白藜芦醇对小鼠胚胎腭突间充质细胞生长的影响

Effect of resveratrol on the growth of mouse embryonic palatal mesenchymal cells

目的 研究白藜芦醇调控亚甲基四氢叶酸还原酶(MTHFR)基因影响小鼠胚胎腭突间充质细胞生长的内在机制。方法 将20只孕鼠使用维甲酸建立腭裂小鼠的模型;于妊娠13天上午处死孕鼠,解剖仔鼠取体内胎鼠腭突,分离培养腭突间充质细胞,将分离得到细胞随机分为对照组、白藜芦醇组、白藜芦醇+si-NC组、白藜芦醇+si-MTHFR组和白藜芦醇+转化生长因子-β1(TGF-β1)组。对照组细胞用正常培养基进行培养;白藜芦醇组细胞加入100μmol·L^(-1)白藜芦醇进行培养;白藜芦醇+si-NC组和白藜芦醇+si-MTHFR组分别使用control siRNA、MTHFR siRNA与脂质体转染试剂盒进行转染,转染24~48 h,然后用100μmol·L^(-1)的白藜芦醇进行培养;白藜芦醇+TGF-β1组将重组质粒pcDNA3.1-TGF-β1转入胚胎腭突间充质细胞中,转染48 h,然后用100μmol·L^(-1)的白藜芦醇对细胞进行培养。以CCK-8法分别检测0,20,60,100μmol·L^(-1)白藜芦醇对MEPM细胞增殖的影响;以流式细胞仪检测各组细胞周期;以蛋白质印迹法检测各组细胞中MTHF的表达及TGF-β1/SMAD3通路相关蛋白表达。结果 CCK-8结果发现白藜芦醇可显著促进胚胎腭突间充质细胞增殖,100μmol·L^(-1)浓度的白藜芦醇组0,24,48,72 h的光密度分别为0.22±0.01,0.37±0.02,0.60±0.04,1.06±0.09。白藜芦醇+si-NC组和白藜芦醇+si-MTHFR组在72 h的光密度分别为1.06±0.06,0.66±0.05,可通过MTHFR增加MEPM细胞增殖能力。对照组和白藜芦醇组的MTHFR表达水平分别为1.00±0.08,1.78±0.09;TGF-β1蛋白表达水平分别为1.03±0.06,0.44±0.05;p-Smad3蛋白表达水平分别为0.96±0.06,0.43±0.06,差异均有统计学意义(均P<0.05)。结论 白藜芦醇可通过调控MTHFR的表达影响MEPM细胞增殖和周期进程,其调控机制可能与TGFβ1/SMAD3信号通路有关。

ObjectiveTo investigate the internal mechanism o resveratrol regulating methylenetetrahydrofolate reductase (MTHFR)gene and affecting the growth of mouse embryonic palatal mesenchyma cells.Methods The cleft palate mice were modeled with retinoic acid from 20 pregnant mice.The pregnant rats were sacrificed on the morning of 13th day of gestation.The fetal rat palatal process was dissected and taken out from the body.The palatal process mesenchymal cells were isolated and cultured.The isolated cells were randomly divided into control group,resveratrol group,resveratrol+si-NC group,resveratro+si-MTHFR group,and resveratrol+TGF-β1 group.The cells in control group were not transfected and cultured in normal medium.Cells in resveratrol group were cultured in 100μmol·L^(-1)resveratrol medium.The control siRNA and the MTHFR siRNA were transfected with the liposome transfection kit in resveratrol+si-NC group and the resveratrol+siMTHFR group respectively,and the transfected cells were transfected for 24 to 48 h,and then the cells were cultured with 100μmol·L^(-1)resveratrol.The recombinant plasmid pcDNA3.1-TGF-β1 was transferred into the embryonic palatal process mesenchymeand then they were cultured with 100μmol·L^(-1)resveratrol.The effects of 0,20,60 and100μmol·L^(-1)resveratrol on the proliferation of MEPM cells were detected by CCK-8 method.The cell cycle of each group was detected by flow cytometry.The expression of MTHF and TGF-β1/SMAD3 pathway related proteins in each group were detected by western blot.Results The results of CCK-8 showed that resveratrol could significantly promote the proliferation of mesenchymal cells in the embryonic palatine process.The absorbance values in the 100μmol·L^(-1)resveratrol group at 0,24,48 and 72 h were 0.22±0.01,0.37±0.02,0.60±0.04,1.06±0.09,respectively.The optical density at 72 h in resveratro+si-NC group and resveratro+si-MTHFR group were1.06±0.06 and 0.66±0.05,respectively,which indicated that MTHFR could increase the proliferation of MEPM cells.The expression levels of MTHFR in control group and resveratrol group were 1.00±0.08,1.78±0.09,respectively,the expression levels of TGF-β1 protein were 1.03±0.06,0.44±0.05,respectively,p-Smad3protein expression levels were 0.96±0.06,0.43±0.06,respectively,the difference were all statistically significant(all P<0.05).Conclusion Resveratrol can affect the proliferation and cycle process of MEPM cells by regulating MTHFR expression,which may be related to TGF-β1/Smad3 signaling pathway.