慢病毒载体介导TLR2基因干扰大鼠角膜上皮细胞和基质细胞的实验研究  

Investigation of gene interference of TLR2 mediated by lentivirus vector in corneal epithelial and stromal cells of rats

作　　者：梁伟怡[1] 白浪[1] 刘晶[1] 苏亚茹 于健[1] 刘琼[1] 杨娟[1] 

机构地区：[1]南方医科大学南方医院眼科,广东省广州市510515

出　　处：《眼科新进展》2015年第12期1101-1104,共4页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助(编号:81170887);南方医科大学南方医院横向课题匹配基金资助(编号:G201202)~~

摘　　要：目的观察慢病毒载体(lentiviral vector,LV)介导TLR2基因干扰大鼠角膜上皮细胞(corneal epithelial cell,CEC)和基质细胞(corneal stromal cell,CSC)的有效性和安全性。方法分别培养大鼠CEC和CSC,转染组用携带绿色荧光蛋白(enhanced green fluorescent protein,eGFP)和TLR2小干扰RNA(small interference RNA,siRNA)的LV转染,空白对照组加入空白培养液,阴性对照组加入不携带目的基因的LV。转染组按最佳感染复数(multiplicity of infection,MOI)分别为10、50、100、200加入LV-TLR2-siRNA-eGFP,选择eGFP表达最强的MOI进行后续实验,观察细胞形态,CCK8检测细胞增殖情况。流式细胞仪检测两种角膜细胞的转染效率,RT-PCR检测转染后TLR2 mRNA的表达情况。结果 MOI=200时转染CEC和CSC荧光表达最高,和空白对照组相比细胞形态未发生明显改变;CCK8结果显示转染组与空白对照组、阴性对照组的IOD值差异均无统计学意义(均为P>0.05);流式细胞仪检测CEC和CSC转染效率分别为77.600% ±1.100%和76.300% ±1.387%,和阴性对照组相比差异均有显著统计学意义(均为P<0.001)。RT-PCR检测转染后CEC和CSC TLR2 mRNA相对表达量均较对照组明显下降,差异均有显著统计学意义(均为P<0.001)。结论 LV-TLR2-siRNA-eGFP可在体外稳定有效转染CEC和CSC,且对细胞安全性无影响,可有效下调TLR2 mRNA的表达。Objective To investigate the effectiveness and safety of TLR2 gene interference mediated by lentiviral vector（LV） in corneal epithelial cell（CEC） and corneal stromal cell（CSC） of rats.Methods CEC and CSC were cultured and divided into 3 groups,TLR2-siRNA transfected group was exposed to LV mediated TLR2 small interference RNA（siRNA） carrying enhanced green fluorescent protein（LV-TLR2-siRNA-eGFP）,blank control group was cultured in the same quality of blank culture medium and the negative control group was exposed to LV without carrying objective gene fragments.The eGFP expression was observed under the fluorescent microscope after transfecting the cells at different multiplicity of infection（MOI：10,50,100,200）,and the M O I with the strongest expression was chosen to carry out the subsequent experiment.The morphology and proliferation of cells were observed and checked by C C K8,and the transfection efficiency of the transfected cells were evaluated by flow cytometery.The expression of TLR2 mRNA in the transfected cells were measured by RT-PCR.Results When transfected at MOI =200,the transfected CEC and CSC showed the strongest expression of eGFP.The morphology of transfected cells showed no difference compared to the blank control group.CCK8 results showed that there was no significant difference in IOD of transfected group compared with other two groups（all P〉0.05）.Transfection efficiency of CEC and CSC reached 77.6%±1.1% and 76.3%± 1.387%,and were significantly higher than that in the control group（all P〈0.001）.TLR2 mRNA expression of transfected CEC and CSC were significantly less than that in the control groups（all P〈0.001）.Conclusion LV-TLR2-siRNA-eGFP can be efficient and safe to transfect CEC and CSC of rats,and down-regulate the expression of TLR2 mRNA.

关 键 词：慢病毒载体 基因转染 角膜细胞 TLR2 

分 类 号：R772.2[医药卫生—眼科]