Establishment of axnic culture of non-invasive Entamoeba dispar and study on its biological characterization

非侵入性迪斯帕内阿米巴轴培养物的建立及其生物学特性研究

• 批准号: 08670287
• 负责人: KOBAYASHI Seiki
• 金额: $ 1.41万
• 依托单位: Keio University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08670287/
• 关键词: Entamoeba dispar; axnic cultivation; growth promoting facoor; Pseudomonas sp.; Crithidia sp.; Entner-Doudoroff pathway; 6-phosphogluconate; dihydroxy acetone; 6ホスホグルコン酸; hexokinase; fructose

We developed an axnic cultivation system for Entamoeba dispar which is difficult to grow in usual axnic medium of Entamoeba histolytica without intestinal bacteria or some protozoa such as Crithidia fasciculata.Since Clark (1995) reported successful axnic cultivation of strain SAW 760 RR clone A of E.dispar, other strains also have been tried for the axnic cultivation. However, E.dispar strain which could be adapted to axnic culture conditions is only the SAW 760 RR clone A,up to the present.In this research project, we found one metabolic intermediate (6-phosphogluconate : 6PG) which could promote the growth of three strains, at least, of E.dispar. This finding followed our study on growth promoting activities in the extract of Pseudomonas aeruginosa. The basic monoxemic culture system of E.dispar with P.aeruginosa needed modification of the medium for axnic Entamoeba histolytica (BI-S-33 medium) as follows : the addition of acetone or dihydroxyacetone, removal or replacement of gluco … More se with maltose or hydrlized starch and sterilization by filtration. In this system, the number of E.dispar ameba isolates (over 12 strains) usually reached 2-3 x 10^5/ml at those growth peaks, constantly.We tried modification of this system and design a new medium for cultivation of E.dispar axnically. Basic medium for the axnic culture was casein-free YI-S (Diamond, 1995) by which Clack (1995) reported successful axnic cultivation of E.dispar by supplementation of gastric mucin.A E.dispar growth promoting activity could be found in the polysaccharide rich fraction of P.aeruginosa, and it was more effective in gluconate and dihydroxyacetone supplemented YI-S medium. Though gluconate kinase and glucose-6-phosphate dehydrogenase (G6PDH) for producing 6PG could not be detected in the extract of E.dispar, gluconate seemed to be metabolized by E.dispar in the presence of P.aeruginosa extract. And, interestingly, it was found that 6PG rich fraction made from glucose-6-phosphate (G6P), NADP and crude extract of Crithidia fasciculata which has extremely high G6PDH ativity and also gluconate kinase activity, has the growth promoting effect. It was further confirmed that this growth promoting effect was attributed to 6PG by using commercial G6PDH and G6P.These results are still preliminary but seem clear and reproducible, so we are now studying the role of 6PG in the axnic growth of E.dispar and the related glycolytic pathway such as Entner-Doudoroff pathway which was reported to be present in xenic E.histolytica (Hilker and White, 1959). Less

本文针对溶组织内阿米巴（Entamoeba dispar）在无肠道细菌和一些原生动物（如束状隐孢子虫（Crithidia fasciculata））的常规培养基中难以生长的问题，建立了一种适合于该阿米巴的培养体系，自Clark（1995）报道了成功培养出的菌株SAW 760 RR clone A以来，其他菌株也进行了培养试验。本研究发现了一种代谢中间产物6-磷酸葡萄糖酸盐（6-phosphogluconate：6PG），它能促进至少3株E. dispar菌株的生长。这一发现是在我们对铜绿假单胞菌提取物的促生长活性进行研究之后得出的。用溶组织内阿米巴（Entamoeba histolytica，简称BI-S-33）作为溶组织内阿米巴的基本单氧培养体系，需要对BI-S-33培养基进行如下改进：添加丙酮或二羟丙酮，去除或替代葡萄糖，加入葡萄糖，或用葡萄糖代替葡萄糖。 ...更多信息 用麦芽糖或水解淀粉乳化，过滤除菌。在该系统中，在这些生长高峰期，阿米巴分离株的数量通常达到2-3 × 10 ~ 5/ml（超过12株），并且一直保持不变。结果表明，在无酪蛋白的YI-S（Diamond，1995）培养基中，铜绿假单胞菌的多糖组分具有明显的促生长作用，而葡萄糖酸盐和二羟丙酮对铜绿假单胞菌的促生长作用更强。虽然在舞毒蛾提取物中未检测到产生6-PG的葡萄糖酸激酶和葡萄糖-6-磷酸脱氢酶（G6 PDH），但在铜绿假单胞菌提取物存在下，葡萄糖酸似乎被舞毒蛾代谢。并且，有趣的是，发现由葡萄糖-6-磷酸（G6 P）、NADP和具有极高G6 PDH活性和葡萄糖酸激酶活性的束状短肢虫粗提取物制成的富含6PG的级分具有生长促进作用。因此，我们目前正在研究6-PG在异源性E.dispar细胞生长过程中的作用，以及相关的糖酵解途径，如Entner-Doudoroff途径（Hilker and白色，1959）。少

项目成果

Hiroshi Tachibana: "Differentiation of Entamoeba histolytica from E.dispar facilitated by monoclonal antibodies against a 150-KDa surface antigen." Parasitology Research. 83. 435-439 (1997)

Hiroshi Tachibana：“针对 150 KDa 表面抗原的单克隆抗体促进了溶组织内阿米巴与迪斯帕阿米巴的分化。”

Seiki Kobayashi: "Entamoeba dispar : Cultivation with sterilized Crithidia fasciculata." J.Euk.Microbiol.45. 173-178 (1998)

Seiki Kobayashi：“迪斯帕内阿米巴：用灭菌的束状短膜虫进行培养。”

Tsutomu Takeuchi: "Entamoeba dispar : Cultivation without viable associate and its characterization." Arch.Med.Res.28. 108-109 (1997)

Tsutomu Takeuchi：“Dispar 内阿米巴：没有可行伙伴的培养及其特征。”

Asao Makioka: "Comparison of DNA polymerase between Entamoeba histolytica and E.dispar" Pavasitology International. (in press). (1997)

Asao Makioka：“溶组织阿米巴和迪斯帕阿米巴 DNA 聚合酶的比较”Pavasitology International。

JUNICHI SANUKI: "Identification of Entamoeba histolytica and E. dispar cysts in stool by polymerase chain reaction" Parasitology Research. 83. 96-98 (1997)

JUNICHI SANUKI：“通过聚合酶链反应鉴定粪便中的溶组织阿米巴和迪斯帕阿米巴包囊”寄生虫学研究。