人膀胱癌荷瘤鼠中shRNA沉默ΔNp63基因对肿瘤的抑制和cyclin D_1表达的影响  

shRNA silencing of delta Np63 inhibits cyclin D_1 expression and tumor growth in xenograft nude mice with human transitional cell carcinoma

作　　者：何云锋[1] 吴小候[1] 唐伟[1] 尹志康[1] 王德林[1] 蒲军[1] 陈刚[1] 

机构地区：[1]重庆医科大学附属第一医院泌尿外科,重庆400014

出　　处：《第三军医大学学报》2010年第1期46-50,共5页Journal of Third Military Medical University

基　　金：重庆市卫生局基金(072032)~~

摘　　要：目的利用RNA干扰(RNA interference,RNAi)技术沉默ΔNp63基因的表达,研究其对人膀胱癌裸鼠皮下移植模型的抑瘤作用,同时检测ΔNp63基因被抑制后对细胞周期素D1(cyclin D1)表达的影响。方法建立人膀胱癌裸鼠皮下移植模型,构建ΔNp63基因特异性shRNA(short hairpin)表达质粒,将表达质粒转染荷瘤鼠瘤体。将符合实验条件的15只裸鼠分为3组:ΔNp63-shRNA组、Control-shRNA组与生理盐水组,每组5只。观察各组肿瘤生长情况、瘤组织超微结构的改变,检测ΔNp63基因和蛋白、增殖细胞核抗原(PCNA)和cyclin D1蛋白表达的变化。结果转染质粒8周后,ΔNp63-shRNA组与生理盐水组瘤体积有显著性差异(P<0.01),抑瘤率为74.44%,Control-shRNA组与生理盐水组体积无显著性差异(P>0.05)。病理结果显示ΔNp63-shRNA组肿瘤生长受到抑制,细胞核分裂象减少,可见大量肿瘤细胞坏死和凋亡、炎症细胞浸润。RT-PCR结果显示ΔNp63-shRNA组ΔNp63基因表达降低。免疫组化结果显示ΔNp63、PCNA、cyc-lin D1蛋白表达均降低。结论ΔNp63特异性shRNA质粒表达载体沉默ΔNp63基因后,可显著抑制人膀胱癌裸鼠肿瘤的生长,ΔNp63可能通过抑制cyclin D的表达而抑制肿瘤的细胞增殖。Objective Delta Np63 （△Np63） is a nuclear transcription factor that maintains epithelial progenitor cell populations, and is over-expressed in several epithelial cancers. However, the expression of △Np63 in transitional cell carcinoma of the bladder （TCCB） is still disputable, and the mechanisms by which △Np63 promotes cell survival are unclear. To explore the inhibitory effect of tumor growth and cyelin Dl expression by silencing △Np63 with short hairpin RNA （shRNA） on human TCCB in xenograft nude mice. Methods Human TCCB 5637 cells were transplanted into nude mice to establish xenograft tumors. △Np63 specific shRNA expression plasmid was constructed and then transfected into the tumor masses. The tumor volume was observed, tumor morphology with HE staining, and uhramicrostructure with transmission electron microscopy. The inhibitory effect of △Np63 was studied with semi-quantitative reverse transeriptase-polymerase chain reaction （RT-PCR）. Protein expressions of △Np63, proliferation cell nuclear antigen （PCNA） and cyclin D1 were examined with the immunohistochemical staining assay. Results After 8 weeks of the transfection of △Np63 specific shRNA plasmid, the tumor volume was significantly smaller in △Np63 shRNA group than in control shRNA and normal saline group （P 〈 0.01 ）, with an inhibitory rate of 74.44% in △Np63 shRNA group, and no significant difference was seen between control shRNA and normal saline group （ P 〉 0. 05 ）. In △Np63 shRNA group, the necrosis and decrease of karyokinesis were observed under light microscope and transmission electron microscope, and apoptosis and inflammatory cells were also found under transmission electron microscope. There was no such change seen in control shRNA and normal saline groups. △Np63 shRNA directly down-regulated the expressions of delta Np63 protein and mRNA, and the protein expression of PCNA and eyelin Dl. Conclusion The Pgenesil-1 △Np63 shRNA plasmid significantly inhibits the growth of TCCB in nude

关 键 词：RNA 短发卡 △NP63 cyclin D1 膀胱癌 

分 类 号：R737.14[医药卫生—肿瘤] R342.2[医药卫生—临床医学]