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目的通过细胞划痕实验观察过量全反式视黄酸(all-trans retinic acid,atRA)对原代腭突中嵴上皮细胞迁移的影响。方法以10 w龄昆明小鼠按雌雄比例2:1合笼,得到妊娠13 d孕鼠。将1只妊娠13 d孕鼠处死,分离得到全部胎鼠腭突,以中性蛋白酶分离腭突中嵴上皮,继以胰酶消化,用DMEM/F-12培养基调整细胞浓度后接种于培养瓶中,CO2培养箱中培养96 h。取同等细胞浓度接种于6孔板中,同样条件下培养24 h。弃去原培养基,在各孔贴壁细胞层划出一条划痕带,分别加入相应培养基后得到3孔5μmol/L atRA实验组和3孔对照组,继续原条件培养72 h。结果:原代腭突中嵴上皮细胞贴壁生长,多角形或卵圆形,成片生长;当细胞融合率>80%,呈现铺路石状外观。在培养过程中,对照组划痕逐渐弥合,培养72 h,爬行细胞几乎覆盖整个划痕带;而atRA实验组未见到向划痕区迁移的细胞,划痕宽度没有改变。结论:过量atRA可抑制腭突中嵴上皮细胞发生迁移。
OBJECTIVE: To investigate the effect of atra on the migration of primary epithelial cells in primary palate process by cell scratch assay. Methods Kunming mice (10 weeks old) were housed in a 2: 1 ratio of male and female, and pregnant rats of 13 d were obtained. One pregnant rat was sacrificed on day 13 of gestation and all fetus was isolated. Neutral protease was used to isolate the midrib of the palate. After trypsin digestion, the cells were cultured in DMEM / F-12 medium Bottle, CO2 incubator 96 h. Take the same cell concentration inoculated in 6-well plates, the same conditions for 24 h. Discard the original culture medium, and draw a scratch band on the adherent cell layer of each well, then add 3μm 5μmol / L atRA experimental group and 3-hole control group respectively after adding corresponding medium, and continue to cultivate for 72 hours. Results: The primary epithelial cells in the primary palate had adherent growth, polygonal or oval shape and formed a piece of growth. When the cell fusion rate was> 80%, the appearance of paving stone appeared. Scratches in the control group gradually closed during the culturing process. Crawling cells almost covered the entire scratched zone after culturing for 72 h. However, no cells migrated to the scratched area were observed in the atRA group, and the width of the scratches did not change. Conclusion: Excess atRA can inhibit the migration of midgut epithelium in the palate.