摘要
目的研究TGF-β/Smad信号对人胚胎性横纹肌肉瘤细胞系RD生长和凋亡的调控作用。方法采用小分子RNA干扰(RNAi)技术,以阳离子脂质体为载体,将生物合成的发夹状短链RNA(shRNA)转染RD细胞,封闭Smad4的表达以阻碍内源性TGF-β/Smad信号的转导;采用逆转录-聚合酶链反应(RT-PCR)和Westernblot检验shRNA干扰Smad4表达的效果;采用免疫荧光化学,通过激光共聚焦显微镜检测Smad4封闭前后RD细胞中Smad2/3表达位置的变化;应用四甲基偶氮唑盐(MTT)和3H-thymidine摄入实验检测阻碍内源性TGF-β/Smad信号转导和以外源性TGF-β1刺激这2种条件下RD细胞的生长状况;采用流式细胞术和荧光化学染色检测上述2种条件下RD细胞的凋亡情况。结果RNAi技术可有效封闭RD细胞中Smad4mRNA和蛋白的表达;Smad4的封闭导致Smad2/3由胞质入核明显减少;内源性TGF-β/Smad信号转导受阻不但抑制RD细胞生长,并且诱导凋亡;外源性、较高浓度的TGF-β1可抑制RD细胞生长,但不诱导凋亡。结论TGF-β/Smad信号在一定的浓度范围内精细调控RD细胞的生长和凋亡。
Objective To study the effects of TGF-β/Smad signaling on the growth and apoptosis of human rhabdomyosarcoma cell line RD. Methods Biosynthesized short hairpin RNA (shRNA) was transfected into RD cells by cation liposome vector to suppress Smad4 expression. The mRNA and protein expression of Smad4 in RD after shRNA-transfection were examined by RT-PCR and Western blot respectively. Immunofluorescent staining was used to detect the location of Smad2/3 in RD by laser scanning confocal microscopy. The viability of RD cells was examined by MTT method and 3H-thymidine incorporation assay. The apoptosis of RD was examined by flow cytometry analysis and fluorescent staining. Results The expression of mRNA and protein of Smad4 in RD were effectively suppressed by shRNA interference. Such suppression effectively interrupted the endogenous TGF-β/Smad signaling and consequently blocked the translocation of Smad2/3. The interruption of endogenous TGF-β/Smad signaling not only inhibited the growth of RD but also induced apoptosis of RD. Exogenous TGF-β1 inhibited the growth of RD but did not influence the apoptosis of RD. Conclusion shRNA interference can effectively interrupt the TGF-β/Smad signaling by suppressing the expression of Smad4. TGF-β/Smad signaling subtly regulates the growth and apoptosis of RD.
出处
《中华病理学杂志》
CAS
CSCD
北大核心
2005年第7期407-412,共6页
Chinese Journal of Pathology
基金
美国中华医学基金会资助项目(00-722)
