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

在没有肠道细菌和一些原生动物（如束状棘虫）的溶组织内阿米巴的常规无菌培养基中，很难生长出一种不同的内阿米巴。自Clark（1995）报道e.d dispar的SAW 760 RR克隆A株成功培养后，其他菌株也进行了盆栽试验。然而，迄今为止，能适应干旱培养条件的e.d dispar菌株只有SAW 760 RR克隆A。在本研究项目中，我们发现了一种代谢中间体（6-磷酸葡萄糖酸盐：6PG），它可以促进至少三株e.d dispar的生长。这一发现与我们对铜绿假单胞菌提取物促进生长活性的研究一致。dipara与P.aeruginosa的基本单氧培养体系需要对axnic型溶组织内阿米巴培养基（BI-S-33培养基）进行如下修改：添加丙酮或二羟基丙酮，用麦芽糖或水解淀粉去除或替代葡萄糖，并通过过滤杀菌。在该系统中，异巴变形虫分离株数（12株以上）在生长高峰时通常达到2-3 × 10^5/ml。对该体系进行了改进，并设计了一种新的培养培养基。axic培养的基本培养基为不含酪蛋白的YI-S (Diamond, 1995), Clack（1995）通过添加胃粘蛋白成功培养了e.d dispar。P.aeruginosa富多糖部位具有促生长活性，在葡萄糖酸盐和二羟丙酮添加的i - s培养基中效果更好。虽然在假铜绿假单胞菌提取物中没有检测到葡萄糖酸激酶和葡萄糖-6-磷酸脱氢酶（G6PDH），但在假铜绿假单胞菌提取物存在的情况下，葡萄糖酸似乎被假铜绿假单胞菌代谢。有趣的是，我们发现由葡萄糖-6-磷酸（G6P）、NADP和麻瓜粗提物制备的富含6PG的组分具有极高的G6PDH活性和葡萄糖酸激酶活性，具有促进生长的作用。利用商用G6PDH和G6P进一步证实了6PG的促生长作用。这些结果仍是初步的，但似乎是清晰和可重复的，因此我们现在正在研究6PG在e.d dispar的体外生长中的作用，以及相关的糖酵解途径，如据报道存在于xenic e.s hihistolytica中的enterner - doudoroff途径（Hilker和White， 1959）。少

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