人毛滴虫膜性细胞器的电镜细胞化学研究

本文应用电镜细胞化学技术,对人毛滴虫膜性细胞器进行研究,结果表明:酸性磷酸酶与胞嘧啶核苷酸酶反应产物均定位于初级溶酶体、消化泡和副基体成熟面囊泡;硫胺素焦磷酸酶和烟酞胺腺瞟呤二核苷酸酶反应分别显示于副基体成熟面膜囊与中间胶囊;消化泡中可见过氧化物酶反应物;而细胞色素氧化酶与过氧化氢酶反应则为阴性,提示虫体缺乏线粒体与微体。铀-铅-铜浸染法可选择性深染氢化酶体、副基体和内质网。对细胞化学反应结合膜性细胞器功能进行了讨论。     

弓形虫的酶细胞化学及蒿甲醚对其影响的研究

小鼠腹腔接种RH株弓形虫速殖子1万个，2h后分别灌服5％淀粉溶液及蒿甲醚200mg／kg，连续8d，取腹腔液沉淀作电镜酶细胞化学。测定弓形虫虫体胞嘧啶单核苷酸酶（CMP酶）和葡萄糖-6-磷酸酶（G－6－P酶）的定位及用药后2种酶超微结构水平上的变化。结果表明，CMP酶定位于虫体的溶酶体，药物对其无明显影响。G－6－P酶定位于虫体的质膜外及围虫泡内，用药后酶反应减弱，说明药物对该酶的活性有一定影响，这可能是药物抗弓形虫的作用机制之一。     

双氢青蒿素与甲硝唑体外抗阴道毛滴虫的电镜观察

目的观察双氢青蒿素与甲硝唑单用和联合用药体外对阴道毛滴虫超微结构的影响。方法在2.5×10~6个/ml滴虫培养液中加入两药合剂(双氢青蒿素0.5 mg/ml和甲硝唑0.002mg/ml),同时设不加药的阴性对照组和单药对照组,即甲硝唑组(5 mg/ml)、双氢青蒿素组(1 mg/ml),37℃培养3.5～5 h,扫描电镜和透射电镜观察阴道毛滴虫超微结构结果扫描电镜观察结果表明,仅用双氢青蒿素作用3.5 h,阴道毛滴虫细胞膜被破坏,部分表膜脱落;仅用甲硝唑作用3.5 h虫体表面结构无变化,作用5 h虫体表面出现许多小泡和小凹,但表膜未见破坏。两药联合作用3.5～4.2 h,阴道毛滴虫表面凹凸不平,出现皱褶、裂隙;细胞膜破损、脱落明显,表面粗糙,呈蜂窝状;细胞内含物从细胞膜裂口处溢出,虫体破裂,其内氢化酶体、核、轴柱和盾等裸露。透射电镜观察结果表明,仅用双氢青蒿素作用3.5 h,阴道毛滴虫膜系统破坏明显,破坏的滴虫细胞质从细胞膜破损处溢出;仅用甲硝唑作用3.5～5 h,细胞质破坏严重,细胞中可见许多空泡、裂隙和无结构区两药联合作用3.5～4.5 h,滴虫表膜破损,细胞内含物破坏严重,出现许多空泡、裂隙,内质网肿胀,氢化酶体膜破损、变形,细胞器大多消失;细胞核变形,核内出现裂隙,核膜受损,甚至消失。结论双氢青蒿素和甲硝唑对阴道毛滴虫的作用部位不同,两药联用可增进对虫体的破坏力。     

双氢青蒿素对体外培养阴道毛滴虫作用的电镜观察

目的 观察双氢青蒿素（DHA）对阴道毛滴虫超微结构的影响。 方法 在2.5×10⁶个／ml滴虫培养液中加入双氢青蒿素1 mg／ml。 37 ℃ 培养3 和3.5 h, 进行扫描电镜观察，培养2.5和4 h进行透射电镜观察。 结果 双氢青蒿素作用滴虫后，虫体细胞膜受损明显，表面皱褶加深、表膜凹陷，并出现裂隙。细胞核膜破损，细胞核和细胞质均出现裂隙。内质网呈囊状肿胀，氢化酶体受损、变形。细胞质从细胞膜破裂处溢出。 细胞膜剥脱后细胞核、氢化酶体和盾等结构裸露。最后虫体变性、坏死。 结论 双氢青蒿素能破坏阴道毛滴虫膜系结构并导致虫体裂解死亡。     

阴道毛滴虫粘附大鼠阴道的超微结构与组织化学研究

目的　研究阴道毛滴虫与生殖道上皮的关系 ,探讨虫体的致病机制。　方法　应用透射与扫描电镜技术 ,结合光镜免疫组织化学等技术 ,观察在动物模型中虫体对大鼠阴道粘膜的粘附作用。　结果　虫体为PAS阳性 ,成群虫体粘附于阴道的中上段表面的富含粘多糖棱柱状上皮细胞 ;虫体组织蛋白酶阳性 ,常见释放水解酶破坏上皮细胞膜 ;虫体肌动蛋白阳性 ,显示微丝束在阿米巴样虫体内呈网络状。虫体可呈阿米巴样穿钻于上皮细胞间 ,其微丝状伪足可伸入上皮细胞微绒毛间包绕蚕食微绒毛 ,其指状伪足可插入上皮细胞间包绕局部。少量虫体附着在阴道下段粘膜褶皱间的角化上皮。　结论　虫体嗜好寄生阴道穹窿等处 ,是因表层上皮细胞内富含粘原颗粒 ,表面有较多微绒毛。虫体在粘附后释放水解酶消化并吞噬上皮细胞 ,可直接损伤生殖道寄生部位的上皮 ,且大量摄取粘多糖 ,影响阴道自洁 ,继而引起组织炎症。虫体的细胞骨架、细胞衣、多形性伪足和溶酶体 ,分别在变形、粘附、包绕、吞噬与消化过程中起重要作用。     

华支睾吸虫乙酰胆碱酯酶定位的电镜观察

用透射电镜对华支睾吸虫乙酰胆碱酯酶（ＡｃｈＥ）的亚显微定位进行了观察，发现该酶的反应产物以微粒形式沉着于实质细胞的核膜和内质网、神经突起的轴突膜及肠绒毛的片层膜结构。同时观察到实质细胞和皮层合胞体内Ａ、Ｂ二型囊泡及神经轴突内的Ａ型囊泡在酶定位过程中有破裂或变形现象，而实质细胞内的Ｃ型大颗粒及Ｄ型分泌小体无明显变化。     

寄生虫细胞外囊泡的研究现状及展望

细胞外囊泡(extracellular vesicles,EVs)是细胞旁分泌释放到细胞外的膜性小囊泡,几乎所有类型的细胞均可以产生并释放细胞外囊泡,外泌体(exosomes,Exo)、微囊泡(microvesicles,MVs)等均在其范畴,作为细胞间信号传导的重要方式,其功能已成为当前生命科学研究中的热点。研究表明,细胞外囊泡在多种寄生虫中均能分泌,且广泛参与虫体感染宿主及致病的生理过程,有望成为相关疾病诊断的生物标志物、治疗靶点或工具,具有广阔的应用前景。本文重点对主要的致病寄生虫分泌的细胞外囊泡及其生理功能进行综述。     

齿龈内阿米巴的超微结构与溶酶体酶细胞化学研究

目的　研究齿龈内阿米巴的细胞器与功能 ,探讨虫体与宿主细胞的关系。方法　用透射电镜结合电镜酶细胞化学技术观察虫体细胞超微结构 ;将虫体与口腔上皮细胞孵育 ,观察虫体对上皮细胞的粘附。结果　虫体及其伪足呈多形态 ;胞质内细胞器主要为泡状和管状的溶酶体、一些滑面内质网 ,以及丰富的糖原颗粒 ;溶酶体标志酶CMPase显示溶酶体酶丰富 ;可见虫体粘附于上皮细胞表面和小伪足伸入上皮细胞微绒毛间。结论　虫体的溶酶体水解酶消化作用及虫体伪足的机械作用对宿主牙周上皮组织的损伤 ,是虫体的致病机制之一。     

约氏疟原虫红内期抗原定位的免疫电镜研究

应用LR White低温包埋法并结合胶体金标记免疫电镜细胞化学技术对保护性单克隆抗体M26-32识别的约氏疟原虫红内期抗原进行了超微结构定位。结果表明与单克隆抗体M26-32发生特异性免疫反应的抗原主要定位于早期滋养体、晚期滋养体、裂殖体和裂殖子的细胞质,为无性期不同发育阶段的共同抗原;在滋养体发育过程中该抗原有所增加,一部分可通过原虫的胞吐作用运出并定位于邻近虫体的感染红细胞细胞质。     

猪带绦虫囊尾蚴发育规律的实验观察

本文报告选择出生不久的仔猪,分别感染不同剂量的猪带绦虫卵,然后定期观察受染猪体各个部位组织内囊尾蚴的发育变化。感染后90d 剖检,中度和重度感染猪有17.65～20.11%的虫体发育不成熟,呈粟粒状,无囊泡形成;已形成囊泡的虫体经胆汁培养,翻出头节较短,约3～4mm,不活动。感染后180d 剖检,中度和重度感染组仍可见到不成熟的虫体(约占12.5～22.23%),虫体较90d 时略大,头部已见吸盘、顶突和头钩,但无囊泡形成;成熟之虫体经胆汁培养,头节翻出较长,可见到摆动。至感染后270d,虫体全部发育成熟。本实验表明,囊尾蚴的生长发育成熟时间应为60～270d。各部位囊虫的成熟率,成活率及密度有明显差别;虫体的大小与生长时间和密度有关;中度组重复感染未见虫体密度明显增加,表明机体受感染后可产生一定的保护性免疫。     

Mitochondrial-type assembly of FeS centers in the hydrogenosomes of the amitochondriate eukaryote Trichomonas vaginalis

Mitochondria are the site of assembly of FeS centers of mitochondrial and cytosolic FeS proteins. Various microaerophilic or anaerobic unicellular eukaryotes lack typical mitochondria ("amitochondriate" protists). In some of these organisms, a metabolically different organelle, the hydrogenosome, is found, which is thought to derive from the same proteobacterial ancestor as mitochondria. Here, we show that hydrogenosomes of Trichomonas vaginalis, a human genitourinary parasite, contain a key enzyme of FeS center biosynthesis, cysteine desulfurase (TviscS-2), which is phylogenetically related to its mitochondrial homologs. Hydrogenosomes catalyze the enzymatic assembly and insertion of FeS centers into apoproteins, as shown by the reconstruction of the apoform of [2Fe-2S]ferredoxin and the incorporation of 35S from labeled cysteine. Our results indicate that the biosynthesis of FeS proteins is performed by a homologous system in mitochondriate and amitochondriate eukaryotes and that this process is inherited from the proteobacterial ancestor of mitochondria.     

阴道毛滴虫双链RNA病毒感染及其对甲硝唑耐药性的影响

目的调查河南地区阴道毛滴虫临床分离株阴道毛滴虫病毒感染情况,探索病毒感染对阴道毛滴虫甲硝唑耐药性的影响。方法 TYM(trypticase-yeast extract-maltose)无菌培养基培养阴道毛滴虫临床分离株,达到纯培养后提取虫体总核酸(DNA和RNA),进行1%琼脂糖凝胶电泳分析;连续稀释法测量每株虫体的甲硝唑最小致死浓度。结果对30株阴道毛滴虫总核酸进行电泳,其中6株有5.5 kb双链RNA病毒带,病毒感染率为20.0%。阴道毛滴虫病毒阴性组甲硝唑最小致死浓度为(24.27±20.899)μg/ml,病毒阳性组为(5.68±3.588)μg/ml,差异有统计学意义(t=2.143,P<0.05)。结论河南地区阴道毛滴虫临床分离株中检测到阴道毛滴虫病毒,无阴道毛滴虫病毒寄生的虫体易发生甲硝唑抵抗。     

An agar culture technique to quantitate Trichomonas vaginalis from women

In subjects with trichomoniasis the number of Trichomonas vaginalis in vaginal discharges or secretions is unknown. The presence of T. vaginalis was evaluated in 177 consecutive female patients attending an inner city sexually transmitted disease clinic by patient history, wet mount, and broth culture. T. vaginalis was quantitated by a novel agar culture technique. Of the 177 women, 86 (49%) were positive for T. vaginalis by either wet mount or culture. Clinical symptoms were not reliable for making an accurate diagnosis of trichomoniasis. Culture on modified Diamond's medium was more sensitive (98%) than the wet mount method (38%) in detecting T. vaginalis. Of the 86 patients who were positive for trichomoniasis, quantitation was obtained for 81 patients, with 70% yielding greater than 10(4) colony-forming units (cfu)/ml. The number of T. vaginalis ranged from 40 to greater than 10(6) cfu/ml. The wet mount method was very insensitive for detecting T. vaginalis and was positive only in patients yielding greater than 10(5) cfu/ml.     

Comparison of conventional testing to polymerase chain reaction in detection of Trichomonas vaginalis in indigenous women living in remote areas

There are high rates of Trichomonas vaginalis in remote areas of Central Australia. Conventional tests for T. vaginalis have low sensitivity in this setting. Aims of the study were to estimate the prevalence of T. vaginalis, to assess the presence of clinical signs and symptoms, to compare a T. vaginalis polymerase chain reaction (PCR) test with conventional methods of diagnosis, and to compare the PCR from different samples, including self-collected swabs (SCS). Of 205 women recruited, the prevalence of T. vaginalis was 24%. The prevalence of T. vaginalis was higher in women under 25 years (33%), compared with those who were 25-34 years (26%) and those over 35 years (15%, P < 0.05). The sensitivity of T. vaginalis PCR detection from SCS (94%) was not statistically different from a practitioner-collected HVS (96%), but was superior to urine PCR (74%) and conventional methods. After multivariate analysis, those women with high pH were almost three times more likely to be positive for T. vaginalis (odds ratio = 2.71 with 95% confidence interval 1.06-6.93, P = 0.037). Superior assays such as PCR should be a diagnostic option to adequately screen and treat women with T. vaginalis, in order to reduce complications, including the increased risk of HIV transmission.     

Use of intravaginal microbicides to prevent acquisition of Trichomonas vaginalis infection in Lactobacillus-pretreated, estrogenized young mice.

D2A21, a novel peptide antibiotic has in vitro activity against a wide spectrum of sexually transmitted diseases (STD). In this study we tested the hypothesis that intravaginal D2A21 would interfere with acquisition of Trichomonas vaginalis infection in a modified mouse model. T. vaginalis infections of estrogenized young mice pretreated with Lactobacillus vaginalis or Lactobacillus rhamnosus were more frequent and persistent than those in mice pre-treated with Lactobacillus gasseri or Lactobacillus acidophilus. One hundred percent T. vaginalis infection was achieved for 2-4 days post-challenge when intravaginal L. rhamnosus pre-treatments were given to estrogenized mice 48 hr prior to a single T. vaginalis challenge. Estrogenized mice pre-treated with L. rhamnosus were pre-medicated with intravaginal placebo gel, 0.5% or 2% D2A21 gel, or 500 microg/mL metronidazole gel prior to T. vaginalis challenge. Both 2% D2A21 and metronidazole gels were significantly more efficacious (10% or none infected) than placebo gel (53% infected) in preventing vaginal T. vaginalis infections in mice.     

硝唑尼特体外抑杀阴道毛滴虫效果观察

目的观察硝唑尼特体外抑杀阴道毛滴虫效果。方法分别以2、1、0.5、0.25、0.125、0.062 5和0.031 25mg/ml,硝唑尼特作用于阴道毛滴虫携病毒株与无病毒株12和24 h,观察体外抑杀效果。实验以甲硝唑作为药物对照。结果随着药物浓度的增加和作用时间的延长,硝唑尼特对两种虫株的抑制率均增高,显微镜下可见滋养体活力下降,培养基底部有死亡虫体沉淀,染色后可见滋养体细胞质内有空泡,鞭毛脱落,虫体变形,细胞核变形,细胞膜破裂,细胞溶解。经统计学分析,硝唑尼特对阴道毛滴虫两种虫株的抑制率差异无统计学意义(χ2=0.12,P>0.05)。在24 h时,甲硝唑对阴道毛滴虫无病毒株抑制率为81.06%,硝唑尼特(1 mg/ml)对阴道毛滴虫两种虫株的抑制率可达100%,硝唑尼特对阴道毛滴虫无病毒虫株的抑制率显著高于甲硝唑(χ2=4.43,P<0.01)。结论硝唑尼特对阴道毛滴虫两种虫株均有较好的抑杀作用,其中对无毒株阴道毛滴虫的抑杀效果硝唑尼特优于甲硝唑。     

Murine models of vaginal trichomonad infections

Trichomonas vaginalis and Tritrichomonas foetus cause common sexually transmitted infections in humans and cattle, respectively. Mouse models of trichomoniasis are important for pathogenic and therapeutic studies. Here, we compared murine genital infections with T. vaginalis and T. foetus. Persistent vaginal infection with T. foetus was established with 100 parasites but T. vaginalis infection required doses of 10(6), perhaps because of greater susceptibility to killing by mouse vaginal polymorphonuclear leukocytes. Infection with T. vaginalis persisted longest after combined treatment of mice with estrogen and dexamethasone, whereas infection was only short-lived when mice were given estrogen or dexamethasone alone, co-infected with Lactobacillus acidophilus, and/or pretreated with antibiotics. Infection rates were similar with metronidazole-resistant (MR) and metronidazole-sensitive (MS) T. vaginalis. High dose but not low dose metronidazole treatment controlled infection with MS better than MR T. vaginalis. These murine models will be valuable for investigating the pathogenesis and treatment of trichomoniasis.