Study on the Development of Free Living Nematodes and Observe on the Parasitic Larval Trematodes

本论文包括两个部分： 1、三种自由生活线虫的发育研究： 本文对在厦门地区采集的三种小杆目自由生活线虫：小杆科中的Rhabditis属Rhabditissp.和Distolabrellus属的Distolabrellusxiamenensisn.sp.，以及头叶科中的Acrobeloides属Acrobeloidessp.进行了鉴定和描述，以这三类小杆目自由生活线虫作为试验材料在实验室进行人工培养，对它们的整体，尤其生殖系统的形态结构、生殖方式、胚胎发育以及胚后生殖系统的发育进行了观察对比与研究。试验结果表明：此三类线虫的生殖系统形态结构不同，Rhabditissp.是双生殖腺；Distol...There are two parts in this article. 1. Studying the development of 3 species of nematodes. We have collected and described 3 species of nematodes( Rhabditida ), Rhabditis sp., Distolabrellus xiamenensis n.sp.and Acrobeloides sp.. These nematodes were cultivated clean-bredly as research materials. We compared the reproductive system, the reproductive type and development of the 3 species wi...学位：理学博士院系专业：生命科学学院生物学系_动物学学号：2012005140312

The reproductive system and gametogenesis in a nematode Distolabrellus veechi

以线虫Distolabrellus veechi的卵巢和精巢为材料,用甲醇固定,DAPI荧光染料对细胞核进行染色,通过Normaski荧光显微镜对其生殖系统结构、性细胞染色体数目、体细胞染色体数目进行了观察。结果显示了该线虫雌虫和雄虫生殖细胞减数分裂Ⅰ前期的细胞核的变化特征;减数分裂Ⅰ前期丝球期雌、雄生殖细胞的二价体数目均为6;卵细胞染色体数目为6,精细胞染色体数目为5或6,其性别决定机制为XX/X0型;体细胞染色体数目为11(雄虫)或12(雌虫)。In this study we analyzed oogenesis and spermatogenesis in the nematode Distolabrellus veechi.The D.veechi germ line can be divided into several regions based upon the state of the DNA in the nuclei visualized by DAPI in situ staining of dissected adult gonadal arms.We identified a mitotic zone,a transition zone between mitosis and meiosis,a pachytene zone and a diakinesis zone.In D.veechi males,the primary spermatocytes initially assemble on the rachis.Upon entering meiosis,the primary spermatocytes develop to the diplotene stage and bud away from the rachis.At diakinesis(which follows the diplotene stage) six spermatocyte bivalents become more apparent due to chromosomal condensation.The spermatocytes then undergo two maturation divisions and produce haploid spermatids,which are stored in the seminal vesicle.Five or six chromosomes can be seen in the spermatids.The male's somatic gonad consists of three tissues:two distal tip cells,the seminal vesicle and the vas deferens.In D.veechi females,oogonia(early oocytes) are produced from the rachis.The oocytes then undergo a growth phase and arrest at diakinesis;further development is halted until fertilization occurs.At this time,six bivalents can be seen.After penetration of the oocyte by a sperm,the oocyte advances to the metaphase stage and passes into the uterus.After two maturation divisions have been completed,two polar bodies are formed.Shortly afterwards,the sperm and egg pronuclei fuse to form the zygote nucleus,and cleavage division begin after some time.The somatic gonad of female D.veechi consists of one distal tip cell,the gonadal sheath,the spermatheca,the oviduct and the uterus.The results of this study also indicate that D.veechi show a 2N condition of 11 chromosomes for males,and 12 for females.国家自然科学基金资助项目(No.30670237)~

日本血吸虫幼虫在先感染外睾吸虫后不同时间钉螺体内被生物控制效果的比较

前此实验显示:21d外睾吸虫阳性钉螺能使再侵入的全部血吸虫幼虫发育停留在异常早期母胞蚴阶段〔1-3〕。本文实验比较观察了感染外睾吸虫后不同时间(21d、35d、55d、70d及85d)的钉螺对再侵入的日本血吸虫幼虫生物控制的情况,发现外睾吸虫和血吸虫双重感染的钉螺,两者间隔时间愈长,后侵入的血吸虫幼虫被击毁程度愈强(图1-20);不同间隔时间双重感染的各组实验钉螺,血吸虫幼虫感染率及幼虫侵入率如下:间隔21d,75%(39/52)和25.5%(398/1560);间隔37d,77.8%(21/27)和35.6%(299/840);间隔55d,44.4%(8/18)和11.9%(19/160);间隔70d,100%(15/15)和10.6%(143/1350);间隔85d,43.3%(13/30)和5.8%(66/1140)

Cloning and Expression of D-like Aspartic Protease of Anisakis simplex

作者单位： 厦门大学生命科学院， 厦门361005 通讯作者， E-mail:[email protected][中文文摘]目的克隆简单异尖线虫Ⅲ期幼虫(L3)的D-天冬氨酸蛋白酶基因(AsAP)全长,研究其表达蛋白的特性。方法根据GenBank中简单异尖线虫D-天冬氨酸蛋白酶基因表达序列标签的部分信息,设计特异引物并用cDNA末端快速扩增技术得到AsAP全长序列,分析推导的蛋白序列特征,并预测其三级结构。用RT-PCR扩增简单异尖线虫L3的AsAP基因编码序列,产物用EcoRⅠ和SalⅠ双酶切,连入表达载体pET32а(+),转化大肠埃希菌(E.coli)BL21(DE3),以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,表达产物经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测。结果简单异尖线虫L3的AsAP基因全长1 753 bp,编码453个氨基酸,与锡兰钩虫(Ancylostoma ceylanicum)的D-天冬氨酸蛋白酶相似性达65%。该蛋白具有两个保守的催化域,1个活性中心翼环,S2和S3亚位点各1个;具有由20个氨基酸组成的N端信号肽,构成疏水性强的跨膜域。不同浓度的IPTG(0.2～1.6 mmol/L)诱导对AsAP表达的影响较小,1.0 mmol/LIPTG诱导2 h后表达量达到最高水平。结论克隆并表达了简单异尖线虫的D-天冬氨酸蛋白酶。[英文文摘]Objective To clone and express the full length of D-like aspartic protease gene(AsAP) of the third stage larvae of Anisakis simplex.Methods According to the partial information of D-like aspartic protease encoding gene of A.simplex from GenBank,specific primers were designed to amplify 3′end and 5′ end of AsAP gene using rapid amplification of cDNA ends(RACE),and the full length of the D-like aspartic protease gene was obtained.Using total RNA of the third-stage larvae of A.simplex,coding sequence of the AsAP gene was amplified by reverse transcription-PCR(RT-PCR).The PCR product was digested by EcoRⅠ and SalⅠ,and cloned into pET32 vector.The recombinant plasmid was checked by double enzyme digestion and sequencing,and the positive recombinant plasmid was transformed into E.coli BL21(DE3).Expression of the protein induced by IPTG under gradient concentration and different time was conducted.Result A 1 753 bp full length of AsAP was obtained,which contained 30 bp 5′UTR,361 bp 3′UTR and a 1 362 bp open reading frame(ORF) encoding 453 amino acids with a predicted molecular mass of Mr 50 726.It showed 65% identity with the D-like aspartic protease of Ancylostoma ceylanicum.The predicted amino acid sequence contains two conserved catalytic motif,an active site flap,an S2 subsite and an S3 subsite.A 20 amino acids signal peptide was found in the N-terminus,with significant hydrophobic property.Different concentration of the IPTG(0.2~1.6 mmol/L) showed little effect on the expression,and the production of the protein was up to maximum after 2 hours induction.Conclusion The AsAP gene has been cloned and expressed.国家自然科学基金(No.81171595);福建省自然科学基金(No.2010J01229

人参皂甙Rg3抗小鼠Lewis肺癌的机制研究

目的以小鼠Lewis肺癌为肿瘤模型探讨人参皂甙Rg3抗肿瘤机制。方法以肺癌细胞系LLC荷瘤C57/BL小鼠建立小鼠肺癌模型,随机分为人参皂甙Rg3组、阳性药顺铂组和模型组,分别给予人参皂甙Rg3、顺铂和生理盐水;同时设立空白对照组。以肿瘤质量、抑瘤率观察人参皂甙Rg3的抗肿瘤效果;以流式细胞术测定脾脏CD4+T细胞、CD8+T细胞数量及比值;以Elispot技术检测机体肿瘤抗原特异性CTL的诱生情况;以肝脏指数、脾脏指数、胸腺指数判定人参皂甙Rg3的毒副作用。结果人参皂甙Rg3组抑瘤率高于顺铂组,差异具有统计学意义(P<0.05)。生理盐水组小鼠脾脏CD4+T细胞、CD8+T细胞百分率比正常小鼠低,而人参皂甙Rg3组和顺铂组高于生理盐水组,差异具有统计学意义(P<0.05)。人参皂甙Rg3组肿瘤特异性IFN-γ斑点数明显增多,与生理盐水组相比具有统计学差异(P<0.05),顺铂组脾脏无肿瘤特异性IFN-γ斑点出现。和模型组相比较,人参皂甙Rg3组小鼠肝脏、脾脏、胸腺指数无明显差异,而顺铂组的肝脏、脾脏、胸腺指数低于人参皂甙Rg3组,差异具有统计学意义(P<0.05)。结论 LLC细胞荷瘤小鼠具有明显的免疫抑制现象,人参皂甙Rg3可显著增加脾脏CD4+T细胞、CD8+T细胞阳性率并促进肿瘤特异性CTL细胞诱生,人参皂甙Rg3无肝脏、胸腺、脾脏毒性

对细胞膜质蛋白共定位染色方法的改良

[目的]将流式细胞术染色方法进行改良用于T细胞膜质蛋白共定位的共聚焦显微镜检测,在共定位表达于细胞膜上的CD4和细胞质内表达的TIPE2蛋白较常规镜检免疫荧光染色法可提高实验效率。[方法]取小鼠脾脏T细胞,用流式细胞术的染色方法代替实验室常规免疫染色法,对细胞膜上的CD4与细胞质内的TIPE2蛋白进行染色,制片并在激光共聚焦显微镜下观察。[结果]小鼠脾脏CD4+T细胞上出现了TIPE2蛋白的共表达,符合实验预期,同时实验效率从原有10%提高到90%。[结论]采用该染色方法后突破了常规免疫荧光染色必须要在载玻片上进行的局限,简化了操作,保证了实验的有效性,提高实验效率。厦门大学本科生创新创业训练计划项目(2018X0584);;\n机能与临床转化福建省高校重点实验室开放基金资助项目(JNYLC1813

湖南目平湖钉螺血吸虫病原生物控制资源调查及感染试验

目的虽然一种贝类可作数种吸虫的中间宿主,但在各吸虫种类都存在的一自然环境中,它们共同贝类宿主的一个体通常只携带一种吸虫幼虫期。作者利用这一规律在湖南汉寿目平湖血吸虫病区进行媒介钉螺的调查,查到钉螺感染有日本血吸虫(Schistosoma japonicum)、外睾类吸虫(Exorchis)、斜睾类吸虫(Plagiorchis)、侧殖类吸虫(Asymphylodora)和背孔类吸虫(Notocotylus)5种吸虫的幼虫期。其中对生物控制媒介钉螺有利用价值的外睾类吸虫,其钉螺感染率为3.298%(96/2911),终宿主鲶鱼(Parasilurus asoyus)的感染率为99.31%(434/437)、平均每尾鲶鱼的感染强度为115.4条。先用外睾类吸虫虫卵感染钉螺后再感染日本血吸虫毛蚴,切片观察不同时间的实验螺,后侵入的血吸虫幼虫,全部受损害不能发育

Studies on the Nematodes in Soil Nearby Root of Three Kind of Wild Plants of Composite Family in Xiamen

2003年12月~2004年12月在厦门大学附近丘陵中,调查了藿香蓟(Ageratum conyzoides)、银胶菊(Partheniumhysterophorus)以及鬼针草(B idens pilosa)根周土壤中的线虫类群(F ilenchus cylindricus,Aphenlenchussp.,Helicotylenchus di-hystera,Criconem ellaspp.,Ogmasp.,Dorylaim ida,无口针自由生活线虫),对线虫密度与季节、土壤深度和植物种类之间的关系进行了分析.结果显示,在小生境中野生菊科植物根周土壤线虫密度在不同季节有变化,但与季节变化之间没有明显的相关关系;线虫密度会随土壤深度的增加而减少,以全年样品的数据分析,0~5 cm土层的线虫密度明显高于5~10 cm土层(p<0.05).不同植物根周土壤线虫的类群和密度都有区别,说明植物种类会影响线虫类群的分布.The density and structure of nematode communities(Filenchus cylindricus,Aphenlenchus sp.,Helicotylenchus dihystera,Criconemella spp.,Ogma sp.,Dorylaimida nematode,free nematodes) in soil nearby root of three common wild plants(Ageratum conyzoides,Parthenium hysterophorus,Bidens pilosa) were studied from Dec.2003 to Dec.2004.The studies include the effects of climate change and soil depth change on soil nematode communities in root soil around different wild plants.The studies have showed some results.There were no correlations between climate and the density of soil nematode in Xiamen.Soil depth and the density of soil nematode have significant negative correlations(p<0.05).And the different species of plants do effect the density and structure of nematode communities in root soil