阳离子脂质肽用于单纯型大疱性表皮松解症基因治疗的初步评价  

Preliminary evaluation of cationic lipopeptide for gene therapy of epidermolysis bullosa simplex

作　　者：胡雪依 路姣[1] 杨焕[1] 郝丹 金蓉蓉 聂宇[1] HU Xueyi;LU Jiao;YANG Huan;HAO Dan;JIN Rongrong;NIE Yu(National Engineering Research Center for Biomaterials,College of Biomedical Engineering,Sichuan University,Chengdu,Sichuan,610064 P.R.China)

机构地区：[1]四川大学生物医学工程学院、国家生物医学材料工程技术研究中心,四川成都610064

出　　处：《华西药学杂志》2023年第2期142-146,共5页West China Journal of Pharmaceutical Sciences

基　　金：国家自然科学基金资助项目(批准号:81873921);四川省科技厅项目(2020YJ0057)。

摘　　要：目的合成两亲性阳离子脂质肽(RLS),通过报告绿色荧光蛋白基因(pEGFP)及治疗基因野生型角蛋白14(KRT14)的表达水平检测,考察其在小鼠成纤维细胞L929、小鼠胚胎成纤维细胞NIH-3T3和人永生化角质形成细胞HACAT中的基因递送效率。方法利用1HNMR和基质辅助激光解吸电离飞行时间质谱表征RLS的化学结构。采用溶剂注入法制备RLS自组装体,检测其与基因复合前后的粒径、电位。将RLS自组装体与pEGFP静电吸附形成的基因复合物转染至L929、NIH-3T3、HACAT,利用EGFP表达平均荧光强度(MFI)半定量法评估复合物的转染效率,并在转染48 h后,采用CCK-8试剂法考察复合物对以上3种细胞的毒性。实时荧光定量PCR(q-PCR)检测KRT14 mRNA的表达水平。结果成功制备了粒径为142.8±0.9 nm,Zeta电位为28.7±0.4 mV的RLS自组装体。将其与DNA复合后,粒径增至167.1±2.3 nm,Zeta电位降至20.2±0.5 mV。RLS基因复合物在3种细胞中均未显示明显毒性。在转染L929、NIH-3T3和HACAT细胞后,RLS组的pEGFP的平均荧光强度分别为96、46、33。q-PCR结果显示:在L929、NIH-3T3、HACAT细胞中,RLS组KRT 14 mRNA的表达水平分别约是Lipo2000的130、100和5倍,是PEI的1.7×10^(5)、120、5倍,证明了阳离子脂质肽具有高的基因递送效率。结论RLS作为一种高效、安全的基因递送载体。在由KRT14基因突变引起的单纯型大疱性表皮松解症遗传性皮肤疾病中,具有一定的应用潜力。OBJECTIVE Amphiphilic cationic lipopeptide(RLS)were synthesized and the efficiency of gene delivery in mouse fibroblasts(L929),mouse embryonic fibroblasts(NIH-3T3)and human immortalized keratinocytes(HACAT)was examined by determining the expression levels of reporter gene(Green fluorescent protein gene,pEGFP)and therapeutic gene(Wild-type keratin 14,KRT 14).METHODS The chemical structure of the synthesized RLS was confirmed by proton nuclear magnetic resonance(1HNMR)and matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS).The self-assembly of RLS was prepared by solvent injection method,and its particle size and potential were characterized before and after complexation with DNA by Malvern laser particle sizer.Gene complexes formed by electrostatic adsorption of RLS self-assembly to pEGFP were transfected into L929,NIH-3T3,and HACAT.pEGFP transfection efficiency was assessed using mean fluorescence intensity(MFI)semi-quantitative method.The cytotoxicity of the RLS gene complexes was investigated by CCK-8 reagent after 48 h transfection.Real-time fluorescence quantitative PCR(q-PCR)was used to detect KRT 14 mRNA expression levels.RESULTS The RLS self-assembly was successfully prepared with a size of 142.8±0.9 nm and a Zeta potential of 28.7±0.4 mV.After complexing with DNA,the size was 167.1±2.3 nm and the Zeta potential was 20.2±0.5 mV.The RLS gene complexes had low cytotoxicity.After transfection in L929,NIH-3T3 and HACAT cells,the mean fluorescence intensities of pEGFP in the RLS group were 96,46 and 33,respectively.Q-PCR results showed that in L929,NIH-3T3 and HACAT cells,the expression levels of KRT 14 mRNA in the RLS group were about 130,100 and 5 times of Lipo2000,while 1.7×10^(5),120 and 5 times of PEI,respectively.It is proved that cationic lipopeptide have high gene delivery efficiency.CONCLUSION RLS show favorable gene delivery efficiency in vitro,and exhibit potential in the hereditary skin disease epidermolysis bullosa simplex with KRT 14 gene mutation.

关 键 词：阳离子脂质肽 两亲性 基因转染 基因递送 绿色荧光蛋白基因 野生型角蛋白14 单纯型大疱性表皮松解症 遗传性皮肤疾病 

分 类 号：R915[医药卫生—微生物与生化药学]