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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 relationship between the expression of hyaluronic acid synthase 3 (HAS3) and the biological behavior of transitional cell carcinoma of bladder in human bladder transitional cell carcinoma by RNA interference. Methods The HAS3 mRNA sequence - specific and nonspecific smallinterference RNA (siRNA) were synthesized and synthesized in vitro. The recombinant plasmid was combined with LipofectamineTM2000 and transfected into human bladder transitional cell carcinoma fibroblasts. The experiment was divided into four groups A, B, C and D. After incubation for 48h, the cell culture medium of each group was used to determine the concentration of hyaluronic acid by radioimmunoassay. The immunocytochemical staining was used to detect the synthesis of hyaluronic acid. The mRNA of HAS3 was detected by RT- expression. Results Compared with group A, the expression of HAS3 mRNA in group D decreased by 78.2% and the HA staining decreased significantly. The concentration of HA in cell culture medium decreased by 60.3% (P <0.01), while the mRNA expression in group B and C decreased by 4.7% , 5.4% respectively. There was almost no change in HA staining, HA concentration decreased by 5.2% and 5.8% respectively (P> 0.05). Conclusion RNAi can significantly reduce the expression of HAS3 in fibroblasts of transitional cell carcinoma of the bladder and thus significantly reduce the intracellular hyaluronic acid synthesis.