Mdr2基因敲除小鼠建立原发性肝癌模型观察

目的 观察C57BL/6背景的Mdr2基因敲除小鼠自发肝肿瘤形成情况。方法 (11.3±4.2)周龄Mdr2基因敲除C57BL/6-Abcb4^tm1小鼠9只和野生型C57BL/6小鼠5只,连续饲养65周后处死小鼠,留取血清及肝标本。检测血清ALT、AST、AFP水平,肝组织石蜡切片做HE、天狼猩红染色,免疫组织化学检测肿瘤及肿瘤旁组织CK-7、CK-19表达情况。结果 9只Mdr2基因敲除小鼠均自发形成肝肿瘤,血清ALT、AST、AFP水平均显著高于野生型小鼠(P<0.01),Mdr2基因敲除小鼠肝肿瘤CK-7、CK-19染色均为阴性。结论 Mdr2基因敲除小鼠连续饲养至(76.3±4.2)周龄时均自发形成肝肿瘤,其病理组织分型为肝细胞癌。     

不依赖IL-15的人类NK细胞的体内稳态

<正>IL-15是一种促炎细胞因子,在NK细胞的发育和活化作用中起重要作用,IL-15是炎性疾病治疗的潜在靶点。用IL-15-和IL-15R基因敲除的小鼠进行研究,证明了IL-15是维持NK细胞稳态的一种重要细胞因子,与基因改良小鼠的信息相吻合,作者证明了用小鼠抗-鼠IL-15单克隆抗体(M96)来中和IL-15的确减少了C57BL/6小鼠的NK细胞数量。针     

纳米氧化锌经口染毒对C57BL/6J小鼠外周脏器的影响*

目的:探讨纳米氧化锌经口染毒60 d对C57BL/6J小鼠多种外周脏器的损伤作用。方法:20只雄性C57BL/6J小鼠随机分为对照组和实验组,每组10只,实验组将纳米氧化锌溶液以20 mg/kg体重的剂量连续灌胃染毒60 d,对照组给予相应量的生理盐水;小鼠每周称重一次,染毒结束后,眼球取血,检测血糖、血脂、肝功能和肾功能相关指标,以及血清中炎症因子PAF、IL-6和TNF-α含量;取心脏、肝脏、脾脏、肺、肾脏和小肠组织制备病理切片,HE染色后,观察组织形态学变化。结果:实验组和对照组之间的体重无显著性差异;与正常对照组比较,实验组大鼠血中白蛋白(ALB)、白蛋白/球蛋白比值(A/G)、碱性磷酸酶(ALP)、谷草/谷丙转氨酶比值(S/L)、尿酸(UA)和尿素氮(BUN)含量明显升高(P＜0.05或 P＜0.01);两组间血清中炎症因子含量无显著差异。病理学检查发现,实验组心肌中部分区域出现浊肿,肝脏出现轻度炎性病变(灶性或小灶性坏死),脾脏色素沉着减少,肺部出现轻或中度间质性炎症,肾脏和小肠未见明显病理改变。结论:纳米氧化锌经口染毒60 d未引起C57BL/6J小鼠血液系统炎症,但可诱导心脏、肝脏、脾脏和肺脏出现轻度的病理变化,并导致肝脏和肾脏的功能异常。     

载脂蛋白E基因敲除小鼠血管外膜成纤维细胞G蛋白信号调节因子3,5的表达变化

目的:检测载脂蛋白E基因敲除小鼠(apoE(-/-))血管外膜成纤维细胞中G蛋白信号调节因子(regulator of G protein signaling,RGS)3,5的变化,探讨其在动脉粥样硬化发病中的作用.方法:体外培养高脂喂养2周的apoE(-/-)小鼠和C57BL/6小鼠动脉外膜成纤维细胞,利用基因芯片技术分析RGS3,RGS5mRNA的变化,通过RT-PCR进一步验证.结果:基因芯片数据分析显示,apoE(-/-)小鼠成纤维细胞中RGS3表达下调,与C57BL/6小鼠比值为0.46.RGS5表达上调,与C57BL/6小鼠比值为2.25.RT-PCR结果显示apoE(-/-)小鼠成纤维细胞RGS3mRNA水平低于对照组小鼠(比值为0.78),RGS5mRNA水平高于对照组小鼠(比值为2.34),与基因芯片检测结果相比,变化方向一致.结论:apoE(-/-)小鼠血管外膜成纤维细胞中选择性RGS3降低和RGS5升高可能与外膜成纤维细胞的激活密切相关,从而参与了动脉粥样硬化的发生与发展,RGS有可能成为动脉粥样硬化药物治疗及基因治疗的新靶点.     

Pd-1基因敲除小鼠构建及初步表型验证

目的:细胞程序性死亡蛋白(programmed death ligand-1,PD-1)是机体T细胞的免疫检查点,也是肿瘤治疗的重要靶点。采用CRISPR/Cas9技术,利用非同源重组修复引入突变的方式,使基因蛋白读码框移码造成PD-1功能缺失,建立Pd-1基因敲除小鼠模型,为深入探究Pd-1基因功能及作用机制提供基础。方法:针对Pd-1基因2-4号外显子设计并合成2对sgRNA片段,与编码Cas9片段共同体外转录,通过受精卵显微注射方法将两者mRNA混合注射到C57BL/6小鼠受精卵中,经PCR产物测序鉴定获得F0代小鼠,之后与野生型C57BL/6小鼠交配获得F1代杂合子小鼠,F1代小鼠自交即获得F2代纯合子小鼠品系(Pd-1^-/-)。刀豆蛋白(concanavalin A,ConA)刺激Pd-1^-/-小鼠后,通过实时荧光定量PCR和流式细胞技术在mRNA和蛋白水平上分别检测Pd-1^-/-小鼠中Pd-1基因在转录和翻译过程中的表达情况,并通过ELISA方法检测Pd-1^-/-小鼠血清中IL-6、IFN-γ、IL12/IL23及TNF-α等因子的表达水平,初步分析Pd-1通路在T细胞反应调控中的作用机制及对免疫刺激的响应情况。结果:PCR及测序结果表明在小鼠基因组中Pd-1基因2-4号外显子被成功敲除;Real-Time PCR实验和流式检测结果显示:与野生型小鼠相比,Pd-1^-/-小鼠脾、肠系膜淋巴结、胸腺和血液各组织中Pd-1表达水平均显著降低;双抗夹心ELISA测定结果显示:Pd-1敲除后经ConA刺激,血清中IL-6和IFN-γ表达上调。结论:成功构建Pd-1基因敲除小鼠模型。Pd-1缺失能够上调IL-6和IFN-γ对ConA刺激的响应,增加ConA引起的炎症反应,为Pd-1的体内基因功能研究提供了新的小鼠模型和研究思路。     

载脂蛋白E基因敲除小鼠血脂及心肌酶学的测定及意义

目的:研究载脂蛋白E基因敲除(ApoE-/-)小鼠血脂及心肌酶学的变化,为利用其探讨动脉粥样硬化的病理生理进程提供实验依据。方法:选取28W龄雄性ApoE-/-小鼠6只和同性别、同周龄的野生型C57BL/6J(WT)小鼠10只,应用日立7600全自动生化分析仪分别测定其血清中甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白(LDL-C)、高密度脂蛋白(HDL-C)、载脂蛋白B(Apo-B)、超敏C反应蛋白(hs-CRP)、游离脂肪酸(NEFA)、肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)、乳酸脱氢酶(LDH)和缺血修饰白蛋白(ACB)的水平。结果:与WT鼠比,ApoE-/-小鼠血清中血脂指标TG、TC、LDL-C、hs-CRP和NEFA水平显著升高(P<0.01),而HDL-C仅为0.46±0.16mmol/L,远低于WT鼠的水平(1.86±0.26mmol/L)。心肌酶学指标CK、LDH明显高于对照组(P<0.01),缺血修饰白蛋白(IMA()55.61±3.50U/mL)明显低于对照组(72.47±4.26U/mL)(P<0.01)。CK-MB与对照组相比无显著性变化。结论:ApoE-/-小鼠血脂水平...     

Fancm基因敲除小鼠构建及其表型分析

目的构建范可尼贫血通路Fancm基因敲除小鼠,研究Fancm基因缺失对小鼠生理功能,特别是雄性生殖器官的影响。方法采用CRISPR/Cas9技术,获得Fancm基因敲除小鼠。分析FANCM蛋白在野生型和Fancm^-/-小鼠睾丸组织中的表达。统计Fancm^-/-小鼠的出生率、体重、性别比例及子代生育情况,分析血液常规指标。组织形态学研究雄性Fancm^-/-小鼠睾丸生理病理表型。结果敲除Fancm基因ATG区域,获得稳定遗传的C57BL/6背景Fancm^-/-小鼠。Fancm^-/-小鼠睾丸中FANCM蛋白表达完全丢失。Fancm^-/-小鼠无明显的胚胎致死现象,但雌性Fancm-/-小鼠数目显著少于雄性Fancm^-/-小鼠。同窝Fancm^-/-小鼠比较野生型体重无明显区别,部分血常规指标有显著性差异。Fancm^-/-小鼠有明显的生殖能力缺陷。雄性Fancm^-/-小鼠睾丸有显著的发育缺陷,其生精细胞凋亡增加、细胞周期阻滞,影响睾丸发育与精子的生成。结论成功获得稳定遗传C57BL/6背景Fancm^-/-小鼠,Fancm基因参与小鼠的生长发育,特别是雄性生殖器官功能的维持及调控。     

PM2.5鼻腔滴注致小鼠海马组织损伤的炎性机制

目的: 探讨鼻腔滴注不同浓度的PM_2.5对小鼠海马组织损伤的炎性机制。方法: 30只C57BL/6J小鼠随机分为3组(n=10):对照组、低剂量组、高剂量组。采用鼻腔滴注方法进行染毒,每次染毒前测量体重,低剂量组和高剂量组PM_2.5染毒剂量分别为1.5 mg/kg BW和7.5 mg/kg BW,对照组给予等体积的生理盐水,隔天染毒一次,共12次。用ELISA法测定血清中肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)和白介素-6(IL-6)水平。HE染色和电镜观察肺和海马组织的病理变化及超微结构。用抗体芯片技术测定海马组织炎性细胞因子水平。结果: PM_2.5鼻腔滴注染毒对小鼠血清中TNF-α、IL-1β和IL-6水平无显著影响(P＞0.05),肺组织结构也无明显病理改变。而在海马组织中,低剂量和高剂量PM_2.5暴露均能导致海马CA3区神经元排列紊乱,并存在神经元细胞周围突触数量减少,小血管周围水肿等超微结构变化。利用抗体芯片检测海马组织炎性细胞因子表达变化,结果显示,与对照组比较,低剂量组海马组织中CX3CL1、CSF2和TECK等炎性细胞因子水平显著升高(P＜0.05),而MIG和sTNFR1显著降低(P＜0.05);高剂量组中炎性因子CX3CL1、CSF2和TCA-3等显著升高(P＜0.05),而Leptin、MIG和FASLG等显著降低(P＜0.05)。结论: PM_2.5鼻腔滴注可诱导小鼠海马组织结构损伤,其作用途径可能为嗅脑通路,引起海马损伤的炎性机制可能与TNF-α和IL-6等促炎因子的显著升高和sTNFR1 、FASLG等炎性疾病标志物的显著降低有关。     

内皮细胞条件性敲除EMCN小鼠模型建立及肿瘤肺转移对比分析

目的 建立内皮细胞条件性敲除EMCN基因小鼠模型,探讨C57BL/6小鼠与内皮细胞条件性敲除EMCN小鼠模型体内肿瘤肺转移存在的差异。方法 将内皮细胞特异性Tek-CreERT2小鼠与EMCN^flox/flox小鼠杂交,将子代雄性EMCN^flox/wt/Tek-CreERT2⁺与雌性EMCN^flox/flox小鼠交配,取得内皮细胞特异性敲除EMCN基因小鼠(EMCN^flox/flox/Tek-CreERT2⁺,EMCN^ecko)。通过在DNA和蛋白水平验证基因敲除准确性及效率,提取小鼠基因组DNA后,PCR扩增检测Tek-CreERT2及FLOX位点,Tamoxifen诱导后,Western Blot检测组织中EMCN蛋白的表达。对正常C57BL/6小鼠与EMCN^ecko小鼠表型进行观察及脏器HE染色。为了证实EMCN敲除对肿瘤转移的影响,将LLC细胞尾静脉注射C57BL/6与EMCN^ecko     

用rAAV8-1.3HBV制备两种品系小鼠乙型肝炎病毒感染模型的比较研究

我们先前用rAAV8-1.3HBV静脉注射C57BL/6小鼠成功地制备了慢性乙型肝炎病毒(Hepatitis B virus,HBV)感染模型。为了探讨不同品系的小鼠对rAAV8-1.3HBV静脉注射是否具有不同反应,本研究比较了C57BL/6和BALB/c小鼠静脉注射重组病毒后外周血中HBV抗原和抗体水平、病毒载量和肝脏组织HBcAg表达情况,以及不同剂量重组病毒注射与这些指标的关系。将低(4×109 Viral genome,vg)、中(4×1010vg)和高(4×1011vg)三种剂量的rAAV8-1.3HBV通过尾静脉注射至C57BL/6和BALB/c小鼠,分别利用ELISA和荧光定量PCR方法检测血清中的HBV抗原、抗体水平以及HBV DNA,利用免疫组化检测肝脏组织HBcAg的表达。结果发现,对于C57BL/6小鼠,三种不同剂量rAAV8-1.3HBV注射均可造成100%小鼠出现HBV持续感染;血清HBsAg、HBeAg和HBV DNA以及肝组织HBcAg稳定表达超过8个月,其表达水平随重组病毒注射剂量的增加而升高,高剂量注射时可造成超过40%的肝细胞感染HBV,血清中HBV DNA可达105 IU/mL以上;未检测到针对HBV的抗体。对于BALB/c小鼠,三种不同剂量rAAV8-1.3HBV注射也可造成100%小鼠出现HBV持续感染;血清HBeAg和HBV DNA以及肝组织HBcAg稳定表达超过8个月,但是血清HBsAg在重组病毒注射2周之后显著下降甚至消失;在中剂量注射组的BALB/c小鼠血清中检测到低水平的Anti-HBs;血清HBeAg和肝组织HBcAg的表达水平随重组病毒注射剂量的增加而增高,并且各剂量组表达水平均高于C57BL/6小鼠,高剂量注射时可造成超过50%的肝细胞感染HBV。本研究表明,低至4×109 vg剂量的rAAV8-1.3HBV注射即可造成C57BL/6和BALB/c两种品系小鼠出现HBV持续感染,并且HBV复制水平随重组病毒注射剂量增加而增高;BALB/c小鼠对HBV的免疫反应强于C57BL/6小鼠,可以产生针对HBsAg的体液免疫反应而使血清HBsAg转阴,但无法清除携带HBV的肝细胞。     

Mechanisms of perpetuation of atrial fibrillation in chronically dilated atria

The progressive nature of atrial fibrillation (AF) has been demonstrated in numerous experimental as well as clinical investigations. Electrical remodeling (shortening of atrial refractoriness) develops within the first days of AF and contributes to the increase in stability of the arrhythmia. However, "domestication of AF" must also depend on other mechanisms since the stability of AF continues to increase after electrical remodeling has been completed. Chronic atrial stretch induces activation of numerous signaling pathways leading to cellular hypertrophy, fibroblast proliferation and tissue fibrosis. The resulting electro-anatomical substrate is characterized by increased non-uniform anisotropy and local conduction heterogeneities facilitating reentry in the dilated atria. Atrial fibrosis may lead to disruption of the electrical side-to-side junctions between muscle bundles. This can result in electrical dissociation between neighboring muscle bundles, i.e. they become activated out-of-phase. Recent mapping studies in goats with persistent AF showed that electrical dissociation can not only occur between neighboring muscle bundles but also in the third dimension, i.e. between the epicardial layer and the endocardial bundle network. Such endo-epicardial dissociation will significantly increase the number of wavefronts which can simultaneously be present in the atrial wall. This article reviews data suggesting a role of endo-epicardial dissociation in dilated and fibrillating atria, for the self-perpetuating nature of AF as well as its possible implications for therapeutic interventions.     

Mouse heart rate in a human: diagnostic mystery of an extreme tachyarrhythmia

We report telemetry recording of an extreme non-fatal tachyarrhythmia noted in a hospitalized quadriplegic male with history of atrial fibrillation where the average ventricular conduction rate was found to be about 600 beats per minute and was associated with transient syncope. A medical literature review suggests that the fastest human ventricular conduction rate reported to date in a tachyarrhythmia is 480 beats per minute. We therefore report the fastest human heart rate noted in a tachyarrhythmia and the most probable mechanism of this arrhythmia being a rapid atrial fibrillation with 1:1 conduction in the setting of probable co-existing multiple bypass tracts.     

Atrial flutter catheter ablation in adult congenital heart diseases

The important increase in life expectancy of adult patients with congenital heart disease (ACHD) has generated new challenges, including arrhythmias that represent one of the main late complications. Reentrant atrial arrhythmias are by far the main mechanism encountered, and catheter ablation has been now presented as a first-line therapy in this patient population. The number of procedures is expected to continuously increase year after year. The heterogeneity and complexity of phenotypes encountered require these cases to be performed by highly experienced operators, in specialized centers with multidisciplinary competencies. A thorough knowledge and understanding of anatomic specificities, vascular access issues, and main circuits encountered according to underlying phenotype is essential. Acute success rates have significantly improved and are now excellent, but recurrences remain a common issue, with different mechanisms or circuits frequently encountered. Observational data have suggested the interest of systematically targeting all inducible atrial arrhythmias, whether previously documented or not, and a lot of hope and research is based on the prediction of arrhythmia substrate before arrhythmia development by imaging or electroanatomic mapping to deliver a prophylactic patient tailored ablation approach. In this review, we summarize those different points in the most common or distinctive defects to offer a didactic overview of atrial flutter catheter ablation in ACHD patients.     

Signal processing methods for information enhancement in atrial fibrillation: Spectral analysis and non-linear parameters

Recently, the research efforts in the context of electrocardiographical recording during atrial fibrillation (AF) has been directed to broaden the understandings on the electrophysiological and structural remodelling occurring during the arrhythmia and on characterizing the different types of AF. Following this line, both surface ECG and endocardial electrograms have been thoroughly studied and a series of linear and non-linear parameters were computed either directly on the electrograms or on the derived activation series.

In this paper, we reviewed some signal processing methods used to characterize surface ECG and endocardial electrograms during AF, focusing on spectral and non-linear analysis. In particular, parametric and non-parametric methods for spectral analysis of the residual ECG, i.e. atrial waves obtained from surface ECG after removing ventricular activity, and endocardial recordings are described. The different purposes of spectral analysis (exploring autonomic functions, analysis of spontaneous AF behaviour and predicting therapeutic effects) are illustrated with some examples. In addition, we described some more recent non-linear methods applied to AF, assessing the organization of atrial signals as well as ventricular response in AF. In particular, methods derived from embedding time series and based on entropy computation are illustrated and exemplified.     

Localization of wheat-germ agglutinin-binding sites in the Golgi complex of cultured rat atrial myocytes

Summary In the Golgi region of cultured rat atrial myocytes, condensed secretory protein was seen in Golgi-associated tubules or cisternae which lay beyond, and often separated from, the remainder of the Golgi stacks. These structures appeared to be involved in packaging of condensed secretory protein into atrial granules. Binding sites of HRP-conjugated wheat-germ agglutinin (WGA) in saponin-treated cultured atrial myocytes were examined by electron microscopy with special reference to atrial granules and the tubular structures associated with the Golgi stacks. HRP reaction products were observed in both trans-cisternae of the Golgi stacks and the associated tubular structures. While the majority of atrial granules were devoid of reaction products, some granules, which were connected to the WGA-positive tubular structures in the vicinity of the Golgi trans-cisternae, showed HRP reaction products at their connected necks. Similar results were obtained when sections of the cells embedded in Lowicryl K4M were labeled with WGA coupled to colloidal gold (G-WGA); the Golgi complex was G-WGA positive, whereas no specific binding of G-WGA to atrial granules was observed. These results suggest that glycoproteins and/or glycolipids with oligosaccharides recognized by WGA in the Golgi transcisternae, may be separated from atrial natriuretic peptides which are packaged into atrial granules.Abbreviations ANP atrial natriuretic peptide - HRP horseradish peroxidase - M199 medium 199 - TGN trans-Golgi network - WGA wheat-germ agglutinin - G-WGA WGA coupled to colloidal gold     

起搏器术后新发房性心律失常的影响因素分析

目的:探讨起搏器术后新发房性心律失常的发生情况及其相关影响因素。方法:选择2006年1月至2007年12月于沈阳军区总医院首次植入永久起搏器的107例患者,男性50例,平均年龄65.0±11.9岁,术前通过追问病史及相关检查均排除房性心律失常(房颤、房扑、房速),术后平均随访3.9年,观察新发房性心律失常情况。按术后是否出现房性心律失常,将患者分为新发房性心律失常组和无房性心律失常组,比较两组患者术前和术后心脏超声结果的变化、心室起搏比例、起搏部位及起搏模式,并通过logistic回归分析起搏器术后发生房性心律失常的影响因素。结果:新发房性心律失常组26例(24.3%),其中房颤17例(15.9%),房扑2例(1.9%),房速7例(6.5%);无房性心律失常组81例。与无房性心律失常组比较,新发房性心律失常组左房内径明显增加(P=0.040)、二尖瓣返流程度较重(P=0.032)及左室射血分数明显下降(P=0.001),心室起搏百分比(VP%)显著升高(P=0.017)。心尖部起搏患者房性心律失常的发生率明显高于间隔部起搏(33.3%vs 16.9%,P<0.05),双腔起搏组患者房性心律失常发生率明显低于单腔起搏器组(18.7%vs 37.5%,P<0.05)。Logistic回归分析显示术后新发房性心律失常的发生与高比例的心室起搏(P=0.006)、VVI(R)起搏模式(P=0.014)及右心室起搏电极导线植于心尖部(P=0.024)显著相关。结论:起搏模式、心室起搏百分比、起搏部位是起搏器术后发生房性心律失常的影响因素。     

Characterization and localization of atriopeptin in rat atrium

An antiserum specific for atriopeptin was used to characterize and localize atriopeptin-like immunoreactive material in rat atrium by radioimmunoassay and immunohistochemical techniques. The antiserum recognizes atriopeptin I, atriopeptin III, and -human atrial natriuretic polypeptide, but does not recognize met-enkephalin, cholecystokinin, dynorphin A (1–13), bradykinin, substance P, or β-endorphin. A high content of atriopeptin was found in crude extracts of rat atria, as compared to ventricles, and the atriopeptin-like immunoreactive material was found to be located exclusively in granules within atrial cardiocytes. Fractionation of the immunoreactive material by gel filtration and reverse-phase HPLC revealed the presence of multiple atriopeptins.     

Potential role of MG53 in the regulation of transforming-growth-factor-β1-induced atrial fibrosis and vulnerability to atrial fibrillation

Atrial fibrosis plays a critical role in atrial fibrillation (AF) by the transforming growth factor (TGF)-β1/Smad pathway. The disordered differentiation, proliferation, migration and collagen deposition of atrial fibroblasts play significant roles in atrial fibrosis. Mitsugumin (MG)53 is predominantly expressed in myocardium of rodents and has multiple biological functions. However, the role of MG53 in cardiac fibrosis remains unclear. This study provided clinical and experimental evidence for the involvement of MG53 in atrial fibrosis in humans and atrial fibrosis phenotype in cultured rat atrial fibroblasts. In atrial tissue from patients we demonstrated that MG53 was expressed in human atrium. Expression of MG53 increased with the extent of atrial fibrosis, which could induce AF. In cultured atrial fibroblasts, depletion of MG53 by siRNA caused down-regulation of the TGF-β1/Smad pathway, while overexpression of MG53 by adenovirus up-regulated the pathway. MG53 regulated the proliferation and migration of atrial fibroblasts. Besides, exogenous TGF-β1 suppressed expression of MG53. In conclusion, we demonstrated that MG53 was expressed in human atrium, and may be a potential upstream of the TGF-β1/Smad pathway in human atrium and rat atrial fibroblasts. This suggests that MG53 is a potential regulator of atrial fibrosis induced by the TGF-β1/Smad pathway in patients with AF.