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。     

丝状真菌基因敲除的研究现状

基因敲除又称目标基因破坏或目标基因替换(Targeted gene replacement,TGR).本文主要对近年来基因敲除在丝状真菌基因功能研究中的应用及优缺点作简要综述,以期为科研工作者提供一定参考.     

RNA干扰在肝病治疗中的研究进展

RNA干扰(RNAi)是双链RNA(dsRNA)分子在mRNA水平关闭相应序列及基因的表达使其沉默的过程[1].RNAi最主要的功能特色在于它可调节和关闭基因的表达,促使mRNA降解或阻止蛋白产物合成,进而调控细胞的各种高级生命活动,其抑制基因表达作用具有高效性和特异性[2].RNAi作为一种快捷方便的工具广泛用于高通量的研究基因功能、基因敲除、基因治疗和基因表达调控领域的研究.现对RNAi在肝病治疗中的最新进展作一综述.     

Cramp基因对小鼠脾脏及胸腺细胞发育的影响

目的研究Cramp基因敲除在衰老过程中对小鼠脾脏及胸腺细胞发育及组成的影响。方法应用流式细胞仪分析不同年龄Cramp基因敲除C57BL/6J小鼠及同龄野生型小鼠的脾脏、胸腺等组织器官中各种细胞的比例。结果与野生型小鼠相比,Cramp基因敲除小鼠的脾脏、胸腺等组织器官中各种细胞的比例均无明显变化。结论本研究中,Cramp基因敲除对不同年龄小鼠脾脏及胸腺等组织器官的细胞发育无明显影响。     

载脂蛋白E基因敲除小鼠主动脉壁胆固醇含量的测定及意义

目的　探讨载脂蛋白E(apoE)基因敲除小鼠主动脉胆固醇含量的检测方法 ,并分析其对apoE基因敲除小鼠动脉粥样硬化斑块面积的评估价值。 方法　分离apoE基因敲除小鼠和正常小鼠主动脉 ,用酶法检测主动脉胆固醇的含量 ;用油红O染色法和图像分析法测量小鼠动脉粥样斑块面积。结果　apoE基因敲除小鼠主动脉胆固醇的含量显著高于正常小鼠 (P <0 .0 1) ,apoE基因敲除小鼠的动脉粥样硬化斑块面积也显著高于正常小鼠 ,且二者之间呈显著性相关 (相关系数r =0 .982 ,P <0 .0 1)。结论　apoE基因敲除小鼠主动脉胆固醇含量与其动脉粥样斑块面积的大小密切相关 ,是apoE基因敲除小鼠动脉粥样硬化斑块形成的一个重要指标。     

VSMC-Cx43-/-条件性基因敲除小鼠模型的构建及基因型鉴定

目的 应用CRISPR-Cas9技术和LoxP-Cre系统构建血管平滑肌缝隙连接蛋白Cx43条件性基因敲除小鼠(VSMC-Cx43-/-)模型.方法 采用受精卵同源重组的方式,对Gja1基因进行flox修饰.通过体外转录的方式,获得Cas9 mRNA和gRNA;通过In-Fusion cloning的方法构建同源重组载...     

小鼠Pkd2基因条件性敲除打靶载体的构建

[目的]构建小鼠Pkd2条件性基因敲除打靶载体,为建立Pkd2基因条件性敲除小鼠模型奠定基础.[方法]以正常小鼠(129×1/SvJ)基因组DNA为模板,扩增小鼠Pkd2基因一段包括第9号外显子的长为9.3kb的序列,通过引入LoxP和Neo基因等步骤,建立条件性敲除Pkd2第9号外显子的条件性基因打靶载体.[结果]经多个限制性核酸酶酶切鉴定和测序证实,所构建的Pkd2基因条件性敲除打靶载体符合设计要求.[结论]成功构建小鼠条件性Pkd2基因敲除打靶载体,为建立Pkd2基因条件性敲除小鼠模型奠定基础.     

生物电阻抗技术在肝病病人人体成分测量中的应用

该文介绍了生物电阻抗技术在肝病中应用的现状,重点介绍了多频节段电阻抗技术的应用.多频节段电阻抗技术可以克服因水肿、腹水而造成的体重对人体组成结果的影响,还可能判定腹水量.相比单频电阻抗技术,其在肝病病人中可能存在较好的应用前景,有助于客观地评价肝病病人的营养状况及能量代谢,但仍需临床进一步研究.     

CRISPR/Cas9基因编辑技术的应用研究进展

近年来,CRISPR/Cas9基因编辑技术经过不断完善和改造,正逐步取代锌指核酸酶(ZFN)技术和转录激活因子样效应物核酸酶(TALEN)技术,成为具有广阔应用前景的第三代基因编辑技术。CRISPR/Cas9技术目前已广泛应用于细胞的基因编辑和基因调节、基因敲除动物模型的构建、人类疾病动物模型的治疗研究等领域。此外,已有相关实验证实该技术能够在人类胚胎中发挥基因编辑的作用,并且国内外已有2项CRISPR/Cas9临床试验正在开展,未来应用于临床靶向治疗前景广阔。现就CRISPR/Cas9系统的基本结构、作用原理及其应用的最新研究进展进行综述。     

敲除Attractin或Mahoganoid基因对雄性小鼠生殖内分泌激素的影响

目的:探讨敲除Attractin(Atrn)或Mahoganoid(Md)基因对雄性小鼠生殖内分泌激素的影响。方法:选取成年雄性Atrn基因敲除纯合子、Md基因敲除纯合子及正常小鼠(均为C3H/Hej种系)各12只设为Atrn基因敲除组、Md基因敲除组和对照组。内眦静脉取血清取外周血,化学发光法检测血清睾酮(T)、雌二醇(E2)浓度,用酶联免疫法(ELISA)检测血清卵泡刺激素(FSH)、黄体生成素(LH)和泌乳素(PRL)浓度,进行组间浓度对比。结果:Atrn基因敲除组和Md基因敲除组的外周血E2、LH、FSH、PRL浓度均低于对照组,差异有统计学意义(P<0.05);血清T浓度Atrn基因敲除组高于对照组,而Md基因敲除组低于对照组,差异无统计学意义(P>0.05)。结论:Atrn或Md基因敲除后雄性小鼠的外周血E2、LH、FSH、PRL浓度均有下降,其具体机制及对外周血T浓度的影响有待进一步研究。     

高脂膳食对SR-AⅠ/Ⅱ基因缺失小鼠脂代谢的影响

目的探讨A类Ⅰ型和Ⅱ型清道夫受体(scavenger receptor class A typesⅠandⅡ,SR-AⅠ/Ⅱ)基因缺失对高脂膳食小鼠脂质代谢的影响及其可能的作用机制。方法以SR-AⅠ/Ⅱ基因敲除与野生型雄性小鼠为对象,分别喂饲普通膳食和高脂膳食12w,应用酶法或油红O染色法检测脂质代谢(包括血脂水平和肝脏脂质水平)的变化,采用RT-PCR法检测肝脏B类Ⅰ型清道夫受体(scavenger receptor class B typeⅠ,SR-BⅠ)和CD36的表达。结果SR-AⅠ/Ⅱ基因敲除鼠与野生型鼠相比,高脂膳食喂饲的第3、6、12w,其血清TG、TC、LDL、HDL均比野生型小鼠下降,肝细胞中脂滴的数量较多,体积较大,SR-BⅠmRNA表达上调,CD36mRNA的表达无差异。结论高脂膳食诱导SR-AⅠ/Ⅱ基因缺失小鼠脂质代谢的变化可能与肝脏SR-BⅠ表达升高及外周脂质向肝脏的逆向转运有关。     

Genetic background of alcoholic liver disease

The genetic background of alcoholic behaviour and alcoholic liver diseases has long been the target of intense research. The current knowledge of this very interesting topic is herewith summarized with special emphasis on findings and facts which might have clinical significance including results of family studies, gene polymorphisms of enzyme families of alcohol metabolism, cytokines as well as HLA antigens.     

肝纤维化血清学无创检测研究进展

肝纤维化指肝脏细胞外基质弥漫性的过度沉积,是机体对于肝实质损伤的一种修复反应及许多慢性肝病共同的病理过程,也是各种慢性肝病向肝硬化发展的重要步骤。迄今为止,临床尚缺乏特异性有效逆转或阻止肝纤维化进展的药物,尽早对肝纤维化进行诊断具有重要意义。目前肝纤维化的诊断主要靠组织病理学、血清学标志物及影像学手段。肝活检被认为是肝纤维化诊断和分期的金标准,但由于肝活检的风险和局限性,无创肝纤维化评价模式的建立成为临床亟待解决的科学问题和研究热点。本文对近年来肝纤维化血清学无创检测研究进展进行综述,为肝纤维化诊断提供参考。     

甘露糖结合凝集素与肝脏疾病

甘露糖结合凝集素作为人类天然免疫系统中重要的一线抗感染分子,在免疫系统清除"异物"中起重要作用,已经证实血清甘露糖结合凝集素的含量及其基因多态性与多种肝脏疾病相关,此文将分两部分对甘露糖结合凝集素及其与肝脏疾病的关系进行综述.     

The role of evolutionary biology in research and control of liver flukes in Southeast Asia

Stimulated largely by the availability of new technology, biomedical research at the molecular-level and chemical-based control approaches arguably dominate the field of infectious diseases. Along with this, the proximate view of disease etiology predominates to the exclusion of the ultimate, evolutionary biology-based, causation perspective. Yet, historically and up to today, research in evolutionary biology has provided much of the foundation for understanding the mechanisms underlying disease transmission dynamics, virulence, and the design of effective integrated control strategies.Here we review the state of knowledge regarding the biology of Asian liver Fluke-host relationship, parasitology, phylodynamics, drug-based interventions and liver Fluke-related cancer etiology from an evolutionary biology perspective. We consider how evolutionary principles, mechanisms and research methods could help refine our understanding of clinical disease associated with infection by Liver Flukes as well as their transmission dynamics.We identify a series of questions for an evolutionary biology research agenda for the liver Fluke that should contribute to an increased understanding of liver Fluke-associated diseases.Finally, we describe an integrative evolutionary medicine approach to liver Fluke prevention and control highlighting the need to better contextualize interventions within a broader human health and sustainable development framework.     

脑钠肽和肝硬化心肌病研究进展

肝硬化心肌病(CCM)是肝硬化并发症之一,近年逐渐受到临床的重视.脑钠肽(BNP)目前已成为心功能不全实验室指标之一,并被视为CCM潜在标志物之一.此文对BNP生物学特性、CCM发病机制以及BNP在CCM中的运用等方面的研究进展作了综述.     

Diagnosis of hepatic fibrosis and cirrhosis

Liver biopsy is the gold standard in the diagnosis of liver diseases, despite its limitations, such as sampling error and its invasive nature. Given the increasing prevalence ofhepatic fibrosis and cirrhosis, new non-invasive methods are being developed as a substitute for liver biopsy. Transient elastography (Fibroscan), which measures the stiffness of the liver by means of ultrasound as a measure of fibrosis and cirrhosis, is simple to perform and the inter- and intra-observer variability is small. The accuracy, in relation to liver biopsy, is high in discriminating between cirrhosis and fibrosis, but lower for discriminating between the different stages offibrosis. The Fibrotest is the most investigated combination of serum markers for fibrosis. Its accuracy is lower than that ofa liver biopsy. In the years to come, more research by various independent research groups is needed with the Fibroscan method and the combination of serum markers in different groups of patients and with standardised cut-off points; these must be compared with liver biopsies of sufficient length. In this way, the value of these non-invasive methods can be evaluated. A possible future role for serum markers is to combine them with the Fibroscan, whereby a discrepancy between the different tests could be an indication for a liver biopsy.     

肝癌基因治疗的基础研究与临床应用

肝癌基因治疗发展至今已有30余年,基因治疗的策略、载体和治疗基因随着科学的发展不断更新。近年来由于基础研究的重要突破,肝癌基因治疗逐步由基础研究走向临床治疗,并在临床实践过程中不断积累经验,不断成熟。本文将结合自身工作就肝癌基因治疗的基础研究与临床应用作一简短综述。     

维生素D受体位点基因多态性与自身免疫性肝病易感性的关系

目的明确维生素D受体位点（Apal、Bsml、Taql）基因多态性与自身免疫性肝病易感性的关系。方法通过Pubmed、Ovid、Medline和Web of science databases数据库检索相关病例对照试验文献，并按照纳入、排除标准纳入本研究。对人选研究中有关试验设计、研究对象的特征、研究结果等内容进行摘录，并用STATA version 12．0软件进行分析，关联强度用比值比（OR）和95％可信区间（ci）表示。结果共收集到2000年1月至2012年2月国内外公开发表的6篇论著包括9项试验研究符合纳入标准，其中7项研究关于原发性胆汁性肝硬化（PBC），2项研究关于自身免疫性肝炎（AIH），累计病例为844例，对照为1522例。总研究人群分析显示Apal位点基因中a等位基因降低自身免疫性肝病的发病风险（等位基因模型a比A：OR值0．85，95％CI0．74～0．96，P=0．058；纯合子模型8a比AA：OR值0．75，95％C10．58～0．97，P=0．212；杂合子模型Aa比AA：OR值0．78，95％C10．63～0．98，P=0．235；显性模型Aa／aa比AA：OR值0．77，95％C10．63～0．94，P=0．231），然而Bsml和Taql位点基因并未发现与自身免疫性肝病具有相关性。结论维生素D受体Apal位点基因中a等位基因可能是自身免疫性肝病的保护基因，而Bsml和Taql位点基因与自身免疫性肝病的易感件无相关件。     

Polyporus and Bupleuri radix effectively alter peripheral circadian clock phase acutely in male mice

In mammals, daily physiological events are precisely regulated by an internal circadian clock system. An important function of this system is to readjust the phase of the clock daily. In Japan, traditional herb medicines, so-called crude drugs (Shoyaku), are widely used for many diseases, and some are reported to affect circadian clock impairment, suggesting that some of them might have an ability to modify clock gene expression rhythms. Therefore, from selected 40 crude drugs, finding candidates that control the circadian clock phases was the first purpose of this study. As there are several crude drugs used for liver- and/or kidney-related diseases, the second aim of the present study was to find some crude drugs affecting liver/kidney circadian clock in vivo. To assess phase changes in the daily circadian rhythm, bioluminescence from the core clock gene product Period 2 was continuously monitored in mouse embryonic fibroblasts in vitro and in some peripheral tissues (kidney, liver, and submandibular gland) of PERIOD2::LUCIFERASE knock-in mice in vivo. In our screening, Polyporus and Bupleuri radix were found to be good candidates to effectively manipulate the peripheral circadian clock phase acutely, with stimulation time-of-day dependency in vitro as well as in vivo. Interestingly, Polyporus and Bupleuri radix are traditional herb medicines use for treating edema and promoting diuresis, and for chronic hepatitis, respectively. These crude drugs may be therefore good modulators of the circadian peripheral clocks including liver and kidney, and circadian clock genes become new molecular targets for these crude drugs.