Electroporation enhances protective immune response of a DNA vaccine against Japanese encephalitis in mice and pigs

Japanese encephalitis virus (JEV) is a pathogenic cause of Japanese Encephalitis (JE), which is a zoonotic disease transmitted by mosquitoes and amplified by pigs. Infection of JEV may lead to severe neurological sequelae, even death in humans and reproductive disorders in pigs. Vaccination is the only way to control JEV infection in humans. For pigs play important role in the JEV transmission cycle, developing a new veterinary vaccine is considered as a useful strategy for cutting off the transmission route of JEV. We have previously reported that DNA vaccine pCAG-JME, expressing prM-E proteins of JEV, is effective in mice through intramuscular injection (IM). However, the poor immunogenicity, due to low expression of immunogen, is the major obstacle for the development of DNA vaccine in large animals. In the present study, therefore, we immunized mice and pigs with pCAG-JME intramuscularly accompanied with electroporation (EP) stimulation, the attractive gene delivery approach. As compared with IM, EP-mediated vaccination markedly increased the expression of immunogen in the injection site and induced a dose- and time-dependent immune response. 100% survival rate was observed in groups vaccinated with doses ranged from 10 to 100μg, indicating that 10μg of DNA with EP for individual was enough for inducing effective protection in mice. Surprisingly, survival rate and end-point titers of anti-JEV antibodies were higher in mice even at lower dose of DNA (5μg) than that in mice inoculated 100μg through IM. Notably, the prM-E antigens also induced high antibody response in pig, while the neutralizing antibody titer achieved 1:320. Our results suggested that EP-mediated DNA immunization might act as an effective strategy against JEV, at least in pig, and that EP has a potential application prospect in DNA vaccination.     

产黄青霉简单高效遗传转化法——电激转化法

目的 研究产黄青霉(P.chrysogenum)电激转化方法,建立产黄青霉快速、高效的遗传转化方法.方法 应用美国Bio-Rad电转仪,将外源DNA转化入产黄青霉菌丝中,研究了菌丝培养时间、电场强度、DNA类型等对遗传转化的影响.结果菌丝培养24h,处于旺盛生长阶段,电场强度为6000V/cm,电阻200Ω,电容20μF,环状质粒DNA较线状能获得转化效率稍高,1μg DNA获得的克隆数可达500个.结论 应用电激转化方法对产黄青霉进行遗传转化属首次报道,特别是可直接将目的片段与T-DNA相融合,转化入产黄青霉中,该方法操作简单,快速高效.     

A novel plasmid DNA electroporation method allows transfection of murine DC

Under steady state conditions dendritic cells (DC) exert tolerogenic function, but acquire potent immunogenic function due to strong upregulation of costimulatory molecules and proinflammatory cytokines. In numerous studies the potential of modified DC to induce tolerance or immune reactions towards a distinct antigen has been demonstrated. However, DC are refractory to transfection with plasmid DNA by non-viral methods. In this study we have tested the suitability of a newly developed electroporation device to transfect immature murine bone-marrow derived DC (BM-DC). Transfected BM-DC expressed reporter molecules at considerable extent which renders this method suitable to perform all kinds of promoter studies. While electroporation did not alter the low allostimulatory capacity of immature BM-DC, it impaired the stimulation-associated increase in allostimulatory potency of transfectants. However, stimulated transfected BM-DC pulsed with myelin oligodendrocyte protein (MOG)-derived peptide induced proliferation of MOG-reactive CD4⁺ T cells as potently as did non-transfected MOG peptide-pulsed BM-DC. BM-DC transfected with an expression construct encoding MOG efficiently stimulated MOG peptide-specific T cell proliferation. Transfection of BM-DC with an IL-10 encoding expression construct resulted in high IL-10 expression and strongly diminished allogeneic T cell proliferation. Therefore, this method also allows to study functional properties of genetically altered DC.     

不同类型的转基因细胞核供体对生产小鼠转基因克隆胚胎的影响

目的 利用体细胞核移植(SCNT)技术生产小鼠转基因克隆胚胎. 方法 利用所构建的含新霉素抗性(Neor)基因和增强型绿色荧光蛋白(EGFP)基因的双标记选择载体, 通过电穿孔的方法, 分别转染小鼠胎儿成纤维细胞和小鼠胚胎干细胞,经G418筛选14d后用于核移植.同时以未转基因小鼠卵丘细胞和成纤维细胞为核供体,进行体细胞核移植. 结果 转基因与未转基因胎鼠成纤维细胞的重构胚的囊胚(154/160)发育率为19.23%和22.91%,差异不显著(P＞0.05);转基因胚胎干细胞与胎鼠成纤维细胞的重构胚的囊胚(152/154)发育率为41.54%和19.23%,差异显著(P＜0.05);未转基因卵丘细胞与成纤维细胞的重构胚的囊胚(171/160)发育率为41.17%和22.91%,差异显著(P＜0.05). 结论 利用体细胞核移植技术,小鼠胎儿成纤维细胞和胚胎干细胞可以有效地生产小鼠转基因囊胚.     

弓形虫转基因学研究进展

转基因技术在弓形虫的运用加速了对弓形虫功能基因组学的研究。本文对转基因弓形虫载体的构建、转基因方法和转基因弓形虫技术在弓形虫研究中的应用进展加以综述。     

电穿孔介导的基因治疗对兔下颌骨牵引区新生骨骨密度与强度的影响

目的 探索电穿孔介导的基因治疗对下颌骨DO过程中牵引间隙新生骨密度与强度的影响,从而为促进下颌骨DO新骨生成,缩短牵引周期,减少并发症提供新思路.方法 以新西兰大白兔为实验动物模型,于术后3 d开始下颌骨牵引,每天0.8 mm,连续牵引7 d后,将实验动物分为5组.A组:在牵引区注射2 μg(O.1μg/μl)重组质粒pIRES-hVEGF165-hBMP2;B组:在牵引区注射2 μg(0.1μg/μl)重组质粒pIRES-hBMP2;C组:在牵引区注射2 μg(0.1μg/μl)重组质粒pIRES-hVEGF165;D组:在牵引区注射2 μg(0.1μg/μl)空质粒pIRES;E组:在牵引区注射相同剂量的生理盐水.5组实验动物均施加电穿孔刺激.各组分别于固定期第1、2、4、8周行X线及QCT检查.选整个牵张间隙新生骨痂部分为兴趣区,测定骨密度.然后处死动物.取材测量牵引区新生骨的三点抗压强度.结果 A、B、C组新生骨痂密度各时相点新生骨痂密度明显高于D、E组(P<0.01).固定2周,A组明显高于各组,但B、c组间比较差异无统计学意义.固定4周,A、B组明显高于C、D、E组(P<0.01).固定8周A组明显高于B、c、D、E组(P<0.01).B、C组间比较差异无统计学意义,但高于D、E组(P<0.01).固定4周,A组新生骨的三点抗压强度明显高于B、C、D、E组(P<0.01).固定8周,A组仍明显高于各组(P<0.01),且B组也明显高于c、D、E组(P<0.05).结论 电脉冲介导的pIRES-hVEGF165-hBMP2重组质粒体内转染可使牵引区获得较满意的骨再生和骨化成熟进程,其新骨骨化、改建过程均超过对照组.提示联合应用BMP与VEGF,可能会实现成骨与血供的联合重建,并且使单一生长因子的效应放大,使骨愈合的速度加快.     

甲状腺癌细胞系方波电脉冲基因转染条件的探讨

目的：探讨方波脉冲基因电转染对人甲状腺癌细胞系的转染条件和效果。方法：选用pEGFP-Cl作为外源基因与方波电脉冲相结合，以人甲状腺癌细胞系TA-K为导入对象，探讨方波脉冲基因电转染对人甲状腺癌细胞系转染的脉冲幅度，脉冲时值，脉冲次数和反应体系大小。结果：当脉冲幅度在60V／mm时，开始出现阳性细胞，脉冲时值20ms时，基因转染效率可高达35％，细胞生存分数55％，脉冲时值20ms、次数1次转染效率高、细胞死亡率低。200μl混合液电转染后观察，对细胞生存影响不大。结论：脉冲幅度在60V／mm，脉冲时值20ms，脉冲次数1次，反应体系200μl，可以为人甲状腺癌细胞系的转染提供良好效果。     

Efficient gene delivery to articular cartilage using electroporation

Effective in vivo gene transfer into articular cartilage has not yet been established. Since chondrocytes are embedded within a rich extracellular matrix, various gene transfer methods have failed to introduce genes into deeper layers of the articular cartilage. In this study, we developed new superfine pointed needle electrodes for in situ electroporation (EP), and investigated the efficiency of gene transfer into articular cartilage with different degrees of degeneration. Full-thickness articular cartilage slices were obtained from the knee joint of a 3–4-month-old rabbit. The cartilage tissues were treated briefly with trypsin to partly remove matrix proteoglycan. Human articular cartilage with different grades of degeneration was also used. For EP, the articular cartilage surface was soaked in a solution containing green fluorescent protein (GFP) plasmid. Then, the superfine pointed 7-needle electrodes were gently stabbed into the surface layer of the articular cartilage and the gene was transfected by an electroporator. GFP expression was examined by immunohistochemical analysis. Cartilage tissue was successfully transfected with the GFP gene by the electrodes and EP. Transfection efficiency was enhanced by depleting the matrix proteoglycan in rabbit articular cartilage. Chondrocytes in the deeper layer of the articular cartilage were also transfected and expressed GFP. In human osteoarthritic cartilage, ca. 30% of the cells in the deeper layer were transfected by selecting optimal EP conditions. No adverse effects of EP on damaged articular cartilage were obvious from histological analysis or TUNEL staining. The results indicated that EP-mediated in vivo gene transfer into articular cartilage may provide a useful therapeutic strategy to treat cartilage degeneration.     

Irreversible electroporation of pancreatic cancer – Effect on quality of life and pain perception

BackgroundMost pancreatic cancer (PC) patients are incurable and may need palliative treatment at some point in time. Irreversible electroporation (IRE) is a novel ablative treatment, which aims to provide local tumor control. The aim of this study was to examine how consolidative treatment with IRE affects quality of life (QOL) and pain perception (PP) in patients with non-metastatic pancreatic cancer.MethodsSecondary outcomes were extracted from a prospective cohort of non-metastatic PC patients treated with IRE from 2013 to 2019. Patients filled in two questionnaires examining QOL and PP at different timepoints during treatment and follow-up. Data from a selected panel of subscales were extracted and analyzed using a mixed random intercept regression model.ResultsSubscales from 41 patients at four different timepoints were included in the model. Global health status, physical functioning, fatigue, nausea and vomiting, appetite loss and mean pain interference were negatively impacted (p < 0.05) in the short- and mid-term, corresponding to a low or moderate clinical effect size. However, all negative effects showed a tendency to dissipate over time.ConclusionsIRE treatment negatively impacted QOL and PP in the short- and mid-term. No positive long-term effects of IRE were found.     

Radiotherapy Enhancement with Electroporation in Human Intestinal Colon Cancer HT-29 Cells

Background: The efficiency of radiotherapy for tumors can be enhanced with different radiosensitizers. Previousstudies have shown that electroporation (EP) can sensitize some cancer cell lines to ionizing radiation (IR). HT-29is a radiation resistant colorectal cancer cell line, representative of a cancer type which is the second cause of cancermortalities in developed countries. The present study aimed to evaluate radiosensitizing effects of EP on HT-29 cellsin vitro exposed to 6 MV X-ray photon beams. Methods: HT-29 cells were exposed to a 6 MV X-ray photon beamas the control or to a combination of electroporation and irradiation. The response of cells was evaluated by colonyformation assay and survival curves. Results: The survival fraction of the HT-29 cells was significantly decreased byelectroporation prior to radiotherapy. A single electric pulse increased colorectal HT-29 cancer cell sensitivity to megavoltageradiation by a factor of 1.36. Conclusion: Our findings showed that EP before radiotherapy can significantlyenhance tumor cell sensitivity. This combined treatment modality should be assessed for its applicability in clinic settingsfor employment against radioresistant cancers. However, to facilitate achieving this goal, many different tumors witha broad range of radiosensitivities should be evaluated.