温度及酸碱度对角膜分离棘阿米巴虫株活性影响的实验研究

Influence of temperatures and pH value on biological activity of acanthamoeba isolated from keratitis

目的观察不同温度及pH条件对角膜分离的棘阿米巴虫株活性的影响,探讨抑制其生长的条件。方法从棘阿米巴性角膜炎患者角膜中分离培养棘阿米巴虫株,置于棘阿米巴液体培养基(PYG)中进行纯培养。利用四甲基偶氮唑盐(MTT)法,观察不同pH值及不同温度下棘阿米巴的增生率及其形态变化。结果在深低温条件下(-80～-20)℃,酸性环境中的棘阿米巴多呈皱缩状,部分虫体破裂;在碱性环境中,棘阿米巴多呈典型的囊前期或包囊状态存在。培养温度为4～35℃,棘阿米巴虫株于酸性环境中,多以滋养体形式存在,其增生率随温度的升高而增加;相反,在pH>8的碱性环境中,棘阿米巴虫株则多以包囊形式存在。28～35℃、pH6.5为棘阿米巴虫株体外生长的最适条件。结论棘阿米巴在酸性环境中适应生长,多以繁殖能力强的滋养体形式存在,而在碱性环境中,棘阿米巴则多以包囊的形式存在。

Objective Acanthamoeba survival at the forms of trophozoit and encyst inwrap, and the change of these two forms is associated with temperature and pH value. The trophozoit of acanthamoeba has the stronger pathogenecity. Present study aimed to observe the biological activity of acanthamoeba isolated from keratitis under different temperatures and pH value. Methods The corneal tissue from penetrating keratoplasty for keratitis was obtained. The acanthamoeba was isolated from the cornea tissue with acanthamoeba keratitis. The cloned isolates were axenized and cultured in proteose-yeast-glucose （PYG） medium and passaged. The proliferation rate and growth statue were subsequently observed under the light microscope and inverted microscope. The growth acanthamoeba cultures were harvested to the density 10^5/mL by centrifugation for 15 minutes, and then the MTT assay was performed using 20 μL（ 5 mg/mL）of 3-（ 4,5-dimethylthiazol-2-yl） 2,5-diphenyl- tetrazolium bromide under the different pH values and temperatures. Initial cultures with 10^4/mL were seeded under the different pH values （6.5, 7.0,7.5,8.0）. Samples were examined for trophozoits and cysts at 12-hour interval by directly counting. All assay was performed in triplicate. Results Under the temperatures from - 20℃ to - 80℃ ,acanthamoeba shrinked or disrupted in pH values of 1.5 and 3.0,however,under the same temperature and alkali conditions with pH values 10 and 12,the acanthamoeba showed the irregular cysts and pre-cysts. Under the temperatures of 4 % to 35 ℃ and pH values 3.0, 6.5, acanthamoeba proned to be trophozoit. On the same temperature with alkali condition （ pH values 7.0,7.5,8.0） , acanthamoeba existed at the fashion of cyst. The optimum temperature range and pH value for acanthamoeba to growth was 4 ℃ to 35 ℃ and 6. 5 respectively Acanthamoeba is tend to be trophozoit and has higher reproducing ability in acid condition and suitable temperature environment,aeanthamoeba exists as cysts and presents lower active ability.