过量全反式视黄酸对腭突中嵴上皮细胞迁移的影响

EFFECT OF EXCESS ALL-TRANS RETINOIC ACID ON MIGRATION OF PALATAL MEDIAL EDGE EPITHELIUM CELLS

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 observe the influence of excessive all-trans retinic acid （atRA） on migration of primary palatal medial edge epithelium （MEE） cells. Methods Kunming mice at 10 w of age were mated at the ratio of 1：2 （male：female）. At day 13, the pregnant rats were killed and the fetuses were dissected out and their palate shelves were isolated. Dispase was applied to separate MEE cells from the underlying basement membrane. These primary MEE cells were inoculated in culture flasks, which contained DMEM/F-12 medium in a cell culture chamber for 96 h. Cell layer scratching experiments were performed in a 6-well-plate under the same cultivation condition for 24 h, of which, 3 wells belonged to control group and 3 belonged to 5 μmol/L atRA group. A scratching line paralleled with the long axis of plate was made in each well to observe the influence of atRA on MEE cell migration. Results After cultivation, primary MEE cells showed normal appearenee, cobble-shaped. When more than 80% of the MEE cells fused together, they looked like an arrangement of slabstone. During the cultivation, MEE cells in the control group grew toward the scratching line from the sides of scratch and almost covered the whole scratch area by the end of culture period. In contrast, no migration growth was seen in the atRA-treated MEE cells. Conclusion Excess atRA could inhibit the migration of primary palatal MEE cells.