泡状棘球蚴病病原生物学研究进展

泡状棘球蚴病(alveolar echinoeoeeosis,AE)是一种重要的人兽共患寄生虫病。本文综述了其病原泡状棘球蚴的地理分布、宿主类别、传播情况及其发育生物学方面的研究情况,指出研究病原生物学的现实意义和今后仍需努力的方向。     

内蒙古西伯利亚棘球绦虫和多房棘球绦虫泡状蚴在小白鼠发育成熟的比较

本文报道人兽共患的泡状肝包虫病原,西伯利亚棘球绦虫Echinococcus sibiricensis (Rausch andSchiller, 1954)泡状蚴和多房棘球绦虫Ech inococcusmultilocularis ( Leuckart, 1863)泡状蚴在KM株小白鼠发育成熟过程比较观察的结果.此两虫种泡状蚴的发育成熟过程仍然和它们早期发育的规律(唐崇惕等,2001)相同.虽然它们成熟的泡囊都被着生在网状结构中的许多原头节所充满,但是在多房棘球绦虫9-14个月的泡状蚴,仍然可以见到它们的原头节和网状结构都是起源于泡囊囊壁内表面的胚细胞层,并且始终保持与该层的联系.而西伯利亚棘球绦虫泡状蚴在鼠肺脏或肝脏的各泡囊中的原头节和网状结构是由可移动的胚细胞团发育生成,它们与泡囊囊壁没有如前者样的联系.西伯利亚棘球泡状蚴在各别小白鼠肝脏也能发育成熟,但不正常,宿主反应异常强烈.     

内蒙古西伯利亚棘球绦虫和多房棘球绦虫泡状蚴在小白鼠发育成熟的比较

The alveolar echinococcus is one of the most dangerous worm parasites in man. Rausch and Schiller reported a new species, Echinococcus sibiricensis n. sp. from arctic fox, Alpex logopus, on St. Lawrence Island of Alaska, USA. According to the view of Vogel, the sibiricensis form is only a geographical race or subspecies of Europe Echinococcus multilocularis. So far, the two names, Echinococcus multiocularis multilocularis and Echinococcus multilocularis sibiricensis, existed in many references and text books. We have found the adults of Echinococcus sibiricensis and Echinococcus multilocularis from sand foxes, Vulpes corsac and their larval stages (alveolar echinococcus) from field voles, Microtus brandti in the Hulunbeier Pasture of Inner Mongolia, northeastern China in 1985 and 1998-1999. Two types of metacestodes with quite different styles of early development of E. sibiricensis and E. multilocularis were found from field voles and laboratory experimental white mice. As one characteristic of alveolar E. multilocularis, the capsules are produced by the exogenous budding of germinal cell layer together with cyst wall. The protoscoleces grow from germinal cells on germinal cell layer. The peduncles of early protoscoleces attached to the germinal cell layer on the inner surface of capsule wall(Plate I, Figs. 1-2). Some protoscoleces in reticular structure were linked with the inner surface of capsule wall (Plate I, Fig. 3) in livers of mice in 9.5th month postinfection. In 14th month old alveolar multilocularis, large number of mature protoscoleces in reticular structure were still linked to the inner surface of capsule wall (Plate I, Figs. 4-8). The cavities of some capsules were filled with protoscoleces in meshes of reticular structure which were also linked around with the inner surface of capsule wall (Plate I, Fig. 9). The superficial surface of livers of positive field voles and experimental mice never showed any hyperemic phenomenon. The superficial surfaces of livers and lungs of positive field voles and experimental mice infected with alveolar E. sibiricensis were highly hyperemic. The metacestodes of E. sibiricensis composed of mother cyst, undifferentiated embryonic cysts and small brood capsules. Cavities of all cysts were fully filled with germinal cell masses. Host reaction appeared to be very strong, all cysts were surrounded by thick connective tissue and dense leukocytes (Plate II, Fig. 10). All alveolar vesicles were found located in lungs tissue of experimental mice. Large germinal cell masses metastasized out from undifferentiated embryonic cysts into host lung tissue, where germinal cell masses developed into accumulation of early protoscoleces (Plate II, Figs. 11-12). Early protoscoleces of alveolar E. sibiricensis were seen earliest in mice lung tissues on 101-104th days after infection. Many small capsules in different sizes and different shapes containing mature protoscoleces and reticular structure (Plate II, Figs. 13-15) were found in lungs of mice in 9th month after infection. Only in one experimental mouse infected with alveolar E. sibiricensis in 8.5th month postinfection, both its lung and liver existed alveolar cysts; the capsules in liver were surrounded by very thick connective tissue of the host, and there were some protoscoleces in their cavities (Plate II, Figs. 16-18).     

细粒棘球绦虫原头蚴组织化学的观察

本文对细粒棘球绦虫原头蚴的组织化学成份进行了较为系统和全面的观察,结果表明,该虫体内存在糖原、RNA、DNA、蛋白质结合的α-氨基、酪氨酸、色氨酸及组氨酸、碱性蛋白质、酸性粘多糖、胶原及网状纤维成分、AKP、ATP酶及ACP等生化物质。并对上述成分的定位,含量及其生理学的意义进行了重点讨论。     

细粒棘球蚴FABP基因的原核表达及蛋白鉴定

构建原核表达载体pET-FABP,优化表达条件,采用免疫学方法鉴定纯化的FABP融合蛋白。载体pMD19-T-FABP和pET-44a(+)经EcoRⅠ和HindⅢ双酶切,将回收的FABP片段与pET-44a(+)连接,构建原核表达载体pET-FABP并在大肠杆菌BL21(DE3)中表达,优化表达条件,纯化FABP融合蛋白,Western blotting鉴定。成功构建了原核表达载体pET-FABP并在大肠杆菌中高效表达。纯化后的蛋白经SDS-PAGE和Western blotting鉴定正确。构建的表达载体pET-FABP可以在大肠杆菌中大量表达Nus-FABP融合蛋白,为进一步研制FABP亚单位疫苗奠定了基础。     

细粒棘球蚴eg95基因的克隆以及原核融合表达

从青海省羊源细粒棘球蚴提取基因组DNA,PCR扩增eg95基因并且克隆。测序结果表明该基因序列全长1262bp,由2个内含子和3个外显子组成,3个外显子分别为70bp、306bp和95bp。因此,推测其ORF为471bp,编码156个氨基酸。用RT—PCK方法扩增该基因的ORF,经克隆、DNAstar比对分析序列,它与预测的ORF序列100％相同。将目的基因克隆到原核表达载体pGEX-5x-1,构建重组表达质粒pGEX-5x-1-eg95,将其转入表达菌株BL21中进行原核表达。表达产物经过SDS—PAGE电泳分析,得到一条特异的蛋白条带;质谱鉴定证明表达的蛋白确实是eg95抗原蛋白;酶联接免疫吸附剂测定表明抗原抗体发生了特异性反应,进一步证明融合蛋白进行了正确表达,从而为囊型包虫病的研究奠定基础。     

棘球蚴囊液对小鼠脾细胞IL-17及Smad2表达的影响

目的:观察细粒棘球蚴囊液对体外培养BALB/c小鼠脾细胞白介素(IL)-17和Smad2基因表达的影响.方法:制备小鼠脾细胞悬液,接种于48孔培养板中进行培养,以不加任何干预为对照组,囊液处理组为实验组.定时取样提取总RNA,经反转录成cDNA后用实时荧光定量PCR(qRT-PCR)检测IL-17和Smad2基因的表达.结果:小鼠脾细胞IL-17表达在囊液处理6h和12h后升高(1.000±0.207 VS 3.672±0.746和1.000±0.154 VS 5.525±0.843),差异有统计学意义(P＜0.05);小鼠脾细胞Smad2表达在培养1h后升高(1.000±0.077 VS 2.069±0.098)在12h后降低(1.000±0.110 VS 0.247±0.011),差异有统计学意义(P＜0.05);协同分析发现IL-17基因与Smad2基因的表达变化呈现负相关(P＜0.01),相关系数r=-1.结论:细粒棘球蚴囊液对小鼠脾细胞IL-17和Smad2基因的表达具有上调作用,推测其可能与宿主抗棘球蚴感染的机制有关.     

细粒棘球蚴AgB8/2基因的原核表达及免疫学鉴定

旨在克隆细粒棘球蚴 AgB8/2基因,优化原核表达体系,鉴定纯化蛋白并初步确定其诊断价值.采用 RT-PCR扩增 AgB8/2基因 cDNA,构建原核表达载体pET-AgB8/2并在大肠杆菌 BL21(DE3)中表达,优化表达条件.纯化的AgB8/2融合蛋白经 Western blot鉴定正确后,采用斑点免疫金渗滤法初步验证其血清学诊断价值.结果显示,克隆的 AgB8/2基因包含了 273 bp的完整ORF,序列分析表明与其它已报道的AgB8/2 cDNA序列的同源性在 99%～100%之间.优化的表达条件为,当菌液 OD600值为0.6时,加入终浓度为0.05 mmol/L的 IPTG,37℃,振荡培养5 h.纯化的蛋白经 Western blot鉴定为目的蛋白.克隆的 AgB8/2基因高度保守,原核表达载体pET-AgB8/2构建正确并高效表达可溶性融合蛋白,可作为特异性抗原应用于斑点免疫金渗滤法检测血清抗体.     

光棘球海胆seali基因克隆及表达特性分析

seali基因隶属于PIWI超家族,其编码的RNA结合蛋白在生殖细胞发育过程中发挥重要作用。研究经同源比对从光棘球海胆(Mesocentrotus nudus)性腺转录组数据库中筛选得到seali基因片段,随后通过cDNA末端快速扩增技术(Rapid amplification of cDNA ends, RACE),获得其全长cDNA序列。光棘球海胆seali基因cDNA全长3462 bp,其中3′UTR长度为416 bp, 5′UTR长度为180 bp,其中3′UTR的加尾信号并非经典的AAUAAA或AUUAAA,而是较少见的AAUACA。开放阅读框(Open Reading Frame, ORF)2862 bp,编码954个氨基酸,具有保守的PIWI和PAZ结构域,多重序列比对和系统进化分析结果表明其属于Argonaute家族的PIWI亚家族成员。荧光定量PCR技术检测结果表明, Mnseali基因在光棘球海胆性腺、肠、管足和体腔细胞中均有表达,在性腺中表达量最高。此外, Mnseali基因为母源因子,在整个胚胎发育时期均有表达。在卵巢中,随着卵母细胞的成熟, Mnseali的表...     

中间球海胆、光棘球海胆及杂交F1代(中间球海胆♀×光棘球海胆♂)群体遗传多样性AFLP分析

应用AFLP技术对中间球海胆、光棘球海胆及杂交F1代(中间球海胆♀×光棘球海胆♂)群体的遗传多样性进行了分析。结果表明, 4对引物共扩增得到272个位点, 其中269个多态位点, 总的多态位点比例为98.89%。3个群体的香农多样性指数分别为: 0.2331 ± 0.1273、0.2005 ± 0.1385和0.2625 ± 0.1067。群体内遗传相似度分别为: 0.6876 ± 0.0523、0.6501 ± 0.0548和0.6552 ± 0.0553。分子方差分析(AMOVA)结果表明, 变异来源有25.39%来自群体间, 有74.61%来自群体内, 群体内的遗传多样性比较丰富。尽管杂交海胆在表型上可以明显分成两种类型, 但是通过AFLP统计的遗传距离进行的个体聚类却随机聚在一起, 不能分成两个群体。     

A Case of Human Hepatic Alveolar Echinococcosis Accompanied by Lung and Brain Metastases

Alveolar echinococcosis (AE) is considered as a fatal zoonosis caused by the larvae of Echinococcus multilocularis. The lungs and brain are the most common metastatic organs. We report a human case of hepatic alveolar echinococcosis accompanied by lung and brain metastasis. In particular, the patient had a history of tuberculosis and the lung lesions were easily misdiagnosed as lung abscesses. The lesions of liver and lung underwent radical resection and confirmed as alveolar echinococcosis by pathological examination. The patient had no surgical complications after operation and was discharged after symptomatic treatment. Unfortunately, the patient later developed multiple intracerebral AE metastases. We required the patient to take albendazole orally for life and follow up.     

肝切除手术治疗肝包虫病的疗效及对患者预后和肝功能的影响

目的:探讨肝切除手术治疗肝包虫病的疗效及对患者预后和肝功能的影响。方法:选取2015年2月-2018年5月期间我院收治的肝包虫病患者103例为研究对象,所有患者根据手术方式的不同分为A组(n=51,行外膜内完整外囊摘除术)和B组(n=52,行肝切除手术),比较两组患者围术期临床指标、肝功能指标、并发症发生情况,随访半年,记录两组患者随访期间死亡及原位复发情况。结果:B组术中出血量少于A组,手术时间、术后拔管时间以及住院时间均短于A组,组间比较差异有统计学意义(P0.05)。与术前比较,两组患者术后3 d、术后7 d谷丙转氨酶、胆红素以及谷草转氨酶均升高,但B组低于A组(P0.05);与术前比较,两组患者术后3 d、术后7 d白蛋白降低,但B组高于A组(P0.05);与术后3 d比较,两组患者术后7d谷丙转氨酶、胆红素以及谷草转氨酶均降低,白蛋白升高(P0.05)。B组术后并发症总发生率为7.69%(4/52),低于A组的23.52%(12/51),组间比较差异有统计学意义(P0.05)。两组随访期间无患者死亡,原位复发率比较差异均无统计学意义(P0.05)。结论:肝切除手术治疗肝包虫病患者安全有效,可减少术中出血量,促进患者术后康复,减轻手术对肝功能的影响,且术后复发率较低,患者预后较好。     

肝包虫病的诊断现状及进展

肝包虫病是畜牧地区常见的寄生虫病,是一种人畜共患病,非牧地区偶尔也可以见到,可以通过与狗等动物密切接触直接感染,还可以通过呼吸道吸入虫卵或经消化道误食含有虫卵感染的食物或水源等方式感染使人患病,并且可以寄生于人体内各个部位,但以肝脏最为多见,严重危害人体健康和畜牧业的发展。加强肝包虫病的诊断是防控本疾病的重要措施之一,在不能排除肝包虫病的情况下,应结合病史、化验室检查、B超、CT等影像学检查、免疫学检查进行综合分析,给出明确诊断,以防止误诊、漏诊。近年来随着分子生物学以及病原学等技术的发展,为包虫病的诊断提供更多的方法和信息,本文将从肝包虫病的病史及临床表现、影像学检查、免疫学检查、分子生物学检查及病原学检查等方面对其诊断现状及进展做一综述。     

A Case of Budd-Chiari Syndrome Associated with Alveolar Echinococcosis

Although alveolar echinococcosis (AE) can cause a serious disease with high mortality and morbidity similar to malign neoplasms. A 62-year-old woman admitted to a hospital located in Sivas, Turkey, with the complaints of fatigue and right upper abdominal pain. On contrast abdominal CT, a 54×70×45 mm sized cystic lesion was detected in the left lobe of the liver that was seen to extend to the posterior mediastinum and invade the diaphragm, esophagus, and pericardium. The cystic lesion was seen to be occluding the inferior vena cava and left hepatic vein at the level where the hepatic veins poured into the inferior vena cava. Bilateral pleural effusion was also detected. We discussed this secondary Budd-Chiari Syndrome (BCS) case, resulting from the AE occlusion of the left hepatic vein and inferior vena cava, in light of the information in literature.     

B超在肝囊型包虫病诊断中的临床价值与意义

目的：评价B超在肝囊型包虫病诊断中的价值与临床意义。方法：回顾性分析我院2000年12月至2011年12月收治的接受B超检查和手术治疗的332肝囊型包虫病例的临床资料。结果：332例肝囊型包虫病患者中,303例经过手术证实;肝囊型包虫病的B超分型可分为单发单囊型、多发单囊型、多子囊型、破裂感染型和坏死钙化型。单发单囊型患者以低年龄组多发,而高年龄组较少;破裂感染型以高年龄组多发;坏死钙化型病人共3例年龄均在60岁以上;破裂感染型占各型中病例数最多。结论：B型超声检查是肝囊型包虫病的主要检查方法,可确定包虫病的分型,了解包虫囊肿的部位、数目、大小和结构,为术前诊断、手术定位、观察治疗效果提供参考依据。     

Echinococcus granulosus Protoscolex DM9 Protein Shows High Potential for Serodiagnosis of Alveolar Echinococcosis

Alveolar echinococcosis (AE) caused by infection with E. multilocularis metacestode, represents one of the most fatal helminthic diseases. AE is principally manifested with infiltrative, proliferating hepatic mass, resembling primary hepatocellular carcinoma. Sometimes metastatic lesions are found in nearby or remote tissue. AE diagnosis largely depends on imaging studies, but atypical findings of imaging features frequently require differential diagnosis from other hepatic lesions. Serological tests may provide further evidence, while obtaining reliable AE materials is not easy. In this study, alternative antigens, specific to AE were identified by analyzing E. granulosus protoscolex proteins. An immunoblot analysis of E. granulosus protoscolex showed that a group of low-molecular-weight proteins in the range from 14 kDa to 16 kDa exhibited a sensitive and specific immune response to AE patient sera. Partial purification and proteomic analysis indicated that this protein group contained myosin, tubulin polymerization promoting protein, fatty-acid binding protein, uncharacterized DM9, heat shock protein 90 cochaperone tebp P-23, and antigen S. When the serological applicability of recombinant forms of these proteins was assessed using enzyme-linked immunosorbent assay, DM9 protein (rEgDM9) showed 90.1% sensitivity (73/81 sera tested) and 94.5% specificity (172/181 sera tested), respectively. rEgDM9 showed weak cross-reactions with patient sera from the transitional and chronic stages of cystic echinococcosis (3 to 5 stages). rEgDM9 would serve as a useful alternative antigen for serodiagnosis of both early- and advanced-stage AE cases.     

肝囊型包虫病非手术治疗方法的研究进展

肝囊型包虫病(简称肝包虫病)是一种由细粒棘球蚴感染肝脏引起的人兽共患寄生虫病,严重危害人类以及家畜的健康。近年来,由于该病术后复发率较高且易出现并发症,非手术治疗方法如介入技术、放射治疗以及药物治疗均得到了不断的发展。介入技术对于肝包虫病的治疗是一种比较新颖的方法,其临床应用时间较短,主要包括经皮穿刺治疗、射频消融、门静脉栓塞,以经皮穿刺治疗最为常见。放射治疗肝包虫病尚处于研究阶段,效果尚不完全明确。苯并咪唑类药物及其新剂型、非苯并咪唑类药物如地塞米松等及中草药治疗肝包虫病也均具有各自的优缺点。本文主要对上述非手术治疗方法及优缺点进行了综述。     

经皮穿刺微波消融术与腹腔镜切除肝癌预后的比较研究

目的:探讨在肝癌治疗中分别应用腹腔镜切除术与经皮穿刺微波消融术对患者预后影响。方法:从我院2010年3月至2012年3月收治的肝癌患者中选取90例为研究对象,经随机数字表法分为A组45例,B组45例,A组行经皮穿刺微波消融术治疗,B组行腹腔镜切除术治疗,观察两组患者手术时间、术中出血量及预后效果。结果:A组患者手术时间及术中出血量情况明显优于B组(P0.05);A组并发症发生为6.67%,同B组比较,明显较低(P0.05);A组局部复发率为20.00%,同B组8.89%比较,明显较高(P0.05),总复发率无明显差异(P0.05);两组患者术后1、2、3年存活率均无明显差异(P0.05)。结论:在肝癌治疗中,经皮穿刺微波消融术与腹腔镜切除远期疗效均较好,经皮穿刺微波消融术创伤小,并发症发生率低,对预后改善具有重要作用。     

不同手术时机治疗颅内前循环动脉瘤破裂疗效及预后分析

目的:比较不同手术时机治疗颅内前循环动脉瘤破裂患者的疗效及对患者远期预后的影响。方法:回顾性分析我院2010年3月~2015年10月收治的120例颅内前循环动脉瘤破裂患者的临床资料,所有患者均接受显微手术夹闭治疗,按手术时机分为超早期组(24 h,n=43)、早期组(24-72 h,n=36)、延期组(≥10 d,n=41),比较各组术后颅内动脉栓塞改善程度,统计各组术中及术后并发症发生情况,采用格拉斯哥量表(GOC)评定患者术后恢复情况,采用改良Rankin(m RS)表评定患者远期预后。结果:超早期组完全栓塞率略高于早期组、延期组,但对比差异无统计学意义(P0.05);超早期组术中、术后各并发症发生率略低于早期组、延期组,但对比差异无统计学意义(P0.05);术后6、12、24个月,超早期组、延期组GOS评分高于早期组、m RS评分低于早期组,超早期组GOS评分高于延期组,m RS评分低于延期组(P0.05)。结论:不同手术时机治疗颅内前循环动脉瘤破裂手术效果无明显差异,但超早期、延期手术患者术后恢复及预后评分稍优于早期手术。