摘要
目的:(1)探讨在体细胞固相转染siRNA的可行性、安全性及有效性。(2)探讨改变宫颈表面黏膜通透性提高转染效果的可能性。方法:(1)设计针对HPV16 siRNA,长度为21nt,采用Cy3荧光标记,制备siRNA-Lipo2000-卡波姆凝胶。(2)选新西兰雌性未孕兔12只,随机分为实验组及对照组,实验组在转染前使用200mmol/L高渗盐水处理兔宫颈表面10min,对照组则使用生理盐水处理兔宫颈,将siRNA-Lipo2000-卡波姆凝胶1.5mL涂于兔宫颈表面,进行siRNA转染,持续24h。24h后将兔处死,取其宫颈组织。(3)肉眼观察宫颈局部有无红肿、破溃等改变,取部分作快速冰冻切片,荧光显微镜检测转染siRNA的转染效果;部分组织进行石蜡包埋切片,普通显微镜观察宫颈局部组织细胞形态,评估siRNA-Lipo2000-卡波姆凝胶的副反应。(4)将12只荷HPV宫颈癌SCID小鼠随机分为siRNA转染组及对照组,转染组使用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,转染后取瘤体,采用PCR检测治疗前后瘤体中HPV16-DNA滴度,鉴定siRNA-Lipo2000-卡波姆凝胶疗效。结果:(1)高渗氯化钠预处理组及对照组兔宫颈局部上皮组织内均可见红色的荧光;被转染组织局部无红肿、破溃等不良反应。宫颈局部组织结果正常,细胞形态完整,局部无炎性细胞浸润。(2)使用siRNA-Lipo2000-卡波姆凝胶治疗后小鼠瘤体中HPV-DNA的滴度较前明显下降(P<0.05)。结论:(1)siRNA可通过Lipo2000成功转染入在体宫颈局部组织细胞中,siRNA-Lipo2000-卡波姆凝胶对宫颈局部组织无明显毒、副反应。(2)siRNA-Lipo2000-卡波姆凝胶可有效降低荷SiHa细胞宫颈癌瘤体中HPV16-DNA滴度,具有抑制HPV复制的作用。(3)目前没有足够的证据表明转染前增加宫颈黏膜的通透性可以提高siRNA的转染效果。
AIM：To investigate the possibility of transfecting siRNA into rabbit cervical cells in transformation zone by the method of solid phase in vivo and to verify the effectivity of siRNA transfection by modifying the permeability of the cervical epithelium. METHODS： A sense strand small-interference RNA （siRNA） for human papillomavirus type 16 （21 bp） was designed and labeled with Cy3. siRNA-Lipo2000-carbomer gum was prepared. Twelve rabbits were included in the study and divided into experimental group and control group. In order to modify the permeability of cervical epithelium,hypertonic saline solution at concentration of 200 mmol/L was used to infuse the cervix in the experimental rabbits for 10 min,and normal saline was used for the control animals. The siRNA-Lipo2000-carbomer gum was applied to the surface of the rabbit cervix. Twenty-four hours later,the rabbits were sacrificed,and the cervix was isolated,cut into 2 parts,one part was for rapid frozen sectioning and the efficiency of transfection was observed under fluorescence microscope,another part was prepared by paraffin embedding and sectioning,and the form of cervical histiocytes was observed. Twelve SCID mice with SiHa cell cervical tumor,divided into experimental group and control group,were also used in the study. The mice in experimental group were treated with siRNA-Lipo2000-carbomer gum for 7 d. The control mice were treated with Lipo2000-carbomer gum only. Five days later,the mice were sacrificed and the tumor was collected,and the HPV16-DNA was measured by PCR. RESULTS： （1） Red fluorescence （Cy3） in cervical epithelium was observed in all rabbits. However,no different effect of siRNA transfection was found between the ways of modifying the cervical epithelium permeability. （2） No abnormal change such as flare,swelling and ulcer at all cervical tissue was observed,the cervical cell form was normal. （3） The titer of HPV16-DNA was decreased significantly after siRNA transfection （P〈0.05）. CONCLUSION： Transfection of siRNA into rabbit cervical epithelium in vivo is successful by using the method of solid phase and inhibits the processes of HPV-DNA,indicating that using RNAi is a practical way to treat HPV infection in human cercix and to decrease the incidence of cercical carcinoma.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2010年第4期695-699,共5页
Chinese Journal of Pathophysiology
基金
广东省医学科研基金资助项目(No.A2008142)
