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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 invasion and development of intestinal epithelial cells by Trichinella spiralis larvae in vitro. Methods Trichinella spiralis larvae were incubated in rat intestinal contents or mouse bile for 2 h and then inoculated into semi-solid medium (DMEM / F12 complete medium + 1.75% agarose), semi-solid medium + T84 cells , DMEM / F12 complete medium + T84 cells and semi-solid medium (high glucose DMEM complete medium + 1.75% agarose) + Caco-2 cells were cultured in 37 ℃ 5% CO2 for 24 h, observed and counted in the cell layer 1 and 2 to 4 larvae. Results The percentages of larvae that developed from intestinal contents and bile to 2-4 larvae in semi-solid medium + Caco-2 cells were 50.00% and 34.78%, respectively (χ2 = 0.836, P> 0.05) Higher than the percentage of larvae incubated with RPMI-1640 (χ21 = 5.978, χ22 = 4.600, P <0.05). The percentages of 2-4 larvae (40.00%) in the semi-solid medium + T84 cells of larvae incubated with intestinal contents were significantly higher than those of the semi-solid medium 4.76% (χ2 = 10.947, P <0.05) ; The percentages of 2-4 stage larvae developing in T84 and Caco-2 cells were 40.00% and 50.00%, respectively, with no significant difference (χ2 = 0.546, P> 0.05); in complete medium + T84 cells Two to four larvae were observed. In semi-solid media, larvae could invade T84 / Caco-2 cells and migrate through the cell monolayer, leaving clear migration trajectories. After incubation for 37 h and 42 h, larvae were found to have one Female with 3 males. Conclusion Trichinella spiralis larvae were inoculated into semi-solid medium after incubation with intestinal contents or bile, which could invade intestinal epithelial cells and grow in vitro.