基于双荧光系统的HepG2肝细胞癌原位移植裸鼠模型建立及活体荧光成像动态定量分析
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摘要
目的:建立一种可以实时、定量、动态监测的肝细胞癌原位移植模型,并利用活体荧光成像系统对裸鼠体内原位肝细胞癌生长进行分析。方法:利用慢病毒包装系统包装pCDH-GFP-Luc质粒,将绿色荧光蛋白(GFP)和萤光素酶(Luc)基因通过病毒感染的方式整合到HepG2肝癌细胞染色体中,利用流式细胞术分选GFP+细胞,扩增培养后,将该细胞注射到裸鼠皮下进行成瘤,成瘤后分离肿瘤组织接种裸鼠肝脏,将造模成功的裸鼠分为对照组和治疗组,分别灌胃给与0.5%羧甲基纤维素钠(CMC-Na)和50 mg/kg索拉非尼,2/d,连续28 d,每7 d利用活体荧光成像系统观察肝癌细胞在对照组和治疗组裸鼠肝脏内的生长情况。实验结束后,分离裸鼠肝脏肿瘤,拍照称重。结果:建立了稳定表达双荧光的人肝癌细胞系HepG2-GFP-Luc,体外发光强度与表达萤光素酶的细胞数量呈正相关(R2=0.9945);建立了肝细胞癌原位移植活体荧光成像模型,对照组和治疗组肝脏内肿瘤细胞荧光强度随时间的延长逐渐增加,治疗组荧光强度明显低于对照组。定量分析结果显示,在第24、31和38 d,治疗组荧光总光子数值显著低于对照组;治疗组平均瘤重显著低于对照组。结论:建立了一种肝细胞癌原位移植荧光成像模型,可通过活体成像系统对肿瘤大小进行动态定量分析,为抗肝癌药物的药效学评价提供了实时定量分析动物模型。
Objective:To establish an orthotopic transplantation model of hepatocellular carcinoma that can be monitored in real time, quantitatively and dynamically, and analyze the growth of orthotopic hepatocellular carcinoma in nude mice by in vivo bioluminescent imaging system.Methods:The pCDH-GFP-Luc plasmid was packaged using lentiviral packaging system, then the green fluorescent protein(GFP) and luciferase(Luc) genes were integrated into the chromosome of HepG2 by viral infection. GFP+cells were sorted by flow cytometry. The cells were injected subcutaneously into nude mice for tumor forming, and tumor tissue was transplanted into liver. After successful modeling, the nude mice were divided into control group and treatment group. 0.5% sodium carboxymethyl cellulose(CMC-Na) or 50 mg/kg sorafenib were intragastrical administration twice a day for 28 days. Hepatocellular carcinoma cells of the control group and the treatment group in liver was observed by in vivo bioluminescent imaging system every 7 days. At the end of the experiment, the liver tumors of nude mice were isolated, photographed and weighed.Results:The human hepatocellular carcinoma cell line HepG2-GFP-Luc expressing dual fluorescence was obtained. In vitro luminescence intensity was positively correlated with the number of cells(R2=0.9945). The bioluminescence intensity of tumor cells in liver of the control group and the treatment group increased with time, while the bioluminescence intensity of the treatment group was significantly lower than that of the control group. Quantitative analysis showed that the total photon flux of the treatment group was significantly lower than that of the control group at day 24, 31 and 38. The average tumor weight of the treatment group was significantly lower than that of the control group.Conclusion:This study established an orthotopic transplantation model of hepatocellular carcinoma, which can be used to quantitatively analyze the tumor growth by in vivo bioluminescent imaging system. It provides a real-time quantitative analysis animal model for the pharmacodynamic evaluation of hepatocarcinoma therapy drugs.
Objective:To establish an orthotopic transplantation model of hepatocellular carcinoma that can be monitored in real time, quantitatively and dynamically, and analyze the growth of orthotopic hepatocellular carcinoma in nude mice by in vivo bioluminescent imaging system.Methods:The pCDH-GFP-Luc plasmid was packaged using lentiviral packaging system, then the green fluorescent protein(GFP) and luciferase(Luc) genes were integrated into the chromosome of HepG2 by viral infection. GFP+cells were sorted by flow cytometry. The cells were injected subcutaneously into nude mice for tumor forming, and tumor tissue was transplanted into liver. After successful modeling, the nude mice were divided into control group and treatment group. 0.5% sodium carboxymethyl cellulose(CMC-Na) or 50 mg/kg sorafenib were intragastrical administration twice a day for 28 days. Hepatocellular carcinoma cells of the control group and the treatment group in liver was observed by in vivo bioluminescent imaging system every 7 days. At the end of the experiment, the liver tumors of nude mice were isolated, photographed and weighed.Results:The human hepatocellular carcinoma cell line HepG2-GFP-Luc expressing dual fluorescence was obtained. In vitro luminescence intensity was positively correlated with the number of cells(R2=0.9945). The bioluminescence intensity of tumor cells in liver of the control group and the treatment group increased with time, while the bioluminescence intensity of the treatment group was significantly lower than that of the control group. Quantitative analysis showed that the total photon flux of the treatment group was significantly lower than that of the control group at day 24, 31 and 38. The average tumor weight of the treatment group was significantly lower than that of the control group.Conclusion:This study established an orthotopic transplantation model of hepatocellular carcinoma, which can be used to quantitatively analyze the tumor growth by in vivo bioluminescent imaging system. It provides a real-time quantitative analysis animal model for the pharmacodynamic evaluation of hepatocarcinoma therapy drugs.
引文
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[7]Mezzanotte L,van′t Root M,Karatas H,et al.In vivo molecular bioluminescence imaging:new tools and applications[J].Trends Biotechnol,2017,35(7):640-652.
[8]Thompson E M,Adenot P,Tsuji F I,et al.Real time imaging of transcriptional activity in live mouse preimplantation embryos using a secreted luciferase[J].Proc Natl Acad Sci USA,1995,92:1317-1321.
[9]Contag C H,Contag P R,Mullins J I,et al.Photonic detection of bacterial pathogens in living hosts[J].Mol Microbiol,1995,18(4):593-603.
[10]Contag C H,Spilman S D,Contag P R,et al.Visualizing gene expression in living mammals using a bioluminescent reporter[J].Photochem Photobiol,2008,66(4):523-531.274
[11]Zinn K R,Chaudhuri T R,Szafran A A,et al.Noninvasive bioluminescence imaging in small animals[J].ILAR J,2008,49(1):103-115.
[12]Gross S,Abraham U,Prior J L,et al.Continuous delivery of D-luciferin by implanted micro-osmotic pumps enables true real-time bioluminescence imaging of luciferase activity in vivo[J].Mol Imaging,2007,[[6(2):121-130.
[13]Klerk C P,Overmeer R M,Niers T M,et al.Validity of bioluminescence measurements for noninvasive in vivo imaging of tumor load in small animals[J].Biotechniques,2007,43(1 Suppl):7-13.
[14]Singer O,Verma I M.Applications of lentiviral vectors for shRNA delivery and transgenesis[J].Curr Gene Ther,2008,8(6):483-488.
[15]Cockrell A S,Kafri T.Gene delivery by lentivirus vectors[J].Mol Biotechnol,2007,36(3):184-204.
[2]Kew M C.Epidemiology of chronic hepatitis B virus infection,hepatocellular carcinoma,and hepatitis B virus-induced hepatocellular carcinoma[J].Pathol Biol,2010,58(4):273-277.
[3]Choi K J,Baik I H,Ye S K,et al.Molecular targeted therapy for hepatocellular carcinoma:present status and future directions[J].Biol Pharm Bull,2015,38(7):986-991.
[4]Belghiti J,Kianmanesh R.Surgical treatment of hepatocellular carcinoma[J].HPB(Oxford),2005,7(1):42-49.
[5]Shang N,Arteaga M,Zaidi A,et al.Fak is required for c-Met/beta-catenin-driven hepatocarcinogenesis[J].Hepatology,2015,61(1):214-226.
[6]Llovet J M,Ricci S,Mazzaferro V,et al.Sorafenib in advanced hepatocellular carcinoma[J].N Engl J Med,2008,359(4):378-390.
[7]Mezzanotte L,van′t Root M,Karatas H,et al.In vivo molecular bioluminescence imaging:new tools and applications[J].Trends Biotechnol,2017,35(7):640-652.
[8]Thompson E M,Adenot P,Tsuji F I,et al.Real time imaging of transcriptional activity in live mouse preimplantation embryos using a secreted luciferase[J].Proc Natl Acad Sci USA,1995,92:1317-1321.
[9]Contag C H,Contag P R,Mullins J I,et al.Photonic detection of bacterial pathogens in living hosts[J].Mol Microbiol,1995,18(4):593-603.
[10]Contag C H,Spilman S D,Contag P R,et al.Visualizing gene expression in living mammals using a bioluminescent reporter[J].Photochem Photobiol,2008,66(4):523-531.274
[11]Zinn K R,Chaudhuri T R,Szafran A A,et al.Noninvasive bioluminescence imaging in small animals[J].ILAR J,2008,49(1):103-115.
[12]Gross S,Abraham U,Prior J L,et al.Continuous delivery of D-luciferin by implanted micro-osmotic pumps enables true real-time bioluminescence imaging of luciferase activity in vivo[J].Mol Imaging,2007,[[6(2):121-130.
[13]Klerk C P,Overmeer R M,Niers T M,et al.Validity of bioluminescence measurements for noninvasive in vivo imaging of tumor load in small animals[J].Biotechniques,2007,43(1 Suppl):7-13.
[14]Singer O,Verma I M.Applications of lentiviral vectors for shRNA delivery and transgenesis[J].Curr Gene Ther,2008,8(6):483-488.
[15]Cockrell A S,Kafri T.Gene delivery by lentivirus vectors[J].Mol Biotechnol,2007,36(3):184-204.