Use of an enzymatic micromethod to quantify amastigote stage of Leishmania amazonensis in vitro
Use of an enzymatic micromethod to quantify amastigote stage of Leishmania amazonensis in vitro
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DOI：
10.1007/s004360050272
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发表时间：
1997-04-01
影响因子：
2
通讯作者：
Lemesre, JL
中科院分区：
文献类型：
--
作者：
Sereno, D;Lemesre, JL
利什曼病是热带和亚热带地区发病和死亡的重要原因。这些原生动物寄生虫在其白蛉载体中以鞭毛胞外原毛菌形式存在。在哺乳动物宿主中，只有无鞭毛的无鞭毛体存在，在巨噬细胞的吞噬体中存活和分裂。利什曼原虫promastigotes通常通过酶促法在体外定量，该方法涉及将四氮唑盐3-（4,5 -二甲基噻唑-2-基）- 2,5 -二苯基溴化四氮唑（MTT）转化为有色的不溶性甲酸产物，其数量取决于存在的活寄生虫的数量（Mossman 1983; Cole 1986； Rabinovitch等人1986；Berg等人1994）。由于难以获得足量的纯化的哺乳动物阶段，无尾体形式的使用受到阻碍。为此，我们和其他研究人员最近开发了一种培养系统，用于在体外培养大量无宿主细胞污染的细胞外无梭菌（Bates 1993; Lemesre，专利PCT/FR 94/00577）。在本文中，我们报告了使用基于mtt的微量测定来量化亚马逊河蛭无尾线虫的增殖和生存能力。将该方法的效率与另一种传统的细胞计数方法进行了比较。在目前的研究中，使用了一种通用的程序来产生大量的每个寄生虫阶段。在25-cm2的烧瓶中，采用MAA（无性系无性系培养基）的无细胞培养基（Lemesre et al. 1994; Lemesre，专利号PCT/FR 94/00577），将无性系无性系L. amazonensis （MHOM/BR/76/LTB-012）的活跃和分裂群体维持在32±1℃。在初始接种量为5× 105株/ml的情况下，第7天的细胞密度约为5× 107株/ml。Promastigote培养物在含有25 mM HEPES和2 mM NaHCO3 （pH 7.2）缓冲的RPMI 1640培养基（Gibco BRL）中保持在25±1℃，并添加20%热灭活胎牛血清（FCS）。初始浓度为5× 105鞭毛虫/ml培养基。为了验证基于mtt的实验与寄生虫数量之间的相关性，增加了后期寄生虫的浓度，从2.5到40× 105个细胞/孔，在96孔的平底微皿中100 l培养基中分布三份。然后将10 l MTT （10 mg/ml）在25或32±1℃下孵育4小时，取决于寄生虫的阶段。加入100 l的50%异丙醇和10%十二烷基硫酸钠（SDS, pH 5.4）溶液停止酶-底物反应。微孔板在室温下进一步孵育30分钟。使用Titertech 96孔扫描仪在570 nm处测定光密度（OD）。为了评估使用基于mtt的检测方法获得生长动力学曲线的可能性，正如细胞计数实验一样，微生物以两个接种剂（1和2× 105寄生虫/孔）播种，并按上述方法每天测量脱氢酶活性。在0.01 M磷酸盐缓冲盐水（PBS, pH 7.2）中充分稀释后，通过400倍放大的托马室中寄生虫的每日计数来测定细胞浓度。每天在显微镜下观察寄生虫的形态学方面，以检测96孔板上可能发生的分化现象。
Leishmaniasis is a significant cause of morbidity and mortality in tropical and subtropical areas. These protozoan parasites exist as a flagellate extracellular promastigote form in their sandfly vectors. In the mammalian hosts, only the nonflagellate amastigote form persists, surviving and dividing in the phagolysosome of macrophages. Leishmania promastigotes have often been quantified in vitro by an enzymatic method that involves the conversion of a tetrazolium salt, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), into a colored, insoluble formazan product, the amount of which depends on the number of viable parasites present (Mossman 1983; Cole 1986; Rabinovitch et al. 1986; Berg et al. 1994). The use of amastigote forms has been hampered by difficulties in obtaining sufficient amounts of the purified mammalian stage. Toward that end, we and other investigators have recently developed culture systems for the in vitro growth of large amounts of extracellular amastigote forms that are free of host-cell contamination (Bates 1993; Lemesre, patent PCT/FR 94/00577). In the present paper we report the use of an MTT-based microassay to quantify the proliferation and the viability of axenically grown amastigote forms of L. amazonensis. The efficiency of the method was compared with that of another conventional method, cell counting. A general procedure was used in the current study to generate large quantities of each of the parasite stages. Active and dividing populations of axenically grown amastigote forms of L. amazonensis (MHOM/BR/76/LTB-012) were maintained at 32±1 C by weekly subpassage in a cell-free medium called MAA (medium for axenically grown amastigotes) in 25-cm2 flasks (Lemesre et al. 1994; Lemesre, patent PCT/FR 94/00577). From a starting inoculum of 5× 105 amastigote forms/ml a cell density of about 5× 107 parasites/ml was obtained on day 7. Promastigote cultures were maintained at 25±1 C in RPMI 1640 medium (Gibco BRL) buffered with 25 mM HEPES and 2 mM NaHCO3 (pH 7.2) and supplemented with 20% heat-inactivated fetal calf serum (FCS). Initial parasite concentrations were 5× 105 flagellates/ml of medium. For verification of the correlation between the MTT-based assay and the number of parasites, increasing concentrations of latelog-phase parasites, ranging from 2.5 to 40× 105 cells/well, were distributed in triplicate in 100 l of media in 96-well flat-bottom microtrays. Plates were then incubated with 10 l of MTT (10 mg/ml) for 4 h at 25 or 32±1 C, depending upon the parasite stage. The enzyme-substrate reaction was stopped by the addition of 100 l of a solution of 50% isopropanol and 10% sodium dodecyl sulfate (SDS; pH 5.4). Microplates were further incubated for 30 min at room temperature. The optical density (OD) was determined at 570 nm with a Titertech 96-well scanner. To evaluate the possibility of using the MTT-based assay to achieve growth kinetics curves as is possible with cell-counting experiments, microorganisms were seeded at two inocula (1 and 2× 105 parasites/well) and the dehydrogenase activities were measured daily as described above. Cell concentrations were determined by daily counting of parasites in a Thoma chamber at 400x magnification after adequate dilution in 0.01 M phosphate-buffered saline (PBS, pH 7.2). Morphological aspects of parasites were determined daily by microscope examination to detect the possible occurrence of differentiation phenomena in 96-well plates.