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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。
Objective: To investigate the mechanism of siRNA inhibiting TGFβ3-induced cleft palate. Methods: Uterine fibroblasts were injected intraperitoneally with different doses (200, 400, 800 pmol) of TGFβ3 StealthTM RNAi encapsulated by LipofectamineTM 2000 in 12 days post mating (days post coitum, dpc). At 14dpc, part of fetal rat palatal process was taken, and the expression of TGFβ3, Smad2 and BMP2 in the palatal process was detected by RT-PCR and Western blotting. At 16dpc, some fetus and palatal fossa were harvested, and the fusion of palatal fossa was observed. Immunohistochemistry and immunofluorescence were used to observe the expression of TGFβ3, Smad2 and BMP2. The experimental data were analyzed by SPSS13.0 software package, and the one-way ANOVA was used to compare the multiple groups. The t-test was used to compare the two groups. RESULTS: Intrauterine injection of 800 pmol of StealthTM RNAi resulted in a significant decrease of TGFβ3 mRNA and protein expression. 400 pmol group significantly decreased Smad2 mRNA and protein expression, but BMP2 expression was not significantly affected. In the experimental group, 20% -60% of mouse embryo palatal fossa could not fuse and form the cleft palate. Compared with the blank control group, the expression of TGFβ3 and Smad2 was low, while the expression of BMP2 was not significantly changed. Conclusion: Intraperitoneal injection of TGFβ3 StealthTM RNAi in mice at 12dpc can effectively silence TGFβ3 gene, and 20% ~ 60% mouse embryos form palatal incisors, while Smad2 gene expression is down regulated, but BMP2 gene expression is not significantly affected.