切换至 "中华医学电子期刊资源库"
高级检索
中华临床医师杂志(电子版)

中华临床医师杂志(电子版) ›› 2017, Vol. 11 ›› Issue (19) : 2273 -2279. doi: 10.3877/cma.j.issn.1674-0785.2017.19.005

所属专题： 文献；

基础论著

构建靶向性高尔基体膜蛋白高尔基体蛋白73修饰脂质体介导单纯疱疹病毒胸苷激酶诱导肝癌细胞凋亡
孟凡秀1, 张琪1, 姚志坚1, 袁洋洋1, 曾思衡1, 刘畅1, 李勇芳1, 张英敏1, 赵娜1, 陆嘉慧2, 张智宣2, 李高凯3, 于保锋1,†(), 郭睿1, 王海龙1, 解军1, 李高鹏4, 徐钧4,†()   
  1. 1. 030001　太原，山西医科大学基础医学院生物化学与分子生物学教研室
    2. 030001　太原，山西医科大学口腔医学院
    3. 030001　太原，山西医科大学公共卫生学院
    4. 030013　太原，山西医科大学附属山西省肿瘤医院普外科
  • 收稿日期:2017-03-14 出版日期:2017-10-01
  • 通信作者: 于保锋, 徐钧
  • 基金资助:
    国家自然科学基金(30901821，81172136); 山西省自然科学基金(2015011113)

Construction of a Golgi protein 73 modified liposome that mediates HSVtk induced liver cancer cell apoptosis

Fanxiu Meng1, Qi Zhang1, Zhijian Yao1, Yangyang Yuang1, Siheng Zeng1, Chang Liu1, Yongfang Li1, Yingmin Zhang1, Na Zhao1, Jiahui Lu2, Zhixuan Zhang2, Gaokai Li3, Baofeng Yu1,†(), Rui Guo1, Hailong Wang1, Jun Xie1, Gaopeng Li4, Jun Xu4,†()   

  1. 1. Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan 030001, China
    2. College of Dental Medicine, Shanxi Medical University, Taiyuan 030001, China
    3. School of Public Health, Shanxi Medical University, Taiyuan 030001, China
    4. Department of General Surgery, Affiliated Tumor Hospital of Shanxi Medical University, Taiyuan 030013, China
  • Received:2017-03-14 Published:2017-10-01
  • Corresponding author: Baofeng Yu, Jun Xu
  • About author:
    Corresponding authors: Yu Baofeng, Email:
    Xu Jun, Email:
全文 ( ) 下载PDF ( ) 导出引用
引用本文：

孟凡秀, 张琪, 姚志坚, 袁洋洋, 曾思衡, 刘畅, 李勇芳, 张英敏, 赵娜, 陆嘉慧, 张智宣, 李高凯, 于保锋, 郭睿, 王海龙, 解军, 李高鹏, 徐钧. 构建靶向性高尔基体膜蛋白高尔基体蛋白73修饰脂质体介导单纯疱疹病毒胸苷激酶诱导肝癌细胞凋亡[J]. 中华临床医师杂志(电子版), 2017, 11(19): 2273-2279.

Fanxiu Meng, Qi Zhang, Zhijian Yao, Yangyang Yuang, Siheng Zeng, Chang Liu, Yongfang Li, Yingmin Zhang, Na Zhao, Jiahui Lu, Zhixuan Zhang, Gaokai Li, Baofeng Yu, Rui Guo, Hailong Wang, Jun Xie, Gaopeng Li, Jun Xu. Construction of a Golgi protein 73 modified liposome that mediates HSVtk induced liver cancer cell apoptosis[J]. Chinese Journal of Clinicians(Electronic Edition), 2017, 11(19): 2273-2279.

目的

构建一种具有靶向性的非病毒基因治疗载体高尔基体蛋白73（GP73）-脂质体lipoplexes，并检测其对肝癌细胞HepG2和正常肝细胞HL-7702的转染效率和凋亡的影响。

方法

以水溶性碳二亚胺为交联剂将GP73与lipoplexes偶联。用GP73-lipoplexes运载标记有绿色荧光蛋白（green fluorescent protein，GFP）荧光报告基因的PGenesil-l质粒转染肝癌细胞组HepG2和肝正常细胞组HL-7702，通过荧光显微镜观察被转染细胞GFP的表达情况，流式细胞术检测转染效率。并用GP73-lipoplexes运载含治疗基因HSVtk的质粒PBI-SUR-TK转染肝癌细胞和肝细胞，经RT-PCR和Western blot检测细胞中HSVtk表达情况，流式细胞术和CCK-8检测细胞凋亡情况。

结果

经GP73修饰的lipoplexes转染的肝癌细胞组[（26.37±0.76）%]比肝细胞组[（9.68±0.57）%]有更高的转染效率和更多的HSVtk基因的表达，肝癌细胞增殖能力明显减弱，凋亡显著。

结论

GP73修饰的lipoplexes介导的HSVtk自杀基因系统对肝癌细胞有治疗作用，可以尝试作为一种靶向性杀伤肝癌细胞的非病毒基因转运载体。

关键词: GP73, 阳离子脂质体, 肝癌细胞, 转染效率, 细胞凋亡
Objective

To construct a non-viral gene therapy vector (GP73 modified lipoplexes) and to assess its delivery efficiency and impact on apoptosis in hepatoma cell line HepG2 and normal liver cell line HL-7702.

Methods

Lipoplexes were conjugated with human GP73 using water-soluble carbodidimide, a crossing agent. Then, GP73-lipoplexes were used to carry pGenesil-l plasmid containing EGFP gene and transfect hepatoma cell line HepG2 and normal liver cell line HL-7702. Fluorescence microscopy and flow cytometry were used to assess the transfection efficiency. GP73-lipoplexes were then used to carry PBI-SUR-TK plasmid containing the therapeutic gene HSVtk and transfect hepatoma cells and normal hepatocytes. RT-PCR and Western blot were used to detect the expression of HSVtk in cells, and flow cytometry and CCK-8 assay were used to detect apoptosis.

Results

Compared with HL-7702 cells, HepG2 cells had higher transfection efficiency [(9.68±0.57)% vs (26.37±0.76)%], obvious HSVtk gene production, and significant apoptosis.

Conclusion

GP73 modified lipoplexes can be an effective tool to improve the transfection efficiency and therapeutic effects of exogenous genes in hepatoma cells.

Key words: GP73, Lipoplexes, Hepatoma cell, Transfection efficiency, Apoptosis
图1 流式检测细胞转染效率　A：HepG2转染组：B：HepG2未转染组；C：HL-7702转染组；D：HL-7702未转染组
图2 琼脂糖凝胶电泳检测TK基因表达　泳道5：pBI-cMV2-AcGFP1-pSurvivin-TK转染的HepG2细胞的目的基因，泳道6：未转染的HepG2细胞的目的基因；泳道7：转染的HL-7702细胞的目的基因；泳道8：未转染的HL-7702细胞的目的基因；泳道1、2、3、4：分别为转染HepG2细胞、未转染HepG2细胞、转染HL-7702细胞、未转染HL-7702细胞的actin
图3 蛋白水平检测TK表达　泳道1：pBI-cMV2-AcGFP1-pSurvivin-TK转染HepG2细胞；泳道2：未转染HepG2细胞；泳道3：pBI-cMV2-AcGFP1-pSurvivin-TK转染HL-7702细胞；泳道4：未转染HL-7702细胞；所有actin均在206 bp有条带
图4 96孔板加药后结果图　A：未转染的HepG2细胞加入0 μg/ml GCV；B：未转染的HepG2细胞加入80 μg/ml GCV；C：转染的HepG2细胞加入0 μg/ml GCV；D：转染的HepG2细胞加入80 μg/ml GCV；E：未转染的HL-7702细胞加入0 μg/ml GCV；F：未转染的HL-7702细胞加入80 μg/ml GCV；G：转染的HL-7702细胞加入0 μg/ml GCV；H：转染的HL-7702细胞加入80 μg/ml GCV；GCV为丙氧鸟苷
图5 流式检测细胞凋亡　5A：转染的HepG2的流式图；5B：未转染的HepG2的流式图；5C：转染的HL-7702细胞流式图：5D：未转染的HL-7702细胞流式图
图6 CCK-8在450 nm处检测不同浓度GCV作用后细胞的吸光度值　6A是在不同浓度GCV作用下的转染与未转染的HepG2细胞的吸光度值；6B是在不同浓度GCV作用下的HL-7702细胞的吸光度值；GCV为丙氧鸟苷
[1]
Bruix J, Sherman M. Management of hepatocellular carcinoma: an update [J]. Hepatology, 2011, 53: 1020-1022.
[2]
Cheng JW, Lv Y. New progress of non-surgical treatments for hepatocellularcarcinoma [J]. Med Oncol, 2013, 30(1): 381-389.
[3]
Zhong ZR, Zhang ZR, Deng Y, et al. Preparation of a TK/GCV admipistration system mediated by transefrrin modified proationic liposomes [J]. Pharmazie, 2007, 62(7): 522-527.
[4]
Kladney RD, Bulla GA, Guo L, et al. GP73, a novel Golgi-localized protein upregulated by viral infection [J]. Gene, 2000, 249(1-2): 53-65.
[5]
Zhang Y, Xi Y, Fang J, et al. Identification and characterization of monoclonal antibodies against GP73 for use as a potential biomarker in liver cancer screening and diagnosis [J]. Immunoassay Immunochem, 2016, 37(4): 390-406.
[6]
Zhao Y, Wang M, Cui C, et al. Significance of combined tests of serum golgi glycoprotein 73 and other biomarkers in diagnosis of small primary hepatocellular carcinoma [J]. Cancer Biomark, 2015, 15(5): 677-683.
[7]
Mao Y, Yang H, Xu H, et al. Golgi protein 73(GOLPH2) is a valuable serum marker for hepatocellular carcinoma [J]. Gut, 2010, 59(12): 1687-1693.
[8]
Zhou Y, Yin X, Ying J, et al. Golgi protein 73 versus alpha-feto protein as a biomarker for hepatocellular carcinoma: adiagnosticmeta-analysis [J]. BMC Cancer, 2012, 12(1): 17.
[9]
Ardito G, RevellL, D′Alatri L, et al. Revisited anatomy ofThe recurrent laryngeal nerves [J]. Am J Surg, 2004, 187(2): 249-253.
[10]
秦琴，莱智勇，高然朋, 等. 载脂蛋白Apo E修饰脂质体对肝癌细胞的靶向转染 [J]. 中华实验外科杂志, 2014, 31(8): 1709-1711.
[11]
Lai Z, Qin Q, Yu B, et al. Construction of plasmid vector p AFP-HSVtk-IRES2- EGFP and its effect on the cytotoxicity of ganciclovirto hepatocellular carcinoma [J]. Chin Med J (Engl), 2014, 127(12): 2337-2341.
[12]
Pardridge WM. Drug and gene targeting to the brain with molecular trojan horses [J]. Nat Rev Drug Discov, 2002, 1(2): 131-139.
[13]
Pathak PO, Nagarsenker MS, Barhate CR, et al. Cholesterol anchored arabinogalactan for asialoglycoprotein receptor targe-ting: synthesis, characterization, and proof of concept of hepato-specific delivery [J]. Carbohyd Res, 2015, 408: 33-43.
[14]
Daniels TR., Delgado T, Helguera G, et al. The transferrin receptor part Ⅱ: targeted delivery of therapeutic agentsinto cancer cells [J]. Clin Immunol, 2006, 121(2): 159-176.
[15]
Feng D, Song Y, Shi W, et al. Distinguishing folate-receptor-positive cells from folate-receptor-negative cells using a fluorescence off-on nanoprobe [J]. Anal chem, 2013, 85(13): 6530-6535.
[16]
Kim IY, Kang YS, Lee DS, et al. Antitumor activity of EGFR targeted pH - sensitive immunoliposomes encapsulating gemcitabine in A549 xenograft nude mice [J]. J Control Release, 2009, 140(1): 55-60.
[17]
Wei H, Li B, Zhang R, et al. Serum GP73, a marker for evaluating progression in patients with chronic HBV infections [J]. PLoS One. 2013, 8: e53862.
[18]
Hu JS, Wu DW, Liang S, et al. GP73, a resident Golgi glycoprotein, is sensibility and specificity for hepatocellular carcinoma of diagnosis in a hepatitis B- endemic Asian population [J]. Med Oncol, 2010, 27(2): 339-345.
[19]
Bachert C, Fimmel C, Linstedt AD. Endosomal trafficking and proprotein convertase cleavage of cis Golgi protein GP73 produces marker for hepatocellular carcinoma [J]. Traffic, 2007, 8(10): 1415-1423.
[20]
Gu Y, Chen W, Zhao Y, et al. Quantitative analysis of elevated serum Golgi protein-73 expression in patients with liver diseases [J]. Ann Clin Biochem, 2009, 46(1): 38-43.
[21]
Riener MO, Stenner F, Liewen H, et al. Golgi phosphoprotein 2 (GOLPH2) expression in liver tumors and its value as a serum marker in hepatocellular carcinomas [J]. Hepatology, 2009, 49(5): 1602-1609.
[22]
谢红彬，宫钰，彭涛. 高尔基体驻膜糖蛋白GP73启动子克隆 [J]. 安徽农业科学, 2008, 36(24): 10359-10361, 10401.
[23]
Kladney RD, Tollefson AE, Wold WS, et al. Upregulation of the Golgi protein GP73 by adenovirus infection requires the E1A Ct BP interaction domain [J].Virology, 2002, 301(2): 236-246.
[24]
Liu Y, Zhang X, Sun T, et al. Knockdown of Golgi phosphoprotein 2 inhibits hepatocellular carcinoma cell proliferation and motility [J]. Oncotarget, 2016, 16 (7): 21404-21415.
[25]
Zhang F, Gu Y, Li X, et al. Up-regulated Golgi phosphoprotein 2(GOLPH2) expression in lung adenocarcinoma tissue [J]. Clin. Biochem, 2010, 43 (12): 983-991.
[26]
Qu L, Wang Y, Gong L, et al. Suicide gene therapy for hepatocellular carcinoma cells by surviving promoter-driven expression of the herpes simplex virus thymidine kinase gene [J]. Oncol Rep, 2013, 29(4): 1435-1440.
[1] 孔莹莹, 谢璐涛, 卢晓驰, 徐杰丰, 周光居, 张茂. 丁酸钠对猪心脏骤停复苏后心脑损伤的保护作用及机制研究[J]. 中华危重症医学杂志(电子版), 2023, 16(05): 355-362.
[2] 张晓燕, 肖东琼, 高沪, 陈琳, 唐发娟, 李熙鸿. 转录因子12过表达对脓毒症相关性脑病大鼠大脑皮质的保护作用及其机制[J]. 中华妇幼临床医学杂志(电子版), 2023, 19(05): 540-549.
[3] 周伟, 蔡恒, 范海迪, 李惠中, 王传霞, 顾茂胜. cblC型甲基丙二酸血症MMACHC基因新突变对小鼠神经细胞凋亡及Wnt/β-catenin信号通路的作用机制[J]. 中华妇幼临床医学杂志(电子版), 2022, 18(05): 528-539.
[4] 徐煜琛, 李璐, 薛冬令, 赵德伟. 外泌体介导股骨头坏死机制与治疗的研究进展[J]. 中华损伤与修复杂志(电子版), 2022, 17(03): 247-252.
[5] 刘硕儒, 王功炜, 张斌, 李书豪, 胡成. 新型溶瘤病毒M1激活内质网应激致前列腺癌细胞凋亡的机制[J]. 中华腔镜泌尿外科杂志(电子版), 2023, 17(04): 388-393.
[6] 邓春文, 陈嵩, 钟裴, 闵师强, 万健. LncRNA CRNDE通过miR-181a-5p/SOX6轴调节脂多糖诱导人肺泡上皮细胞的炎症反应和细胞凋亡[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(03): 129-136.
[7] 田鹏飞, 王丽娟, 肖圣超. 黄芪总黄酮通过调控miR-190a-5p对缺氧/复氧诱导的心肌细胞损伤的影响[J]. 中华细胞与干细胞杂志(电子版), 2022, 12(06): 346-352.
[8] 郭周威, 屈艳玲, 李永慧. 类叶升麻苷通过上调miR-204对缺氧/复氧诱导H9C2细胞凋亡的抑制作用[J]. 中华细胞与干细胞杂志(电子版), 2022, 12(03): 145-152.
[9] 李翠平, 陈晓燕, 钱师宇, 林惠珠, 曾彩辉, 阳莉, 卢建溪. 不同抗凝剂保存液对脐血培养的NK细胞增殖及杀伤效应的影响[J]. 中华肝脏外科手术学电子杂志, 2023, 12(05): 572-576.
[10] 王楚风, 蒋安. 原发性肝癌的分子诊断[J]. 中华肝脏外科手术学电子杂志, 2023, 12(05): 499-503.
[11] 张师垚, 徐岩岩, 张琦, 李春强, 赵智成, 刘刚. m6A结合蛋白YTHDC2调节p38MAPK信号通路影响结直肠癌细胞凋亡[J]. 中华结直肠疾病电子杂志, 2023, 12(02): 117-124.
[12] 姚尧, 杨新明, 杜雅坤, 朱宁, 阴彦林, 贾永利, 张瑛, 张培楠, 田野, 陈丽星. 雷公藤甲素与甲泼尼龙调节细胞自噬和凋亡促进脊髓损伤修复的比较研究[J]. 中华神经创伤外科电子杂志, 2022, 08(03): 132-140.
[13] 于迪, 于海波, 吴焕成, 李玉明, 苏彬, 陈馨. 发状分裂相关增强子1差异表达对胆固醇刺激下血管内皮细胞的影响[J]. 中华脑科疾病与康复杂志(电子版), 2023, 13(05): 264-270.
[14] 郭德华, 贺迎坤, 白卫星, 何艳艳, 李天晓. 脑动静脉畸形部分栓塞术后血管组织增殖与凋亡的变化[J]. 中华介入放射学电子杂志, 2022, 10(02): 152-157.
[15] 邱甜, 杨苗娟, 胡波, 郭毅, 何奕涛. 亚低温治疗脑梗死机制的研究进展[J]. 中华脑血管病杂志(电子版), 2023, 17(05): 518-521.
阅读次数
全文


摘要

版权所有 中华医学会 中华医学电子音像出版社有限责任公司  京ICP备14006079号-1  网络出版服务许可证：(署)网出证(京)字第075号