摘要
目的探讨RNA干扰技术阻断人膀胱移行细胞癌组织中成纤维细胞内透明质酸合成酶3(hyaluronicacidsynthase3,HAS3)的表达与膀胱移行细胞癌生物学行为之间的关系。方法设计、体外化学合成HAS3mRNA序列特异性、非特异性小干涉双链RNA(smallinterferencingRNA,siRNA),分别与脂质体LipofectamineTM2000结合后转染体外培养的人膀胱移行细胞癌组织中的成纤维细胞。实验分为A、B、C、D四组:孵育48h后,取各组细胞培养液进行放免法透明质酸浓度测定、免疫细胞化学染色了解透明质酸的合成、RT-PCR法检测HAS3mRNA的表达。结果与A组相比,D组细胞HAS3mRNA表达减少了78.2%,HA染色明显变淡,细胞培养液中HA浓度下降了60.3%(P<0.01);B、C组mRNA表达分别减少了4.7%、5.4%,HA染色几乎无变化,HA浓度分别下降了5.2%、5.8%(P>0.05)。结论RNA干扰技术可以显著地减少膀胱移行细胞癌组织中成纤维细胞的HAS3的表达,从而大幅度地降低该细胞内透明质酸的合成。
Objective To investigate the inhibitory effect of RNA interference technique on the expression of the hyaluronic acid synthase 3 （HAS3） in the fibroblasts of human bladder transitional cell carcinoma tissues. Methods The 4 specific and one non- specific small interfering RNA （siRNA） ,according to human HAS3 mRNA were designed and chemosynthesized in vitro. These siRNA were transfected into the cultured fibroblasts of human bladder transitional cell carcinoma tissue after combined with liposome LipofectamineTM 2000. The trial had been divided into 4 groups： A,B,C and D group. After being incubated for 48 h, the HA concentrations in each group were detected by radioimmunoassay. The HA stain was obtained by immunocytochemistry and the expression of the HAS3 mRNA was detected by RT- PCR. Results The expression of the HAS3 mRNA in group D was reduced by 78.2 % as compared with that in group A,the HA cstaining almost disappeared and the HA concentration in nutrienr fluid decreased by 60.3 %. However, the reduction of the expression of HAS3 mRNA in groups B and C was 4.7 % and 5.4 %, respectively, and their HA staining nearly had no change and their HA concentrations reduced by 5.2% and 5.8%, respectively. Conclusion RNA interference technique can strongly silence HAS3 gene in the fibroblasts of human bladder cell carcinoma tissue and significantly reduce its HA svnthesis.
出处
《临床外科杂志》
2006年第6期366-368,403,共4页
Journal of Clinical Surgery
基金
湖北省科技攻关计划基金资助项目(2003AA301C29)
关键词
RNA干扰技术
膀胱移行细胞癌
成纤维细胞
透明质酸合成酶3
RNA interference technique
bladder transitional cell carcinoma
fibroblast
hyaluronic acid synthase 3
作者简介
杨为民（1956-），男，教授，硕士生导师。
