CRISPR技术在蚊媒传染病检测中的研究进展

蚊媒传染病威胁着世界上近一半的人口,开发快速灵敏的检测技术是及时管控感染者及遏制蚊媒传染病传播的关键,但目前常用的检测技术多存在检测时间长、资源耗费多、条件要求高等缺点,基于CRISPR-Cas系统的检测技术准确度高、灵敏性强。本文重点关注CRISPR技术在疟疾、登革热、寨卡病毒病等蚊媒传染病检测中的研究进展。     

CRISPR技术在蚊媒传染病防控中的应用

蚊媒会传播多种疾病,为全球存在极大危害的一类公共卫生问题.这些年,不断有新的虫媒传染病出现,极大提高了蚊媒传染病的控制难度.受到基因编辑技术出现、发展的影响,CRISPR技术也随之出现,为蚊媒生长、发育、繁殖、传播疾病等多个方面的基础性分子生物研究配备了靶标特异性的修饰工具,实现了对蚊媒传染病防控技术的创新.文章围绕当...     

基因敲除技术及其在肝病研究中的应用

基因敲除技术是研究基因功能的重要手段,是探讨基因在疾病中作用机制的一个非常有效的研究工具.近年来该技术在肝病研究领域得到普遍应用,尤其是条件性基因敲除技术及RNA干扰的出现,进一步深化了肝病的研究.此文介绍基因敲除的基本原理及其在肝病研究中的应用.     

CRISPR/Cas9基因编辑技术对抗病毒潜伏感染的应用进展

目前抗病毒治疗主要靶向产毒阶段的病毒蛋白酶系统,而对于具有潜伏感染特征的病毒无效,因此能够靶向病毒基因组的抗病毒药物将是未来对抗病毒潜伏感染的研究策略.CRISPR/Cas9作为一种近年快速发展的基因编辑技术,在对抗病毒潜伏感染方面取得一定的成果,如HIV、人致病疱疹病毒、HBV等.此文对其应用进展进行综述,并总结了逃逸突变体的产生、脱靶效应、载体安全性等问题,以期为研制有效的抗潜伏感染病毒的治疗方法提供参考依据.     

腺相关病毒载体在基因治疗中的应用及研究进展

腺相关病毒对人没有致病性,并且能够进行特殊位点的整合,在多种组织、细胞中均证实可以有效转染,因此,在临床基因治疗方面日益受到重视。目前通过不断的发展、改造病毒载体的基因结构,扩大其载体容量,提高病毒产率和转染效率,克服体内的免疫反应,以使腺相关病毒载体更加符合实际需要。     

基因敲除在雌性小鼠生殖系统研究中的应用

基因敲除技术是20世纪80年代后半期应用DNA同源重组技术发展起来的,在对基因功能、发育生物学和疾病的研究中发挥了重要作用。直到现在,运用同源重组实现基因敲除依然是构建基因敲除动物模型中最普遍最经典的方法。基因敲除动物模型中应用最广泛的是基因敲除小鼠模型。综述目前国内外实现基因敲除的几种重要方法以及利用同源重组实现的基因敲除技术在雌性小鼠生殖系统研究中的应用。这些应用主要包括研究性激素对生殖器官及激素依赖性疾病的影响、细胞分泌因子在发育期调控生殖器官组织的机制及胚胎形成后的性腺发育与成熟缺陷等。     

通过CRISPR Cas/9系统敲除ICP227蛋白基因抑制单纯孢疹病毒HSV-1的复制

CRISPR-Cas9基因编辑技术是基于细菌或古细菌CRISPR介导的获得性免疫系统衍生而来,由一段RNA通过碱基互补配对识别DNA,指导Cas9核酸酶切割识别的双链DNA,诱发同源重组或非同源末端链接,进而实现在目的 DNA上进行编辑.病毒通过特异的受体侵染细胞[1],其基因组在细胞内发生复制、转录、翻译等过程完成其生活周期,某些DNA病毒或逆转录病毒基因组会整合到宿主基因组中.基因治疗是病毒感染疾病治疗的新趋势,因此,基因编辑技术在持续感染的病毒或潜伏感染病毒疾病治疗中具有重大的潜在意义.本文我们利用CRISPR-Cas9敲除HSV-1病毒复制过程中重要基因,从而抑制病毒的复制,对病毒感染的治疗中有重要意义.     

携带人肿瘤抑素Tumstatin基因的重组腺相关病毒载体的构建及其在乳腺癌细胞中的表达研究

目的构建携带人肿瘤抑素Tumstatin基因的重组腺相关病毒载体,研究其在乳腺癌细胞MCF-7中的表达。方法以人胚肾293细胞为材料,用RT-PCR方法获取人肿瘤抑素Tumstatin基因,将其克隆入pAAV-MCS载体中形成重组载体pAAV-Tum质粒。用脂质体介导法将重组载体导入乳腺癌细胞株(MCF-7)建立一株稳定的表达Tumstatin基因的乳腺癌细胞株MCF/AAV-Tum,并用RT-PCR检测该细胞系中Tumstatin基因的表达。结果成功构建了pAAV-Tum重组腺相关病毒载体,筛选出乳腺癌细胞株MCF/AAV-Tum,该细胞能表达Tumstatin基因。结论构建的pAAV-Tum重组载体能在MCF-7细胞中表达,为其后的肿瘤抗血管生成治疗研究奠定了基础。     

AAV介导NGF过表达载体转染BMSCs对高强度聚焦超声致活体SD大鼠坐骨神经损伤模型保护作用

目的干细胞是当前研究的热门领域,利用干细胞定向分化作用可以促进神经损伤修复,但是干细胞具有分化方向不定和易老化的特点。本研究探究腺相关病毒(adeno associated virus,AAV)介导神经生长因子(nerve growth factor,NGF)过表达载体转染骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)对高强度聚焦超声致活体SD大鼠坐骨神经损伤模型的保护作用。方法分离和培养人源BMSCs,采用流式细胞仪检测BMSCs的CD29,CD45和CD90等表面蛋白表达。采用免疫组织化学染色鉴定BMSCs表面CD34和CD44表达。AAV腺病毒构建NGF过表达质粒,通过高强度聚焦超声制备SD大鼠坐骨神经损伤模型,根据处理方式不同,将SD大鼠分成空白对照组,模型组,BMSCs组和NGF过表达组。利用HE染色,电镜方法观察坐骨神经形态,示踪金染色观察神经接头的聚集,TUNEL染色检测细胞凋亡,RT-qPCR检测NGF和caspase-3mRNA表达。结果流式细胞仪检测第2代BMSCs表面蛋白,CD29阳性率为98.03%,CD90阳性率为96.92%,CD45阴性率为77.2%,共表达CD29、CD90且CD45表达阴性的细胞数占总细胞数的96.89%。BMSCs细胞表面CD34和CD44免疫组织化学染色呈阳性。慢病毒转染效率>90%,转染效率较高。HE染色结果中模型组大鼠坐骨神经有明显断裂,BMSCs组大鼠坐骨神经较模型组裂痕缩小,而NGF过表达组断裂处几乎不可见。电镜结果显示,模型组大鼠坐骨神经密度少,且形态不规则,BMSCs组大鼠坐骨神经较模型组密度大,且周边为纤维组织填充,NGF过表达组大鼠坐骨神经密度大,排列较为规则。示踪金染色显示NGF过表达组神经接头处可见神经聚集。RT-qPCR结果显示空白对照组、模型组、BMSCs组和NGF过表达组NGF mRNA相对表达量分别为1.18±0.05、0.77±0.06、1.75±0.07和3.09±0.08,F=9.082,P=0.019;空白对照组、模型组、BMSCs组和NGF过表达组caspase-3mRNA表达量分别为2.03±0.09、5.05±0.23、3.12±0.09和1.23±0.22,F=9.177,P=0.011。空白对照组、模型组、BMSCs组和NGF过表达组细胞凋亡率分别为(9.34±0.32)%、(31.24±3.43)%、(22.09±2.66)%和(4.97±1.05)%,F=9.664,P=0.014。结论腺病毒介导NGF过表达质粒转染BMSCs可以促进坐骨神经损伤大鼠坐骨神经修复,其机制可能是通过升高NGF mRNA,降低caspase-3mRNA水平实现。     

终末期肝病模型在肝病中的应用进展

终末期肝病模型(modelforend_stageliverdisease,MELD)是主要应用血清胆红素、凝血酶原时间国际标准化比值和血清肌酐指标来评价的系统。其在预测终末期肝病死亡率及肝移植中的应用已渐趋成熟,应用范围也开始扩大到重型肝炎、肝癌中。部分学者针对腹水等严重的肝病并发症对MELD评分的影响做了相应研究,并对血钠浓度在预测肝病患者死亡率中的作用作了进一步的研究。文中对MELD作了回顾,并对最新进展进行阐述。     

全肠外营养相关肝损害小鼠模型的建立

目的:为进一步研究肠外营养(PN)相关肝损害(PNALD)的患病机制及干预措施,建立有效的小鼠模型。方法:将20只小鼠随机分为对照组和模型组,对照组小鼠正常饮食、颈静脉置管后微量泵持续输注等渗盐水;模型组小鼠禁食后颈静脉置管,用微量泵持续输注PN液。1周后比较两组小鼠体重变化,血清生化指标以及肝组织学改变。结果:模型组小鼠体重明显低于对照组(P<0.05),直接胆红素、总胆红素和胆固醇均显著高于对照组(P<0.05),且肝组织在光镜下可见广泛性肝细胞脂肪变性,细胞质内出现大小不一的空泡,主要集中于中央静脉周围。脂肪变性评分为(3.1±0.5)分,显著高于对照组(1.0±0.0)分。结论:模型组小鼠与成人PNALD病人初期的临床和病理改变相似,可用于该病的患病机制,药物治疗疗效以及具体机制的观察和研究。     

Development of a vectored vaccine against Hepatitis E virus

Hepatitis E virus is a non-enveloped ssRNA virus [1] that causes human acute hepatitis through primarily fecal and oral transmission [2]. Currently, no commercial hepatitis E (HEV) vaccine is available. In the absence of an appropriate cell culture system for HEV propagation, HEV pseudocapsids (ORF2 protein) have been produced either in Escherichia coli or in insect cells and they have been shown to protect monkeys against virus challenge and to be effective in the prevention of natural HEV infection of humans.     

Evaluation of Lassa virus vaccine immunogenicity in a CBA/J-ML29 mouse model

Lassa fever (LF) is one of the most prevalent viral hemorrhagic fevers in West Africa responsible for thousands of deaths annually. The BSL-4 containment requirement and lack of small animal model to evaluate Lassa virus (LASV)-specific cell-mediated immunity (CMI) complicate development of effective LF vaccines. Here we have described a CBA/J-ML29 model allowing evaluation of LASV-specific CMI responses in mice. This model is based on Mopeia virus reassortant clone ML29, an attractive immunogenic surrogate for LASV. A single intraperitoneal (i.p.) immunization of CBA/J mice with ML29 protected animals against a lethal homologous intracerebral (i.c.) challenge with 588 LD(50). The ML29-immunized mice displayed negligible levels of LASV-specific antibody titers, but LASV-specific CMI responses were detectable early and peaked on day 8-10 after immunization. A T cell cytotoxicity assay in vivo showed a correlation between LASV-specific cytotoxicity and the timing of protection induced by the ML29 immunization. Notably, CBA/J mice that received CD8+ T cell-depleted splenocytes from ML29-immunized donors all succumbed to a lethal i.c. challenge, demonstrating that CD8+ T cells are critical in protection. The CBA/J-ML29 model can be useful immunological tool for the preliminary evaluation of immunogenicity and efficacy of vaccine candidates against LASV outside of BSL-4 containment facilities.     

MELD分级与Child分级对328例肝硬化患者临床评估的比较

目的比较MELD和Child分级评估差异,分析影响预后的危险因素。方法回顾328例肝硬化患者2.5年,计算入院当天MELD和Child分值,通过ROC曲线及截断值比较分析,利用Spearman等级相关检验研究两体系相关性。结果3个月生存期的患者两体系ROC曲线面积有显著差异(P<0.05),而生存期超过1年的患者MELD较Child分级无明显差异(P>0.05)。短期内MELD>13,敏感性为74.7%,特异性97.8%;胆红素>2.8 mg/dl,敏感性为53.7%,特异性93.5%;INR>1.53,敏感性为48.8%,特异性93.9%;有腹水,敏感性为67.1%,特异性58.9%。两体系显著相关(r=0.785,P<0.01)。结论MELD和Child均精确评估各生存期预后;MELD适宜评估急、危重患者,预后超过1年的患者无明显优势。MELD>13,胆红素>2.8 mg/dl,INR>1.53及有腹水均提示应尽快治疗以提高患者短期预后。     

Development of a technique for efficient gene transfer to antral follicular cells in the mouse ovary

Ovarian follicle development is a complex process mediated by interactions between oocytes and surrounding follicular cells. In an ovary, oocytes are ultimately released from Graafian follicles, which develop from antral follicles localized near the surface of an ovary. To examine the molecular interaction between these 2 cell types, direct gene transfer to follicular cells as well as oocytes appears to be a promising approach, but few studies have applied this technique. The aim of the present study was to develop a technique for gene transfer to antral follicle cells based on their accessibility near the surface of an ovary. B6C3F1 (a hybrid between C57BL6/N and C3H/HeN) female mice aged 4 or 8 w were anesthesized and their ovaries were exposed. About 100 nl of a solution containing reporter plasmid DNA (0.5?µg/μl) and 0.1% trypan blue was injected into a follicle using a glass micropipette attached to the mouthpiece. A total of 6 follicles were injected per ovary. After injection, the ovary was immediately subjected to in vivo electroporation (EP) using an electroporator with 8 square electric pulses of 50?ms and 50 V. After 24?h, the treated ovaries were excised to examine the expression of reporter constructs by histochemistry. All the injected follicles expressed reporter genes to different extents. Inspection of cryostat sections of ovaries injected with the lacZ expression plasmid demonstrated that 50–100% of follicular cells within a follicle were successfully transfected. However, there were no oocytes within the antral follicles that were negative for such staining (15 follicles tested). Similar results were obtained when the enhanced green fluorescent protein expression plasmid was introduced. The present method based on in vivo EP was found to be very effective for transfection of follicular cells. This approach might be useful to explore the roles of genes related to oogenesis/folliculogenesis, and for reproductive manipulation targeted to antral follicles.     

Acetyl-l-carnitine and lipoic acid improve mitochondrial abnormalities and serum levels of liver enzymes in a mouse model of nonalcoholic fatty liver disease

Mitochondrial abnormalities are suggested to be associated with the development of nonalcoholic fatty liver. Liver mitochondrial content and function have been shown to improve in oral feeding of acetyl-l-carnitine (ALC) to rodents. Carnitine is involved in the transport of acyl-coenzyme A across the mitochondrial membrane to be used in mitochondrial β-oxidation. We hypothesized that oral administration ALC with the antioxidant lipoic acid (ALC + LA) would benefit nonalcoholic fatty liver. To test our hypothesis, we fed Balb/C mice a standard diet (SF) or SF with ALC + LA or high-fat diet (HF) or HF with ALC + LA for 6 months. Acetyl-l-carnitine and LA were dissolved at 0.2:0.1% (wt/vol) in drinking water, and mice were allowed free access to food and water. Along with physical parameters, insulin resistance (blood glucose, insulin, glucose tolerance), liver function (alanine transaminase [ALT], aspartate transaminase [AST]), liver histology (hematoxylin and eosin), oxidative stress (malondialdehyde), and mitochondrial abnormalities (carbamoyl phosphate synthase 1 and electron microscopy) were done. Compared with SF, HF had higher body, liver, liver-to-body weight ratio, white adipose tissue, ALT, AST, liver fat, oxidative stress, and insulin resistance. Coadministration of ALC + LA to HF animals significantly improved the mitochondrial marker carbamoyl phosphate synthase 1 and the size of the mitochondria in liver. Alanine transaminase and AST levels were decreased. In a nonalcoholic fatty liver mice model, ALC + LA combination improved liver mitochondrial content, size, serum ALT, and AST without significant changes in oxidative stress, insulin resistance, and liver fat accumulation.     

多基因表达系统在口岸流感病毒检测中的应用

目的 多基因表达分析系统(Gene expression Profiler genetic analysis system, GeXP)是一种全新的基因表达分析平台,检测特异性强,检测速度和灵敏度明显提高。本研究利用GeXP系统和多重PCR技术,结合我国口岸检疫查验主要呼吸道传染病现状,建立甲型流感病毒、乙型流感病毒、甲型H1N1病毒的检测技术方法。方法 制备病毒标准品质粒,收集病原体样本,利用GenBank下载3种病毒的核苷酸序列,根据3种病毒的序列特征,利用GeXP eXpress Profiler设计特异性引物,优化条件使GeXP系统能够特异性的检测对应病毒。结果 优化后的引物能够检测3种病毒,产物经GeXP检测,3个峰值位于166.04 bp、193.33 bp、251.42 bp,符合甲型流感病毒、乙型流感病毒、甲型H1N1病毒病原体质粒PCR产物条带大小,特异性强,无交叉反应。对已知临床样本的检测中,检出率高且特异性强。结论 GeXP系统能够用于病原体高通量检测工作,有益于扩展检测途径,提高检测效率。