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Objective To investigate the effects of cryptotanshinone and tanshinone Ⅱ A,two major monomer components of tanshinone,on the proliferation,differentiation and lipid synthesis of rat meibomian gland epithelial cells in vitro.Methods The eyelid meibomian gland tissue was isolated from 2-month-old SD rats and co-cultured with 3T3 trophoblasts for 5 days.Cryptotanshinone and tanshinone Ⅱ A were prepared with DMSO into different concentrations.The cells were grouped to 0.125 μmol/L,0.250 μmol/L,0.500 μmol/L,1.250 μmol/L and 2.500 μmol/L drug groups and treated for 48 hours,respectively.Only dimethyl sulfoxide (DMSO) was added in medium in the DMSO control group.The expressions of keratin 14 (K14) and p63 in frozen sections of meibomian gland tissue and clones of meibomian gland cells were detected by immunofluorescence.Real-time fluorescence quantitative PCR was used to assay the relative expression of K16,cell proliferation related antigen 67 (Ki67) and CCAAT enhancer binding protein α (C/EBPcα) in meibomian gland cell clones.Crystal violet staining and oil red staining were used to evaluate the colony formation rate and lipid synthesis of meibomian gland epithelial cells.Results Primary cultured meibomian gland cells were cloned on day 4-5 in vivo,and the cloning area was increased on day 7 after culture,p63 and K14 were positively expressed in clones.Compared with the DMSO control group,the relative expression levels of Ki67 mRNA were significantly elevated in the 0.500 μmol/L,1.250 μmol/L and 2.500 μmol/L cryptotanshinone groups (all at P ＜ 0.05).The relative expressions of Ki67 mRNA in the 0.500 μmol/L and 1.250 μmol/L tanshinone Ⅱ A groups were significantly higher than those in the DMSO control group (all at P＜0.05).No significant difference was found in the relative expression of K16 and C/EBPα mRNA among different concentrations of cryptotanshinone or tanshinone Ⅱ A group (all at P＞0.05).No lipid drop was found in the tarsal gland cell clones;however,the accumulation of lipid was seen in the cell clusters at the margin of the clones by oil red O staining.The average clone formation rate of tarsal gland cells in the 1.250 μ mol/L cryptotanshinone group was (2.55±0.20)％,which was significantly higher than (2.05±0.13)％ in the DMSO control group (t =4.379,P＜0.05).The average clone formation rate of tarsal gland cells in the 1.250 μmol/L tanshinone Ⅱ A group was (2.25±0.20)％,there was no significant difference between 1.250 μmol/L tanshinone Ⅱ A group and DMSO control group (t=1.616,P＞0.05).Conclusions Cryptotanshinone and tanshinone Ⅱ A promote the proliferation of meibomian gland epithelia cells,but play less impacts to lipid synthesis of meibomian gland epithelia cells in vitro.cryptotanshinone promote the clone tormation of meibomian gland epithelia cells.
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Objective To construct engineered corneal epithelium from embryonic stem cells (ESCs) using Rock inhibitor combined with hypoxia-normoxia culture condition. Methods Human ESC line H1 was induced to differentiate into epithelial-like cells by addition of retinoic acid (RA) and bone morphogenetic protein 4(BMP4) in the differentiation medium under the adherent culture condition. The ESCs derived epithelial-like cells were expanded in the mixed medium of SHEM and KSFM with the mixture ratio of 1 : 2 with or without Rock inhibitor Y27632. The H1 derived epithelial-like cells were seeded on the denuded ammonic membrane to construct engineered corneal epithelium under hypoxia,normoxia and hypoxia-normoxia culture conditions,respectively. The inducted effect of ESCs into epithelial-like cells,the expansion ability of the epithelial-like cells and the characteristics of the constructed engineered corneal epithelium were evaluated by morphological observation, real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) and immunofluorescence technology. Results Compared with the control group,the relative expressions of ESCs marker Oct4 mRNA, Notch signaling pathway related factors Notch1 and Jagged1 mRNA,and Wnt signaling pathways related factors c-myc and Cyclin D1 mRNA were significantly reduced, and the relative expressions of cutaneous ectoderm markers p63 and K18 mRNA were significantly increased at day 8 after induction in the induced group,with significant differences between them (t =14.63,20.15,93.50,11.60, 19.30,18.44,22.63;all at P<0.05). Compared with the without Y27632 group,the relative expressions of p63 and K14 mRNA,Notch signal pathway receptor Notch1 and Jagged1 mRNA were significantly increased,and Wnt signaling pathways downstream targeted gene c-myc and CylinD1 mRNA were significantly decreased at day 8 after induction in the Y27632 group,with significant differences between them (t =20.29,59.22,2.90,39.59,5.32,10.14;all at P<0.05),and the relative expression of K18 mRNA in the two groups was not significantly changed(t=1.38,P>0.05). The ESCs derived epithelium and constructed under hypoxia-normoxia culture condition showed more obvious stratification and tighter cell arrangement in comparison with those cells cultured in consistent hypoxia culture condition or normoxia culture condition. Epithelial markers Pan-CK and K18 as well as epithelial progenitor cell markers p63 and K14 expressed in the whole cell layers of the ESCs derived epithelium constructed under hypoxia-normoxia culture condition. Conclusions The addition of Y27632 enhances the proliferation ability of H1 derived epithelial cells and actives Notch signaling pathway and inhibits Wnt signaling pathway. The culture and construction in the expansion medium with Y27632 under the hypoxia-normoxia culture condition can promote the stratification of H1 derived engineered corneal epithelium.