旋毛虫幼虫体外对肠上皮细胞侵入及发育的观察

Observation of the in vitro invasion of intestinal epithelial cells by Trichinella spiralis larvae and their development in those cells

目的观察旋毛虫幼虫在体外对肠上皮细胞的侵入及发育情况。方法将旋毛虫肌幼虫在大鼠肠内容物或小鼠胆汁中孵育2 h后,分别接种至半固体培养基（DMEM/F12完全培养基＋1.75%琼脂糖）、半固体培养基＋T84细胞、DMEM/F12完全培养基＋T84细胞及半固体培养基（高糖DMEM完全培养基＋1.75%琼脂糖）＋Caco-2细胞中,37℃5%CO2培养24 h,观察并计数细胞层中的1期及2~4期幼虫。结果经肠内容物与胆汁孵育后的幼虫在半固体培养基＋Caco-2细胞中发育为2~4期幼虫的百分比分别为50.00%和34.78%（χ2=0.836,P〉0.05）,两者均高于经RPMI-1640孵育的幼虫百分比8.70%（χ21=5.978,χ22=4.600,P〈0.05）。经肠内容物孵育后的幼虫在半固体培养基＋T84细胞中2~4期幼虫的百分比（40.00%）显著高于半固体培养基中的幼虫百分比4.76%（χ2=10.947,P〈0.05）;在T84和Caco-2细胞中发育为2~4期幼虫的百分比分别为40.00%和50.00%,差异无统计学意义（χ2=0.546,P〉0.05）;在完全培养基＋T84细胞中未观察到2~4期幼虫;在半固体培养基中,幼虫可侵入T84/Caco-2细胞并在细胞单层中移行,留下明显的移行轨迹,培养37 h与42 h后分别发现1条雌虫与3条雄虫。结论旋毛虫幼虫经肠内容物或胆汁孵育后再接种至半固体培养基,可在体外侵入肠上皮细胞并能生长发育。

Objective To observe the in vitro invasion of intestinal epithelial cells by Trichinella spiralis larvae and their development in those cells.Methods T.spiralis muscle larvae were incubated in rat intestinal contents or mouse bile for 2 h and then inoculated into semisolid medium（DMEM/F12 complete medium ＋1.75% agarose）,semisolid medium＋T84 cells,DMEM/F12 complete medium＋T84 cells,or semisolid medium（high glucose DMEM complete medium ＋1.75% agarose） ＋ Caco-2 cells.Cultures were incubated at 37 ℃ in 5% CO2 for 24 h,and then the L1 and L2-L4 larvae in the cell monolayer were observed and counted.Results When the larvae incubated in gut contents and bile were inoculated into semisolid medium ＋Caco-2 cells,the percentages of L2-L4 larvae were 50% and 34.78%,respectively（χ2=0.836,P0.05）;both were higher than the percentages incubated in RPMI-1640（8.70%）（χ12=5.978,χ22=4.600,P0.05）.After incubation in gut contents,the percentage（40%） of L2-L4 larvae cultured in semisolid medium＋T84 cells was higher than that（4.76%） cultured in semisolid medium（χ2=10.947,P0.05）.The difference between the percentage of the L2-L4 larvae in the T84（40%） and Caco-2（50%） monolayer was not statistically significant（χ2=0.546,P0.05）.The L2-L4 larvae were not observed in complete medium ＋T84 cells.Larvae invaded the T84 and Caco-2 monolayer,migrated through the monolayer,and left obvious trails.After larvae were cultured for 37 h and 42 h,one female adult and three male adults were observed.Conclusion After incubation in intestinal contents or bile,T.spiralis larvae inoculated into semisolid medium invaded intestinal epithelial cells and developed in vitro.