EntransterTM纳米载体介导大鼠角膜CD25siRNA转染的效果及安全性评估  

The efficiency and safety assessment of EntransterTM nanoparticle carrier for CD25 siRNA transfection in rat cornea

作　　者：秦琴[1] 石韵洁 赵敏[1] 

机构地区：[1]重庆医科大学附属第一医院眼科重庆市眼库,400016

出　　处：《中华实验眼科杂志》2016年第10期888-895,共8页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目（81170822）

摘　　要：背景 基因转染是多种眼病基因治疗的有效方法,理想的非病毒载体是研究角膜基因治疗的关键因素,选择高转染率、基因高表达、低毒性的非病毒载体是成功实施基因治疗的前提. 目的 探讨EntransterTM、脂质体非病毒载体对正常SD大鼠角膜转染CD25 siRNA的转染率和安全性,筛选角膜基因转染的最佳载体.方法 应用随机数字表法将80只雄性SPF级成年SD大鼠随机分为EntransterTM-CD25 siRNA 组、脂质体-CD25 siRNA组、单纯CD25 siRNA组和生理盐水组,每组20只,均以右眼作为实验眼.实验眼眼表麻醉后刮除角膜上皮,按照分组不同分别用EntransterTM-CD25 siRNA、脂质体-CD25 siRNA、单纯CD25siRNA和生理盐水各50μl点眼.于点眼后12h、24 h、3d和7d在裂隙灯显微镜下观察大鼠眼表组织反应,检查各组大鼠角膜表面绿色荧光个数.分别于上述时间点处死各组大鼠各4只并获取角膜组织,采用苏木精-伊红染色法行角膜组织病理学检查;采用罗丹明染色行荧光检测,评估各组大鼠角膜基因转染的转染率;采用TUNEL染色法检测实验眼角膜细胞的凋亡情况以评估各种转染载体的安全性;采用免疫荧光技术检测角膜组织中CD11b的表达. 结果 EntransterTM-CD25 siRNA组大鼠角膜表面的荧光染色数量及强度明显高于脂质体-CD25 siRNA组,单纯CD25 siRNA组大鼠角膜荧光染色出现早,但转染后24 h角膜荧光染色消失.角膜组织病理学检查显示,各组大鼠行基因转染后脂质体-CD25 siRNA组大鼠角膜上皮水肿和角膜炎性细胞浸润程度较EntransterTM-CD25 siRNA组、单纯CD25 siRNA组和生理盐水组严重,角膜基质层和内皮层未发现异常,脂质体-CD25 siRNA组大鼠角膜炎性细胞数明显多于EntransterTM-CD25 siRNA组、单纯CD25 siRNA组和生理盐水组,差异均有统计学意义（均P=0.00）.TUNEL检测发现,基因转染后12h和3d,脂质体-CD25siRNA组大鼠角膜细胞凋亡数明显多于EntransterTM-CD25Background Gene transfection is an effective therapeutic avenue to target many kinds of eye diseases.Non-viral vectors with high transfection efficiency,long-term expression,low toxicity and high expression levels are pivotal in gene therapy of corneal disease.Objective This study was to evaluate and compare the safety and efficiency between EntransterTM and liposome vectors for transfer of CD25 siRNA in rat cornea.Methods Eighty male SPF SD rats were randomly divided into EntransterTM-CD25 siRNA group,liposome-CD25 siRNA group,simple CD25 siRNA group and normal saline solution （NSS） group with the right eye as experimental eyes.Corneal epithelia of the rats were completely removed after ocular surficial anesthesia,and 50 μl EntransterTM-CD25 siRNA,liposome-CD25 siRNA,CD25 siRNA solution and NSS were topical administered in the eyes respectively.Ocular response and green fluorescence number on the corneas were examined under the slit lamp assisted microscope 12 hours,24 hours,3 days and 7 days after use of the drugs.The rats were sacrificed and the corneas were obtained,and corneal histopathological examination was performed by using hematoxylin eosin stain.The gene transferred efficiency in the corneas was evaluated by fluorescence technology,and the safety of EntransterTM and liposome carriers was assessed using TUNEL stain.The expression and location of CD11b in the corneas were detected by immunofluorescence technology.The use and care of the experimental animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Committee.Results The quantity and intensity of fluorescence staining in the corneas were significantly increased in the EntransterTMCD25 siRNA group in comparison with the liposome-CD25 siRNA group,and the corneal fluorescence appeared earlier in the simple CD25 siRNA group,but it disappeared in 24 hours after transfection.Corneal histopathological examination revealed that the corneal edema and inflammatory cell infiltrati

关 键 词：基因转染 小干扰RNA 角膜 基因治疗/方法 凋亡/药物作用 纳米聚合物 脂质体 大鼠 

分 类 号：R77[医药卫生—眼科] R450[医药卫生—临床医学]