表皮生长因子对绵羊子宫内膜上皮原代细胞增殖的影响

通过建立和完善绵羊子宫内膜上皮原代细胞体外培养技术，为研究绵羊母体和孕体之间的相互作用机制建立体外着床模型。采用组织块法分离培养子宫内膜上皮细胞，观察其生长情况，并比较不同表皮生长因子（EGF）浓度对子宫内膜上皮细胞增殖的作用效果。结果显示，组织块培养1~2 d后，组织周围迁出子宫内膜上皮细胞，长满60 mm培养皿需9~12 d；经差时消化法纯化后，F1代绵羊子宫内膜上皮细胞纯度可达90%以上，表明所获细胞可用于后续试验；F2代细胞在不同浓度EGF（0、12.5、25、50、75、100 ng/mL）下培养144、168 h，经检测在144 h，12.5、25和100 ng/mL浓度下D_{450 nm}值极显著高于对照组（P<0.01），50 ng/mL浓度下显著高于对照组（P<0.05），在168 h，12.5 ng/mL浓度下显著高于对照组（P<0.05），因此，在12.5 ng/mL EGF作用下效果最佳；F3代细胞在0、12.5 ng/mL浓度下培养不同时间（24、48、72、96、120、144、168、192、216 h），经检测D_{450 nm}值在144 h差异显著（P<0.05），168 h后差异极显著（P<0.01）。因此，在培养液中添加12.5 ng/mL EGF可以更加高效、快速得到子宫内膜上皮细胞。

Effect of EGF on the Proliferation of Primary Cells of Sheep Endometrial Epithelium

In order to study the interaction mechanism between maternal and conceptus of sheep,an in vitro implantation model was established by establishing and improving the in vitro culture technology of primary sheep endometrial epithelial cells.Endometrial epithelial cells were isolated and cultured by tissue block method.The growth of endometrial epithelial cells was observed,and the effects of different EGF concentrations on the proliferation of endometrial epithelial cells were compared.The results showed that after 1-2 days of tissue culture,the endometrial epithelial cells migrated from the peri-tissue,and it took about 9-12 days to grow the 60 mm culture dish.After purification by differential digestion,the purity of the endometrial epithelial cells of F1 generation sheep could reach more than 90%,which indicated that the obtained cells could be used in subsequent experiments.F2 cells were cultured at different EGF concentrations (0,12.5,25,50,75 and 100 ng/mL) for 144 and 168 hours.The D_{450 nm} values were extremely significant higher in 12.5,25 and 100 ng/mL EGF treatment group (P<0.01),and significantly higher in 50 ng/mL EGF treatment group (P<0.05) compared with control group at 144 h.At 168 h,the D_{450 nm} values were significantly higher in 12.5 ng/mL EGF treatment group than those in control group (P<0.05).Therefore,at 12.5 ng/mL EGF concentration,the effect was the best.At 0 and 12.5 ng/mL EGF concentrations,F3 cells were cultured for different time (24,48,72,96,120,144,168,192 and 216 h).The D_{450 nm} values were significantly different at 144 h (P<0.05),and extremely significant at 168 h (P<0.01).Consequently,the culture medium with 12.5 ng/mL EGF would obtain the endometrial epithelial cells more efficiently and rapidly.