中心组合设计法优化载基因壳聚糖纳米粒的最佳转染制备区域

目的：采用中心组合设计法优化载基因壳聚糖纳米粒的最佳转染制备区域。方法采用复凝聚法制备载质粒基因的壳聚糖纳米粒,选择壳聚糖浓度和质粒基因浓度作为实验考察因素,应用两因素五水平中心组合设计优化最佳转染制备区域,优化指标选择平均粒径和基因转染率。通过透射电镜观察纳米粒的形态;通过动态光散射和电泳光散射技术分别测量纳米粒的粒径和Zeta电位;通过凝胶电泳分析考察质粒在纳米粒制备过程中的稳定性;通过倒置荧光显微镜观察质粒基因在细胞内的表达;通过流式细胞技术测定纳米粒的转染效率。结果成功优化了载基因壳聚糖纳米粒的最佳转染制备区域。优选条件下制备的纳米粒大多呈球形,纳米粒平均粒径为217．6 nm,粒径多分散系数为0．241,表明粒径分布较窄。纳米粒zeta电位为＋22．4 mV,表明纳米粒表面带有正电荷,可以增加纳米粒混悬液的稳定性。凝胶电泳分析结果表明质粒基因在纳米粒制备过程中没有遭到破坏。纳米粒的细胞转染效率比较高,能够高效地将绿色荧光蛋白质粒基因递送到细胞内,并且基因表达产生绿色荧光蛋白。结论本研究建立的数学模型具有良好的预测性。在优化的制备区域内制备的载基因壳聚糖纳米粒的转染性能比较理想。     

鼠疫菌9.5kb质粒对假结核菌粘附、侵袭上皮细胞能力的影响

用电穿孔方法把克隆的鼠疫菌９．５ｋｂ质粒转移到不同血清型的假结核菌中，研究了不同实验条件下９．５ｋｂ质粒对假结核粘附、侵袭上皮细胞（Ｈｅｐ－２）能力的影响。结果显示，尽管９．５ｋｂ质粒不能明显改变假结核菌的粘附、侵袭特征，但相对于亲本株而言，转化后的菌体在所有实验条件下侵袭上皮细胞的能力确定有所下降，表明９．５ｋｂ质粒介导的粘附、侵袭机制有别于假结核菌。由此，分析和评价了９．５ｋｂ质粒在粘附、侵袭     

日本血吸虫(大陆株)14-3-3 epsilon同型信号转导蛋白诱导小鼠保护性免疫的研究

目的：探索日本血吸虫新的疫苗候选分子对小鼠的保护性免疫效果。方法：利用已构建的真核表达质粒pBK-Sj14-3-3在大肠杆菌ＢＬ２１内表达的日本血吸虫（大陆株）14－3－3epsilon同型融合蛋白质，免疫6周龄BALB／c雌性小鼠，免疫3次，每次间隔2周，第3次免疫后2周感染尾蚴。42d后剖杀小鼠，计算其减虫率和肝减卵率。结果：各免疫组与对照组相比，均获得了部分抗血吸虫保护性免疫力，第1、2、3各免疫组的减虫率分别为32．7％（P＜0．05）、30．7％（P＜0．05）、27．5％（P＜0．05）；其肝减卵率分别是48．5％（P＜0．05）、43．1％（P＜0．05）、28．2％（P＜0．05）。结论：首次证明日本血吸虫14－3－3epsilon同型重组融合蛋白可诱导小鼠抗血吸虫感染的部分保护性免疫力。     

Mash1 efficiently reprograms rat astrocytes into neurons简

To date, it remains poorly understood whether astrocytes can be easily reprogrammed into neurons. Mashl and Brn2 have been previously shown to cooperate to reprogram fibroblasts into neurons. In this study, we examined astrocytes from 2-month-old Sprague-Dawley rats, and found that Brn2 was expressed, but Mashl was not detectable. Thus, we hypothesized that Mashl alone could be used to reprogram astrocytes into neurons. We transfected a recombinant MSCV-MASH1 plasmid into astrocytes for 72 hours, and saw that all cells expressed Mashl. One week later, we observed the changes in morphology of astrocytes, which showed typical neuro- nal characteristics. Moreover, β-tubulin expression levels were significantly higher in astrocytes expressing Mashl than in control cells. These results indicate that Mashl alone can reprogram astrocytes into neurons.     

Toxin Kid uncouples DNA replication and cell division to enforce retention of plasmid R1 in Escherichia coli cells

Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid–bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum β-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs.Plasmids serve as extrachromosomal DNA platforms for the reassortment, mobilization, and maintenance of antibiotic resistance genes in bacteria, enabling host cells to colonize environments flooded with antimicrobials and to take advantage of resources freed by the extinction of nonresistant competitors. Fueled by these selective forces and aided by their itinerant nature, plasmids disseminate resistance genes worldwide shortly after new antibiotics are developed, which is a major clinical concern (1–3). However, in antibiotic-free environments, such genes are dispensable. There, the cost that plasmid carriage imposes on cells constitutes a disadvantage in the face of competition from other cells and, because plasmids depend on their hosts to survive, also a threat to their own existence.Many plasmids keep low copy numbers (CNs) to minimize the problem above, because it reduces burdens to host cells. However, this also decreases their chances to fix in descendant cells, a new survival challenge (4). To counteract this, plasmids have evolved stability functions. Partition systems pull replicated plasmid copies to opposite poles in host cells, facilitating their inheritance by daughter cells (5). Plasmids also bear postsegregational killing (PSK) systems, which encode a stable toxin and a labile antitoxin (TA) pair that eliminates plasmid-free cells produced by occasional replication or partition failures. Regular production of the labile antitoxin protects plasmid-containing cells from the toxin. However, antitoxin replenishment is not possible in cells losing the plasmid, and this triggers their elimination (5).TA pairs are common in plasmids disseminating antibiotic resistance in bacterial pathogens worldwide (2, 6–10). The link of these systems to PSK and the exiguous list of alternatives in the pipeline have led some to propose that chemicals activating these TA pairs may constitute a powerful antibiotic approach against these organisms (5, 11–13). However, the appropriateness of these TA pairs as therapeutic targets requires unequivocal understanding of their function in plasmids. Although PSK systems encode TA pairs, not all TA pairs might function as PSK systems, as suggested by their abundance in bacterial chromosomes, where PSK seems unnecessary (14–16). Moreover, the observation that many plasmids bear several TA pairs (6–10) raises the intriguing question of why they would need more than one PSK system, particularly when they increase the metabolic burden that plasmids impose on host cells (17). Because PSK functions are not infallible, their gathering may provide a mechanism for reciprocal failure compensation, minimizing the number of cells that escape killing upon plasmid loss (5). Alternatively, some TA pairs may stabilize plasmids by mechanisms different from PSK, and their grouping might not necessarily reflect functional redundancy (18).This may be the case in plasmid R1, which encodes TA pairs hok-sok (host killing-suppressor of killing) and kis(pemI)-kid(pemK) (19–23). Inconsistent with PSK, we had noticed that activation of toxin Kid occurred in cells that still contained R1, and that this happened when CNs were insufficient to ensure plasmid transmission to descendant cells. We also found that Kid cleaved mRNA at UUACU sites, which appeared well suited to trigger a response that prevented plasmid loss and increased R1 CNs without killing cells, as suggested by our results. In view of all this, we argued that Kid and Kis functioned as a rescue system for plasmid R1, and not as a PSK system (24). This proposal cannot be supported by results elsewhere, suggesting that Kid may cleave mRNA at simpler UAH sites (with H being A, C, or U) (25, 26), a view that has prevailed in the literature (14, 16, 27–29). Moreover, other observations indicate that our past experiments may have been inappropriate to conclude that Kid does not kill Escherichia coli cells (30, 31). Importantly, Kid, Kis, and other elements that we found essential for R1 rescue are conserved in plasmids conferring resistance to extended-spectrum β-lactamases, a worrying threat to human health (1, 6–10, 32). Therapeutic options to fight pathogens carrying these plasmids are limited, and activation of Kid may be perceived as a good antibiotic alternative. Because the potential involvement of this toxin in plasmid rescue advises against such approach, we aimed to ascertain here the mode of action; the effects on cells; and, ultimately, the function of Kid (and Kis) in R1.     

Trends and molecular characteristics of carbapenemase-producing Enterobacteriaceae in Japanese hospital from 2006 to 2015

BackgroundThe increasing number of carbapenemase-producing Enterobacteriaceae (CPE) has become a global problem. Most carbapenemases detected in Japan are imipenemase, which is an imipenem-degrading enzyme with low ability; thus, CPE could have been overlooked. Therefore, this study aimed to detect and analyze CPE, without overlooking CPE showing the low minimum inhibitory concentration phenotype.MethodsCPE screening was conducted on 531 ceftazidime-resistant Enterobacteriaceae isolated from Kitasato University Hospital during 2006–2015. We confirmed the presence of the carbapenemase genes (bla_IMP, bla_VIM, bla_KPC, bla_NDM, and bla_OXA-48) by multiplex polymerase chain reaction. The detected CPE strains were analyzed by antimicrobial susceptibility testing, multilocus sequence typing, conjugal experiments, replicon typing, and plasmid profiling by restriction enzyme treatment.ResultsThe CPE detection rate in Kitasato University Hospital within the past 10 years was 0.0003% (nine CPE strains). These nine CPE strains were identified to harbor 8 bla_IMP-1 or 1 bla_NDM-5. The CPE strains consisted of five species including Klebsiella pneumoniae and Citrobacter freundii. Six of eight bla_IMP-1 were coded by IncHI2 plasmid, and the other two were coded by IncA/C plasmid. Plasmid profiling revealed that K. pneumoniae and C. freundii isolated from the same patient harbored the same plasmid.ConclusionThe CPE detection rate in this study was significantly lower than those previously reported in Japan. In one case, IncA/C plasmid transmission through different bacterial species within the body was speculated. Although the number of CPE detected was low, these results indicated that the resistance plasmid could spread to other bacterial species.     

携带融合基因穿梭质粒的构建及在人肝癌细胞中的表达

构建携带胸苷激酶(tk)与增强型绿色荧光蛋白(EGFP)融合基因穿梭质粒,并观察其在肝癌细胞系HepG2细胞中的表达。应用基因重组技术,构建EGFP与tk的融合表达穿梭质粒载体,经限制酶切鉴定和测序分析,脂质体转染将其导入HepG2中,48h后用荧光显微镜观察荧光的表达,RT-PCR法检测融合蛋白tk和EGFP的mRNA表达,四甲基偶氮唑蓝(MTT)实验检测转染融合蛋白载体后不同浓度的更昔洛韦(GCV)对HepG2细胞的细胞毒作用。酶切鉴定和测序分析证实重组质粒中插入融合目的基因片断及载体DNA大小、方向和插入位点正确,在转染此穿梭质粒的HepG2细胞中检测到绿色荧光蛋白的表达,RT-PCR检测到融合蛋白tk和EGFP的mRNA表达;GCV对转染了携带tk与EGFP融合基因穿梭质粒载体有明显的细胞毒作用。成功构建携带tk与EGFP融合基因穿梭质粒载体,转染人肝癌细胞株HepG2中tk和EGFP的独立表达没有受到影响,该载体可利用绿色荧光蛋白作为报告基因监测tk的表达并可用于肝癌的自杀基因治疗。     

细粒棘球蚴2HSP70重组质粒的构建、原核表达、纯化和初步鉴定

目的构建细粒棘球蚴（Echinococcus granulosus，EG）2热休克蛋白70（heat shock protein，2HSPT0）表达重组质粒，原核表达及纯化重组蛋白，并对重组蛋白进行免疫学特性的初步鉴定。方法从EG2HSP70／pGEM-T／JM109质粒中分离EG2HSP70目的基因，将此片段重组入表达载体pGEX-6P-1后转化入大肠杆菌BL21，鉴定后以IPTG诱导表达，通过SDS-PAGE检测其表达水平，用亲和层析纯化并分离重组蛋白，利用Western blot来鉴定重组蛋白的免疫学特性。结果酶切鉴定及测序分析表明，成功构建细粒棘球蚴pGEX-6P-1／EG2HSP70重组质粒；IPTG诱导表达后，融合蛋白占菌体总蛋白的39％，占裂解物上清总蛋白的70．4%，表明重组的EG2HSP70基因能在BL21中高效表达，表达的蛋白大部分以可溶性状态存在。亲和层析法纯化重组蛋白EG2HSP70，通过Western blot证实该重组蛋白能被细粒棘球蚴囊壁免疫兔血清识别。结论成功构建细粒棘球蚴中国大陆株pGEX-6P-1／EG2HSP70重组质粒，重组的EG2HSP70能够高效表达，表达的EG2HSP70具有免疫学活性。     

Effects of intramyocardial injection of phVEGF-A165 as sole therapy in patients with refractory coronary artery disease--12-month follow-up: angiogenic gene therapy

OBJECTIVE: To test the safety and bioactivity of phVEGF-A165 after intramyocardial injection during 12-month follow-up. DESIGN: Open-labelled study. SUBJECTS: Inclusion criteria were angina pectoris, Canadian Cardiovascular Society (CCS) class III-IV, unamenable to further revascularization, ejection fraction (EF) >30%, perfusion defects extending over >10% of the anterolateral left ventricle wall detectable with adenosine single photon emission computerized tomography (SPECT) and at least one patent vessel visible by coronary angiography. Seven of 39 patients referred for gene therapy were included. INTERVENTION: Via a mini-thoracotomy under general anaesthesia. phVEGF-A165 was injected directly into the myocardium at four sites in the anterolateral region of the left ventricle. RESULTS: Operative procedures were uneventful. Perioperative release of myocardial markers and electrocardiogram (ECG) changes were detected in two patients. There were no perioperative deaths but one patient died 7 months postoperatively because of myocardial infarction. Plasma vascular endothelial growth factor (VEGF)-A levels increased two to threefold peaking 6 days postoperatively (P < 0.004) and returning to baseline by day 30. A significant reduction in angina pectoris was reported. The CCS class improved from 3.3+/-0.2 to 1.9+/-0.3 (P < 0.01) and nitroglycerine intake decreased from 39+/-15 to 12+/-5 tablets week(-1) (P < 0.001) 2 months after gene transfer. Improvements remained after 12 months when nitroglycerine consumption approached zero. Improved myocardial function in the phVEGF-A165 injection region was documented in all patients (P < 0.016) by tissue velocity imaging (TVI). Reduced reversible ischaemia was detected by adenosine SPECT in four patients. Improved collateralization was detected in four patients with coronary angiography. CONCLUSION: Intramyocardial injection of phVEGF-A165 is safe and may lead to improved myocardial perfusion and function with longstanding symptomatic relief in end-stage angina pectoris. Based on these results this therapeutic potential is being tested in a double-blind placebo controlled multicentre trial.     

脑胶质瘤组织中R132H突变型hIDH1基因双顺反子真核表达载体的构建及鉴定

目的以pcDNA3.1(+)为骨架,构建并鉴定人脑胶质瘤组织中含R132H突变的人源异柠檬酸脱氢酶1基因(hIDH1R132H)和增强型绿色荧光蛋白(EGFP)序列的双顺反子真核表达载体。方法以内部核糖体进入位点(IRES)/EGFP片段为模板,扩增出IRES/EGFP片段,并在5’末端引入3×Flag标签;通过PCR介导的定点突变法,以pCMV-sports6-hIDH1为模板,扩增hIDH1R132H基因片段,并在3’末端引入3×Flag标签;通过PCR连接两种产物后经双酶切定向克隆入pcDNA3.1(+)骨架;转化大肠杆菌后挑取阳性克隆,提取质粒进行PCR检测及基因序列测定。结果 PCR检测7个菌落中有6个阳性克隆,DNA测序发现引入了hIDH1基因。结论成功构建了双顺反子真核表达载体pcDNA3.1(+)-hIDH1R132H/3×Flag/IRES/EGFP,为研究人胶质瘤组织中R132H突变的作用机制奠定了基础。