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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: To silence the expression of ΔNp63 gene by RNAi (RNAi) technique and study its antitumor effect on human bladder cancer xenografts in nude mice. At the same time, to detect the expression of cyclin D1 Impact. Methods Human bladder cancer xenograft model was established, and the expression vector of ΔNp63 gene shRNA (short hairpin) was constructed. The expression plasmid was transfected into the tumor-bearing mouse tumor. Fifty nude mice were divided into 3 groups according to the experimental conditions: ΔNp63-shRNA group, Control-shRNA group and saline group, 5 rats in each group. The changes of tumor growth and the ultrastructure of tumor tissue in each group were observed. The expression of ΔNp63 gene and protein, PCNA and cyclin D1 protein were detected. Results After 8 weeks of transfection, the tumor volume of ΔNp63-shRNA group was significantly different from that of saline group (P <0.01), and the inhibition rate was 74.44%. There was no significant difference between the control-shRNA group and the saline group (P > 0.05). The pathological results showed that the tumor growth was inhibited in the ΔNp63-shRNA group and the mitotic figures were reduced, showing a large number of tumor cell necrosis and apoptosis, inflammatory cell infiltration. RT-PCR results showed that ΔNp63-shRNA group decreased ΔNp63 gene expression. The results of immunohistochemistry showed that the expressions of ΔNp63, PCNA and cyclin D1 were all decreased. Conclusion The ΔNp63 specific shRNA plasmid expression vector silenced ΔNp63 gene can significantly inhibit the growth of human bladder cancer in nude mice. ΔNp63 may inhibit the proliferation of tumor cells by inhibiting the expression of cyclin D.