siRNA抑制TGFβ3诱导小鼠腭裂机制的研究

siRNA suppress TGF β3 gene expression and establish murine embryonic cleft palate model

目的:探讨siRNA抑制TGFβ3诱发腭裂的机制。方法:使用不同剂量(200、400、800pmol)经脂质体LipofectamineTM2000包裹的TGFβ3 StealthTMRNAi于交配后(days post coitum,dpc)12天做孕鼠宫腔注射,等体积生理盐水注射作为空白对照。14dpc时取部分胎鼠腭突,RT-PCR及Western印迹检测腭突中TGFβ3、Smad2、BMP2的表达情况。16dpc时再取部分胎鼠腭突,观察腭突融合情况,免疫组织化学及免疫荧光观察胚鼠腭突的TGFβ3、Smad2、BMP2的表达情况。实验数据均以SPSS13.0软件包进行分析,多组间比较采用单因素方差分析,两组间比较采用t检验。结果:宫腔注射800pmol的StealthTMRNAi,可引起TGFβ3基因mRNA和蛋白表达水平明显降低,400pmol组能显著降低Smad2基因的mRNA和蛋白表达水平,但BMP2的表达情况未受明显影响。20%～60%的实验组小鼠胚胎腭突不能融合,形成腭隐裂,与空白对照组相比,其TGFβ3、Smad2的表达量较低,而BMP2的表达未见显著变化。结论:在12dpc给予小鼠宫腔注射TGFβ3 StealthTMRNAi,能有效沉默TGFβ3基因,20%～60%小鼠胚胎形成腭隐裂,同时Smad2基因表达下调,但对BMP2基因表达无明显影响。

PURPOSE：This study was designed to suppress TGFβ3 gene expression by small interfering RNA（siRNA） in vivo and establish murine embryonic cleft palate model.METHODS：Different dosages（200,400,800pmol）of TGFβ3 siRNA warped by the LipofectamineTM2000 were injected into pregnant mouse uterine on 12 dpc and the control animals were given the equivalent volume of N.S.The embryonic palatine processes were collected on 14 dpc and 16dpc in order to examine the morphological changes in palates.Immunohistochemistry,immunofluorescence and Western blot were applied to examine the expressions of TGFβ3,Smad2,and BMP2 gene.Statistically significant differences were calculated after analysis of variance（ANOVA） and Student＇s t tests by SPSS software package.RESULTS： After using 800pmol StealthTMRNAi,the TGFβ3 gene expression was down-regulated significantly on 14dpc in mRNA and protein level.The Smad2 gene expression was down-regulated significantly after 400pmol injection,but BMP2 gene expression showed no significant change.The expression of TGFβ3 and Smad2 was weaker in siRNA group than in control group.CONCLUSION： TGFβ3 StealthTMRNAi could silence TGFβ3 gene effectively in the murine embryonic palatine process by injection into uterine cavity on 12dpc and could induce cleft palate in 20%-60% of the viable fetuses.Smad 2 gene mRNA and the protein expression decreased along with TGFβ3 gene silence,but BMP2 gene expression had no significant change.Dr.CHEN Mu and CHEN Yi-yang contributed equally to the study.