxide to water. Primary human neurons were transduced with SV40-derived vectors carrying SOD1 and GPx1, then HIV-1 Tat protein was added. Both SV(SOD1) and SV(GPx1) delivered substantial transgene expression. Tat decreased endogenous cellular, but not transduced, SOD1 and GPx1. Tat rapidly increased neuron [Ca(2+)](i), which effect was not altered by SV(SOD1) or SV(GPx1). However, both vectors together blocked Tat-induced [Ca(2+)](i) fluxes. Similarly, neither SV(SOD1) nor SV(GPx1) protected neurons from Tat-induced apoptosis, but both vectors together did. Tat therefore activates multiple signaling pathways, in one of which superoxide acts as an intermediate while the other utilizes peroxide. Gene delivery to protect neurons from Tat must therefore target both.