重组p16腺病毒的构建及其对人白血病细胞的抑制作用

为了探讨腺病毒载体用于基因治疗的可行性及野生型 p1 6基因的抗肿瘤特性 ,构建了复制缺陷型重组 p1 6腺病毒 .首先将 p1 6全长 c DNA插入穿梭质粒 p Ad CMV产生重组质粒 p Ad-CMV- p1 6,然后通过脂质体介导与 p JM1 7共转染 2 93细胞 ,经同源重组产生 E1区缺失的重组腺病毒空斑 .用纯化后的腺病毒感染人白血病细胞株 HL- 60后 ,PCR及 Western blot分析显示在感染细胞中有外源性 p1 6 c DNA存在和 p1 6蛋白表达 ;被感染的 HL- 60细胞的生长受到明显抑制 ,而未感染细胞及对照腺病毒感染的细胞没有受到抑制 .结果表明 ,腺病毒作为一种新型基因转移载体 ,可有效地介导肿瘤抑制基因 p1 6的表达 ,在肿瘤基因治疗方面具有很大的应用前景 .     

Evaluation of a Serum-free Medium for the Production of rAAV-2 using HeLa Derived Producer Cells

During the last decade, recombinant AAVs have become of increasing interest for gene therapy. Clinical trials have been conducted following promising in vivo evaluations, thus leading laboratories to adapt their production systems for larger and higher quality demands. Classical transfection protocols seem difficult and cumbersome to adapt to a bioreactor scale. The use of stable producer cells appears as an attractive alternative, as this system requires only a single infection step to induce rAAV production. Furthermore, the switch to a serum-free medium is an interesting strategy to increase the biosafety level to satisfy clinical grade requirements for gene therapy products. Here, we have combined both approaches and evaluated different rAAV producer clones in a serum-free medium. We first evaluated the cell growth in a serum-free medium and then did a partial optimisation of the medium composition to obtain vector yields as close as possible to the yields obtained in a classical serum containing medium. Different helper viruses, multiplicity of infection, times of infection and harvest have been compared in small scale cultures in order to determine the optimal settings which were then transferred and evaluated in suspension cultures in spinner flasks. The yields obtained in this system were similar to or at most 2 times lower than those obtained in a serum-containing medium. The scale-up of such a production system as well as the use of high cell density perfusion culture systems will probably lead to considerably higher yields than those obtained in a classical process.     

Development and improvement of a serum-free suspension process for the production of recombinant adenoviral vectors using HEK293 cells

Human Embryonic Kidney 293 (HEK293) cells were adapted into a serum-free suspension medium through steps of gradual serum weaning for the production of adenoviral (AdV) gene therapy vectors. The presence of sodium heparin in the medium formulation reduced cell clumping dramatically in suspension culture. The adapted cells were ready to grow either in serum-containing medium as an attached culture or in serum-free medium in suspension culture. A scalable production process was developed in shake flasks and was then evaluated in stirred tank bioreactors. This process includes a growth phase in batch-mode followed by a production phase involving medium perfusion and supplementation. Fortification with calcium chloride post viral inoculation resulted in an increase in virus production by at least one fold. Addition of stimulating agents such as sodium butyrate, N-acetyl-L-cysteine (NAC), dimethyl sulfoxide(DMSO), or ethyl alcohol post infection was shown to further improve virus production in a dose-dependent manner. The serum-free suspension process described here should be suitable for the manufacturing of other E1-deleted AdV vectors and could potentially be used for the production of recombinant proteins by HEK293 cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

一种新的重组腺病毒的构建及其对人肿瘤细胞的影响

腺病毒E1A基因诱导细胞凋亡．E1B19K基因及E1B55K基因抑制细胞凋亡,前者被克隆到腺病毒转移载体pCA13的HCMVIE启动子下游．构建成转移载体pCAE1A。采用lipofectin法将PCAE1A和含腺病毒基因组（E1、E3区缺失）的质粒pBHG11共转染293细胞,7～10d后得到重组病毒v5Ad4。用v5Ad4感染人肺腺癌细胞系A549,结果表明v5Ad4有明显杀伤和裂解肿瘤细胞功能。在人胚肺正常二倍体细胞中,v5Ad4没有表现出可见的细胞毒效应。     

肺腺癌A549/DDP细胞周期变化及其多药耐药性

用Fura 2 /AM标记药物敏感的肺腺癌细胞A54 9和抗顺铂药物的肺腺癌细胞A54 9/DDP两种细胞胞内游离Ca2 ,用碘化丙锭 (PI)标记细胞DNA ,检测其胞内Ca2 的变化及两种细胞增殖能力和细胞周期 .实验结果表明 ,抗药性细胞株A54 9/DDP胞浆内游离Ca2 的浓度仅为药物敏感细胞株A54 9的 1/ 3左右 ,同时前者的细胞增殖能力较后者明显增强 ,而且细胞周期也明显缩短 .当用BAPTA AM和EGTA或A2 3187和Thapsigargin处理细胞以降低或升高其胞内自由Ca2 浓度时可改变细胞的生长周期 ,二者也呈现明显差别 .这些结果表明 ,对顺铂产生耐药性的人肺腺癌A54 9/DDP细胞胞内Ca2 浓度的降低 ,可能影响细胞的增殖 ,缩短细胞的生长周期 ,特别是影响起决定作用的G1期 ,从而有利于肿瘤细胞多药耐药特性的维持     

Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media

The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown.     

A549/DDP细胞的抗凋亡特性与其pHi和［Ca2+］i变化相关

以临床用药剂量(30 μmol/L)的顺铂处理敏感的人肺腺癌细胞A549和抗顺铂的A549/DDP细胞,在相同条件下培养.对培养不同时间的两株细胞DNA琼脂糖凝胶电泳分析的结果表明，前者在培养12 h后即呈现DNA梯子，而后者在培养48 h后却未见凋亡特征.用流式细胞计检测其凋亡峰也获得相似的结果.当测定与凋亡相关的生物化学和生物物理变化时发现对顺铂敏感的A549细胞的线粒体膜电势显著降低，胞内更加酸化且胞内钙离子浓度升高；而抗顺铂药物的A549/DDP细胞的线粒体膜电势和pH值却维持在相对较高的水平，而其胞内钙离子浓度随培养时间的延长下降.这些结果表明，抗顺铂的人肺腺癌A549/DDP细胞的抗凋亡特性与其胞内的相对碱化和较高的线粒体膜电势,以及胞内钙离子浓度的降低相关.这些特性可能参与了A549/DDP的顺铂耐药性的调节.     

IL-8对肺腺癌A549细胞迁移的影响及其机制探讨

探讨IL-8对肺腺癌A549细胞迁移的影响及其可能机制。用MTT法选择了合适的IL-8使用浓度。分别用划痕试验及Transwell试验证明了IL-8可以促进肺腺癌A549细胞的迁移。Westernblot结果表明:(1)IL-8可以促进MMP-2蛋白的表达,而对MMP-9的表达无明显影响;(2)IL-8可促进JNK/SAPK磷酸化蛋白的表达;(3)抑制剂(SP600125)可以阻断IL-8对MMP-2蛋白表达的影响。划痕试验从反面验证了低表达的MMP-2可以抑制A549细胞的迁移。表明IL-8可通过JNK/SAPK信号通路调控MMP-2蛋白的表达,进一步促进肺腺癌A549细胞的迁移。     

Development and characterization of a cell line for large-scale, serum-free production of recombinant adeno-associated viral vectors

BACKGROUND: One of the major limitations to the use of adeno-associated virus (AAV) vectors for gene therapy has been the difficulty in producing enough vector to supply a clinical trial. More than 20 000 roller bottles may be required to generate AAV by the traditional transient transfection process to treat 50 patients. A scalable AAV producer cell line grown in serum-free media will meet the needs for the manufacture of AAV gene therapeutics. METHODS: A packaging cell line was generated by introducing the AAV rep and cap genes into A549 cells. From this packaging cell line, a number of producer cell lines were generated by infecting the packaging cell with the appropriate AAV vector. Producer cell lines were then adapted to serum-free suspension conditions for growth in bioreactors. RESULTS: We report here the development of six AAV producer cell lines that generate > 10(4) particles/cell. The rAAV vector preparations from these cell lines have physical and functional characteristics similar to rAAV vectors prepared by transient transfection. To enable large-scale production, producer cell lines were adapted to serum-free suspension and we demonstrate production of AAV at the 15 L scale. In addition, vector preparations from these cell lines were shown to be free of wild-type AAV. CONCLUSIONS: AAV producer cell lines can be readily scaled to meet the needs of clinical trials. One 500 L bioreactor of these producer cells can produce the equivalent of 2500 high capacity roller bottles or 25 000 T-175 tissue culture flasks.     

利用CRISPRi技术沉默MRP1基因增强A549/DDP细胞对顺铂的敏感性*

目的: 采用CRISPRi技术沉默人肺癌A549/DDP细胞MRP1基因表达,观察细胞对顺铂敏感性的变化。方法: 采用生物信息学软件分析MRP1启动子序列,设计合成3对sgRNA干扰片段,定向克隆到pSPgRNA载体中,构建靶向MRP1的干扰表达载体,分别与dCas9表达载体共转染A549/DDP细胞。实验共分为5组,分别为A549/DDP细胞组,Scrambled组,sgRNA-MRP1-1组,sgRNA-MRP1-2组和sgRNA-MRP1-3组,每组设置3个复孔,处理48 h后进行后续实验。通过qRT-PCR和Western blot检测MRP1 mRNA和蛋白表达水平,MTT法检测细胞对药物的敏感性,激光共聚焦显微镜下观察细胞的形态变化。结果: 成功构建了sgRNA-MRP1-1、sgRNA-MRP1-2和sgRNA-MRP1-3 3种干扰载体,分别与dCas9表达载体共转染A549/DDP细胞后,均能显著降低细胞MRP1基因表达(P<0.01),其中sgRNA-MRP1-2干扰效果最好,MRP1 mRNA水平降低了72%,蛋白水平降低了53%。将sgRNA-MRP1-2转染细胞后,细胞对顺铂的敏感性显著增加,IC₅₀值由74.26±3.71降低至34.29±2.51,细胞形态由梭形变为椭圆形,染色质高度凝聚、边缘化,产生凋亡小体。结论: 成功构建3种靶向MRP1的干扰表达载体,均能有效沉默A549/DDP细胞中MRP1基因表达,可增强细胞对顺铂的敏感性。     

不同强度高压电场对A549肺癌细胞肿瘤生物学特性的影响

目的：探讨不同强度高压电场对A549肺癌细胞肿瘤转移生物学特性的影响。方法：选择处于生长周期的A549细胞,共分为7个组进行研究,其中A-F组为实验组,G组为不施加电场的空白对照组,A施加500V/cm强度高压电场,F组施加1750V/cm的高压电场,电压场强间隔为250V/cm。采取粘附实验、侵袭及转移实验,检验A549细胞在不同强度高压电场中,其肿瘤生物学转移特性的改变。结果：①各实验组与对照组、各实验组之间的细胞粘附能力,均存在显著性差异（P〈0.05）;②电场强度≥750V/cm时,各实验组之间、及其与对照组之间的细胞迁移能力,存在显著性差异（P〈0.05）;③电场强度≥1000V/cm时,各组与对照组间的细胞侵袭能力,存在显著性差异（P〈0.05）;④电场强度为1000-1250V/cm的各组与1500-1750V/cm各组间的细胞侵袭能力,存在显著性差异,有统计学意义（P〈0.05）。结论：不同强度的电场抑制A549肺癌细胞的程度不同,随着强度的增加,A549细胞粘附、迁移和侵袭能力的抑制现象依次出现,并随着电场强度的增加其抑制程度也持续增加。     

Inhibition of cellular proliferation and induction of apoptosis in human lung adenocarcinoma A549 cells by T-type calcium channel antagonist

The anti-proliferative and apoptotic activities of new T-type calcium channel antagonist, 6e (BK10040) on human lung adenocarcinoma A549 cells were investigated. The MTT assay results indicated that BK10040 was cytotoxic against human lung adenocarcinoma (A549) and pancreatic cancer (MiaPaCa2) cells in a dose-dependent manner with IC₅₀ of 2.25 and 0.93 μM, respectively, which is ca. 2-fold more potent than lead compound KYS05090 despite of its decreased T-type calcium channel blockade. As a mode of action for cytotoxic effect of BK10040 on lung cancer (A549) cells, this cancer cell death was found to have the typical features of apoptosis, as evidenced by the accumulation of positive cells for annexin V. In addition, BK10040 triggered the activations of caspases 3 and 9, and the cleavages of poly (ADP-ribose) polymerase (PARP). Moreover, the treatment with z-VAD-fmk (a broad spectrum caspase inhibitor) significantly prevented BK10040-induced apoptosis. Based on these results, BK10040 may be used as a potential therapeutic agent for human lung cancer via the potent apoptotic activity.     

The ubiquitin-interacting motif of 26S proteasome subunit S5a induces A549 lung cancer cell death

The subunit S5a is a key component for the recruitment of ubiquitinated substrates to the 26S proteasome. When the full-length S5a, the N-terminal half of S5a (S5aN) containing the von Willebrand A (vWA) domain, and the C-terminal half of S5a (S5aC) containing two ubiquitin(Ub)-interacting motifs (UIMs) were ectopically expressed in HEK293 cells, Ub-conjugates accumulated most prominently in S5aC-expressing cells. In addition, S5aC induced A549 lung cancer cell death but not non-cancer BEAS-2B cell death. Similar effects were observed using only S5a-UIMs. Our data therefore suggest that S5a-UIMs can be used as upstream inhibitors of the proteasome pathway.     

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these traditionally modified Ad5 vectors in clinical trials could be primarily correlated with Ad5 pre-existing immunity (PEI) among the majority of the population. To promote Ad5-vectored vaccine development by solving the concern of Ad5 PEI, the innovative Antigen Capsid-Incorporation strategy has been employed. By merit of this strategy, Ad5-vectored we first constructed the hexon shuttle plasmid HVR1-KWAS-HVR5-His₆/pH5S by subcloning the hypervariable region (HVR) 1 of hexon into a previously constructed shuttle plasmid HVR5-His₆/pH5S, which had His₆ tag incorporated into the HVR5. This HVR1 DNA fragment containing a HIV epitope ELDKWAS was synthesized. HVR1-KWAS-HVR5-His₆/pH5S was then linearized and co-transformed with linearized backbone plasmid pAd5/∆H5 (GL) , for homologous recombination. This recombined plasmid pAd5/H5-HVR1-KWAS-HVR5-His₆ was transfected into cells to generate the viral vector Ad5/H5-HVR1-KWAS-HVR5-His₆. This vector was validated to have qualitative fitness indicated by viral physical titer (VP/ml), infectious titer (IP/ml) and corresponding VP/IP ratio. Both the HIV epitope and His₆ tag were surface-exposed on the Ad5 capsid, and retained epitope-specific antigenicity of their own. A neutralization assay indicated the ability of this divalent vector to circumvent neutralization by Ad5-positive sera in vitro. Mice immunization demonstrated the generation of robust humoral immunity specific to the HIV epitope and His₆. This proof-of-principle study suggested that the protocol associated with the Antigen Capsid-Incorporation strategy could be feasibly utilized for the generation of Ad5-vectored vaccines by modifying different capsid proteins. This protocol could even be further modified for the generation of rare-serotype adenovirus-vectored vaccines.     

Autoxidation of extracellular hydroquinones is a causative event for the cytotoxicity of menadione and DMNQ in A549-S cells

Cytotoxicity of 1,4-naphthoquinones has been attributed to intracellular reactive oxygen species (ROS) generation through one-electron-reductase-mediated redox cycling and to arylation of cellular nucleophiles. Here, however, we report that in a subclone of lung epithelial A549 cells (A549-S previously called A549-G4S (Watanabe, et al., Am. J. Physiol. 283 (2002) L726-736), the mechanism of ROS generation by menadione and by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and therefore that of cytotoxicity, differs from the paradigm. Ninety percent of H(2)O(2) generation by both the quinones can be prevented by dicumarol, an inhibitor of NAD(P)H quinone oxidoreductase (NQO1), at the submicromolar level, regardless of the quinone concentrations. Exogenous SOD also inhibits H(2)O(2) production at low but not high concentrations of the quinones, especially DMNQ. Thus, at low quinone concentrations, superoxide-driven hydroquinone autoxidation accounts for more than half of H(2)O(2) generation by both quinones, whereas at high quinone concentrations, especially for DMNQ, comproportionation-driven hydroquinone autoxidation becomes the predominant mechanism. Hydroquinone autoxidation appears to occur predominantly in the extracellular environment than in the cytosol as extracellular catalase can dramatically attenuate quinone-induced cytotoxicity throughout the range of quinone concentrations, whereas complete inactivation of endogenous catalase or complete depletion of intracellular glutathione has only a marginal effect on their cytotoxicity. Finally, we show evidence that ROS production is a consequence of the compensatory defensive role of NQO1 against quinone arylation.     

关于微生物燃料电池底物的研究进展

近年来,微生物燃料电池已引起了广泛关注,它将低能量废水和木质纤维素生物质等有机废物转化为电能。在将来,微生物电能将成为一种重要的生物能源,因为微生物燃料电池提供了一种复合有机物和可再生生物能源中提取电能的可行性。人们研究了许多物质,以考察其是否能作为微生物电能转化的底物。这些物质包括人工的和天然废物,以及木质纤维素生物质。尽管现在微生物燃料电池提供的电流和功率较低,但是随着技术的发展和对微生物燃料电池系统的深入了解,微生物燃料电池转化的电流和电力将极大增加,从而向世人提供了一种可以将纤维素生物质和废水直接转化为有用能源的有效方法。本文介绍了迄今为止在微生物燃料电池中用到的各种反应底物,并对它们的应用效率和存在的不足进行了分析。     

A New Pathway for Poly(3-hydroxybutyrate) Production in Escherichia coli and Corynebacterium glutamicum by Functional Expression of a New Acetoacetyl-coenzyme A Synthase

A biosynthetic pathway for poly(3-hydroxybutyrate) [P(3HB)] was developed in Escherichia coli and Corynebacterium glutamicum by an acetoacetyl-coenzyme A (CoA) synthase (AACS) recently isolated from terpenoid-producing Streptomyces sp. strain CL190. Expression of AACS led to significant productions of P(3HB) in E. coli (10.5 wt %) and C. glutamicum (19.7 wt %).