目的

探讨在胚鼠腭突培养细胞中应用Stealth^TM RNAi 转染有效沉默转化生长因子β3（TGFβ3）基因的可行性。

方法

采用Bal b/c 近交系小鼠在交配后第14 天取胚鼠腭突，离散后进行细胞培养，应用相差显微、MTT 法观察传代细胞的形态和生物学特性。 采用化学合成的Stealth^TM RNAi 抑制原代胚鼠腭突上皮细胞和间充质细胞内源性TGFβ3 mRNA表达，检测作用24、48、72 h 后，TGFβ3、Smad2、BMP2 三种基因的表达情况。

结果

经过本实验设计的Stealth^TM RNAi 干扰的胚鼠腭突培养细胞中，TGFβ3 基因的mRNA 和蛋白表达明显降低，但沉默效果随时间有减弱趋势。 Smad2 基因的mRNA 和蛋白表达均随着TGFβ3 基因的沉默而减低；但BMP2 基因的表达无显著性的改变。

结论

Stealth^TM RNAi 能有效地沉默TGFβ3基因，并下调Smad2 基因的表达，但对BMP2 基因表达无明确影响。

Objectives

This study was designed to evaluate the probability of applying TGFβ3 small interfering RNA （siRNA）to transfect cultured palatal process cells.

Methods

Chemically synthetical TGFβ3 siRNA warped by Lipofectamine^TM2000 were used to transfect primary cultured murine embryonic epithelial and mesenchymal cells， which were derived from gestational day 14 Bal b/c mouse embryos and examined by phase-contrast microscope and MTT.Semi-quantitative RT-PCR and western blot were used to exam the gene expression of TGFβ3，Smad2， BMP2 at 24， 48， 72 hours after using standard dosage Stealth^TM RNAi.

Results

After using standard dosage of Stealth^TM RNAi 24， 48， 72 hours， the TGFβ3 gene expression was down regulated significantly at 24， 48， 72 hours after treatment. The Smad2 gene expression was down regulated significantly at 24 hours after treatment. However， the expression increased but not recovered at 48 hours and 2 hours after. But BMP2 gene expression showed no significant change at all three time points.

Conclusions

TGFβ3 Stealth^TM RNAi could silence TGFβ3 gene effectively in the murine embryonic palate cells. However， the silencing effect had tendency to attenuate. Smad2 gene mRNA and the protein expression decreased along with TGFβ3 gene silence， but BMP2 gene expression had non-significance change.