铜绿假单胞菌噬菌体PaP3基因组电转化宿主菌的条件初探

目的:研究将铜绿假单胞菌噬菌体PaP3基因组DNA电转入其宿主菌并获得噬斑的基本条件。方法:以铜绿假单胞菌PA3作受体菌,将噬菌体PaP3基因组DNA通过电转化导入受体菌中,研究细胞生长状态、感受态细胞的制备方式、电场强度、DNA浓度和细胞密度等条件对转化效率的影响。结果:在含50μg/ml红霉素的LB培养液中培养宿主菌14~16 h,以100 mmol/L蔗糖溶液为介质,在25℃条件下制备感受态并使其浓度达到1011/ml,电转参数为12 kV/cm,300Ω,25μF,在此组条件下能获得较高的转化效率,最高可达2.1×103pfu/μg的DNA。结论:确定了一组电转条件,成功地将铜绿假单胞菌噬菌体完整基因组导入PA3中,并形成噬斑,为铜绿假单胞菌噬菌体的深入研究奠定了基础。     

利用电磁脉冲提高抗癌药物治疗肿瘤细胞疗效的实验研究

本文采用一种新的方法—电穿孔结合化学药物进行肿瘤治疗的电化学疗法。电穿孔由于能使细胞膜出现瞬时微孔,从而能大大提高癌细胞对药物的吸收率、促进了药物等大分子进入细胞。这里利用电穿孔现象结合抗癌药物治疗昆明小鼠身上的S-180肉瘤,从实验数据可看出,这种方法取得了很好的效果。这种癌症治疗新技术具有易于控制、便于操作等优点,特别适用浅表肿瘤的治疗,为临床治疗肿瘤提供了一条新的途径。     

Effect of the electroporation in the field calculation in biological tissues

This article presents results from the field calculation in a model of biological tissue taking into account the electroporation process. The electroporation model used in this study was proposed and experimentally verified by Glaser et al. for planar membranes. The numerical method for field calculation is based on a two-step process: (1) a cell-scale analysis, used for obtaining the field and current in a small volume containing only one cell and its nearest neighbors; and (2) a tissue-scale analysis, based on averaged values of conductivity and permittivity (obtained from the cell-scale analysis) and used for field calculation in a large volume of the tissue. The results for a high voltage applied between two electrodes in a two-dimensional analysis show that the electric field in the porated tissue extends beyond the limits obtained when the electroporation process is not taken into account. Furthermore, due to the electroporation, the tissue conductivity increases in the space between the electrodes. These results show that the electroporation process cannot be ignored in the field calculation in biological tissues when high strength electric fields are present.     

A phase 1, randomized,controlled dose-escalation study of EP-1300 polyepitope DNA vaccine against Plasmodium falciparum malaria administered via electroporation

Plasmodium falciparum malaria is one of the leading infectious causes of childhood mortality in Africa. EP-1300 is a polyepitope plasmid DNA vaccine expressing 38 cytotoxic T cell epitopes and 16 helper T cell epitopes derived from P. falciparum antigens expressed predominantly in the liver phase of the parasite’s life cycle. We performed a phase 1 randomized, placebo-controlled, dose escalation clinical trial of the EP-1300 DNA vaccine administered via electroporation using the TriGrid Delivery System device (Ichor Medical Systems). Although the delivery of the EP-1300 DNA vaccine via electroporation was safe, tolerability was less than that usually observed with standard needle and syringe intramuscular administration. This was primarily due to acute local discomfort at the administration site during electroporation. Despite the use of electroporation, the vaccine was poorly immunogenic. The reasons for the poor immunogenicity of this polyepitope DNA vaccine remain uncertain.Clinical Trials Registration: ClinicalTrials.gov NCT01169077.     

Overexpression of Ten-m3 in the retina alters ipsilateral retinocollicular projections in the wallaby (Macropus eugenii)

Retinal projections to the superior colliculus are organised into retinotopic maps. Binocular vision requires that inputs from the two eyes map in register with each other. Studies in mice lacking Ten-m3, a homophilic transmembrane protein, indicate that it plays a key role in this process by influencing ipsilateral projections. The postnatal, ex utero development of the wallaby allows the targeted manipulation of molecules of interest during development. The distribution of mRNA for Ten-m3 in the retina and superior colliculus of the wallaby, and the effects of its spatiotemporally restricted retinal overexpression was investigated, in particular on the mapping of ipsilateral projections. Quantitative polymerase chain reaction found that Ten-m3 mRNA is expressed at relatively higher levels in the retina and colliculus early in development. Further, it is higher in ventral than dorsal retina, and increased in the retinotopically corresponding medial compared to lateral superior colliculus. In situ hybridisation demonstrated an increasing dorsoventral gradient in retinal ganglion cells was matched to an increasing lateromedial gradient in the superior colliculus. Overexpression of Ten-m3 by in vivo retinal electroporation produced an increase in ipsilateral projections to the binocular rostromedial colliculus, fitting with the proposal that Ten-m3 mediates mapping by attractant homophilic interactions. Retrograde labelling of the projection from this region suggested that overexpression produces a shift in the axons of existing ipsilaterally projecting ganglion cells rather than a rerouting of the axons of contralaterally projecting cells. Retinal manipulation of Ten-m3 levels produces changes in ipsilateral mapping, supporting a role for it in binocular mapping.     

MUC1 mRNA体外负载树突状细胞诱导细胞毒性T细胞对非小细胞肺癌的杀伤作用

 目的：将体外扩增的黏蛋白1 （MUC1） mRNA转染入成熟的树突状细胞（DCs）,观察其体外诱导的特异性抗肿瘤效应。方法：将分离提纯的单核细胞培养诱导为DC并用流式细胞术鉴定。构建pcDNA3.1（+）-MUC1质粒,体外转录为mRNA,电穿孔法转染DCs。定量 PCR检测转染的DCs中MUC1的表达;MTT法检测T细胞增殖情况;流式细胞术检测CD8+ 在T细胞的表达;LDH释放法测定细胞毒性,ELISA检测IFN-γ分泌水平。结果：流式细胞术结果表明成熟DCs标志表型的表达明显高于对照组。定量PCR结果说明转染后的DCs MUC1 mRNA相对表达量增高。转染组DCs与T细胞按1∶10共培养时,刺激增殖能力明显高于未转染组,且CD8+  T细胞表达率高于未转染组,诱导产生特异性的细胞毒性T细胞杀伤表达MUC1蛋白的靶细胞,而未转染组的杀伤作用较弱。转染组DCs与T细胞共培养的上清中IFN-γ的分泌水平高于未转染组。结论：电穿孔法可以将MUC1 mRNA成功转染至DCs,产生特异性杀伤效应,为以MUC1为靶点的非小细胞肺癌的免疫治疗提供实验和理论依据。     

Immunogenicity of Multi-Epitope DNA and Peptide Vaccine Candidates Based on Core,E2, NS3 and NS5B HCV Epitopes in BALB/c Mice

Background:

Hypervariability of HCV proteins is an important obstacle to design an efficient vaccine for HCV infection. Multi-epitope vaccines containing conserved epitopes of the virus could be a promising approach for protection against HCV.

Objectives:

Cellular and humoral immune responses against multi-epitope DNA and peptide vaccines were evaluated in BALB/c mice.

Materials and Methods:

In this experimental study, multi-epitope DNA- and peptide-based vaccines for HCV infection harboring immunodominant CD8+ T cell epitopes (HLA-A2 and H2-Dd) from Core (132-142), NS3 (1073-1081) and NS5B (2727-2735), a Th CD4+ epitope from NS3 (1248-1262) and a B-cell epitope from E2 (412-426) were designed. Multi-epitope DNA and peptide vaccines were tested in two regimens as heterologous DNA/peptide (group 1) and homologous peptide/peptide (group 2) prime/boost vaccine in BALB/c mice model. Electroporation was used for delivery of the DNA vaccine. Peptide vaccine was formulated with Montanide ISA 720 (M720) as adjuvant. Cytokine assay and antibody detection were performed to analyze the immune responses.

Results:

Mice immunized with multi-epitope peptide formulated with M720 developed higher HCV-specific levels of total IgG, IgG1 and IgG2a than those immunized with multi-epitope DNA vaccine. IFN-γ levels in group 2 were significantly higher than group 1 (i.e. 3 weeks after the last immunization; 37.61 ± 2.39 vs. 14.43 ± 0.43, P < 0.05). Moreover, group 2 had a higher IFN-γ/IL-4 ratio compared to group 1, suggesting a shift toward Th1 response. In addition, in the present study, induced immune responses were long lasting and stable after 9 weeks of the last immunization.

Conclusions:

Evaluation of multi-epitope DNA and peptide-vaccines confirmed their specific immunogenicity in BALB/c mice. However, lower Th1 immune responses in mice immunized with DNA vaccine suggests further investigations to improve the immunogenicity of the multi-epitope DNA vaccine through immune enhancers.     

Review of current thermal ablation treatment for lung cancer and the potential of electrochemotherapy as a means for treatment of lung tumours

Lung cancer remains the most common cancer diagnosed worldwide and has one of the lowest survival rates of all cancers. Surgery remains the only curative treatment option but because most patients are either diagnosed at advanced stages or are unfit for surgery, less than a third of all lung cancer patients will undergo a surgical resection. Thermal ablation has emerged as an alternative option in patients who are unfit to undergo surgery. Thermal ablative therapies used in clinical practice to date include Radiofrequency Ablation (RFA), Microwave Ablation (MWA) and Cryoablation This article will focus on the advantages and limitations of thermal ablative therapy and investigates the potential of a relatively new treatment modality, Electrochemotherapy (ECT), as a novel treatment for lung cancer.     

电脉冲疗法治疗恶性肿瘤的研究进展

肿瘤治疗方法主要有外科、化疗、放疗、免疫治疗及它们的联合使用。近20年来,电脉冲疗法因其抑瘤效率高、参数容易控制、无残余毒性等特点,受到国内外专家学者的广泛关注。