ZHX3基因沉默对BMSCs中BMP2/smad通路的影响

Influence of silencing ZHX3 on BMP2/smad pathway in BMSCs

目的:探讨转录抑制因子——锌指蛋白和同源框3(zinc fingers and homeoboxes 3,ZHX3)基因沉默对骨髓间充质干细胞(bone marrow stem cells,BMSCs)中BMP2/smad通路相关因子骨形成蛋白2(bone morphogenetic protein 2,BMP2)、磷酸化smad1(phospho-smad1(或pho-smad1))表达的影响。方法:构建ZHX3低表达慢病毒载体转染大鼠BMSCs,同时设阴性对照空载病毒转染BMSCs及空白对照不做任何处理的BMSCs。采用荧光显微镜下测定细胞转染率、免疫印迹技术鉴定转染效果、免疫细胞化学和免疫印迹技术定性定量检测ZHX3沉默时BMP2、pho-smad1和smad4的表达情况。细胞经成骨诱导液处理后进行碱性磷酸酶和茜素红染色检测各组细胞成骨能力。结果:1复苏培养的细胞具有BMSCs形态。2转染后,ZHX3低表达组和空载体对照组均表达绿色荧光,转染率分别为(93.18±4.41)%和(92.56±4.35)%,空白对照组不表达荧光,未见转染阳性细胞(F=1 123.464,P<0.05)。3免疫细胞化学及免疫印迹结果显示,BMP2、pho-smad1、smad4于3组细胞内均可见阳性表达,但ZHX3低表达组的BMP2、pho-smad1水平(0.383 0±0.006 3,0.329 1±0.025 8)明显高于空白对照组(0.306 2±0.011 6,0.209 0±0.017 0)和空载体对照组(0.291 2±0.022 2,0.180 0±0.018 7),P<0.05;而ZHX3低表达组smad4的表达(0.107 4±0.004 3)显著低于空白对照组(0.380 1±0.023 6)和空载体对照组(0.353 7±0.016 7),P<0.05。4空白对照组、空载体对照组、ZHX3低表达组均可见碱性磷酸酶阳性表达的细胞及矿化结节,但后者数量均小于前两者。结论:ZHX3基因低表达可延迟BMSCs体外成骨能力,可能通过调控BMP2/smad通路中的smad4来发挥作用。

Objective：To investigate the effects of silencing zinc fingers and homeoboxes 3（ZHX3） on expressions of bone morphogenetic proteins 2（BMP2）,phospho-smad1（or pho-smad1）in BMP2/smad pathway in bone marrow mesenchymal stem cells（BMSCs）.Methods：ZHX3 low expression vector was constructed to transfect the rat BMSCs. At the same time,BMSCs transfected with no-load virus was taken as negative control group and BMSCs without any treatment as blank control group. Fluorescence microscope was used to determinate cell transfection rate. Immunocytochemistry method was used to identify the success of the transfection. Immunocytochemistry and Western blot were used to detect the protein expressions of BMP2,phospho-smad1,smad4 when silencing ZHX3 gene. Osteogenesis ability of cell after inducement of osteogenesis solution was detected by alkaline phosphatase and alizarin red staining. Results：1 Cells expressing BMSCs phenotype could be obtained by recovery culturing. 2After transfection,transfection rate of the ZHX3 low expression group and negative control group was（93.18±4.41）% and（92.56±4.35）% respectively,but no transfection cell was observed in blank control group（F=1 123.464,P〈0.05）. 3 According to the results of immunocytochemistry and Western blot,positive expressions of BMP2,pho-smad1,smad4 were observed in three groups,but their expressions were higher in ZHX3 low expression group（0.383 0±0.006 3,0.329 1±0.025 8）than in blank control group（0.306 2±0.011 6,0.209 0±0.017 0）group and negative control group（0.291 2 ±0.022 2,0.180 0 ±0.018 7,P〈0.05）. Expression of smad4 was lower in ZHX3 low expression group（0.107 4±0.004 3）than in blank control group（0.380 1±0.023 6）and negative control group（0.353 7±0.016 7,P〈0.05）. 4Positive expression of nalkaline phosphatase was observed in cells in all groups,however,its quantity was higher in blank control group and negative control group than in ZHX3 low expression group. Conclusion ：ZHX3 gene silencing may delay BMSCs in vitro osteogenesis,which may through the regulation of smad4 in BMP/smad pathway.