被动免疫预防犊牛轮状病毒感染的研究

目的 轮状病毒灭活疫苗免疫孕牛后，研究产下犊牛通过母源抗体获得的被动免疫在预防牛轮状病毒感染中的作用。方法 孕牛产前2月和1月分别接种轮状病毒灭活疫苗，检测产牛后乳汁和犊牛血样中BRV特异性IgG、IgA及中和抗体滴度。犊牛21日龄时口服2mL10^4,59 TCID50／ml的牛轮状病毒，观察犊牛腹泻及排毒情况。结果 免疫组乳清和犊牛血清中产生了高水平的牛轮状病毒特异性IgG、IgA及中和抗体，与对照组差异极显著（P〈0．01）；攻毒后，对照组6头犊牛全部发生腹泻，免疫组6头中有1头腹泻，发病的潜伏期及发病率差异显著。结论 被动免疫可显著提高犊牛血清中牛轮状病毒特异性抗体水平，使犊牛获得一定的免疫保护力抵抗轮状病毒的感染。     

凸隆病毒科——一类引起腹泻的新病毒

<正> 1982年Woode等在美国爱俄华州Breda市腹泻的犊牛粪中发现一种新的传染性的致病因子,它具有病毒形态,能凝结大鼠红细胞,并能在所接种的悉生犊或普通犊体内复制。它有两个血清型,但它与已知的病毒如牛冠状病毒(BCV)、副流感Ⅲ型病毒、牛轮状病毒、牛细小病毒及引起牛病毒性腹泻的粘膜病病毒(BVD-MD)均不相同,将此种未分类的新病毒暂定名为Breda因子或Breda病毒,简称BRV[9]。此后不久在美国俄亥俄州的犊牛群中再次发现,其血清学反应属Ⅱ型BRV。1983年法国Moussa等在里昂的牛群中也发现一种引致腹泻的病毒,称之为Lyon病毒,简称LYV,随后证实LYV与BRV抗原性一致[1]。     

人类组织器官中轮状病毒受体分布初探

目的检测人类绒毛以及胎儿其他肠道外器官轮状病毒（RV）受体的存在情况,初步探讨RV宫内感染及肠道外感染病毒吸附的生物学基础。方法以单克隆抗体免疫组化方法检测上述组织RV受体存在情况。结果受检的10例胎儿中,8例小肠绒毛上皮细胞、8例心肌细胞、9例肺泡上皮细胞、4例肝细胞、5例肾集合管上皮细胞均存在RV受体,脑组织均阴性。受检15例绒毛上皮细胞均存在RV受体。5例正常儿童和5例成人单个核细胞表面均有RV受体表达。结论胎儿绒毛组织及肠道外器官广泛存在RV受体,提示RV宫内感染和肠道外感染存在一定的物质基础。     

半巢式RT-PCR快速检测犊牛轮状病毒方法的建立

目的根据牛轮状病毒的VP7基因设计了一对可以检测牛轮状病毒G6血清型的引物,成功地检测了牛轮状病毒G6血清型。使用该方法检测牛病毒性腹泻-粘膜病病毒,检测结果为阴性。敏感性试验表明本实验方法可以检测10-4稀释的样品。通过对23份临床样本的检测证实,PCR方法比聚丙烯酰胺凝胶电泳和病毒的分离试验灵敏度高,可以快速诊断轮状病毒病,并且能够同时鉴定轮状病毒主要血清型。     

禽多杀性巴氏杆菌外膜蛋白H受体的初步鉴定

目的鉴定鸡胚成纤维(CEF)细胞膜禽多杀性巴氏杆菌外膜蛋白H(OmpH)受体。方法用膜蛋白提取试剂盒制备CEF细胞的膜蛋白,通过SDS-PAGE和配体印迹检测CEF细胞膜蛋白中的OmpH受体,采用MALDI-TOF质谱技术鉴定蛋白种类,并用配体印迹、ELISA和免疫荧光技术检测OmpH受体在不同宿主食管黏膜细胞表面的分布情况。结果在转印CEF细胞膜蛋白的NC膜上有一条明显的疑似受体条带,分子量约为49kDa。MALDI-TOF质谱鉴定结果表明该受体蛋白是ATP合成酶β亚基。在转印鸡和兔食管黏膜细胞膜蛋白的NC膜上检测到能够与OmpH结合的蛋白条带,但在牛和猪食管黏膜细胞膜蛋白中未见任何印迹条带,而OmpH与鸡和兔食管黏膜细胞膜蛋白的结合力高于猪和牛食管黏膜细胞膜蛋白。结论禽多杀性巴氏杆菌OmpH受体可能是CEF细胞膜表面的ATP合成酶β亚基。     

2019-2020年湖北部分地区G9P[8]型人轮状病毒基因特征北大核心CSCD

目的对人A组轮状病毒进行检测及分离鉴定,并研究其各基因片段的遗传进化关系。方法2019-2020年对湖北武汉市和襄阳市临床腹泻病人的粪便样品进行采集,共319份。设计特异性轮状病毒VP6基因引物,RT-PCR检测轮状病毒的感染情况。将阳性样品接种于MA104细胞进行轮状病毒的分离。RT-PCR特异性扩增VP6基因和特异性间接免疫荧光对其进行病毒鉴定及病毒增殖检测;并进一步通过RT-PCR扩增轮状病毒的11个基因片段,在线工具Rota C V2.0对测序结果进行分型分析。Mega软件对其全基因组序列进行遗传进化分析。结果轮状病毒感染阳性标本共69份,阳性率为21.63%。成功分离获得11株人轮状病毒,主要衣壳蛋白VP7和VP4基因型均为G9P[8]型。其中3株轮状病毒归属于类Wa株毒株,基因型图谱为G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1。8株毒株在Wa-like的基因型中具有DS-1-like的NSP4为E2基因型特征。基因型图谱为G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1。结论G9P[8]型人轮状病毒毒株在2019-2020年湖北部分地区占主导趋势,且其NSP4基因以E2基因型为主要流行形式。     

抗血管紧张素Ⅱ-1型受体抗体对在体大鼠心功能的影响

目的观察抗血管紧张素Ⅱ-1型受体（AT1-R）抗体对在体大鼠心功能的影响。方法健康Wistar大鼠随机分为5组：对照组、抗AT1-R抗体组、抗AT1-R抗体＋AT1受体阻断剂Losartan处理组、血管紧张素Ⅱ组、血管紧张素Ⅱ＋Losartan组。通过MS2000生物信号记录分析系统记录左心室收缩压（LVsP）、左心室内压最大上升速率和最大下降速率（±dP／dtmax）。结果抗AT1-R抗体可显著升高大鼠LVSP、±dP／dtmax（P〈0．05）,且这种增强心功能的效应可被Losartan逆转。结论抗AT1-R抗体可能通过激活ATl受体而发挥正性变力效应。     

姜黄素水解衍生物对血管平滑肌细胞增殖及低密度脂蛋白受体表达的影响

目的研究姜黄素水解衍生物对氧化型低密度脂蛋白促牛主动脉平滑肌细胞增殖和对牛血管平滑肌细胞膜低密度脂蛋白受体表达的影响。方法用盐酸加三氯化铝水解姜黄素,获得姜黄素水解衍生物;用氧化型低密度脂蛋白培养牛血管平滑肌细胞建立促增殖模型,MTT比色法观察姜黄素水解衍生物对牛血管平滑肌细胞增的抑制作用;流式细胞仪检测血管平滑肌细胞膜上低密度脂蛋白受体表达的影响;采用逆转录聚合酶链反应检测c-myc的表达。结果姜黄素水解衍生物对10mg/L氧化型低密度脂蛋白培养的血管平滑肌细胞增殖有明显的抑制作用,高中低剂量组的抑制率分别为46%、37%和22%(P<0.01或0.05);对血管平滑肌细胞膜上的低密度脂蛋白受体的表达均有明显的上调作用,高中低剂量组低密度脂蛋白受体表达率分别比对照组高33.85%、17.95%和5.75%(P<0.01或0.05);能够下调c-myc的表达。结论姜黄素水解衍生物能明显抑制牛主动脉平滑肌细胞增殖,上调低密度脂蛋白受体的表达,从而延缓动脉粥样硬化的发生发展。     

茉莉花叶水提物对小肠平滑肌的影响

目的观察茉莉花叶水提物对小肠平滑肌的作用。方法采用常规离体灌注小肠平滑肌标本方法,观察给药前后离体家兔小肠平滑肌平均张力、振幅及收缩频率变化,并检测其对在体小鼠小肠推进功能的影响。结果与给药前相比,茉莉花叶水提物槽内终浓度(2.0~6.0)×10-2g生药/ml可使家兔离体十二指肠平滑肌平均张力及振幅显著降低,并随着给药剂量增加,抑制作用增强,当终浓度为4.0×10-2g生药/ml时抑制效应达到最大,但各浓度组对收缩频率均无明显影响。40 g生药/kg、20 g生药/kg剂量组对小鼠小肠推进运动亦有显著抑制作用。结论茉莉花叶水提物对家兔离体小肠平滑肌活动及小鼠小肠的推进运动有抑制作用,其作用机制可能与平滑肌细胞膜上的M受体有关,而与α,β受体无关。     

猪轮状病毒OSU株的培养特性与致病性研究

摘 要：目的 开展猪轮状病毒OSU株的细胞培养特性及致病性研究,确定轮状病毒培养的关键技术与致病规律。 方法 以MA104细胞培养病毒,对预处理病毒的胰酶浓度与时间、维持液中最佳胰酶浓度等关键条件进行优化,透射电镜观察病毒粒子形态,测定病毒TCID50,口服感染3日龄仔猪进行致病性试验。结果 病毒经20μg/mL胰酶预处理1h,37℃吸附细胞2h,维持液最佳胰酶浓度为4μg/mL,病毒典型细胞病变为病变细胞葡萄串状堆积、胞浆相连、拉网等病变。透射电镜下病毒粒子呈圆形车轮状,直径约80nm。病毒感染3日龄仔猪10h后出现典型的黄色水样腹泻,感染42h后死亡,剖检可见胃内有凝乳块、肠壁变薄充满液体,盲肠、结肠充气。主要病变为：肠上皮细胞变性、坏死、脱落;固有膜高度扩张、充血和伴有轻微出血;粘膜上皮脱落,粘膜下层水肿、、炎性细胞浸润等。结论 研究阐明了轮状病毒OSU株的培养特性与致病特征,为后续开展轮状病毒的病原学研究奠定了基础。     

Affinities of pirenzepine for muscarinic cholinergic receptors in membranes isolated from bovine tracheal mucosa and smooth muscle

Muscarinic cholinergic receptors have been classified into subtypes based on their high (M-1 subtype) or low (M-2 subtype) affinities for the nonclassic antagonist pirenzepine, and this classification has important experimental and therapeutic implications. Because muscarinic receptors are abundant in the airways where they mediate several different cellular responses, the goal of this study was to characterize the affinities of pirenzepine for the muscarinic receptors in bovine tracheal mucosa and smooth muscle. After isolating membrane particulates from mucosa and smooth muscle, as well as from bovine cerebral cortex (a known source of M-1 receptors), we used 3H-quinuclidinyl benzilate to label muscarinic receptors in the particulates and performed competition radioligand binding assays in the presence of either atropine or pirenzepine. Receptors from all 3 tissues (mucosa, smooth muscle, and cerebral cortex) were of a relatively uniform affinity for atropine (range of KI values: 0.8 +/- 0.4 X 10(-9) to 2.4 +/- 1.7 X 10(-9) M), as would be predicted for this classic muscarinic antagonist. By contrast, affinities for pirenzepine differed depending on the tissue. In cerebral cortex, the majority of receptors were of high affinity for pirenzepine (KI = 1.8 +/- 1.4 X 10(-8) M). In both mucosa and smooth muscle, receptors were of low affinity for pirenzepine (Kl = 4.8 +/- 0.4 to 6.9 +/- 3.8 X 10(-7) M). We conclude that muscarinic cholinergic receptors in bovine tracheal mucosa and smooth muscle are predominantly of the M-2 subtype.     

IFN-lambda determines the intestinal epithelial antiviral host defense

Type I and type III IFNs bind to different cell-surface receptors but induce identical signal transduction pathways, leading to the expression of antiviral host effector molecules. Despite the fact that type III IFN (IFN-λ) has been shown to predominantly act on mucosal organs, in vivo infection studies have failed to attribute a specific, nonredundant function. Instead, a predominant role of type I IFN was observed, which was explained by the ubiquitous expression of the type I IFN receptor. Here we comparatively analyzed the role of functional IFN-λ and type I IFN receptor signaling in the innate immune response to intestinal rotavirus infection in vivo, and determined viral replication and antiviral gene expression on the cellular level. We observed that both suckling and adult mice lacking functional receptors for IFN-λ were impaired in the control of oral rotavirus infection, whereas animals lacking functional receptors for type I IFN were similar to wild-type mice. Using Mx1 protein accumulation as marker for IFN responsiveness of individual cells, we demonstrate that intestinal epithelial cells, which are the prime target cells of rotavirus, strongly responded to IFN-λ but only marginally to type I IFN in vivo. Systemic treatment of suckling mice with IFN-λ repressed rotavirus replication in the gut, whereas treatment with type I IFN was not effective. These results are unique in identifying a critical role of IFN-λ in the epithelial antiviral host defense.     

猴轮状病毒的灭活、纯化及抗原性鉴定

目的探讨经β丙内酯灭活的猴轮状病毒SA11的抗原性。方法将猴轮状病毒适应到vero细胞上,用β丙内酯灭活后,接种MA104细胞,鉴定灭活效果,经超滤浓缩、凝胶过滤后,加入弗氏完全佐剂免疫豚鼠,确定豚鼠产生的中和抗体效价。结果利用β丙内酯在4℃作用24、48、72或96h,可以有效的灭活轮状病毒SA11,通过凝胶过滤可得到初步纯化的轮状病毒,以1.33mg/只的剂量与弗氏完全佐剂混合免疫豚鼠后,中和抗体的滴度为1∶1024。结论用β丙内酯可以有效的灭活轮状病毒。     

Direct observation of substance P-induced internalization of neurokinin 1 (NK1) receptors at sites of inflammation.

Substance P (SP) can cause plasma leakage at sites of inflammation by binding to neurokinin type 1 (NK1) receptors on the surface of endothelial cells. Internalization after ligand binding could reduce the number of NK1 receptors on the cell surface and thus participate in the desensitization and resensitization of the inflammatory response to SP. By using an antibody to the receptor, we directly observed SP-induced internalization of NK1 receptors into endosomes in endothelial cells of postcapillary venules in the rat tracheal mucosa. In the absence of SP, an average of 15 immunoreactive endosomes were present per endothelial cell. After an intravenous injection of SP, the number of immunoreactive endosomes peaked at 107 per cell at 3 min and gradually returned to the baseline by 120 min. In parallel experiments we observed that when cultured cells transfected with the NK1 receptor were exposed to rhodamine-SP and an antibody to an extracellular Flag epitope of the NK1 receptor, the SP was internalized with the receptor antibody. Both in the cultured cells and in the endothelial cells of intact animals, the prompt SP-induced internalization was accompanied by rapid, long-lasting desensitization to SP. These studies suggest that internalization of NK1 receptors by endothelial cells may be one of the mechanisms that limit the amount of plasma leakage at sites of inflammation.     

Evaluation of RIT 4237 bovine rotavirus vaccine in newborn infants: correlation of vaccine efficacy to season of birth in relation to rotavirus epidemic period

A single oral dose of bovine rotavirus vaccine RIT 4237 or placebo was given to 2 groups of 5-day-old infants, born in October 1984 (n = 244) and June 1985 (n = 245), who remained in follow-up for 2.8 and 2.0 years, respectively. The vaccine had no effect on the total number of detectable episodes of rotavirus diarrhoea: there were 22 cases in the vaccinees and 24 in the placebo recipients in the October group and 18 and 16 respectively in the June group. However, vaccination decreased significantly the clinical severity of rotavirus diarrhoea, as assessed by a numerical score 0-20; this vaccine effect was much greater in the infants born in October. The mean severity scores for vaccine and placebo recipients were 4.55 and 10.75 respectively in the October group (p less than 0.0001, t-test) and 8.2 and 11.6 respectively in the June group (p = 0.010, t-test). Vaccine-induced clinical protection against rotavirus diarrhoea did not correlate well with serological response after vaccination, but showed good correlation to the presence of rotavirus antibodies before the rotavirus epidemic season. It is concluded that bovine rotavirus vaccine is more efficacious when given immediately before the rotavirus epidemic season: the vaccine effect may be amplified by exposure to wild rotaviruses during the season.     

Airway epithelial cells are the site of expression of a mammalian antimicrobial peptide gene.

We previously reported the isolation and characterization of a broad-spectrum antimicrobial peptide from the bovine tracheal mucosa, which we called tracheal antimicrobial peptide (TAP). We now show the TAP gene is expressed throughout the adult conducting airway, from nasal to bronchiolar tissue, but not in tissues other than airway mucosa, as determined by Northern blot analysis. In situ hybridization of airway sections localizes TAP mRNA to columnar cells of the pseudostratified epithelium. We report the structural organization of the TAP gene and show that TAP is a member of a large family of related sequences with high nucleotide identity in the 5' exon. The data support the hypothesis that antimicrobial peptides contribute to host defense of the respiratory tract.     

Characterization of specific pancreatic polypeptide receptors on basolateral membranes of the canine small intestine.

We have identified specific binding sites for pancreatic polypeptide (PP) on the mucosal lining of canine small intestine. The present study was undertaken to further characterize these binding sites (receptors) on purified intestinal membranes and to establish their location on the brush border or basolateral surface of the intestinal enterocyte. Basolateral and brush border membranes were prepared by sorbitol density centrifugation. PP receptors were localized predominantly to the vascular surface, and thus binding of PP 125I-labeled on Tyr-27 to the basolateral preparation was used to evaluate receptor characteristics. Binding of PP was calcium, time, temperature, and pH dependent. Maximum specific binding of labeled PP occurred after an 8-hr incubation at 4 degrees C with 5 mM calcium at pH 6.8. Data analysis by Scatchard plot showed high- and low-affinity binding sites with relative affinities of 1.5 x 10(-9) M and 2.6 x 10(-8) M and with corresponding binding capacities of 0.23 pmol/mg and 0.84 pmol/mg of protein, respectively. This receptor was specific for PP since peptide YY and neuropeptide Y, peptides of the PP family, cross-reacted by less than 3%, as judged from comparisons of half-maximal displacement of label. Structurally dissimilar peptides, insulin and glucagon, did not compete for binding. Specific 125I-labeled PP binding was localized primarily to basolateral membranes (9.8 +/- 0.8%) with little binding by brush border membranes (0.8 +/- 0.2%). Thus, we have identified highly specific receptors for PP, located predominantly on the vascular surface of the small intestinal mucosa. These data suggest that the mucosal lining of the small intestine is a target tissue for PP and that PP participates in the hormonal regulation of fuel metabolism and substrate transport in the small intestinal mucosa.     

肠黏膜雌孕激素受体表达在肠易激综合征发病机制中的作用研究

目的 探讨雌、孕激素受体在肠易激综合征（IBS）患者肠道内分布、变化及其临床意义，并对在消化道的作用靶点和可能的细胞学机制做出合理解释和推断。方法 经结肠镜钳取24名正常人和59例肠易激综合征患者的回肠末端、盲肠和降结肠黏膜，分别采用抗人雌、孕激素受体抗体标识肠黏膜上雌、孕激素受体，并应用免疫组化方法检测了雌、孕激素受体阳性细胞数目改变。结果 1、IBS患者回肠末端和盲肠黏膜雌激素受体阳性细胞数目增多（P＜0.01)，降结肠黏膜与正常组无显著差别；2、肠道黏膜未见孕激素受体表达 结论 IBS患者肠黏膜雌激素受体高表达在IBS病理过程中发挥一定作用，并认为肥大细胞为雌激素在消化道的重要靶点，其对于揭示IBS在女性中的高发病率和月经期加重的临床现象有重要意义；同时提示雌激素受体拮抗剂可能对部分患者有效。