Components of the mitogen-activated protein kinase cascade are activated in hepatic cells by Echinococcus multilocularis metacestode

AIM： To explore the effect of Echinococcusmultilocularis on the activation of mitogen-activated protein kinase （MAPK） signaling pathways and on livercell proliferation.METHODS： Changes in the phosphorylation of MAPKs and proliferating cell nuclear antigen （PCNA）expression were measured in the liver of patients withalveolar echinococcosis （AE）. MAPKs, MEK1/2 [MAPK/extracellular signal-regulated protein kinase （ERK）kinase] and ribosomal S6 kinase （RSK） phosphorylationwere detected in primary cultures of rat hepatocytesin contact in vitro with （1） E. multilocu/aris vesicle fluid（EmF）, （2）E. multilocularis-conditioned medium （EmCM）.RESULTS： In the liver of AE patients, ERK 1/2 andp38 MAPK were activated and PCNA expression wasincreased, especially in the vicinity of the metacestode.Upon exposure to EmF, p38, c-Jun N-terminal kinase（JNK） and ERK1/2 were also activated in hepatocytesin vitro, as well as MEK1/2 and RSK, in the absenceof any toxic effect. Upon exposure to EmCM, only JNKwas up-regulated.CONCLUSION： Previous studies have demonstratedan influence of the host on the MAPK cascade inE. multilocularis. Our data suggest that the reverse,i.e. parasite-derived signals efficiently acting onMAPK signaling pathways in host liver ceils, is actuallyoperating.

EFFECT OF ANTIHYDATID DRUGS ON CARBOHYDRATE METABOLISM OF METACESTODE OF ECHINOCOCCUS GRANULOSUS

A biochemical some enzymes of glycolysis and a partial reversed tricarboxylic acid cycle together with hydrolytic enzymes in the cyst wall of Echinococcus granulosus was carried out. Lactate dehydrogenase (LDH), pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (PEPCK), and adenosine triphosphatase (ATPase) showed their high level of activity, suggesting that the proliferation of E. granulosus cyst wall is an energy-dependent process and the major pathways for glucose metabolism is glycolysis. Treatment of E. granulosus-in-fected mice with mebendazole and albendazole resulted in marked inhibition of PK, PEPCK and ATPase of E. granulosus cyst wall, whereas praziquantel had no effect, indicating that PK, PEPCK, and ATPase might be chemotherapeutic targets and the differences in the inhibitory effects might account for the efficacy of the three antihydatid drugs.

THE FIRST REPORT OF ECHINOCOCCUS MULTILOCULARIS IN DOMESTIC DOGS IN ZHANG COUNTY OF GANSU PROVINCE

In Zhang County of Gansu Province, 13 human cases of alveolar hydatid disease were reported up to 1990. However, the causative organism has not yet been described in animal hosts. In July 1990, postmortem examinations were made on 59 dogs (Canis familiaris) from the Caotan region of Zhang County. Of them, 6 were found to be infected with the adult worms of Echinococcus multilocularis. The morphological characteristics of the adult were compared in detail with those reported from different parts of China. Our adult specimens were closely resembling those of E. multilocularis reported from Ningxia. The role of domestic dogs in the epidemiology of alveolar echinococcosis in Zhang County was discussed.

Impact of overgrazing on the transmission of Echinococcus multilocularis in Tibetan pastoral communities of Sichuan Province,China

Background Overgrazing was assumed to increase the population density of small mammals that are the intermediate hosts of Echinococcus multilocularis, the pathogen of alveolar echinococcosis in the Qinghai Tibet Plateau. This research tested the hypothesis that overgrazing might promote Echinococcus multilocularis transmission through increasing populations of small mammal, intermediate hosts in Tibetan pastoral communities. Methods Grazing practices, small mammal indices and dog Echinococcus multilocularis infection data were collected to analyze the relation between overgrazing and Echinococcus multilocularis transmission using nonparametric tests and multiple stepwise logistic regression. Results In the investigated area, raising livestock was a key industry. The communal pastures existed and the available forage was deficient for grazing. Open （common） pastures were overgrazed and had higher burrow density of small mammals compared with neighboring fenced （private） pastures; this high overgrazing pressure on the open pastures measured by neighboring fenced area led to higher burrow density of small mammals in open pastures. The median burrow density of small mammals in open pastures was independently associated with nearby canine Echinococcus multilocularis infection （P=-0.003, OR=1.048）. Conclusion Overgrazing may promote the transmission of Echinococcus multilocularis through increasing the population density of small mammals.

Suppression of acute rejective response following orthotopic liver transplantation in experimental rats infected with Echinococcus multilocularis

Background Hepatic alveolar echinococcosis （AE） is a parasitic disease in humans and caused by the Echinococcus multilocularis （Em）. Orthotopic liver transplantation （OLT） may be the only effective treatment for end-stage hepatic AE. However, in some AE patients, extrahepatic Em can not be completely eliminated after OLT. We aimed to study whether the immunological changes caused by Em evasion may influence the rejective response. Methods Rat modles of AE were established by injecting the Em suspension into abdomen of Brown Norway （BN） rats. Three months later, in the experimental group, the liver was transplanted from Lewis （LEW） rats to Em-infected BN rats. In the control group, transplantation was from LEW rats to healthy BN rats. Liver tissue and peripheral blood （PB） samples were collected on days 1, 3, 5, and 7 after OLT. Liver tissue was analyzed after hematoxylin and eosin （H＆E） staining; numbers of CD4, CD8, and CD28 on peripheral blood cells were detected by flow cytometry; and expression of the chemokine fractalkine （Fkn） was detected by reverse transcription PCR （RT-PCR）. Interleukin-10 （IL-10） was measured in the serum by enzyme-linked immunosorbent assay （ELISA）. In every group, eight BN rats were retained for observing survival time. Results The survival times of recipients in the experimental group were prolonged compared with those in the control group. The rejective response occurred later and was milder in the experimental group, percentage of CD4, CD8, CD28 T-cells and Fkn mRNA expression were lower in the experimental group. While the serum IL-10 levels were higher in the experimental group than those in the control group. Conclusions Acute rejective response after OLT was attenuated in the rats with Em infection, and the recipients＂ survival time was prolonged. Em may play a role in this process by elevating IL-10 secretion, decreasing the effector T cells, inhibiting the expression of Fkn, which lead to reduce the inflammatory cells infiltration into the liver.

Distribution of the intermediate hosts of Echinococcus multilocularis in Shiqu County, Sichuan, China

Background The rodentia and lagomorpha animals are the intermediate hosts of Echinococcus multilocularis, their distribution and infection of this parasite may facilitate the infection of definitive hosts such as dogs. The aim of this study was to determine the distribution of the intermediate hosts of Echinococcus multilocularis in Shiqu County, Sichuan, China. Methods A systematic sampling method was used to investigate the density of burrows of rodents and lagomorphs at 97 pasture sites in winter and summer pastureland and remote sensing （RS） techno（ogy was used to correlate their densities to the distribution of these animals in different landscape types. Results Based on the densities of Ochotona curzoniae, Microtus fuscus （dependent variable） and their burrow densities （independent variable） in survey points, regression equations were fitted respectively （Ochotona curzoniae, P 〈0.0001, R2=0.8705; Microtus fuscus, P 〈0.0001,R2=0.9736）. Their burrow density in summer pastureland was higher than in winter pastureiand （F=36.65, P 〈0.0001）. The burrow densities of Ochotona curzoniae and Microtus fuscus in bareland and half-bareland are higher than in grassland （F=7.73, P〈0.001）. Conclusions The regression relationship between the densities of Ochotona curzoniae and Microtus fuscus and their burrow densities indicate that the burrow densities could reflect the animal densities and that the burrow density was greater in summer pastureland than in winter pastureland. The main distribution areas of the intermediate hosts were in bareland and half-bareland.

Lasiopodomys fuscus as an important intermediate host for Echinococcus multilocularis:isolation and phylogenetic identification of the parasite

Background:Echinococcus multilocularis causes alveolar echinococcosis(AE)and is widely prevalent in Qinghai Province,China,where a number of different species have been identified as hosts.However,limited information is available on the Qinghai vole(Lasiopodomys fuscus),which is hyper endemic to Qinghai Province and may represent a potential intermediate host of E.multilocularis.Thus,L.fuscus could contribute to the endemicity of AE in the area.Methods:Fifty Qinghai voles were captured from Jigzhi County in Qinghai Province for the clinical identification of E.multilocularis infection via anatomical examination.Hydatid fluid was collected from vesicles of the livers in suspected voles and subjected to a microscopic examination and PCR assay based on the barcoding gene of cox 1.PCR-amplified segments were sequenced for a phylogenetic analysis.E.multilocularis-infected Qinghai voles were morphologically identified and subjected to a phylogenetic analysis to confirm their identities.Results:Seventeen of the 50 Qinghai voles had E.multilocularis-infection-like vesicles in their livers.Eleven out of the 17 Qinghai voles presented E.multilocularis infection,which was detected by PCR and sequencing.The phylogenetic analysis showed that all 11 positive samples belonged to the E.multilocularis Asian genotype.A morphological identification and phylogenetic analysis of the E.multilocularis-infected Qinghai voles confirmed that all captured animals were L.fuscus.Conclusions:L.fuscus can be infected with E.multilocularis and plays a potential role in the life cycle and epidemiology of E.multilocularis in the Qinghai-Tibetan Plateau of China.

Infection of sheep by Echinococcus multilocularis in Gansu,China:evidence from mitochondrial and nuclear DNA analysis

BackgroundIn the normal life cycle of the parasite(Echinococcus multilocularis)that causes alveolar echinococcosis,domestic and wild carnivores act as definitive hosts,and rodents act as intermediate hosts.The presented study contributes to the research on the distribution and transmission pattern of E.multilocularis in China having identified sheep as an unusual intermediate host taking part in the domestic transmission of alveolar echinococcosis in Gansu Province,China.MethodsFrom 2020 to 2021,nine whitish different cyst-like were collected from the liver of sheep in Gansu Province for examination.A near complete mitochondrial(mt)genome and selected nuclear genes were amplified from the cyst-like lesion for identification.To confirm the status of the specimen,comparative analysis with reference sequences,phylogenetic analysis,and network analysis were performed.ResultsThe isolates displayed≥98.87%similarity to E.multilocularis NADH dehydrogenase sub-unit 1(nad1)(894 bp)reference sequences deposited in GenBank.Furthermore,amplification of the nad4 and nad2 genes also confirmed all nine samples as E.multilocularis with>99.30%similarity.Additionally,three nuclear genes,pepck(1545 bp),elp-exons VII and VIII(566 bp),and elp-exon IX(256 bp),were successfully amplified and sequenced for one of the isolates with 98.42%similarity,confirming the isolates were correctly identified as E.multilocularis.Network analysis also correctly placed the isolates with other E.multilocularis.ConclusionsAs a result of the discovery of E.multilocularis in an unusual intermediate host,which is considered to have the highest zoonotic potential,the result clearly demonstrated the necessity for expanded surveillance in the area.

骨桥蛋白和p38MAPK信号通路在多房棘球蚴原头节发育中的作用

为明确骨桥蛋白(OPN)和多房棘球蚴(Echinococcus multilocularis,Em)p38丝裂原活化蛋白激酶(p38MAPK)信号通路在Em发育过程的作用,剖检感染Em 4~6月的长爪沙鼠,提取Em原头节后,分别用不同浓度的p38MAPK抑制剂(SB202190)处理Em原头节,选取合适的SB202190浓度后在体外将原头节分为DMSO组、anti-p38MAPK组、PBS组、OPN组、OPN+anti-p38MAPK组,采用伊红染色、caspase-3试剂盒检测caspase-3、EDU和Hoechst染色、活性氧(ROS)检测探针检测原头节的活性、凋亡和增殖情况。结果显示,抑制Em的SB202190适宜浓度为20μmol/L,SB202190增加Em的伊红染色率、caspase-3水平,减少EDU阳性、ROS水平,OPN可减少Em伊红染色率、caspase-3水平,增加EDU阳性率、ROS水平,且SB202190可逆转OPN对Em的结果,这些结果表明SB202190可抑制Em的活性和增殖、促进凋亡并与浓度正相关,而OPN可促进Em的活性和增殖、抑制凋亡,且SB202190可逆转OPN对Em的作用。研究结果为OPN和p38MAPK信号通路可能成为治疗多房棘球蚴病的新分子靶点提供证据。

我国内蒙古大兴安岭北麓泡状肝包虫种类的研究 III.苏俄棘球绦虫(Echinococcus russicensis sp.nov.)

在我国内蒙古大兴安岭北麓草场流行有与在前苏联广泛分布的泡状棘球蚴其成虫具球形子宫相似的病原,过去称之为苏俄的多房棘球绦虫。我们在比较研究了它与欧洲的多房棘球绦虫的全程生活史的发育规律,及成虫和幼虫期的结构之后,发现两者之间有极大差异,认为不能再用多房棘球绦虫称呼之。最近,我们用内蒙古的苏俄型泡状棘球蚴人工感染4只羊羔,全部阴性不能发育,说明内蒙古的本虫种不同于所谓幼虫期可以在牛羊发育的"哈萨克亚种"。为了纪念前苏联学者最早发现和叙述本种成虫,兹暂定名为苏俄棘球绦虫新种(Echinococcus russicensis sp.nov.)。

我国内蒙古大兴安岭北麓泡状肝包虫种类的研究 II.西伯利亚棘球绦虫(Echinococcus sibiricensis Rauschet Schiller,1954)

目的Rausch and Schiller(1954)在美国阿拉斯加白令海峡St.Lawrence岛从北极狐(Alopex lagopus)和雪撬狗(sledge dogs)发现西伯利亚棘球绦虫新种(Echinococcus sibiricensis sp.nov.),但诸多学者认为E.sibiricensis Rausch et Schiller,1954是欧洲的多房棘球绦虫(Echinococcus multilocularis Leuckart,1863)的地理株(或亚种)(Vogel,1957;Skrjabin and Abuladze,1964;Kumaratilake and Thompson,1982;Meyajaki,1991等)。于1985-2002年间,作者在我国内蒙古大兴安岭北麓草场多年进行泡状棘球蚴(alveolar Echinococcus)病原的野外调查和实验室鼠类的人工感染试验,结果发现该地区同地点存在有北美西伯利亚棘球绦虫、欧洲多房棘球绦虫及苏俄多房棘球绦虫三种不同"地理株",而且三种成虫常混合感染于同一终宿主沙狐(Vulpes corsac)的体内,它们的幼虫期也存在于当地的布氏田鼠(Microtus brandti)。2001年,作者再次检查当地沙狐151只,从中检出19只感染有泡状棘球绦虫成虫。含成熟虫体的13只阳性沙狐中,感染西伯利亚棘球绦虫有11只,其中2只单独感染此虫种,另9只还混合感染了欧洲多房棘球绦虫及苏俄多房棘球绦虫三虫种。在呼伦贝尔草原的泡状棘球蚴病原中,西伯利亚棘球绦虫是优势种,它在终宿主沙狐和中间宿主的感染率都高过其他虫种。本文着重介绍西伯利亚棘球绦虫的成虫子宫结构和它幼虫期在野外布氏田鼠及人工感染实验鼠体内的发育情况。

Long-term follow-up of liver alveolar echinococcosis using echinococcosis multilocularis ultrasound classification

BACKGROUND When Echinococcus multilocularis infects humans as a false intermediate host,alveolar echinococcosis(AE)usually manifests primarily intrahepatically and is initially asymptomatic.If the disease remains undiagnosed and untreated,progressive growth occurs,reminiscent of malignant tumours.The only curative therapy is complete resection,which is limited to localised stages,and palliative drug therapy is used otherwise.Consequently,early diagnosis and reliable detection of AE lesions are important.For this reason,abdominal ultrasonography,as the most common primary imaging for AE,relies on classification systems.AIM To investigate how hepatic AE lesion sonomorphology changes over time in the Echinococcosis Multilocularis Ulm Classification(EMUC)-ultrasound(US)classification.METHODS Based on data from Germany’s national echinococcosis database,we evaluated clinical and US imaging data for 59 patients according to the AE case definition in our preliminary retrospective longitudinal study.There had to be at least two liver sonographies≥6 mo apart,≥1 hepatic AE lesion,and complete documentation in all US examinations.The minimum interval between two separately evaluated US examinations was 4 wk.The AE reference lesion was the largest hepatic AE lesion at the time of the first US examination.To classify the sonomorphologic pattern,we used EMUC-US.In addition to classifying the findings of the original US examiner,all reference lesions at each examination time point were assigned EMUC-US patterns in a blinded fashion by two investigators experienced in US diagnosis.Statistical analysis was performed using SAS version 9.4(SAS Institute Inc.,Cary,NC,United Stated).P values<0.05 were considered statistically significant.RESULTS The preliminary study included 59 patients,38(64.5%)women and 21(35.6%)men.The mean age at initial diagnosis was 59.9±16.9 years.At the time of initial ultrasonography,a hailstorm pattern was present in 42.4%(25/59)of cases,a hemangioma-like pattern in 16.9%(10/59),a pseudocystic pattern in 15.3%(9/59),and a metastasis-like pattern in 25.4%(15/59).For the hailstorm pattern,the average lesion size was 67.4±26.3 mm.The average lesion size was 113.7±40.8 mm with the pseudocystic pattern and 83.5±27.3 mm with the hemangioma-like pattern.An average lesion size of 21.7±11.0 mm was determined for the metastasis-like pattern.Although the sonomorphologic pattern remained unchanged in 84.7%(50/59)of AE reference lesions,15.3%(9/59)showed a change over time.A change in pattern was seen exclusively for AE lesions initially classified as hemangioma-like or pseudocystic.A total of 70%(7/10)of AE lesions initially classified as hemangioma-like showed a relevant change in pattern over time,and 85.7%(6/7)of these were secondarily classified as having a hailstorm pattern,with the remainder(1/7;14.3%)classified as having a pseudocystic pattern.A total of 22.2%(2/9)of AE lesions initially classified as pseudocystic showed a relevant change in pattern over time and were classified as having a hailstorm pattern.For AE lesions initially classified as having a hailstorm or metastatic pattern,no pattern change was evident.All patients with pattern change were on continuous drug therapy with albendazole.CONCLUSION The sonomorphology of hepatic AE lesions may change over time.The hemangioma-like and pseudocystic patterns are affected.

肝泡型包虫病的诊治进展

肝包虫病为慢性寄生虫病,由多房棘球蚴的幼虫引发,此病具有相当高的致残率和死亡风险,又被称作“虫癌”。临床上将肝包虫病分为肝泡型包虫病和肝囊型包虫病,肝包虫病在世界各地广泛流行,我国主要在西部农牧区居民群体中发生,对该地区居民的生存质量构成了巨大的威胁。目前临床上治疗肝包虫病以手术治疗方式为主,伴随着外科诊治技术的迅速发展和对肝包虫病研究不断深入,其诊治方案也不断完善。本文将主要综述肝泡型包虫病的诊断和治疗进展,旨在为临床医务人员提供参考,以尽早发现和治疗,降低疾病对患者产生的不良影响,改善患者预后。

LAG3分子对泡球蚴感染小鼠肝脏B淋巴细胞亚群及其功能的影响

目的 探究淋巴细胞活化基因-3(Lymphocyte activation gene-3,LAG3)分子对泡球蚴感染小鼠肝脏B淋巴细胞亚群及其功能的影响。方法 将野生型(C57-wild-type, WT)和LAG3基因敲除(LAG3-knockout, LAG3-KO)小鼠经肝门静脉注射泡球蚴原头节,感染12周免疫组织化学染色观察小鼠肝脏CD19和α-SMA表达及定位,流式细胞术检测小鼠肝脏B细胞及其各亚群和表面CD80、CD86、MHC-Ⅱ分子的表达情况。结果 泡球蚴感染12周小鼠肝脏LAG3-KO组CD19阳性区及面积占比略高于WT组,但差异均无统计学意义。α-SMA在LAG3-KO组阳性区及面积占比显著高于WT组(t=-3.224、-3.227,P均<0.05)。LAG3-KO组肝脏B细胞表达CD80和MHC-Ⅱ分子的比例显著上调,差异具有统计学意义(t=-2.379、-3.321,P均<0.05)。LAG3-KO组B2细胞占比显著高于WT组,差异有统计学意义(t=-2.695,P<0.05)。LAG3-KO组B1b细胞表达CD80比例亦显著上调,差异具有统计学意义(t=-5.315,P<0.001),而B2细胞表达CD80比例显著低于WT组,且差异具统计学意义(t=2.806,P<0.05)。LAG3-KO组B2细胞表达MHC-Ⅱ分子显著上调,且具统计学差异(t=-4.227,P<0.01)。结论 泡球蚴感染中期12周,LAG3分子影响小鼠肝脏B细胞亚群及功能,并以B2型淋巴细胞尤为显著,LAG3对B细胞的活化以及MHC-Ⅱ类分子表达产生抑制作用,提示其可能参与泡球蚴感染下的B细胞耗竭。

经门静脉注射慢病毒调控目标蛋白表达治疗小鼠肝泡球蚴病方法的建立

经小鼠肝门静脉注射针对EmOPN的慢病毒构建OPN低表达的小鼠肝泡球蚴病治疗模型。将腹腔感染泡球蚴6月的沙鼠剖检,提取泡球蚴原头蚴,肝脏被膜下注射原头蚴法建立C57BL小鼠肝泡球蚴病模型,饲养1月后麻醉下将针对EmOPN的慢病毒Lv-EmOPN-734注射入门静脉,无菌棉按压后电凝穿刺点止血、关腹。普通环境饲养,5 d后随机选取5只小鼠取其肝脏组织,冰冻切片后在激光共聚焦显微镜下观察验证病毒是否进入泡状棘球蚴组织。慢病毒注射2月后对比肝脏及泡球蚴组织生长情况,Western blot检测OPN表达情况。结果门静脉注射慢病毒处理5 d后,慢病毒在正常肝脏组织、囊泡生发层、囊壁、囊外炎细胞层均有分布,其中生发层、囊壁分布较多。Western blot结果表明门静脉注射慢病毒2月后OPN的表达较空白对照组明显降低(P<0.01)。本研究通过门静脉注射慢病毒成功构建低表达OPN的小鼠肝泡球蚴病治疗模型,为通过调控目标蛋白表达治疗小鼠肝泡球蚴病提供建模方法。

多房棘球蚴囊泡来源EVs及其emu-miR-10-5p促进小鼠肝星状细胞活化

目的明确多房棘球绦虫囊泡来源胞外囊泡(Extracellular vesicles,EVs)及其emu-miR-10-5p在体外调节HSCs活化中的作用。方法小鼠腹腔注射原头节构建继发性多泡型包虫病模型,以小鼠体内病灶为材料培养多房棘球绦虫囊泡并进行PCR鉴定。透射电镜观察培养囊泡组织中胞外囊泡结构。差速-超速离心法分离囊泡培养上清中EVs,透射电镜、纳米粒径以及Western blot对分离EVs进行鉴定。CCK-8检测不同浓度EVs作用后HSCs活力。EVs作用于HSCs后RT-qPCR和Western blot检测α-SMA、CollagenⅠ和CollagenⅢ表达水平。生物信息学分析确定emu-miR-10-5p为特异性miRNA。过表达emu-miR-10-5p后EdU检测HSCs增殖,RT-qPCR个Western blot检测细胞中α-SMA、CollagenⅠ和CollagenⅢ表达水平。干扰囊泡组织中emu-miR-10-5p后提取培养上清液中EVs作用于HSCs后,检测HSCs细胞中α-SMA、CollagenⅠ和CollagenⅢ表达水平。结果成功通过小鼠体内病灶材料培养出多房棘球蚴囊泡,经PCR扩增特异性基因证实为多房棘球绦虫来源。TEM观察培养囊泡表面有较多的囊状结构。通过差速-超速离心分离出多房棘球蚴囊泡来源EVs,并经TEM证实为球形、膜状结构。囊泡来源EVs能被HSCs内化,并促进HSCs增殖和活化。通过综合分析多房棘球绦虫感染小鼠血清、棘球蚴组织、棘球蚴囊泡来源EVs、生发层细胞、原头节miRNA测序结果,发现emu-miR-10-5p在上述成分中均存在表达,并通过RT-qPCR证实emu-miR-10-5p在多房棘球蚴囊泡来源EVs中存在的量较高。进一步研究证实过表达emu-miR-10-5p能促进HSCs增殖,增加HSCs中α-SMA、Collagen I和CollagenⅢmRNA及蛋白表达水平。而干扰囊泡中emu-miR-10-5p后从培养上清液中提取的EVs未增加α-SMA、Collagen I和CollagenⅢmRNA及蛋白表达水平。结论多房棘球蚴囊泡来源EVs及emu-miR-10-5p能够促进HSCs活化。

七种蛋白酶抑制剂对多房棘球蚴DNA损伤诱导样1蛋白活性的影响

目前,多房棘球蚴病(alveolar echinococcosis, AE)尚无有效药物治疗手段,迫切需要开发新型治疗药物。前期研究表明,HIV蛋白酶抑制剂(HIV protease inhibitors, HIV PIs)具有潜在抗寄生虫功能。本文旨在研究HIV PIs对Echinococcus multilocularis(Emu)DNA损伤诱导样1蛋白(DNA damage inducible 1 protein, Ddi1)活性的影响。本研究通过构建真核表达重组载体pFastBac1-Emu Ddi1,在昆虫细胞系Sf9细胞中表达筛选出P1代和P2代,纯化出可溶性Ddi1重组蛋白,然后与目的蛋白的荧光底物检测纯化蛋白的活性,进一步检测沙奎那韦(saquinavir, SQV)、利托那韦(ritonavir, RTV)、安普那韦(amprenavir, APV)、阿扎那韦(atazanavir, ATV)、洛匹那韦(lopinavir, LPV)、福沙那韦(fosamprenavir, Fos)、达芦那韦(darunavir, DRV)等7种HIV PIs对Emu Ddi1重组蛋白活性的抑制能力。结果显示:细胞系内真核表达产物的酶活Km为1.422μmol·L^(-1),具有良好的亲和力和活性,最终筛到沙奎那韦对Ddi1蛋白二聚体活性位点的抑制率达67%,其IC_(50)为34,说明沙奎那韦对Emu Ddi1重组蛋白酶活性具有良好的抑制效果。以上结果提示:沙奎那韦抑制重组蛋白Ddi1的活性,可能成为Ddi1的靶向药物,以期为替代药物或开发联合用药提供基础。

LAG3对多房棘球蚴感染小鼠模型CD8^(+)T细胞免疫功能调节作用的研究

目的研究淋巴细胞激活基因3(Lymphocyte activation gene 3,LAG3)对多房棘球蚴慢性感染小鼠CD8^(+)T细胞免疫功能的调节作用。方法取C57BL/6野生型(Wild-type,WT)和LAG3缺陷型(Knock-out,KO)小鼠各10只,每只小鼠经肝门静脉接种3000个多房棘球蚴原头节建立多房棘球蚴感染模型。感染12周后,分别取两组小鼠肝脏和脾脏组织,采用苏木精-伊红(Hematoxylin-eosin,HE)染色观察肝脏病灶周围炎性细胞浸润和病理表现,通过免疫组织化学观察肝脏病灶周围“炎症微环境”与脾脏中CD8^(+)T的比例。收集两组小鼠肝脏与脾脏淋巴细胞,采用流式细胞术筛选不同CD8^(+)T细胞亚群,检测CD8^(+)T细胞、效应记忆CD8^(+)T细胞(Effector memory CD8^(+)T cell,CD8^(+)Tem)、中心记忆CD8^(+)T细胞(Central memory CD8^(+)T cell,CD8^(+)Tcm)、初始CD8^(+)T细胞(Naive CD8^(+)T cell,CD8^(+)Tn)比例与绝对数,以及分泌细胞因子γ干扰素(IFN-γ)、肿瘤坏死因子α(TNF-α)、白细胞介素10(IL-10)和白细胞介素17A(IL-17A)的比例。通过多房棘球蚴虫体蛋白体外刺激两组小鼠肝脏淋巴细胞,采用流式细胞术检测CD8^(+)T细胞分泌细胞因子IFN-γ、IL-10和IL-17A的比例。结果HE染色结果显示,与野生型小鼠比较,LAG3缺陷型小鼠肝脏形成的炎性病灶数量增多,差异有统计学意义(P<0.05)。免疫组织化学结果显示,与野生型小鼠比较,LAG3缺陷型小鼠肝脏病灶周围CD8^(+)T细胞募集升高,差异有统计学意义(P<0.05);LAG3缺陷型小鼠脾脏CD8^(+)T细胞募集有降低的趋势,差异无统计学意义(P>0.05)。流式细胞术结果显示,与野生型小鼠比较,LAG3缺陷型小鼠肝脏效应记忆型CD8^(+)T细胞比例有升高的趋势,差异无统计学意义(P>0.05);脾脏CD8^(+)T细胞比例降低,脾脏CD8^(+)Tem表型比例升高,差异均有统计学意义(P<0.01)。LAG3缺陷型小鼠肝脏和脾脏CD8^(+)T细胞分泌TNF-α和IL-10能力均增强,差异均有统计学意义(P<0.05)。多房棘球蚴的虫体蛋白体外刺激肝脏淋巴细胞实验结果显示,与野生型小鼠比较,LAG3缺陷型小鼠CD8^(+)T细胞分泌IFN-γ、IL-10比例升高,差异均有统计学意义(P<0.05);LAG3缺陷型小鼠CD8^(+)T细胞分泌IL-17A的能力有升高趋势(P>0.05)。结论在小鼠多房棘球蚴慢性感染中,LAG3可抑制肝脏和脾脏CD8^(+)T细胞免疫应答能力,调节炎症反应。

具有M细胞靶向性的多房棘球蚴表位疫苗GILE口服乳酸菌载体的构建

目的构建具有M细胞靶向性的多房棘球蚴表位疫苗GILE口服乳酸菌载体。方法本研究在前期构建的多表位疫苗GILE的基础上,通过添加SAM基因序列,设计、合成SAM-GILE序列,构建乳酸菌表达质粒pNZ8148-SAM-GILE,并电转至乳酸乳球菌NZ9000中建立乳酸菌表达体系。用酶切与基因测序双重实验验证乳酸菌表达质粒pNZ8148-SAM-GILE是否构建成功,用PCR验证乳酸菌表达质粒pNZ8148-SAM-GILE是否成功电转至乳酸乳球菌NZ9000,用Western Blot实验检测重组蛋白SAM-GILE是否表达,用全细胞ELISA法检测SAM-GILE的表面展示情况,用免疫荧光染色法鉴定LL-plSAM-GILE的M细胞靶向性。结果酶切与基因测序双重实验结果显示,乳酸菌表达质粒pNZ8148-SAM-GILE构建成功。经PCR验证,pNZ8148-SAM-GILE成功电转至乳酸乳球菌NZ9000。经Western Blot验证,通过Nisin诱导,成功表达约45 KD的重组蛋白。经全细胞ELISA检测,SAM-GILE可展示于LL-plSAM-GILE表面。经免疫荧光染色验证,重组抗原与M细胞高度重合,表明重组乳酸菌疫苗LL-plSAM-GILE具有M细胞靶向性。结论成功构建具有M细胞靶向性的多房棘球蚴表位疫苗GILE口服乳酸菌载体。

敲低多房棘球蚴let-7-5p可抑制BALB/c小鼠腹腔巨噬细胞极化和虫体生长

旨在明确敲低多房棘球蚴emu-let-7-5p对小鼠腹腔巨噬细胞极化和虫体生长的影响。构建表达多房棘球蚴emu-let-7-5p海绵序列的重组腺相关病毒载体,并在293T细胞上验证其抑制效果和特异性。70只清洁级BALB/c小鼠分别经尾静脉注射AAV8-si-let-7-5p、AAV8-control和PBS后两周,每组随机选取2只小鼠分别用Western blot和qRT-PCR分析肝组织中GFP、let-7-5p及其靶基因C/EBP δ的表达。随后,所有小鼠每只腹腔接种1 000个原头蚴,3个月后qRT-PCR检测腹腔巨噬细胞emu-let-7-5p、C/EBP δ、M1型(IL-1β、iNOS和IL-6)和M2型(Arg-1、IL-4和IL-10)相关分子的转录水平,并统计各组小鼠多房棘球蚴的包囊重量和原头蚴数量。结果表明,注射后第15天,AAV8-si-let-7-5p成功靶向小鼠肝脏,并引起emu-let-7-5p的敲低和C/EBP δ的上调表达。感染后3个月,AAV8-si-let-7-5p组小鼠腹腔巨噬细胞emu-let-7-5p和M2型相关分子Arg-1均显著下调,而C/EBP δ和M1型相关分子IL-1β和iNOS均显著上调。此外,AAV8-si-let-7-5p组IL-4和IL-6下调表达,而IL-10上调表达,且小鼠肝脏包囊重量和原头蚴数量显著低于对照组。敲低多房棘球蚴感染小鼠体内emu-let-7-5p,可促进C/EBP δ的表达,进而抑制M2型极化和虫体生长。