柯萨奇B_3病毒感染的受体学进展

最近相继揭示了柯萨奇 B3 病毒 (CVB3 )感染的细胞受体 ,本文对其结构、功能及与病毒的作用机制作一综述。

柯萨奇病毒B_3感染的受体学进展

最近,众多的研究陆续揭示了柯萨奇B_3病毒感染的细胞受体,本文对其结构、功能及与病毒的作用机制等进展作了综述。

细胞分子生物学技术在靶向药物筛选中的应用

随着细胞及分子生物学的发展,新技术方法越来越多地用于新靶点建立和药物筛选研究,为药物设计、靶点的选择和用药方案的确定提供理论依据,同时使药物筛选有了更高的特异性,对药物筛选和药理学研究起到了极大的促进作用。论述了功能基因的筛选、高通量细胞筛选和高内涵筛选技术、反义核酸技术、转基因/基因敲除技术、基因芯片、蛋白质芯片、组织芯片、酶免疫分析、荧光免疫分析、流式细胞技术等方法在靶向药物筛选中的应用。

去势大鼠骨质疏松与股骨甲状旁腺激素受体1相关性研究

目的:观测去势大鼠骨质疏松时血清雌二醇(E2)水平与甲状旁腺激素受体1(PTHR1)表达的关系。方法:雌性大鼠随机分为去势和假去势(即对照组)两组,大鼠切除双侧卵巢诱发骨质疏松症。术后3个月腹主静脉取血,测定各组大鼠血清E2水平;分别用小动物骨骼强度测定仪和双能X线骨密度仪检测右股骨生物学力学和骨密度;从左股骨中提取总RAN和总蛋白,分别检测PTHR1在转录和翻译水平上的变化。结果:去势大鼠血清E2含量为13.80±1.10pmol/L,对照组血清E2含量为23.51±1.20pmol/L;去势组右股骨生物学力度:折断力为9.89±0.65N/kg,压碎力为16.10±1.23N/kg,骨密度为0.0730±0.0075gms/cm2;对照组右股骨生物学力度:折断力为11.74±0.95N/kg,压碎力为19.38±1.84N/kg,骨密度为0.0839±0.0097gms/cm2。与对照组相比,去势大鼠PTHR1基因和蛋白水平表达量均明显降低。结论:去势不仅可引起大鼠E2水平明显降低,并诱发骨质疏松进而导致PTHR1基因和蛋白表达水平均显著下降,提示PTHR1表达下降可能与去势诱导大鼠骨质疏松密切相关。

白细胞介素2受体单克隆抗体预防肾移植术后急性排斥反应

目的:观察白细胞介素2受体单克隆抗体在预防肾移植术后急性排斥反应中的作用。方法:选择2004-01/2005-12在武装警察部队安徽省总队医院接受同种异体肾移植的患者47例,患者均知情同意。根据是否应用白细胞介素2受体单克隆抗体分为2组,白细胞介素2受体单克隆抗体组25例;对照组22例,为同期未应用白细胞介素2受体单克隆抗体的患者。两组均采用“环孢素A+霉酚酸酯+强的松”的三联免疫抑制治疗方案。白细胞介素2受体单克隆抗体组在此基础上于术前2h和术后第4天分别静脉滴注白细胞介素2受体单克隆抗体20mg。观察术后6个月移植肾急性排斥反应、药物不良反应及并发症发生情况,并通过检测血清肌酐水平评估肾功能恢复情况。结果:47例患者全部进入结果分析,无脱落。①两组患者的急性排斥反应发生率比较:白细胞介素2受体单克隆抗体组显著低于对照组(12%,41%,P<0.05)。②两组患者的不良反应及并发症发生情况:白细胞介素2受体单克隆抗体组患者发生肺部感染2例,肝功能损害1例,胃肠道反应6例;对照组发生肺部感染2例,肝功能损害3例,胃肠道反应4例,两组差异无显著性意义(P>0.05)。两组均未发生白细胞减少、切口感染、尿路感染、高血糖等并发症。③两组患者的血清肌酐水平比较:术后1,2周,白细胞介素2受体单克隆抗体组的血清肌酐水平低于对照组[(104.7±10.1,103.1±7.7;128.3±28.2,120.0±8.5)μmol/L(P<0.05)];术后1-6个月,两组间血清肌酐水平差异无显著性意义(P>0.05),提示术后2周两组移植肾功能均基本恢复正常。结论:白细胞介素2受体单克隆抗体能有效预防肾移植术后急性排斥反应的发生,且使用安全,不增加术后的不良反应。

病原菌对NOD样受体及Toll样受体信号通路介导的固有免疫逃逸机制研究进展

作为机体抵抗病原微生物的第一道防线,固有免疫细胞通过模式识别受体(PRR)识别病原体相关模式分子(PAMP)继而启动下游信号通路,以发挥固有免疫效应,清除入侵的病原体和异物。固有免疫细胞主要的信号通路有NOD样受体(NLR)及Toll样受体(TLR)信号通路,病原菌经过长期的选择进化产生了针对NLR及TLR信号通路的对抗机制,以利于其在宿主体内的生存增殖。病原菌主要通过产生毒力因子或降低刺激炎症小体活化的PAMP的表达,干扰、抑制或避免固有免疫细胞内炎症小体的活化,实现对NLR介导的信号通路的免疫逃逸。而对TLR信号通路的免疫逃逸主要通过产生毒力因子,抑制丝裂原活化蛋白激酶级联反应、抑制NF-kB活化以及通过产生含有TIR结构域的蛋白,直接与TLR或者TLR信号通路中的接头蛋白结合,干扰下游信号转导三种机制。

异基因造血干细胞移植受者T细胞受体β链CDR3谱型表达与巨细胞病毒激活

目的：探讨T细胞受体（TCR）β链可变区域（BV）的互补决定区（CDR3）谱型表达与异基因造血干细胞移植（HSCT）受者CMV激活的关系。方法：采用荧光定量PCR熔解曲线技术，扩增测序7例HSCT受者和3名健康对照者外周血单个核细胞的TCRBV家族CDR3谱型；采用免疫组织化学法检测外周血白细胞中的CMV—pp65抗原；应用ELISA法检测HSCT受者血清中的CMV—IgM。分析TCRBV家族CDR3表达与CMV激活的相关性。结果：3名健康对照者24个TCRBV家族均表达。7例HSCT受者术后TCRBV家族CDR3测序结果显示为BV9、BV11、BV17、BV20等BV家族序列；TCRBV9含“QVRGGTDTQ”，TCRBV11含“VATDEQ”和“LGDEQ”，TCRBV17含“IGQGNTEA”，TCRBV20含“VGLAANEQ”等共有氨基酸序列。抗原检测结果显示：术后3个月7例受者中有5例受者抗原血症阳性，CMV—pp65阳性细胞数为（2—15）个／5×10^4白细胞。抗体检测结果显示：术后3个月7例受者中3例受者CMV-IgM阳性。TCRBV家族CDR3的表达在CMV抗原血症阳性受者与抗原血症阴性受者之间差异无统计学意义（均P〉0．05）；但TCRBV11家族的表达在CMV-IgM阳性受者与CMV-IgM阴性受者之间差异有统计学意义（P〈0．05）。结论：HSCT受者在T细胞免疫应答中有特定的TCRBV家族CDR3谱型，其中TCRBV11的表达可能与CMV激活有关。

表皮生长因子受体单抗对胰腺癌化疗的增敏作用

【目的】用胰腺癌细胞株，建立胰腺癌荷瘤裸鼠模型，探讨表皮生长N子受体（EGFR）单抗联合吉西他滨、奥沙利铂对胰腺癌化疗的增敏作用。【方法】将胰腺癌细胞株细胞悬液注射于裸鼠背部皮下，建立肿瘤模型。每周观察肿瘤生长情况，并记录；当肿瘤生长到0．15cm^3左右时，将裸鼠随机分组，称量每只荷瘤裸鼠的体重，并记录；干预组腹腔分别注射EGFR抗体（MMAb-2）、吉西他滨、奥沙利铂及其联合用药；对照组注射生理盐水。每周测量肿瘤大小。干预4周称量荷瘤裸鼠体重后处死裸鼠。切取肿瘤，测量瘤体积、称瘤重。肿瘤组织送病理学检查，评价干预效果。【结果】肿瘤细胞移植成功率近90％（62／70），瘤体生长迅速，其他组织和器官内未见转移癌灶。组织学检查符合胰腺低分化腺癌；MMAb-2、吉西他滨、奥沙利铂干预肿瘤有效，瘤体缩小。MMAb-2组裸鼠白细胞计数明显高于其他实验组。【结论】EGFR抗体可有效抑制肿瘤细胞的生长，其效果优于吉西他滨、奥沙利铂，并且联合应用效果更好。

生长抑素Ⅱ型受体在上皮性卵巢癌中的表达

目的:探讨生长抑素型受体在上皮性卵巢癌中的表达及意义。方法:采用免疫组化SP法检测48例上皮性卵巢肿瘤中生长抑素受体蛋白的表达水平。结果:卵巢癌组中晚期生长抑素型受体的阳性表达率为71.4%(10/14),早期生长抑素受体的阳性表达率为23.5%(4/17),两组的阳性表达率间有显著性差异。组织学分化1级阳性表达率为18.2%(2/11),分化2级阳性表达率为50%(7/14),分化3级阳性表达率为83.3%(5/6),三者间有显著性差异。生长抑素Ⅱ型受体表达有腹水组明显高于无腹水组。结论:随着卵巢癌病变进展,肿瘤细胞表面生长抑素Ⅱ型受体表达增强,相应的生长抑素类似物可望为生长抑素受体阳性表达的上皮性卵巢癌的诊治提供新的方向。

The selective 5-HTIA receptor antagonist WAY-100635 inhibits neuronal activity of the ventromedial prefrontal cortex in a rodent model of Parkinson's disease

Objective The ventral part of the medial prefrontal cortex（mPFC）plays an important role in initiation and control of voluntary movement,mood and cognition.However,after the degeneration of the nigrostriatal pathway,the neuronal activity of the ventral mPFC and the role of serotonin1A（5-hydroxytryptamine,5-HT1A）receptors in the firing of the neurons are still unknown.The present study is to investigate the change of neuronal activity in the ventral mPFC and the effect of systemic administration of the selective 5-HT1Areceptor antagonist WAY-100635 on the activity of the neurons in normal and 6-hydroxydopamine（6-OHDA）-lesioned rats.Methods Single unit responses were recorded extracellularly with glass microelectrodes from ventral mPFC neurons in normal rats and 6-OHDA unilaterally lesiond rats in vivo.Results 6-OHDA lesion of the substantia nigra pars compacta（SNc）significantly increased the firing rate with no change in the firing pattern of neurons of the ventral mPFC in rats.Systemic administration of WAY-100635（0.1 mg/kg,i.v.）did not change the mean firing rate and firing pattern of ventral mPFC neurons in normal rats.In contrast,WAY-100635 signifi- cantly decreased the mean firing rate of the neurons in rats with 6-OHDA lesion of the SNc.Conclusion These data suggest that the degeneration of the nigrostriatal pathway results in an increase of neuronal activity of ventral mPFC and dysfunction of 5-HT1Areceptor.

COL1A1-shRNA表达载体的构建及对胃癌细胞增殖迁移的影响

目的:构建COL1A1特异小干扰RNA(siRNA)表达载体,体外评价其对胃癌细胞BGC-823增殖迁移的影响。方法:根据文献获得3个COL1A1的siRNA靶序列,设计能转录短发夹状RNA(short hairpin RNAs,shRNA)的DNA序列,并与pSilencerTM4.1-CMV neo线性质粒载体连接,连接产物转化感受态大肠杆菌DH5α,筛选获得重组质粒;经DNA测序鉴定重组体DNA序列正确后转染胃癌BGC-823细胞,利用G418筛选稳定表达COL1A1-shRNA的BGC-823细胞株。通过实时荧光定量RT-PCR及Western blot检测转染后细胞COL1A1在mRNA及蛋白质水平的表达;MTT法及Transwell法检测COL1A1干扰后胃癌细胞增殖及迁移能力的变化。结果:序列测定表明成功构建3个COL1A1-shRNA表达载体。实时荧光定量RT-PCR及Western blot检测显示,COL1A1-shRNA转染胃癌BGC-823细胞的COL1A1 mRNA及蛋白表达水平明显降低(P<0.05);MTT试验及Transwell迁移试验显示,转染后细胞增殖及迁移能力明显降低(P<0.05)。结论:COL1A1-shRNA转染胃癌BGC-823细胞能有效抑制细胞COL1A1的表达及癌细胞的增殖迁移。

Clinicopathological significance of altered Notch signaling in extrahepatic cholangiocarcinoma and gallbladder carcinoma

AIM:To investigate the role and clinicopathological significance of aberrant expression of Notch receptors and Delta-like ligand-4 (DLL4) in extrahepatic cholangiocarcinoma and gallbladder carcinoma.METHODS:One hundred and ten patients had surgically resected extrahepatic cholangiocarcinoma (CC) and gallbladder carcinoma specimens examined by immunohistochemistry of available paraffin blocks.Immunohistochemistry was performed using anti-Notch receptors 1-4 and anti-DLL4 antibodies.We scored the immunopositivity of Notch receptors and DLL4 expression by percentage of positive tumor cells with cytoplasmic expression and intensity of immunostaining.Coexistent nuclear localization was evaluated.Clinicopatho-logical parameters and survival data were compared with the expression of Notch receptors 1-4 and DLL4.RESULTS:Notch receptor proteins showed in the cytoplasm with or without nuclear expression in cancer cells,as well as showing weak cytoplasmic expression in non-neoplastic cells.By semiquantitative evaluation,positive immunostaining of Notch receptor 1 was detected in 96 cases (87.3%),Notch receptor 2 in 97 (88.2%),Notch receptor 3 in 97 (88.2%),Notch receptor 4 in 103 (93.6),and DLL4 in 84 (76.4%).In addition,coex- istent nuclear localization was noted [Notch receptor 1;18 cases (18.8%),Notch receptor 2;40 (41.2%),Notch receptor 3;32 (33.0%),Notch receptor 4;99 (96.1%),DLL4;48 (57.1%)].Notch receptor 1 expression was correlated with advanced tumor,node,metastasis (TNM) stage (P=0.043),Notch receptor 3 with advanced T stage (P=0.017),tendency to express in cases with nodal metastasis (P=0.065) and advanced TNM stage (P=0.052).DLL4 expression tended to be related to less histological differentiation (P=0.095).Coexistent nuclear localization of Notch receptor 3 was related to no nodal metastasis (P=0.027) and Notch receptor 4 with less histological differentiation (P=0.036),while DLL4 tended to be related inversely with T stage (P=0.053).Coexistent nuclear localization of DLL4 was related to poor survival (P=0.002).CONCLUSION:Aberrant expression of Notch receptors 1 and 3 play a role during cancer progression,and cytoplasmic nuclear coexistence of DLL4 expression correlates with poor survival in extrahepatic CC and gallbladder carcinoma.

A guide to diagnosis of iron deficiency and iron deficiency anemia in digestive diseases

Iron deficiency （ID）, with or without anemia, is often caused by digestive diseases and should always be investigated, except in very specific situations, as its causes could be serious diseases, such as cancer. Diagnosis of ID is not always easy. Low serum levels of ferritin or transferrin saturation, imply a situation of absolute or functional ID. It is sometimes difficult to differentiate ID anemia from anemia of chronic diseases, which can coexist. In this case, other parameters, such as soluble transferrin receptor activity can be very useful. After an initial evaluation by clinical history, urine analysis, and serological tests for celiac disease, gastroscopy and colonoscopy are the key diagnostic tools for investigating the origin of ID, and will detect the most important and prevalent diseases. If both tests are normal and anemia is not severe, treatment with oral iron can be indicated, along with stopping any treatment with non-steroidal anti-inflammatory drugs. In the absence of response to oral iron, or if the anemia is severe or clinical suspicion of important disease persists, we must insist on diagnostic evaluation. Repeat endoscopic studies should be considered in many cases and if both still show normal results, investigating the small bowel must be considered. The main techniques in this case are capsule endoscopy, followed by

Expression of the P2X_2 receptor in different classes of ileum myenteric neurons in the female obese ob/ob mouse

AIM:To examine whether the ob/ob mouse model of obesity is accompanied by enteric nervous system ab-normalities such as altered motility METHODS:The study examined the distribution of the P2X 2 receptor (P2X 2 R) in myenteric neurons of female ob/ob mice. Specifically, we used immunohistochemistry to analyze the co-expression of the P2X 2 R with neuronal nitric oxide synthase (nNOS), choline acetyltrans-ferase (ChAT), and calretinin (CalR) in neurons of the small intestine myenteric plexus in ob/ob and control female mice In these sections, we used scanning confocal microscopy to analyze the co-localization of these markers as well as the neuronal density (cm 2 ) and area profile (μm2) of P2X 2 R-positive neurons In addition, enteric neurons were labeled using the nicotinamide adenine dinucleotide (NA H) diaphorase method and analyzed with light microscopy as an alternate means by which to analyze neuronal density and areaRESULTS:In the present study, we observed a 29 6% increase in the body weight of the ob/ob animals (OG) compared to the control group (CG) In addition, the average small intestine area was increased by approxi-mately 29 6% in the OG compared to the CG Immu-noreactivity (IR) for the P2X 2 R, nNOS, ChAT and CalR was detectable in the myenteric plexus, as well as in the smooth muscle, in both groups This IR appeared to be mainly cytoplasmic and was also associated with the cell membrane of the myenteric plexus neurons, where it outlined the neuronal cell bodies and their processes P2X 2 R-IR was observed to co-localize 100% with that for nNOS, ChAT and CalR in neurons of both groups In the ob/ob group, however, we observed that the neuronal density (neuron/cm 2 ) of P2X 2 R-IR cells was in-creased by 62% compared to CG, while that of NOS-IR and ChAT-IR neurons was reduced by 49% and 57%, respectively, compared to control mice The neuronal density of CalR-IR neurons was not different between the groups Morphometric studies further demonstrated that the cell body profile area (μm2) of nNOS-IR, ChAT-IR and CalR-IR neurons was increased by 34%, 20% and 55%, respectively, in the OG compared to controls Staining for NA H diaphorase activity is widely used to detect alterations in the enteric nervous system; however, our qualitative examination of NA H-diaphorase positive neurons in the myenteric ganglia revealed an overall similarity between the two groups CONCLUSION:We demonstrate increases in P2X2R expression and alterations in nNOS, ChAT and CalR IR in ileal myenteric neurons of female ob/ob mice compared to wild-type controls.

Role of cannabinoids in chronic liver diseases

Cannabinoids are a group of compounds acting pri-marily via CB1 and CB2 receptors.The expression of cannabinoid receptors in normal liver is low or absent.However,many reports have proven up-regulation of the expression of CB1 and CB2 receptors in hepatic myofibroblasts and vascular endothelial cells,as well as increased concentration of endocannabinoids in liver in the course of chronic progressive liver diseases.It has been shown that CB1 receptor signalling exerts profibrogenic and proinflammatory effects in liver tis-sue,primarily due to the stimulation of hepatic stellate cells,whereas the activation of CB2 receptors inhibits or even reverses liver fibrogenesis.Similarly,CB1 re-ceptor stimulation contributes to progression of liver steatosis.In end-stage liver disease,the endocannabi-noid system has been shown to contribute to hepatic encephalopathy and vascular effects,such as portal hypertension,splanchnic vasodilatation,relative pe-ripheral hypotension and probably cirrhotic cardiomy-opathy.So far,available evidence is based on cellular cultures or animal models.Clinical data on the effects of cannabinoids in chronic liver diseases are limited.However,recent studies have shown the contribution of cannabis smoking to the progression of liver fibrosis and steatosis.Moreover,controlling CB1 or CB2 signal-ling appears to be an attractive target in managing liver diseases.

Insights into erlotinib action in pancreatic cancer cells using a combined experimental and mathematical approach

AIM:To gain insights into the molecular action of erlotinib in pancreatic cancer (PC) cells. METHODS:Two PC cell lines, BxPC-3 and Capan-1, were treated with various concentrations of erlotinib, the specific mitogen-activated protein kinase kinase (MEK) inhibitor U0126, and protein kinase B (AKT) inhibitor XIV. DNA synthesis was measured by 5-bromo-2'-deoxyuridine (BrdU) assays. Expression and phosphorylation of the epidermal growth factor receptor (EGFR) and downstream signaling molecules were quantified by Western blot analysis. The data were processed to calibrate a mathematical model, based on ordinary differential equations, describing the EGFRmediated signal transduction. RESULTS:Erlotinib significantly inhibited BrdU incorporation in BxPC-3 cells at a concentration of 1 mol/L, whereas Capan-1 cells were much more resistant. In both cell lines, MEK inhibitor U0126 and erlotinib attenuated DNA synthesis in a cumulative manner, whereas the AKT pathway-specific inhibitor did not enhance the effects of erlotinib. While basal phosphorylation of EGFR and extracellular signal-regulated kinase (ERK) did not differ much between the two cell lines, BxPC-3 cells displayed a more than five-times higher basal phospho-AKT level than Capan-1 cells. Epidermal growth factor (EGF) at 10 ng/mL induced the phosphorylation of EGFR, AKT and ERK in both cell lines with similar kinetics. In BxPC-3 cells, higher levels of phospho-AKT and phospho-ERK (normalized to the total protein levels) were observed. Independent of the cell line, erlotinib efficiently inhibited phosphorylation of EGFR, AKT and ERK. The mathematical model successfully simulated the experimental findings and provided predictions regarding phosphoprotein levels that could be verified experimentally. CONCLUSION:Our data suggest basal AKT phosphorylation and the degree of EGF-induced activation of AKT and ERK as molecular determinants of erlotinib efficiency in PC cells.

Changing the treatment of heart failure with reduced ejection fraction： clinical use of sacubitril-valsartan combination

Despite significant therapeutic advances, patients with chronic heart failure （HF） remain at high risk of morbidity and mortality. Sacubitill valsartan （previously known as LCZ696） is a new oral agent approved for the treatment of symptomatic chronic heart failure in adults with reduced ejection fraction. It is described as the fast in class angiotensin receptor neprilysin inhibitor （ARNI） since it incorporates the neprilysin inhibitor, sacubitril and the angiotensin Ⅱ receptor antagonist, valsartan. Neprilysin is an endopeptidase that breaks down several vasoactive peptides including natriuretic peptides （NPs）, bradykinin, endothelin and angiotensin II （Ang-II）. Therefore, a natural consequence of its inhibition is an increase of plasmatic levels of both, NPs and Ang-Ⅱ （with opposite biological actions）. So, a combined inhibition of these both systems （Sacubitril / valsartan） may enhance the benefits of NPs effects in HF （natriuresis, diuresis, etc） while Ang-Ⅱ receptor is inhibited （reducing vasoconstriction and aldosterone release）. In a large clinical trial （PARADIGM-HF with 8442 patients）, this new agent was found to significantly reduce cardiovascular and all cause mortality as well as hospitalizations due to HF （compared to enalapril）. This manuscript reviews clinical evidence for sacubitril valsartan, dosing and cautions, future directions and its considered place in the therapy of HF with reduced ejection fraction.

Epidemiology and clinical course of Crohn's disease:Results from observational studies

The authors review the clinical outcome in patients with Crohn's disease(CD)based on studies describing the natural course of the disease.Population-based studies have demonstrated that the incidence rates and prevalence rates for CD have increased since the mid-1970s.The authors search for English language articles from 1980 until 2011.Geographical variations,incidence,prevalence,smoking habits,sex,mortality and medications are investigated.An increasing incidence and prevalence of CD have been found over the last three decades.The disease seems to be most common in northern Europe and North America,but is probably increasing also in Asia and Africa.Smoking is associated with an increased risk of developing CD.Age <40 at diagnosis,penetrating/stricturing complications,need for systemic steroids,and disease location in terminal ileum are factors associated with higher relapse rates.A slight predominance of women diagnosed with CD has been found.Ileocecal resection is the most commonly performed surgical procedure,and within the first five years after the diagnosis about one third of the patients have had intestinal surgery.Smoking is associated with a worse clinical course and withincreased risk of flare-ups.In most studies the overall mortality is comparable to the background population.To date,the most effective treatment options in acute flares are glucocorticosteroids and tumor necrosis factor(TNF)-α-blockers.Azathioprine/methotrexate and TNF-α-blockers are effective in maintaining remission.