芽囊原虫体外培养特性的研究

Study on the characteristics of Blastocystis cultured in vitro

探究体外培养的芽囊原虫的形态、体外增殖规律及不同温度保存的降解情况。从腹泻患者芽囊原虫阳性粪便中分离芽囊原虫,在IMDM培养基中培养,观察虫体形态和生殖方式。采用显微镜计数法和荧光定量PCR法(qPCR)测定芽囊原虫18S小亚基核糖体DNA(SSU rDNA)的拷贝数分析体外培养芽囊原虫的增殖情况,绘制增殖曲线,分析两种方法的相关性。芽囊原虫分别置4℃、-20℃、-80℃保存,第1~7天和第1~5周,提取虫体DNA,qPCR法检测芽囊原虫SSU rDNA,分析芽囊原虫的降解情况。形态学研究结果显示,IMDM培养基培养的芽囊原虫,可观察到空泡形、颗粒形、阿米巴形和包囊等4种常见形态和分裂生殖、出芽生殖和胞内生殖等3种生殖方式。芽囊原虫体外培养的第3~7天为快速增长期,第7天虫体密度达到峰值,显微镜计数和qPCR法测定结果分别为2.5×10^(6)个/ml和1.1×10^(6)拷贝/μl。相关性分析显示,显微镜计数和qPCR法测定结果绘制的增殖曲线的皮尔逊相关系数为0.95,呈高度相关。在4、-20和-80℃保存第7天,芽囊原虫SSU rDNA拷贝数分别为(2.75±0.20)×10^(4)、(6.84±1.33)×10^(4)、(1.39±0.06)×10^(5)拷贝/μl,为第1天拷贝数[(2.36±0.06)×10^(5)、(2.39±0.06)×10^(5)、(2.23±0.21)×10^(5)拷贝/μl)]的11.6%、28.3%和63.6%,差异有统计学意义(F=130.67,P<0.05);在4、-20和-80℃保存第5周时,芽囊原虫SSU rDNA拷贝数分别为(3.77±0.23)×10^(4)、(4.37±0.59)×10^(4)、(3.86±0.26)×10^(5)拷贝/μl,为第1周拷贝数[(2.23±0.21)×10^(5)、(2.23±0.21)×10^(5)、(2.23±0.21)×10^(5)拷贝/μl]的2.98%、3.41%和28.74%,差异有统计学意义(F=500.51,P<0.05)。体外培养的芽囊原虫高峰期虫体形态多样,显微镜计数法和qPCR法均可用于芽囊原虫的定量,芽囊原虫在4、-20和-80℃保存均会发生降解,-80℃保存的降解程度小于4、-20℃。

The morphology,proliferation and stability of in vitro cultured Blastocystis were studied.Blastocystis were isolated from Blastocystis positive feces of patients with diarrhea and cultured in IMDM medium to observe the parasite morphology and proliferation.The density of Blastocystis was quantified by microscopic counting and the copy number of Blastocystis 18S small subunit ribosomal DNA(SSU rDNA)was measured by fluorescence quantitative PCR(qPCR)to analyze the proliferation of Blastocystis in the in vitro culture,and the proliferation curve was plotted to analyze the correlation between the two methods.Blastocystis were stored at 4,-20,and-80℃for 1 to 7 days and 1 to 5 weeks respectively,and the Blastocystis SSU rDNA was detected by qPCR to analyze the stability.Morphological studies showed that the four common morphological forms were observed in the IMDM medium,including vacuolar form,granular form,ameboid form and cyst form;and the three reproductive modes including fission,gemmation and endodyogeny were observed.The rapid growth period of Blastocystis was observed from day 3 to 7 from the in vitro culture,and the Blastocystis density peaked on day 7.The microscopic counting and qPCR results were(2.55±0.22)×10^(6)/ml and(1.06±0.10)×10^(6) copies/μl,respectively.Correlation analysis showed that the Pearson correlation coeffi⁃cient of proliferation curve generated by microscopic counting and qPCR was 0.95,which was highly correlated.The degradation of Blastocystis began on the second day after storage at 4,-20 and-80℃,and the copy numbers of SSU rDNA on the seventh day were(2.75±0.20)×10^(4),(6.84±1.33)×10^(4)and(1.39±0.06)×10^(5) copies/μl,respec⁃tively,which accounted for 11.65%,28.67%and 62.42%of the copy volume(2.36±0.06)×10^(5),(2.39±0.06)×10^(5),(2.23±0.21)×10^(5) copies/μl on the first day.The difference was statistically significant(F=130.67,P<0.05).At the fifth week,the copy numbers of SSU rDNA were(3.77±0.23)×103,(4.37±0.59)×103 and(3.86±0.26)×10^(4)copies/μl,accounted for 2.98%,3.41%and 28.74%of the copy volume(2.23±0.21)×10^(5),(2.23±0.21)×10^(5),(2.23±0.21)×10^(5) copies/μl in the first week.The difference was statistically significant(F=500.51,P<0.05).The mor⁃phology of in vitro cultured Blastocystis showed diversive forms at the rapid proliferation stage.Both microscopic count⁃ing and qPCR can be used for the quantification of Blastocystis and the stability of Blastocystis stored at-80℃is less than that stored at 4 and-20℃.