携带人TRAIL基因的双靶向溶瘤腺病毒与X线联合抑瘤效应

目的探讨携带人TRAIL基因的溶瘤腺病毒CNHK500-hTRAIL,联合X线照射对A549细胞生长的影响。方法将质粒pPE3-hTRAIL与pSG500用Lipofectamine2000共转染至293细胞内同源重组,包装出的溶瘤腺病毒CNHK500-hTRAIL,采用PCR法进行鉴定,并在293细胞中大量扩增病毒,采用50%组织培养感染计量法测定病毒滴度,以MOI为5的溶瘤腺病毒CNHK500-hTRAIL感染A549细胞24 h,用RT-PCR法检测hTRAIL基因在肿瘤细胞中的表达。用不同MOI值(0.01～40)的CNHK500-hTRAIL感染A549细胞,感染5 d,通过四甲基偶氮唑蓝(MTT)比色法检测其对A549细胞的细胞存活率,以便确定适宜的感染滴度。用CNHK500-hTRAIL感染A549细胞,感染后48 h,用不同剂量的X线(1和2 Gy)照射,照射后3 d,通过MTT比色法检测CNHK500-hTRAIL联合X线照射下A549细胞的细胞存活率。结果 PCR法鉴定表明溶瘤腺病毒CNHK500内携带hTRAIL基因,制备的CNHK500-hTRAIL病毒滴度为3.25×109PFU/ml,且CNHK500-hTRAIL携带的hTRAIL基因在A549细胞中有效表达。经不同MOI值CNHK500-hTRAIL感染A549细胞,细胞存活率为64.26%～96.02%。CNHK500-hTRAIL感染联合不同剂量X线照射组细胞存活率较单纯X线照射(1和2 Gy)和单纯CNHK500-hTRAIL感染组明显降低(P<0.01),细胞存活率分别为50.77%和45.56%,增敏效应比值(E/O)为1.5和1.6,均大于1.4。结论成功构建了携带hTRAIL基因的溶瘤腺病毒CNHK500-hTRAIL,CNHK500-hTRAIL感染与X射线联合照射对A549细胞生长有协同抑制作用。     

重组腺病毒载体介导HOSM基因对肝癌细胞体外增殖的抑制作用

目的:构建含人抑瘤素M(human oncostatin M,HOSM)基因的复制缺陷型重组腺病毒载体AD-HOSM,观察其对人肝癌细胞株HepG2在体外生长抑制情况.方法:通过同源重组方法构建含HOSM基因的缺陷型重组腺病毒载体AD-HOSM,用报告基因AD-GFP检测腺病毒的对人肝癌细胞系的转染效率:人肝癌细胞株HepG2转染含HOSM基因的缺陷型腺病毒载体AD-HOSM后,用RT-PCR法检测外源HOSM基因在其中的表达;台盼蓝染色、细胞计数法检测AD-HOSM对HepG2的体外增殖抑制情况.结果:成功构建了重组腺病毒载体AD-HOSM,AD-HOSM对肝癌细胞HepG2有较高的转染效率,并且转染效率与病毒的剂量呈正相关.AD-HOSM感染HepG2细胞48h,HOSM基因在转染细胞能有效的表达.体外试验示,AD-HOSM转染的细胞的增殖却受到明显的抑制.结论:重组腺病毒介导HOSM表达能有效抑制HepG2在体外的增殖,提示重组腺病毒介导的HOSM基因治疗可能成为肝癌治疗的候选方案.     

蛋白激酶SGK3基因的克隆及其在乳腺癌细胞中的表达

目的探讨通过克隆蛋白激酶(SGK)3基因并将其转染乳腺癌MCF-7细胞建立高表达SGK3的MCF-7细胞系的稳态性。方法利用真核表达载体N-端增强型绿色荧光蛋白(p EGFP-N1)通过基因克隆技术构建重组质粒,通过菌落PCR和DNA测序方法对重组质粒进行鉴定。通过PEI介导将重组质粒p EGFP-N1-SGK3转染至乳腺癌MCF-7细胞中,荧光显微镜和Western印迹方法对基因转染效率以及融合蛋白GFP-SGK3的表达进行鉴定。结果成功构建了真核表达质粒,将其命名为p EGFP-N1-SGK3;在荧光显微镜下可见多数细胞发出绿色荧光,表明获得了较高的转染效率;Western印迹结果显示,在转染p EGFP-N1-SGK3的细胞中可见阳性条带,而MCF-7亲本细胞和转染空载体的对照组细胞无阳性条带。结论成功构建了重组质粒p EGFP-N1-SGK3,获得了稳定表达融合蛋白GFP-SGK3的MCF-7细胞系,可用于后续SGK3细胞内功能的研究。     

弓形虫ROP18重组腺病毒载体的构建及其对神经干细胞C17.2凋亡的影响

目的 构建弓形虫棒状体蛋白ROP18重组腺病毒载体,研究其对神经干细胞C17.2凋亡的影响.方法 将弓形虫ROP18从PEGFP-G2-ROP18重组质粒上亚克隆至腺病毒载体pHBAd-MCMV-GFP,重组腺病毒载体pHBAd-MCMV-GFP- ROP18经PCR,测序鉴定正确后,与骨架质粒pHBAd-BHG共转染HEK293细胞进行包装,收获,扩增重组腺病毒并对其滴度进行测定.以MOI=70的重组腺病毒感染神经干细胞C17.2,转染不同时间荧光显微镜观察基因的表达情况.转染48 h用CCK-8法检测C17.2的增殖情况;转染24 h采用Annexin V-APC/PI双染流式细胞术检测细胞凋亡情况;Western blot法检测半胱天冬氨酸蛋白酶3(caspase-3)的蛋白水平.结果 重组ROP18过表达腺病毒在C17.2神经干细胞内能有效表达.转染48 h,与空载体对照组相比,C17.2细胞增殖活性明显降低(P<0.05);转染24 h,流式细胞术检测结果显示,ROP18基因转染组C17.2细胞凋亡率明显增加(P<0.001);Western blot检测显示caspase-3活性片段的表达明显增加(P<0.05).结论 成功构建了弓形虫ROP18重组腺病毒并在神经干细胞C17.2中表达,ROP18重组腺病毒能诱导C17.2神经干细胞凋亡.     

X射线对转染Smac基因重组质粒的MCF-7细胞表达及增殖和凋亡的影响

目的探讨Egr-1启动子介导Smac基因的重组质粒pshuttle-Egr1-hSmac的辐射诱导特性,以及联合X射线照射后对MCF-7细胞增殖和凋亡的影响。方法将构建完全正确的pshuttle-Egr-1-hSmac质粒转染到人乳腺癌MCF-7细胞中,24h后给予X射线照射,24h后提取总RNA和蛋白,分别采用RT-PCR和Western印迹法检测细胞中SmacmRNA和蛋白的表达;分别采用MTT法和流式细胞术检测细胞增殖和凋亡的变化。结果未照射组及转染和空载体pshuttle组未见SmacmRNA和蛋白表达;而转染pshuttle-Egr-1-hSmac的MCF-7细胞在0.5Gy照射后未见其mRNA和蛋白表达;1、2和5Gy照射后明显增高,尤其以5Gy表达最高;2Gy照射后4h未见表达,而8～48h则逐渐增加,尤其以48h最高。MTT结果显示,转染了pshutlle质粒后存活率稍有降低,而凋亡未见明显变化;转染pshuttle-Egr-1-hSmac质粒后细胞存活率未见明显降低,而凋亡百分率稍有增加(P0.05);经过2Gy照射后各组存活率降低,凋亡百分数明显增加(P0.05,P0.01),尤其以转染pshuttle-Egr-1-hSmac质粒更明显。结论X射线照射联合pshuttle-Egr1-hSmac能通过提高细胞内Smac的表达,并有效抑制MCF-7细胞增殖,促进细胞凋亡,提示Smac基因联合放射治疗可能成为肿瘤治疗的新策略。     

重组腺病毒介导的缺氧诱导因子-1α对脑缺氧后神经元中缺氧诱导因子-1α的调控

目的探讨重组腺病毒介导的缺氧诱导因子-1α(HIF-1α)转染对缺氧后的大鼠大脑皮层神经元中HIF-1α的调控作用及可能机制。方法 (1)制备大鼠大脑皮层神经元原代培养模型及缺氧模型。重组腺病毒缺氧诱导因子-1α(AdHIF-1α)转染和重组腺病毒空载体(Ad)转染正常及缺氧细胞;将其分为正常对照组(Normal)、缺氧组(Hypoxia)、AdHIF-1α基因转染组、Ad组。(2)荧光显微镜下观察AdHIF-1α/Ad转染情况;采用蛋白印迹(Western blot)法分别在12 h、24 h、48 h及72 h观察各实验组HIF-1α蛋白的表达。结果用Ad/AdHIF-lα转染缺氧的神经元后, 在荧光显微镜下观察转染48 h时荧光表达最明显。Western blot检测经AdHIF-1α基因转染组各时间点HIF-1α的表达明显增加, 12 h有阳性表达, 24 h表达有所增加, 48 h达高峰, 持续到72 h开始下降, 与缺氧组相比差异均有统计学意义(P<0.01, P<0.05), Ad组与缺氧组相比无统计学意义。结论重组腺病毒介导的HIF-1α基因转染能明显提高缺...     

Cyclin G_2表达载体的构建及其对乳腺癌MCF-7细胞的作用

目的构建包含Cyclin G2基因的真核表达载体,探讨其对乳腺癌MCF-7细胞体外增殖的作用。方法运用RT-PCR和基因克隆技术构建pDsRed2-Cyclin G2重组质粒,脂质体法转染至MCF-7细胞中,荧光显微镜下观察其蛋白的表达及定位;AnnexinⅤ-FITC染色流式细胞术检测细胞凋亡率。结果成功构建了pDsRed2-Cyclin G2融合表达质粒,转染MCF-7细胞后散在分布于细胞质和细胞,核转染重组质粒的MCF-7细胞凋亡率明显增高（P〈0.01）。结论 Cyclin G2重组质粒转染至MCF-7细胞后能有效促进细胞凋亡。     

4T42肽腺病毒载体的构建包装及抗SKBR3乳腺癌细胞的效果

目的构建携带有肿瘤抑素相关T42肽(以下简称T42肽)基因的重组腺病毒载体,并转染HEK293细胞中进行病毒包装,最终感染SKBR3乳腺癌细胞,体外四倍体T42肽(4T42)抗SKBR3乳腺癌细胞的效果。方法利用同尾酶连接技术将T42肽进行两次克隆形成4T42,并将其亚克隆至穿梭质粒pEC3.1(+)-EGFP中,获得重组质粒pEC3.1(+)-EGFP-4T42。体外重组至骨架载体pAd/BLOCK-iTTM-Dest(pAd-BL-Dest),获得重组质粒pAd-EGFP-4T42。将该重组骨架质粒转染HEK293细胞,利用RT-PCR方法和荧光显微镜观察标志蛋白-绿色荧光蛋白的表达情况以判断T42转染成功与否。利用病毒倍比稀释法利用荧光显微镜检测病毒滴度,用重组腺病毒感染SKBR3乳腺癌细胞。采用流式细胞仪检测感染重组腺病毒的SKBR3胞凋亡率,MTT实验检测感染重组腺病毒的MCF细胞的生长情况。结果重组腺病毒载体经酶切鉴定证明构建正确,重组腺病毒获得成功包装,纯化的病毒滴度为4*108 DRP/ml。重组腺病毒rAd-EGFP-4T42感染MCF细胞后较对照组未转染空白组细胞凋亡率高(P<0.05);Ad-EGFP-4T42质粒转染组与对照组相比,明显抑制MCF细胞生长(P<0.05)。结论体外4T42肽具有显著促进SKBR3乳腺癌细胞凋亡和抑制SKBR3乳腺癌细胞生长的作用。     

磷酸酶与张力蛋白同源物基因过度表达抑制乳鼠心脏成纤维细胞增殖

目的将携带有张力蛋白同源物基因(PTEN)的重组腺病毒载体转染乳鼠心脏成纤维细胞,观察心脏成纤维细胞过度表达野生型PTEN对其增殖的影响。方法分离培养心脏成纤维细胞,以携带有绿色荧光蛋白(GFP)的重组腺病毒Ad-PTEN转染上述细胞,荧光倒置显微镜下观察GFP表达情况,确定最佳感染倍数(MOI)。原代心脏成纤维细胞接种至96孔板,转染Ad-PTEN[Ad-PTEN转染组(MOI根据生长曲线确定)],并设正常对照组(加入等体积培养基),以MTT的方法检测其吸光度(A),以反映细胞增殖情况,将转染效率达到90%以上成纤维细胞及正常对数生长期成纤维细胞消化,制成单细胞悬液,流式细胞仪分别检测细胞周期,以吖啶橙/溴乙锭(AO/EB)法分别检测Ad-PTEN转染组和正常对照组细胞凋亡情况;结果MTT测定的结果:48 h后Ad-PTEN转染组的A显著低于该时间点正常对照组(P<0.05)。流式细胞仪检测结果发现Ad-PTEN转染的成纤维细胞出现G1期阻滞;AO/EB法检测发现Ad-PTEN转染的成纤维细胞其细胞凋亡率与正常对照组细胞无显著差异;结论PTEN基因过度表达将引起乳鼠心脏成纤维细胞增殖受抑制,该现象与细胞周期G1期阻滞有关。     

hVEGF165四环素调控腺病毒载体的构建及其在大鼠成肌细胞表达

目的构建表达人血管内皮生长因子165基因(hVEGF165)的四环素调控重组腺病毒,并测定其在原代培养大鼠成肌细胞的表达水平。方法亚克隆hVEGF165全长cDNA到穿梭质粒pTREShuttle2,获得四环素可调控的目的基因表达盒;亚克隆表达盒到BDAdeno-XTMViralDNA,获得重组腺病毒质粒;重组质粒转染HEK293细胞以包装产生具有感染能力的重组腺病毒颗粒AdTRE.VEGF165。hVEGF165重组腺病毒和Tetoff调节病毒共感染原代培养的成肌细胞,酶联免疫法检测目的基因的表达。结果成功构建了能表达hVEGF165的四环素调控的重组腺病毒载体。hVEGF165重组腺病毒(20pfu/cell)和Tetoff调控病毒(20pfu/cell)共感染骨骼肌成肌细胞后5d,ELISA检测显示培养上清中VEGF165蛋白浓度显著高于未感染的细胞培养上清[(14.80±1.15ng/ml)×105/(cell·24h)vs(1.39±0.36ng/ml)×105/(cell·24h),P<0.01)]。结论四环素调控表达的腺病毒系统可高效地转导VEGF基因进入哺乳动物细胞;原代培养的骨骼肌成肌细胞感染AdTRE.VEGF165后可高水平地分泌VEGF165蛋白。     

Latest insights on adenovirus structure and assembly

Adenovirus (AdV) capsid organization is considerably complex, not only because of its large size (~950 Å) and triangulation number (pseudo T = 25), but also because it contains four types of minor proteins in specialized locations modulating the quasi-equivalent icosahedral interactions. Up until 2009, only its major components (hexon, penton, and fiber) had separately been described in atomic detail. Their relationships within the virion, and the location of minor coat proteins, were inferred from combining the known crystal structures with increasingly more detailed cryo-electron microscopy (cryoEM) maps. There was no structural information on assembly intermediates. Later on that year, two reports described the structural differences between the mature and immature adenoviral particle, starting to shed light on the different stages of viral assembly, and giving further insights into the roles of core and minor coat proteins during morphogenesis [1,2]. Finally, in 2010, two papers describing the atomic resolution structure of the complete virion appeared [3,4]. These reports represent a veritable tour de force for two structural biology techniques: X-ray crystallography and cryoEM, as this is the largest macromolecular complex solved at high resolution by either of them. In particular, the cryoEM analysis provided an unprecedented clear picture of the complex protein networks shaping the icosahedral shell. Here I review these latest developments in the field of AdV structural studies.     

Disruption of 3D tissue integrity facilitates adenovirus infection by deregulating the coxsackievirus and adenovirus receptor

The human coxsackievirus and adenovirus receptor (CAR) represents the primary cellular site of adenovirus attachment during infection. An understanding of the mechanisms regulating its expression could contribute to improving efficacy and safety of adenovirus-based therapies. We characterized regulation of CAR expression in a 3D cell culture model of human breast cancer progression, which mimics aspects of the physiological tissue context in vitro. Phenotypically normal breast epithelial cells (S1) and their malignant derivative (T4-2 cells) were grown either on tissue culture plastic (2D) or 3D cultures in basement membrane matrix. S1 cells grown in 3D showed low levels of CAR, which was expressed mainly at cell-cell junctions. In contrast, T4-2 cells expressed high levels of CAR, which was mainly in the cytoplasm. When signaling through the epidermal growth factor receptor was inhibited in T4-2 cells, cells reverted to a normal phenotype, CAR protein expression was significantly reduced, and the protein relocalized to cell-cell junctions. Growth of S1 cells as 2D cultures or in 3D in collagen-I, a nonphysiological microenvironment for these cells, led to up-regulation of CAR to levels similar to those in T4-2 cells, independently of cellular growth rates. Thus, expression of CAR depends on the integrity and polarity of the 3D organization of epithelial cells. Disruption of this organization by changes in the microenvironment, including malignant transformation, leads to up-regulation of CAR, thus enhancing the cell's susceptibility to adenovirus infection.     

腺病毒载体的研究进展

腺病毒(adenovirus,Ad)作为基因表达载体的研制起始于20世纪60年代初,当时病毒学家观察到腺病毒基因组可与猴类病毒40(SV40)基因组杂交,说明腺病毒基因组可承载异源性基因.此后腺病毒逐步发展成一种重要的载体系统[1-9],并成功地用于基因治疗的体内外实验和临床试验[10-21].目前运用最广的腺病毒载体(adenovirus vector,Adv)是血清5型腺病毒.     

Increasing the efficacy of oncolytic adenovirus vectors

Oncolytic adenovirus (Ad) vectors present a new modality to treat cancer. These vectors attack tumors via replicating in and killing cancer cells. Upon completion of the vector replication cycle, the infected tumor cell lyses and releases progeny virions that are capable of infecting neighboring tumor cells. Repeated cycles of vector replication and cell lysis can destroy the tumor. Numerous Ad vectors have been generated and tested, some of them reaching human clinical trials. In 2005, the first oncolytic Ad was approved for the treatment of head-and-neck cancer by the Chinese FDA. Oncolytic Ads have been proven to be safe, with no serious adverse effects reported even when high doses of the vector were injected intravenously. The vectors demonstrated modest anti-tumor effect when applied as a single agent; their efficacy improved when they were combined with another modality. The efficacy of oncolytic Ads can be improved using various approaches, including vector design, delivery techniques, and ancillary treatment, which will be discussed in this review.     

Chromatin-like organization of the adenovirus chromosome.

Staphylococcal nuclease (nucleate 3'-oligonucleotidohydrolase; EC 3.1.4.7) cleaved DNA within disrupted adenovirus particles into a regular series of fragments with a repeat unit of 200 base pairs. Since this pattern did not eppear when DNA alone was digested, we postulate that the orderly arrangement of core polypeptides protects discrete regions of DNA from nuclease attack. The 23 X 10(6) dalton adenovirus DNA molecule can accommodate 180 units of roughly 200 base pairs. Based on the stoichiometry of core polypeptides, we calculate that each repeat unit contains six copies of polypeptide VII and a single copy of polypeptide V. This model is bases on proposals for the structure of eukaryotic chromatin. Very brief nuclease digestion generated 1800 base pair fragments (1/20 of the adenovirus chromosome). This result is discussed in terms of a higher order folding of viral DNA within the virus particle.     

腺病毒介导的凋亡素、内皮抑素双基因对食管癌的实验研究

目的探讨携带有凋亡素(vp3、Apoptin)、内皮抑素(Endostatin)双基因的重组腺病毒载体在食管癌等多种肿瘤细胞内的表达情况及致凋亡作用,为进一步的食管癌治疗应用研究打下基础。方法将已纯化的携带凋亡素和内皮抑素基因的腺病毒Ad-vp3-IRES-sEndo-His感染食管癌细胞Eca-109、小鼠肝癌细胞Hepa1-6、结肠癌细胞LoVo提取各种细胞RNA,针对凋亡素、内皮抑素基因的表达进行RT-PCR检测,同时对感染腺病毒Ad-vp3-IRES-sEndo-His、Ad-vp3的食管癌细胞通过流式细胞仪、Hoechst33258染色等方式进行细胞凋亡率的检测。结果 RT-PCR检测显示被感染的多种肿瘤细胞均可表达凋亡素、内皮抑素基因的mRNA,Ho-echst33258染色显示被Ad-vp3-IRES-sEndo-His感染的食管癌细胞出现凋亡形态学改变,用流式细胞仪测定时段最高凋亡率达47.7%,与对照组相比具有统计学意义(P<0.05)。结论携带凋亡素及内皮抑素双基因的腺病毒载体能在多种肿瘤细胞中表达,感染食管癌细胞后,能有效诱导其凋亡且其致凋亡率高于仅携带凋亡素的单基因腺病毒载体。     

Clinical characteristics of human adenovirus type 7 pneumonia and genetic characteristics of human adenovirus type 7

<正>Objective To better understand the clinical features of human adenovirus type 7 (h AdV7) pneumonia and to identify whether there is a variation in the genome of the strain (CHN/BeiJing/2018) isolated during the smallscale epidemic. Methods Forty-two patients were diagnosed with hAdV7 pneumonia between October 27th,