固相转染HPV16-siRNA对荷宫颈癌细胞SiHa小鼠瘤体体积及P53、P16表达的影响

siRNA inhibits HPV16-DNA replication,tumor volume and expression of P16 and P53 in SCID mice with SiHa cell cervical carcinoma in vivo

目的:观察靶向基因治疗后不同时点荷宫颈癌细胞小鼠HPV16-DNA、瘤体体积、P53及P16蛋白的变化,探讨在体固相转染HPV16-siRNA的有效性。方法:采用SiHa细胞皮下注射建立荷宫颈癌细胞SCID小鼠模型。制备HPV16靶向siRNA-Lipo2000-卡波姆凝胶。将40只荷宫颈癌细胞SCID小鼠随机分为对照组8只和研究组32只和研究组使用HPV16靶向siRNA-Lipo2000-卡波姆凝胶进行基因治疗,对照组仅使用Lipo2000-卡波姆凝胶处理;分别于治疗后4 d、8 d、12 d、16 d处死小鼠,每个时点研究组处死8只、对照组处死2只。PCR检测两组小鼠瘤体中HPV16-DNA滴度,测各时点的瘤体体积,常规病理切片检测肿瘤细胞形态,免疫组化检测P16及P53的表达。结果:(1)研究组治疗后8 d及12 d时点瘤体内HPV16-DNA明显降低,与对照组之间的差异显著(P<0.05),4 d及16 d 2个时点与对照组之间的差异不显著(P>0.05);(2)治疗后12 d瘤体平均体积明显缩小,与对照组之间的差异显著(P<0.05);(3)转染后12 d肿瘤细胞核异质性较对照组减轻,核浆比例减小;(4)P16的表达强度在各时点研究组与对照组之间的差异不显著(P>0.05);治疗后8 d及12 d研究组P53的表达强度明显降低,与对照组之间的差异显著(P<0.05)。结论:SCID小鼠所荷宫颈癌细胞瘤体经靶向转染HPV16-siRNA后8 d及12 d时点瘤体内HPV16-DNA和P53均较对照组明显下降,12 d瘤体体积较对照组明显下降;在体固相转染siRNA可以在一定时段内有效抑制HPV-DNA的复制及肿瘤的生长速度。

AIM： To investigate the effect of targeting gene therapy on mouse SiHa cell cervical carcinoma by transfecting siRNA into the tumor with solid-phase method in vivo. METHODS： A sense strand siRNA（21 nt） for human papilloma virus type 16（HPV16） was designed.siRNA-Lipo2000-carbomer gum was prepared.Forty SCID mice with SiHa cell cervical carcinoma were divided into experimental group（n=32） and control group（n=8）.The diameter and volume of the tumors were measured before treatment.The mice in experimental group were treated with siRNA-Lipo 2000-carbomer gum for 7 d.The control mice were treated with Lipo 2000-carbomer gum for 7 d.The mice in experimental group and control group were sacrificed at the time points of 4 d,8 d,12 d and 16 d after treatment.The diameter and volume of the tumors were measured again.The HPV16-DNA in the tumor tissues was measured by PCR.The protein levels of P16 and P53 in the tumors were determined by the method of immunohistochemistry. RESULTS： Compared with control group,the titer of HPV16-DNA decreased significantly at the time points of 8 d and 12 d after transfection in experimental group（P〈0.05）.The protein expression of P16 in experimental group showed decreased tendency 8 d and 12 d after transfection,but without statistical difference.The protein expression of P53 significantly decreased 8 d and 12 d after transfection.The tumor volume in experimental group was significantly decreased as compared to that in control group at the time point of 12 d（P〈0.05）.Transfection of siRNA for 12 d resulted in attenuating dyskaryosis and karyoplasmic ratio of the tumor cells.CONCLUSION： Solid-phase transfaction of siRNA in vivo inhibits HPV-DNA replication and growth of SiHa cell cervical carcinoma.