| Purpose To investigate the impact of two main monomer components of Tanshinone(Cryptotanshinone,Tanshinone ⅡA)with different concentrations on meibomian gland epithelial cells’ proliferation、differentiation as well as lipid synthesis in SD rats primary cultured in vitrol.And to explore whether Tanshinone can be used for clinical treatment of Meibomian gland dysfunction(MGD)and its mechanism of action.Method The meibomian gland tissues in SD rats were digested into single cells with collagenase Ⅳ,and were primary cultured with 3T3 in vitro.After a seven-day cloning,the cloned cells were incubated with different final concentration(0.125、0.25、0.5、1.25、2.5、5.0 umol/L)of Cryptotanshinone and TanshinoneⅡA which were diluted with DMSO for 48 h,while in control group the cloned cells were incubated with 5.0 umol/L DMSO for 48 h.Real-time quantitative polymerase chain reaction were used to detect the gene expression of Keration 16(K16)、 nuclear antigen associated with cell proliferation(Ki67)and CCAAT/enhancer binding protein(CEBP/α),used Crystal Violet staining to detect the cloned rate of meibomian gland epithelial cells and Oil Red staining to detect the lipid synthesis.Result 1、Primary cultivation of meibomian gland epithelial was observed under inverted microscope,they began to have a clone formation in fourth to fifth days,the cloned cells growthed exponentially in sixth to seventh days.PCR detection results:(1)The gene experession of Krt16 in experimental group were all increased compared to their corresponding control group,but showed no statistically significant except the group with a concentration of 1.25umol/L(p = 0.001,p < 0.05);The gene experession of Ki67 in the group with a concentration of 0.125 umol/L was descreased while the other groups were increased compared to the control group,and a statistically significant difference were observed in the concentration of 0.5,1.25 umol/L Group when comparaed with the control group(p1= 0.001,p2 = 0.001,p < 0.05);the expression of C/EBP alpha gene in the experimental groups were increased compared to the control group,but show no statistical difference.(2)The gene experession of Krt16 in experimental group were increased compared to the control group,but show no statistically significant except the group with a concentration of 0.5、1.25umol/L(p1=0.042,p2=0.043,p<0.05);The gene experession of Ki67 in the group with a concentration of 0.125 umol/L was decreased while the other groupes were increased compared to the control group,and a statistically significant difference were observed only in the group with a concentration of 0.5,1.25 umol/L comparaed to the control group((p1=0.001,p2=0.000,p<0.05);the gene expression of C/EBP alpha were increased compared to the control group,but show no statistical difference(p>0.05).3、Oil red staining results: meibomian gland cell oil red staining showed negative results,and no lipid synthesis was observed within the cell.On the borders of cloning cell cluster,oil red staining showed positive results,and lipid accumulation was available.The lipid drops were considered to be from the 3T3 cells which were co-cultured with meibomian gland cells.4、Crystal violet staining results: Meibomian gland cell clone formation rate of DMSO control group is about 2.05%,average clone formation rate of of meibomian gland cells in 1.25 umol/L ctyptotanshinone treatment group is 2.55%,higher than that of DMSO Group(p =0.041,p < 0.05),the difference is statistically significant;The average clone formation rate of meibomian galnd cells in 1.25 umol/L tanshinone ⅡA treatment group is 2.25%,slightly higher than the DMSO control group,(p = 0.429,p > 0.429),with no statistically significant difference.Conclution Ctyptotanshinone and tanshinoneⅡA,two main monomer composition of tanshinone can promote the proliferation and clone of meibomian gland epithelia cells without affect the meibomian gland epithelia cells differentiation and lipid synthesis in vitrol. |
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