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目的:应用小干扰RNA(si RNA)技术抑制裸鼠喉癌皮下移植瘤人端粒酶逆转录酶(hTERT)基因表达,探讨移植瘤中增殖细胞核抗原(PCNA)及p53蛋白表达的变化。方法:根据hTERT cDNA序列构建表达短发夹RNA(shRNA)的、靶向hTERT mRNA的真核表达质粒pshRNA,其载体质粒含荧光素报告基因。建立人喉鳞状细胞癌Hep-2细胞株裸鼠皮下接种模型,将pshRNA转染入荷瘤裸鼠瘤体内,观察肿瘤生长情况。以激光共聚焦显微镜观察质粒在瘤体内的表达;以免疫组织化学SP法检测PCNA及p53蛋白在肿瘤内的表达。结果:所有裸鼠均接种成功,5d后可见皮下肿瘤形成,14d左右肿瘤直径达5~7mm。pshRNA及空质粒载体转染入瘤体后,共聚焦显微镜下见癌组织中有绿色荧光表达。病理学检查发现:pshRNA组肿瘤生长受到抑制,细胞分裂少见,可见大量癌细胞坏死。与注射生理盐水的对照组比较,转染质粒完毕后7d抑瘤率为76.50%,P<0.01。pshRNA治疗后瘤体内PCNA蛋白表达显著下调(P<0.05),p53蛋白表达显著上调(P<0.05)。结论:靶向hTERT mRNA的shRNA可显著抑制人喉癌裸鼠移植瘤的生长,其机制可能是诱导PCNA下调,促进p53蛋白表达所致。
OBJECTIVE: To suppress the expression of human telomerase reverse transcriptase (hTERT) in subcutaneously transplanted laryngeal carcinoma of nude mice with small interfering RNA (si RNA) and to investigate the expression of proliferating cell nuclear antigen (PCNA) and p53 in transplanted tumor. Methods: The eukaryotic expression plasmid pshRNA targeting short hairpin RNA (shRNA) targeting hTERT mRNA was constructed based on the hTERT cDNA sequence. The vector contains luciferase reporter gene. Human laryngeal squamous cell carcinoma Hep-2 cells were subcutaneously inoculated into nude mice and the pshRNA was transfected into tumor-bearing nude mice to observe the tumor growth. The expression of plasmids in the tumor was observed by laser confocal microscopy. The expression of PCNA and p53 protein in tumor was detected by immunohistochemical SP method. Results: All nude mice were inoculated successfully. Subcutaneous tumors formed after 5 days. The diameter of tumors reached 5 ~ 7mm after 14 days. pshRNA and empty plasmid vector transfected into the tumor, the confocal microscope, see the cancer tissue with green fluorescent expression. Pathological examination found: pshRNA tumor growth was inhibited, rare cell division, showing a large number of cancer cell necrosis. Compared with the control group injected with normal saline, the tumor inhibition rate was 76.50% at 7 days after transfection of plasmid, P <0.01. The expression of PCNA protein was significantly downregulated (p <0.05) and the expression of p53 protein was significantly up-regulated after pshRNA treatment (P <0.05). Conclusion: shRNA targeting hTERT mRNA can significantly inhibit the growth of human laryngeal carcinoma xenografts in nude mice, which may be due to the down-regulation of PCNA and the promotion of p53 protein expression.