siRNA沉默OPN基因治疗喉鳞癌的实验研究
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摘要
目的:通过体外实验和动物模型观察OPN-siRNA对喉鳞癌的治疗效应,探索一条喉鳞癌基因治疗的新途径。方法:OPN基因被OPN-siRNA沉默后,RT-PCR检测OPNmRNA的表达水平;Western-blot检测OPN蛋白质的表达水平,PI Staining检测凋亡细胞数目,MTT Assay检测凋亡细胞水平;建立人喉鳞癌裸鼠种植瘤模型;裸鼠瘤体内注射治疗结束前后分别称量裸鼠体重、离体肿瘤的重量和测量肿瘤体积大小。结果:OPN基因被OPN-siRNA沉默后,western blot检测Hep-2细胞的OPN基因剪切体1蛋白表达明显减弱;RT-PCR技术检测OPN mRNA表达量明显降低。PI染料染色后,OPN-RNAi组30%-35%的肿瘤细胞凋亡;MTT Assay结果显示,OPN-siRNA慢病毒转染Hep2细胞后,Hep2细胞细胞活力明显下降;肿瘤生长曲线可知,OPN-siRNA慢病毒转染组的肿瘤生长明显受到抑制;OPN-siRNA慢病毒转染组的裸鼠瘤重和体积均明显低于对照组(P<0.05),抑瘤率达65.37%。结论OPN在Hep-2细胞中表达明显上调可能与喉鳞癌发生、发展相关;OPN-siRNA慢病毒在Hep-2细胞中可获得高效转染,并能产生特异性的基因沉默效应;siRNA介导的OPN基因沉默在体外实验中显著抑制了Hep-2细胞的生长,30%-35%的肿瘤细胞凋亡:在体内实验中对裸鼠喉鳞癌皮下种植瘤产生明显的抗瘤效应。
objective :We try to look for a new approach of gene therapy of LSCC by synthe -sizing and filtrating effective OPN-siRNA in vitro, and checking the inhibitory effect of gene silencing mediated by OPN-siRNA for LSCC in vivo. Methods: emi-quantita -tive analysis of the level of OPN-mRNA expression was performed using the RT PCR technique. The level of OPN protein expression was analyzed with Western - Bl -oting technique, the proliferation index (PI) was calculated. The cell growth and vig -our was detected by MTT Assay .The models of subcutaneous LSCC tumor in nude mi -ce were established with Hep-2 cells. The OPN-siRNA lentivirus have been discontin -ueously injected directly into the established tumor, The changes of weight and size of the tumors and the mice body weight during the therapy were calculated respective-ly. Results: The content of protein of OPN-1 in the OPN-siRNA lentivirus transfect -ed Hep-2 cells was obviously reduced compared with blank control cells.The content of mRNA of OPN in the OPN-siRNA lentivirus transfected Hep-2 cells was obvious -ly reduced compared with blank control cells.PI staining in the OPN-siRNA lentivir -us transfected cell showed 30%-35% of apoptotic cells in Hep-2 cells.MTT Assay res -ult display, Hep-2 cells' vigour decrease obviously after be transfected by OPN-siR -NA lentivirus. The growth of Tumors in the OPN-siRNA lentivirus injected nude mi -ce being inhibitted obviously through tumor growth curve. The weight and volume of transplant tumors in the OPN-siRNA lentivirus injected nude mice were significantly reduced compare with negative siRNA control nude mice and blank control nude mic -e. The weight of transplant tumors was reduced 65.37% compared with blank contro -l.Conclusion:The OPN protein remarkably up regulated in the Hep-2 cells may invol -ve in LSCC carcinogenesis.Eligible 21nt siRNAs synthesized in vivo with lentiviral vector mediated can be highly effectively transfected into LSCC cell, and induced po -st-transcriptional gene silencing.OPN gene silencing mediated by siRNA significant ly suppresses the growth of Hep-2 cell arrest in vitro,30%-35% of apoptotic cells in Hep-2 cells ;and significantly achieve anti-tumor effect in subcutaneous transplant LSCC tumor in nude mice in vivo.
objective :We try to look for a new approach of gene therapy of LSCC by synthe -sizing and filtrating effective OPN-siRNA in vitro, and checking the inhibitory effect of gene silencing mediated by OPN-siRNA for LSCC in vivo. Methods: emi-quantita -tive analysis of the level of OPN-mRNA expression was performed using the RT PCR technique. The level of OPN protein expression was analyzed with Western - Bl -oting technique, the proliferation index (PI) was calculated. The cell growth and vig -our was detected by MTT Assay .The models of subcutaneous LSCC tumor in nude mi -ce were established with Hep-2 cells. The OPN-siRNA lentivirus have been discontin -ueously injected directly into the established tumor, The changes of weight and size of the tumors and the mice body weight during the therapy were calculated respective-ly. Results: The content of protein of OPN-1 in the OPN-siRNA lentivirus transfect -ed Hep-2 cells was obviously reduced compared with blank control cells.The content of mRNA of OPN in the OPN-siRNA lentivirus transfected Hep-2 cells was obvious -ly reduced compared with blank control cells.PI staining in the OPN-siRNA lentivir -us transfected cell showed 30%-35% of apoptotic cells in Hep-2 cells.MTT Assay res -ult display, Hep-2 cells' vigour decrease obviously after be transfected by OPN-siR -NA lentivirus. The growth of Tumors in the OPN-siRNA lentivirus injected nude mi -ce being inhibitted obviously through tumor growth curve. The weight and volume of transplant tumors in the OPN-siRNA lentivirus injected nude mice were significantly reduced compare with negative siRNA control nude mice and blank control nude mic -e. The weight of transplant tumors was reduced 65.37% compared with blank contro -l.Conclusion:The OPN protein remarkably up regulated in the Hep-2 cells may invol -ve in LSCC carcinogenesis.Eligible 21nt siRNAs synthesized in vivo with lentiviral vector mediated can be highly effectively transfected into LSCC cell, and induced po -st-transcriptional gene silencing.OPN gene silencing mediated by siRNA significant ly suppresses the growth of Hep-2 cell arrest in vitro,30%-35% of apoptotic cells in Hep-2 cells ;and significantly achieve anti-tumor effect in subcutaneous transplant LSCC tumor in nude mice in vivo.
引文
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