可活化细胞穿膜肽-聚N-（2-羟丙基）甲基丙烯酰胺-Ad.mda-7.egfp接合物的合成及跨膜运输途径检测  

Synthesis and transmembrane transport pathway of activable cell penetrating peptide-poly N-（2- hydroxypropyl）methylacrylamide-Ad. mda-7. egfp conjugate

作　　者：李书华[1] 许泽鹏 陈艺瑛[1] 陈婷婷[1] 刘谕昆 李灏[1] 马少丹[1] 张雅洁[1] 

机构地区：[1]广州医科大学基础医学院病理学教研室,510182

出　　处：《中华生物医学工程杂志》2015年第1期1-7,共7页Chinese Journal of Biomedical Engineering

基　　金：广东省医学科研基金项目（A2013244）;广州市科技计划项目（2014Y2-00171）;广州市教育系统创新学术团队项目（13C06）

摘　　要：目的：合成一种由可活化细胞穿膜肽（ACPP）、水溶性高分子聚合物聚N-（2-羟丙基）甲基丙烯酰胺（pHPMA）修饰腺病毒的接合物，并初步探索该接合物的跨膜运输途径。方法通过化学合成的方法合成ACPP。将肺腺癌细胞株A549、乳腺癌细胞株MDA-MB-231、肺支气管上皮细胞株HBE和肝癌细胞株HepG2均分为两组，分别加入培养基及终浓度为10 mg/L基质金属蛋白酶抑制剂强力霉素，12 h后加入ACPP，通过荧光显微镜观察并鉴定ACPP对各细胞的穿膜活性。通过化学修饰法合成接合物pc-Ad. mda-7.egfp及ACPP-pc-Ad.mda-7.egfp，通过荧光显微镜观察并初步鉴定接合物的合成和对A549细胞的穿膜活性。应用流氏细胞术检测接合物感染A549细胞的能力。通过荧光显微镜观察并鉴定接合物ACPP-pc-Ad. mda-7. Egfp在A549细胞内定位及穿膜活性。结果合成了ACPP 100 mg。ACPP对A549、MDA-MB-231、HepG2细胞株具有靶向性穿膜活性，而HBE细胞内未见绿色荧光，强力霉素组细胞经过强力霉素作用后与ACPP-FITC孵育，4种细胞内均未见明显绿色荧光反应，强力霉素阻断了ACPP的肿瘤选择性穿膜作用。合成了两种接合物pc-Ad.mda-7.egfp（粒径420.6 nm）及ACPP-pc-Ad.mda-7.egfp（粒径462.2 nm）。将ACPP-pc-Ad.mda-7.egfp、pc-Ad.mda-7.egfp接合物、Ad.mda-7.egfp与A549细胞共培养后，可见空白对照组（培养基）无绿色荧光蛋白表达，pc-Ad.mda-7.egfp接合物仅见微量绿色荧光蛋白表达，ACPP-pc-Ad.mda-7.egfp、Ad.mda-7.egfp组所有细胞均可见强烈绿色荧光蛋白表达。经流氏细胞仪检测，ACPP-pc-Ad.mda-7.egfp组A549细胞PI荧光值明显高于pc-Ad.mda-7. egfp组。经荧光显微镜观察证实接合物ACPP-pc-Ad.mda-7.egfp大量分布于A549细胞胞质，而pc-Ad. mda-7.egfp仅出现少量内吞性囊泡。结论成功合成了ACPP-pc-Ad.mda-7.egfp接合物，并证实ACPP-pc-Ad.mda-7.egfp以非内吞途径进行跨膜运输。Objective To synthesize an adenovirus conjugate modified by activable cell penetrating peptide（ACPP）and water-soluble polymer,poly N-（2-hydroxypropyl）methylacrylamide（pHPMA）,and to preliminarily investigate the transmembrane transport pathway of the conjugate. Methods ACPP was chemically synthesized. The lung adenocarcinoma cell line A549,breast cancer cell line MDA-MB-231, lung bronchial epithelial cell line HBE,and hepatoma cell line HepG2 were divided into two groups,added＆amp;nbsp;with medium and the 10 mg/L （final concentration） matrix metallo-proteinase inhibitor,Doxycycline, respectively. After 12 h,the both groups were added with ACPP. Then,fluorescent microscope was used to determine and identify the transmembrane activity of ACPP on all the included cells. The conjugates pc-Ad. mda-7. egfp and ACPP-pc-Ad. mda-7. egfp were synthesized by chemical modification. Fluorescent microscope was used to preliminarily determine and identify the synthesis of the conjugates,and their transmembrane activities on A549 cells. The infecting abilities of the conjugates were measured by flow cytometry. The fluorescent microscope was used to determine and identify the location and transmembrane activity of conjugate ACPP-pc-Ad. mda-7. egfp in A549 cells. Results 100 mg ACPP was synthesized. ACPP exhibited target transmembrane activity on A549,MDA-MB-231,and HepG2 cell lines,whereas no green fluorescence was found in HBE cells. The cells in the Doxycycline group were incubated with ACPP-FITC after treated with Doxycycline,but no significant green fluorescence was found in the four cell lines. The tumor-selective transmembrane effect of ACPP was blocked by Doxycycline. Then,the two conjugates, pc-Ad. mda-7. egfp（particle size 420.6 nm）and ACPP-pc-Ad. mda-7. egfp（particle size 462.2 nm）were synthesized. After ACPP-pc-Ad. mda-7. egfp,pc-Ad. mda-7. egfp conjugate and Ad. mda-7. egfp were co-cultured with A549 cells,no green fluorescent protein expression was found in the blank control group （medium�

关 键 词：可活化细胞穿膜肽 N-(2-羟丙基)甲基丙烯酰胺 腺病毒 蛋白质转运 

分 类 号：R135.14[医药卫生—劳动卫生]