PBCA纳米粒载体介导的肝癌HSV-TK/GCV基因治疗的体外实验研究
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摘要
近年来，随着基因转移技术的日趋成熟，肝癌的基因治疗成为生物科学和临床医学的研究热点之一，其中作用直接、合理、有效的自杀基因疗法备受关注。目前研究最多、进展最快、作用最为确切的肝癌自杀基因治疗系统是单纯疱疹病毒胸苷激酶/丙氧鸟苷(HSV-TK/GCV)系统。
HSV-TK单磷酸化GCV的能力较哺乳动物细胞内的TK强1000倍以上，然后在哺乳动物细胞内鸟苷酸激酶作用下将单磷酸化的GCV(GCV-MP)代谢为双磷酸化(GCV-DP)的形式，并进一步在细胞内酶的作用下转化为三磷酸化(GCV-TP)的产物。GCV-TP对细胞具有剧毒性，可抑制DNA聚合酶的活性，并可掺入到DNA链的末端作为终止剂。
然而，肝癌的发生多来源于慢性肝炎及肝硬化，其预后主要取决于被保留的有功能的肝组织的多少，转基因中潜在的毒性效应会对预后造成不良影响。因此，肝癌自杀基因治疗的靶向性调控，即调控自杀基因特异性导入肝癌细胞或在肝癌细胞中实现特异性表达，最大限度地杀伤肝癌细胞的同时并不损伤正常肝细胞，已经成为决定其疗效及可行性的关键。
研究表明，肝癌细胞特异性合成甲胎蛋白(AFP)，同时特异性合成的转录调节蛋白(TRP)通过与AFP基因的转录调控元件(TRS)结合促进AFP的表达。因此将AFP基因的TRS与自杀基因结合，使
其调控自杀基因的表达，可实现对肝癌组织的靶向性，且自杀基因的
表达量与肝癌细胞中AFP的表达量呈正相关。
研究证实，AFP增强子/启动子可调控其后续基因在AFP阳性肿
瘤细胞中特异性表达。此外，单独应用AFP增强子，不仅可增强其
调控基因的表达，而且可诱导非特异性启动子调控的后续基因仅表达
于/妞P阳性肝癌细胞，从而实现肿瘤细胞特异性。然而，AFP启动
子单独应用时其调控转录的活性较弱。
根据以上研究进展，我们将AFP基因增强子的核心序列与CMV
启动子调控的HSV-TK基因联合应用于AFP阳性肝癌的治疗中，探
讨提高HSV-TE了GCV系统靶向性的可行性。由于70%的肝癌细胞中
有ZJP的表达，这为我们建立以AFP阳性细胞为靶的肝癌自杀基因
治疗策略提供了可能。
在本研究中，首先，我们使用间接免疫荧光法和westem blot方
法检测各实验细胞系中AFP的表达状态。然后，采用PCR方法扩增
出/汗P增强子核心序列后，构建含AFP增强子、CMV启动子调控
的Luciferase报道基因的表达质粒pAFP一LUC。通过Luciferase报道
基因在各细胞系中表达状态的分析证实，在AFP阳性细胞中，AFP
增强子序列能显著增强Luciferase的表达，表现出良好的肝癌细胞特
异性转录活性。在此基础上，我们成功构建了AFP增强子、CM[V启
动子调控的pAFP一TE了Gcv自杀基因治疗系统，进行肝细胞癌的自杀
基因靶向治疗的实验研究。
基因治疗的载体问题，以及载体相关的免疫反应、细胞毒性与安
全性问题是限制基因治疗发展和临床应用的瓶颈，而目前基因治疗的
研究和临床应用中常用的病毒载体和非病毒载体都存在无法克服的
局限性。纳米粒基因转运体是近年发展起来的一种新型的非病毒基因
转运载体。它是将DNA、RNA等基因治疗分子包裹在纳米颗粒之中
或吸附在其表面，同时也在颗粒表面藕联特异性的靶向分子，如特异
性配体、单克隆抗体等，通过靶向分子与细胞表面特异性受体结合，
在细胞摄取作用下进入胞内，实现安全有效的靶向性基因治疗。
纳米粒材料的选择是成功进行纳米基因转运和治疗的关键。所选
择的材料必须是生物可降解型或者易于从体内排泄，而不产生有害的
降解产物，且无免疫原性，不引起机体的免疫排斥反应。高分子生物
降解材料制备的纳米颗粒具有稳定、无毒、无抗原性、生物相容性好、
对所转运基因的表达有控释作用及对基因有保护作用等优点，是良好
的纳米基因转运载体材料。
研究证实，纳米粒与DNA的连接多通过静电吸引作用来完成。
DNA的磷酸骨架所带的负电荷只能与表面带正电性的载体结合。因
此，需要利用生物活性分子对纳米颗粒的表面进行改性，使其表面携
带阳离子物质，起到防止颗粒团聚，并有利于结合DNA分子的作用。
在本研究中，我们选择聚氰基丙烯酸正丁酷(PBCA)纳米颗粒
作为基因转运载体。该纳米颗粒具有良好的生物降解性、生物相容性，
并无免疫原性。PBCA纳米颗粒表面带有负电性，因此我们采用阳离
子表面活性剂十六烷基三乙基滨化按(CTAB)对其进行表面改性，以使
其表面带上正电荷而获得结合带负电DNA的能力及某些胶体特性。
同时，实验也证实，PBCA纳米颗粒心NA复合物能保护所携带的DNA
免受核酸酶的降解和超声剪切作用。
本研究发现，在适当浓度下PBCA纳米颗粒与C工AB修饰的
PBCA纳米颗粒对肝癌细胞(HePG2)及正常的成纤维细胞(3T3)
均无明显的细胞毒性，只在高浓度下才会表现出一定的细胞毒性作
用。卜在体外基因转运中，该纳米颗粒可有效转运EGFP报道基因表达
质粒进入HePGZ及3T3细胞，其转运效率达47%左右，强于相同条
件下脂质体的转染效率。
因此，我们以CLAB修饰的高分子降解型PBCA纳米颗粒作为
基因转运载体，以AFP增强子、CMV启动子调控的HSV-TK基因表
达质粒pAFP一TK作为治疗基因，并加用GCV处理进行AFP阳性
Hepatocellular carcinoma (HCC) is one of the most common malignancies with poor prognosis. It causes nearly 1 million deaths annually worldwide. But only a minority of patients is eligible for surgical therapies due to advanced tumors or extrahepatic diseases at primary diagnosis. Recently, the methods of gene delivery are gradually developing and have made great progresses. So the gene therapy of HCC is a hot spot of the biology and clinic medical currently. Suicide gene therapy can be envisioned as a powerful therapeutical approach in the treatment of hepatocellular carcinoma due to direct, reasonable and effective effects. HSV-TK/GCV system is a method that applied extensively and achieved favorable effects.
The HSV-TK enzyme is almost 1000-fold more efficient at monophosphorylating ganciclovir(GCV) than the mammalian cellular thymidine kinase. Normal mammalian guanylate kinase metabolizes the monophosphate to the diphosphate form (GCV-DP) and then to the triphosphate form (GCV-TP). The triphophate form is highly toxic to cells by inhibiting DNA polymerase and by incorporation into DNA with chain termination.
However, HCC frequently occurs in patients with chronic hepatitis and liver cirrhosis, and prognosis of patients is influenced by hepatic
reserve. The potentially toxic effects of gene transfer may have marked deleterious consequences. So the target regulation of suicide gene therapy of HCC, that is, the target gene transfer or the specific expression of transfected genes to hepatoma cells, which can kill HCC cells furthest but not damage hepatic function, is vital for curative effect and feasibility.
HCC cells are known to express specifically an oncofetal antigen called a-fetoprotein (AFP). The expression of AFP is promoted by combining the specific transcriptional regulation protein (TRP) and transcriptional regulation elements of AFP. Therefore, combining the transcriptional regulation elements of AFP and suicide gene can induce the specific expression of suicide gene only in HCC cells, and the level of expression of the suicide gene is in direct proportion to the level of AFP expression in HCC cells.
Several investigators have used the enhancer/promoter of AFP to achieve tumor-specific expression of foreign gene in AFP-producing carcinoma cells. Moreover, when the enhancer of AFP was used alone to regulate the foreign gene driven by non-specific promoter, the expression of foreign gene was not only increased but also specific only to HCC cells. However, the transcriptional activity of the AFP promoter was low.
Based on the above progresses, we combined AFP enhancer core sequences and HSV-TK gene in the AFP positive HCC gene therapy and examined the feasibility of increasing the target of HSV-TK/GCV system.
AFP is over expressed in about 70% of HCC cases, so it is potential for constructing the suicide gene therapy strategy of HCC targeting AFP-producing cells.
In this study, we firstly used indirect immune-fluorescence assay and Western blot to analyze the expression of AFP in the experimental cells. After amplifying the AFP enhancer core sequences, we constructed recombinant plasmid pAFP-LUC that expressed the Luciferase driven by AFP enhancer and CMV promoter. We found that AFP enhancer could greatly increase the expression of Luciferase in the AFP positive cells and well conserve the HCC cell-specific transcriptional activity using report genes analysis. Then we constructed pAFP-TK in which the expression of TK was driven by AFP enhancer and CMV promoter, and hoped that it could provide the experimental data for targeting the gene therapy of HCC.
The gene delivery vectors and vectors associated immune response, cell toxicity and safety are the bottleneck of research and clinic application of gene therapy. The traditional gene delivery vectors including viral vectors and non-viral vectors have some disadvantages that are hard to overcome. Nanoparticle is developing as a new non-viral gene delivery vector. Gene therapeutical molecules including DNA, RNA, and so on are encapsulated into or
HSV-TK单磷酸化GCV的能力较哺乳动物细胞内的TK强1000倍以上，然后在哺乳动物细胞内鸟苷酸激酶作用下将单磷酸化的GCV(GCV-MP)代谢为双磷酸化(GCV-DP)的形式，并进一步在细胞内酶的作用下转化为三磷酸化(GCV-TP)的产物。GCV-TP对细胞具有剧毒性，可抑制DNA聚合酶的活性，并可掺入到DNA链的末端作为终止剂。
然而，肝癌的发生多来源于慢性肝炎及肝硬化，其预后主要取决于被保留的有功能的肝组织的多少，转基因中潜在的毒性效应会对预后造成不良影响。因此，肝癌自杀基因治疗的靶向性调控，即调控自杀基因特异性导入肝癌细胞或在肝癌细胞中实现特异性表达，最大限度地杀伤肝癌细胞的同时并不损伤正常肝细胞，已经成为决定其疗效及可行性的关键。
研究表明，肝癌细胞特异性合成甲胎蛋白(AFP)，同时特异性合成的转录调节蛋白(TRP)通过与AFP基因的转录调控元件(TRS)结合促进AFP的表达。因此将AFP基因的TRS与自杀基因结合，使
其调控自杀基因的表达，可实现对肝癌组织的靶向性，且自杀基因的
表达量与肝癌细胞中AFP的表达量呈正相关。
研究证实，AFP增强子/启动子可调控其后续基因在AFP阳性肿
瘤细胞中特异性表达。此外，单独应用AFP增强子，不仅可增强其
调控基因的表达，而且可诱导非特异性启动子调控的后续基因仅表达
于/妞P阳性肝癌细胞，从而实现肿瘤细胞特异性。然而，AFP启动
子单独应用时其调控转录的活性较弱。
根据以上研究进展，我们将AFP基因增强子的核心序列与CMV
启动子调控的HSV-TK基因联合应用于AFP阳性肝癌的治疗中，探
讨提高HSV-TE了GCV系统靶向性的可行性。由于70%的肝癌细胞中
有ZJP的表达，这为我们建立以AFP阳性细胞为靶的肝癌自杀基因
治疗策略提供了可能。
在本研究中，首先，我们使用间接免疫荧光法和westem blot方
法检测各实验细胞系中AFP的表达状态。然后，采用PCR方法扩增
出/汗P增强子核心序列后，构建含AFP增强子、CMV启动子调控
的Luciferase报道基因的表达质粒pAFP一LUC。通过Luciferase报道
基因在各细胞系中表达状态的分析证实，在AFP阳性细胞中，AFP
增强子序列能显著增强Luciferase的表达，表现出良好的肝癌细胞特
异性转录活性。在此基础上，我们成功构建了AFP增强子、CM[V启
动子调控的pAFP一TE了Gcv自杀基因治疗系统，进行肝细胞癌的自杀
基因靶向治疗的实验研究。
基因治疗的载体问题，以及载体相关的免疫反应、细胞毒性与安
全性问题是限制基因治疗发展和临床应用的瓶颈，而目前基因治疗的
研究和临床应用中常用的病毒载体和非病毒载体都存在无法克服的
局限性。纳米粒基因转运体是近年发展起来的一种新型的非病毒基因
转运载体。它是将DNA、RNA等基因治疗分子包裹在纳米颗粒之中
或吸附在其表面，同时也在颗粒表面藕联特异性的靶向分子，如特异
性配体、单克隆抗体等，通过靶向分子与细胞表面特异性受体结合，
在细胞摄取作用下进入胞内，实现安全有效的靶向性基因治疗。
纳米粒材料的选择是成功进行纳米基因转运和治疗的关键。所选
择的材料必须是生物可降解型或者易于从体内排泄，而不产生有害的
降解产物，且无免疫原性，不引起机体的免疫排斥反应。高分子生物
降解材料制备的纳米颗粒具有稳定、无毒、无抗原性、生物相容性好、
对所转运基因的表达有控释作用及对基因有保护作用等优点，是良好
的纳米基因转运载体材料。
研究证实，纳米粒与DNA的连接多通过静电吸引作用来完成。
DNA的磷酸骨架所带的负电荷只能与表面带正电性的载体结合。因
此，需要利用生物活性分子对纳米颗粒的表面进行改性，使其表面携
带阳离子物质，起到防止颗粒团聚，并有利于结合DNA分子的作用。
在本研究中，我们选择聚氰基丙烯酸正丁酷(PBCA)纳米颗粒
作为基因转运载体。该纳米颗粒具有良好的生物降解性、生物相容性，
并无免疫原性。PBCA纳米颗粒表面带有负电性，因此我们采用阳离
子表面活性剂十六烷基三乙基滨化按(CTAB)对其进行表面改性，以使
其表面带上正电荷而获得结合带负电DNA的能力及某些胶体特性。
同时，实验也证实，PBCA纳米颗粒心NA复合物能保护所携带的DNA
免受核酸酶的降解和超声剪切作用。
本研究发现，在适当浓度下PBCA纳米颗粒与C工AB修饰的
PBCA纳米颗粒对肝癌细胞(HePG2)及正常的成纤维细胞(3T3)
均无明显的细胞毒性，只在高浓度下才会表现出一定的细胞毒性作
用。卜在体外基因转运中，该纳米颗粒可有效转运EGFP报道基因表达
质粒进入HePGZ及3T3细胞，其转运效率达47%左右，强于相同条
件下脂质体的转染效率。
因此，我们以CLAB修饰的高分子降解型PBCA纳米颗粒作为
基因转运载体，以AFP增强子、CMV启动子调控的HSV-TK基因表
达质粒pAFP一TK作为治疗基因，并加用GCV处理进行AFP阳性
Hepatocellular carcinoma (HCC) is one of the most common malignancies with poor prognosis. It causes nearly 1 million deaths annually worldwide. But only a minority of patients is eligible for surgical therapies due to advanced tumors or extrahepatic diseases at primary diagnosis. Recently, the methods of gene delivery are gradually developing and have made great progresses. So the gene therapy of HCC is a hot spot of the biology and clinic medical currently. Suicide gene therapy can be envisioned as a powerful therapeutical approach in the treatment of hepatocellular carcinoma due to direct, reasonable and effective effects. HSV-TK/GCV system is a method that applied extensively and achieved favorable effects.
The HSV-TK enzyme is almost 1000-fold more efficient at monophosphorylating ganciclovir(GCV) than the mammalian cellular thymidine kinase. Normal mammalian guanylate kinase metabolizes the monophosphate to the diphosphate form (GCV-DP) and then to the triphosphate form (GCV-TP). The triphophate form is highly toxic to cells by inhibiting DNA polymerase and by incorporation into DNA with chain termination.
However, HCC frequently occurs in patients with chronic hepatitis and liver cirrhosis, and prognosis of patients is influenced by hepatic
reserve. The potentially toxic effects of gene transfer may have marked deleterious consequences. So the target regulation of suicide gene therapy of HCC, that is, the target gene transfer or the specific expression of transfected genes to hepatoma cells, which can kill HCC cells furthest but not damage hepatic function, is vital for curative effect and feasibility.
HCC cells are known to express specifically an oncofetal antigen called a-fetoprotein (AFP). The expression of AFP is promoted by combining the specific transcriptional regulation protein (TRP) and transcriptional regulation elements of AFP. Therefore, combining the transcriptional regulation elements of AFP and suicide gene can induce the specific expression of suicide gene only in HCC cells, and the level of expression of the suicide gene is in direct proportion to the level of AFP expression in HCC cells.
Several investigators have used the enhancer/promoter of AFP to achieve tumor-specific expression of foreign gene in AFP-producing carcinoma cells. Moreover, when the enhancer of AFP was used alone to regulate the foreign gene driven by non-specific promoter, the expression of foreign gene was not only increased but also specific only to HCC cells. However, the transcriptional activity of the AFP promoter was low.
Based on the above progresses, we combined AFP enhancer core sequences and HSV-TK gene in the AFP positive HCC gene therapy and examined the feasibility of increasing the target of HSV-TK/GCV system.
AFP is over expressed in about 70% of HCC cases, so it is potential for constructing the suicide gene therapy strategy of HCC targeting AFP-producing cells.
In this study, we firstly used indirect immune-fluorescence assay and Western blot to analyze the expression of AFP in the experimental cells. After amplifying the AFP enhancer core sequences, we constructed recombinant plasmid pAFP-LUC that expressed the Luciferase driven by AFP enhancer and CMV promoter. We found that AFP enhancer could greatly increase the expression of Luciferase in the AFP positive cells and well conserve the HCC cell-specific transcriptional activity using report genes analysis. Then we constructed pAFP-TK in which the expression of TK was driven by AFP enhancer and CMV promoter, and hoped that it could provide the experimental data for targeting the gene therapy of HCC.
The gene delivery vectors and vectors associated immune response, cell toxicity and safety are the bottleneck of research and clinic application of gene therapy. The traditional gene delivery vectors including viral vectors and non-viral vectors have some disadvantages that are hard to overcome. Nanoparticle is developing as a new non-viral gene delivery vector. Gene therapeutical molecules including DNA, RNA, and so on are encapsulated into or
引文
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[19] Guo W, Gosselin MA, Lee RJ. Characterization of a novel diolein-based LPDⅡ vector for gene delivery. J Control Release. 2002, 83(1): 121-132.
[20] Prabha S, Zhou WZ, Panyam J, et al. Size-dependency of nanoparticle-me
diated gene transfection: studies with fractionated nanoparticles. Int J Pharm. 2002, 244(1-2): 105-115.
[21] 杜仕国，施冬梅，韩其文。纳米颗粒的液相合成技术。粉末冶金技术。2000，18(1)：46-50。
[22] Moghimi SM, Hunter AC, Murray JC. Long-Circulating and Target-Specific Nanoparticles: Theory to Practice. Pharmacol Rev. 2001, 53(2): 283-318.
[23] Mulle RH. Colloidal carriers for controlled drug deliverly and targeting. Wissenschaftliche Verlagsgesellschaft[M]. Stuttgraft. 1991.
[24] Prabha S, Zhou WZ, Panyam J, et al. Size-dependency of nanoparticle-me diated gene tansfection: studies with fractionated nanoparticles. Int J Pharm. 2002, 244(1-2): 105-115.
[25] Bellocq NC, Pun SH, Jensen GS, et al. Transferrin-containing, cyclodextrin polymer-based particles for tumor-targeted gene delivery. Bioconjug Che m. 2003; 14(6): 1122-1132.
[26] Chen Y, Xue Z, Zheng D, et al. Sodium chloride modified silica nanopart icles as a non-viral vector with a high efficiency of DNA transfer into cells. Curr Gene Ther. 2003, 3(3): 273-279.
[1] Watanabe,K., Saito,A. and Tamaoki,T. Cell-specific enhancer activity in a far upstream region of the human alpha-fetoprotein gene. J. Biol. Chem. 1987; 262 (10): 4812-4818.
[2] Qian C, Bilbao R, Bruna O, Prieto J. Induction of sensitivity to ganciclovir in human hepatocellular carcinoma cells by adenovirus-mediated gene transfer of herpes simplex virus thymidine kinase. Hepatology. 1995; 22(1): 118-123.
[3] Gerolami R, Cardoso J, Lewin M, et al. Evaluation of HSV-tk gene therapy in a rat
model of chemically induced hepatocellular carcinoma by intratumoral and intrahepatic artery routes. Cancer Res.2000; 60(4): 993-1001.
[4] Kwon HC, Kim JH, Kim KC, et al. In vivo antitumor effect of herpes simplex virus thymidine kinase gene therapy in rat hepatocellular carcinoma: feasibility of adenovirus-mediated intra-arterial gene delivery. Mol Cells.2001; 11(2): 170-178.
[5] Matono S, Tanaka T, Sueyoshi S, et al. Bystander effect in suicide gene therapy is directly proportional to the degree of gap junctional intercellular communication in esophageal cancer. Int J Oncol. 2003; 23(5): 1309-1315.
[6] Van Dillen IJ, Mulder NH, Vaalburg W, et al. Influence of the bystander effect on HSV-TK/GCV gene therapy. A review. Curr Gene Ther. 2002; 3: 307-322.
[7] Ishikawa H, Nakata K, Mawatari F, et al. Retrovirus-mediated gene therapy for hepatocellular carcinoma with reversely oriented therapeutic gene expression regulated by alpha-fetoprotein enhancer/promoter. Biochem Biophys Res Commun. 2001; 4: 1034-1040.
[8] Qiao J, Doubrovin M, Sauter BV, et al. Tumor-specific transcriptional targeting of suicide gene therapy. Gene Ther. 2002; 3:168-175.
[9] Kitazono M, Chuma Y, Aikou T, et al. Adenovirus HSV-TK construct with thyroid-specific promoter:erthancerment of activity and specificity with histone deacetylase inhibitors and agents modulating the camp pathway. Int J Cancer.2002; 3: 453-459.
[10] Chen XP, Zhao H, Zhao XP. Alternation of AFP-mRNA level detected in blood circulation during liver resection for HCC and its significance. World J Gastroenterol. 2002; 8:818-821.
[11] Lu SY, Sui YF, Li ZS, et al. Construction of a regulable gene therapy vector targeting for hepatocellular carcinoma. World J Gastroenterol.2003; 9(4): 688-691.
[12] Nakaya H, Ishizu A, Ikeda H,et al. In vitro model of suicide gene therapy for alpha-fetoprotein-producing gastric cancer. Anticancer Res. 2003; 23(5A): 3795-3800.
[13] Cao G, Kuriyama S, Gao J, et al. Gene therapy for hepatocellular carcinoma based on tumour-selective suicide gene expression using the alpha-fetoprotein (AFP) enhancer and a housekeeping gene promoter. Eur J Cancer.2001; 37(1): 140-147.
[14] He P, Tang ZY, Ye SL, et al. The targeted expression of interleukin-2 in human hepatocellular carcinoma cells. J Exp Clin Cancer Res.2000; 19(2): 183-187.
[15] Ido A, Nakata K, Kato Y, et al. Gene therapy for hepatoma cells using a retrovirus vector carrying herpes simplex virus thymidine kinase gene under the control of human α-fetoprotein gene promoter. Cancer Res. 1995; 55: 3105-3109.
[16] Ido A, Uto H, Moriuchi A, et al. Gene therapy targeting for hepatocellular carcinoma: selective and enhanced suicide gene expression regulated by a hupoxia-inducible enhancer linked to a human α-fetoprotein promoter. Cancer Research. 2001; 61: 3016-3021.
[17] Yakikawa H, Mafune K, Hamada H,et al. An advanced strategy of enhanced specific gene expression for hepatocellular carcinoma. Int J Oncol. 2003; 22(5): 1051-1056.
[18] Kazutada Watanabe, Akira Saito, Taiki Tamaoki. Cell-specific enhancer activity in a far upstream region of the human α-fetoprotein gene. J Biol Chem. 1987; 262(10): 4812-4818.
[19] Cui J, Yang D. Construction of c-fms antisense eukaryotic expressing vector beating AFP enhancer and its clinical significance. Zhonghua Gan Zang Bing Za Zhi. 2001; 9(5): 297-299.
[1] Andreas Zimmer. Antisense oligonucleotide delivery with polyhexylcyanoacrylate nanoparticles as carries. METHODS: A companion to methods in enzymology.
1999, 18: 286-295.
[2] 基因表达技术。李育阳主编。科学出版社，2001。
[3] 基因治疗。顾健人，曹雪涛主编。科学出版社，2001。
[4] Wu Q, Moyana T, Xiang J. Cancer gene therapy by adenovirus-mediated gene transfer. Curr Gene Ther 2001; 1(1): 101-122.
[5] Rainov NG, Ren H. Clinical trials with retrovirus mediated gene therapy--what have we learned? J Neurooncol. 2003; 65(3): 227-236.
[6] Watanabe T. Adenovirus-mediated CTLA4Ig gene therapy in cardiac xenotransplantation. Hokkaido Igaku Zasshi. 2004; 79(1): 47-53.
[7] Buning H, Nicklin SA, Perabo L,et al. AAV-based gene transfer. Curr Opin Mol Ther. 2003; 5(4): 367-375.
[8] Goins WF, Wolfe D, Krisky DM, et al. Delivery using herpes simplex virus: an overview. Methods Mol Biol. 2004; 246: 257-299.
[9] Marti WR, Schutz A, Oertli D, et al. Recombinant vaccinia viruses as efficient vectors of biologically active, human B7 costimulation molecules. Langenbecks Arch Chir Suppl Kongressbd. 1998; 115(Suppl Ⅰ): 131-136.
[10] Lai CM, Lai YK, Rakoczy PE. Adenovirus and adeno-associated virus vectors. DNA Cell Biol. 2002; 21(12): 895-913.
[11] Fox JL. Gene therapy safety issues come to fore. Nat Biotechnol. 1999; 17(12): 1153.
[12] Wu X, Li Y, Crise B, Burgess SM. Transcription start regions in the human genome are favored targets for MLV integration. Science. 2003; 300(5626): 1749-1751.
[13] Sun X, Zhang HW, Zhang ZR. Transfection efficiency of pORF lacZ plasmid lipopolyplex to hepatocytes and hepatoma cells. World J Gastroenterol. 2004; 10(4): 531-534.
[14] Kwoh DY, Coffin CC, Lollo CP, et al. Stabilization of poly-L-lysine/DNA polyplexes for in vivo gene delivery to the liver. Biochim Biophys Acta. 1999, 1444(2): 171-190.
[15] Golan R, Pietrasanta LI, Hsieh W, et al. DNA toroids: stages in condensation.
Biochemistry. 1999, 38(42): 14069-14076.
[16] J unghans M, Kreuter J, Zimmer A. Antisense delivery using prtamine-oligonucleotide particles. Nucleic Acids Res. 2000, 28: E45.
[17] Jeong JH, Park TG. Ploy(L-lysine)-g-poly(D,L-lactic-co-glycolic acid) micelles for low cytotoxic biodegradable gene delivery carriers. J Control Release. 2002, 82(1): 159-166.
[18] Jeong JH, Song SH, Lira DW. DNA transfection using linear poly(ethylenimine) prepared by controlled acid hydrolysis of poly(2-ethyl-2-oxazoline). J Control Release. 2001, 73(2-3): 391-399.
[19] Lee JH, Lim YB, Choi JS, et al. Polyplexes assembled with internally quaternized PAMAM-OH dendrimer and plasmid DNA have a neutral surface and gene delivery potency. Bioconjug Chem. 2003; 14(6): 1214-1221.
[20] Maruyama K, Iwasaki F, Takizawa T, et al. Novel receptor-mediated gene delivery system comprising plasmid/protamine/sugar-containing polyanion ternary complex. Biomaterials. 2004; 25(16): 3267-3273.
[21] Thoren PE,Persson D,Karlsson M,et al.The antennapedia peptide penetratin translocates across lipid bilayers - the first direct observation[J]. FEBS Lett, 2000, 482(3): 265-268.
[22] 张中太，林元华，唐子龙等，纳米材料及其技术的应用前景。材料工程，2000，3：42-48。
[23] Kneuer C, Sameti M, Haltner EG; et al. Silica nanoparticles modified with aminosilanes as carriers for plasmid DNA. Int J Pharm. 2000; 196(2): 257-261.
[24] Chen Y, Xue Z, Zheng D, et al. Sodium chloride modified silica nanoparticles as a non-viral vector with a high efficiency of DNA transfer into cells. Curr Gene Ther. 2003, 3(3): 273-279.
[25] Zimmer A. Antisense oligonucleotide delivery with polyhexylcyanoacrylate nanoparticles as carriers. Methods: A Companion to Methods in Enzymology. 1999, 18: 286-295.
[26] Zobel HP, Kreuter J, Werner D, et al. Cationic polyhexylcyanoacrylate nanoparticles as carriers for antisense oligonucleotides. Antisense Nucleic Acid
Drug Dev. 1997; 7(5): 483-493.
[27] Ilya Koltover, Kathrin Wagner, Cyrus R. Safinya. DNA condensation in two dimensions. Proc Natl Acad Sci U S A. 2000, 97(26): 14046-14051.
[28] Vijayanathan V, Thomas T, Thomas TJ. DNA nanoparticles and development of DNA delivery vehicles for gene therapy. Biochemistry. 2002, 1(48): 14085-14094.
[29] De Smedt SC, Demeester J, Hennink WE. Cationic polymer based gene delivery systems. Pharm Res. 2000; 17(2): 113-126.
[30] Kircheis R, Wightman L, Wagner E. Design and gene delivery activity of modified polyethylenimines. Adv Drug Deliv Rev. 2001; 53(3): 341-358.
[31] Zhu SG, Lu HB, Xiang JJ, et al. A new non-virus DNA delivery: poly-L-lysine-modified silica nanoparticles. Chinese Science Bulletin. 2002, 47 (3): 193-197.
[1] Qian C, Bilbao R, Bruna O, Prieto J. Induction of sensitivity to ganciclovir in human hepatocellular carcinoma cells by adenovirus-mediated gene transfer of herpes simplex virus thymidine kinase. Hepatology. 1995, 22(1): 118-123.
[2] Gerolami R, Cardoso J, Lewin M, et al. Evaluation of HSV-tk gene therapy in a rat model of chemically induced hepatocellular carcinoma by intratumoral and intrahepatic artery routes. Cancer Res.2000, 60(4): 993-1001.
[3] Kwon HC, Kim JH, Kim KC, et al. In vivo antitumor effect of herpes simplex virus thymidine kinase gene therapy in rat hepatocellular carcinoma: feasibility of adenovirus-mediated intra-arterial gene delivery. Mol Cells. 2001, 11(2): 170-178.
[4] Sun XY, Wu ZD, Hu JB. Suicide gene therapy of hepatocellular carcinoma and
delivery procedure and route of therapeutic gene in vivo. Hepatobiliary Pancreat Dis Int. 2002; 1(3): 373-377.
[5] Gerolami R, Lewin M, Faivre J, et al. Herpes simplex virus thymidine kinase-mediated suicide gene therapy for hepatocellular carcinoma using HIV-1-derived lentiviral vectors. J Hepatol. 2004, 40(2): 291-297.
[6] Uch R, Gerolami R, Faivre J, et al. Hepatoma cell-specific ganciclovir-mediated toxicity of a lentivirally transduced HSV-TkEGFP fusion protein gene placed under the control of rat alpha-fetoprotein gene regulatory sequences. Cancer Gene Ther. 2003; 10(9): 689-695.
[7] Hasegawa H, Shimada M, Yonemitsu Y, et at. Preclinical and therapeutic utility of HVJ liposomes as a gene transfer vector for hepatocellular carcinoma using herpes simplex virus thymidine kinase. Cancer Gene Ther. 2001; 8(4): 252-258.
[8] Cao G, Kuriyama S, Gao J, et al. Gene therapy for hepatocellular carcinoma based on tumour-selective suicide gene expression using the alpha-fetoprotein (AFP) enhancer and a housekeeping gene promoter.Eur J Cancer. 2001; 37(1): 140-147.
[9] Ye X, Liang M, Meng X,et al. Insulation from viral transcriptional regulatory elements enables improvement to hepatoma-specific gene expression from adenovirus vectors. Biochem Biophys Res Commun. 2003 8; 307(4): 759-764.
[10] Song JS, Kim HP, Yoon WS,et al. Adenovirus-mediated suicide gene therapy using the human telomerase catalytic subunit (hTERT) gene promoter induced apoptosis of ovarian cancer cell line. Biosci Biotechnol Biochem. 2003; 67(11): 2344-2350.
[11] Lehtimaki KK, Valonen PK, Griffin JL,et al. Metabolite changes in BT4C rat gliomas undergoing ganciclovir-thymidine kinase gene therapy-induced programmed cell death as studied by 1H NMR spectroscopy in vivo, ex vivo, and in vitro. J Biol Chem. 2003; 278(46): 45915-45923.
[12] Krohne TU, Shankara S, Geissler M,et al. Mechanisms of cell death induced by suicide genes encoding purine nucleoside phosphorylase and thymidine kinase in human hepatocellular carcinoma cells in vitro. Hepatology. 2001; 34(3): 511-518.
[13] Engelmann C, Heslan JM, Fabre M,et al. Importance, mechanisms and limitations of the distant bystander effect in cancer gene therapy of experimental liver tumors. Cancer Lett. 2002; 179(1): 59-69.
[14] Asklund T, Appelskog IB, Ammerpohl O, et al. Gap junction-mediated bystander effect in primary cultures of human malignant gliomas with recombinant expression of the HSVtk gene. Exp Cell Res. 2003; 284(2): 185-195.
[15] 杨淑英，段芳龄，鲁凤民，等。自杀基因表达产生旁观者效应的初步研究。胃肠病学和肝病学研究。1997，6(4)：301-306。
[16] Kawamura K, Bahar R,Namba H, et al. Bystander effect in uracil phosphoribosyltransferase/5-fluorouracil mediated suicide gene therapy is correlated with the level of intercellular communication. Int J Oncol. 2001, 18(1): 117-120.
[17] Moolten FL. Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy. Cancer Res. 1986; 46(10): 5276-5281.
[18] Matono S, Tanaka T, Sueyoshi S,et al. Bystander effect in suicide gene therapy is directly proportional to the degree of gap junctional intercellular communication in esophageal cancer. Int J Oncol. 2003; 23(5): 1309-1315.
[19] Kuriyama S, Tsujinoue H, Yoshiji H. Immune response to suicide gene therapy. Methods Mol Med. 2004; 90: 353-69.
[20] Cho HS, Lee HR, Kim MK. Bystander-Mediated Regression of Murine Neuroblastoma via Retroviral Transfer of the HSV-TK Gene. J Korean Med Sci. 2004; 19(1): 107-112.
[2] Singhal S, Kaiser LR. Cancer chemotherapy using suicide genes. Surg Onc ol Clin N Am. 1998, 3: 505-536.
[3] Dillen IJ, Mulder NH, Vaalburg W, et al. Influence of the bystander effect on HSV-TK/GCV gene therapy. Curr Gene Ther. 2002, 3: 307-322.
[4] Havlik R, Kral V, Habib N. Gene therapy of liver tumors: results of the fi rst clinical studies. Cas Lek Cesk. 2003; 142(9): 528-529.
[5] Germano IM, Fable J, Gultekin SH, et al. Adenovirus/herpes simplex-thymi dine kinase/ganciclovir complex: preliminary results of a phase Ⅰ trial in p atients with recurrent malignant gliomas. J Neurooncol. 2003; 65(3): 279-2 89.
[6] Huber BE, Richards CA, Krenitsky TA. Retroviral-mediated gene therapy f or the treatment of hepatocellular carcinoma: an innovative approach for c ancer therapy. Proc Natl Acad Sci U S A. 1991; 88(18): 8039-8043.
[7] Fuerer C, Iggo R.5-Fluorocytosine increases the toxicity of Wnt-targeting re plicating adenoviruses that express cytosine deaminase as a late gene. Gen eTher. 2004; 11(2): 142-151.
[8] Hay MP, Anderson RF, Ferry DM, et al. Synthesis and evaluation of nitro heterocyclic carbamate prodrugs for use with nitroreductase-mediated genedirected enzyme prodrug therapy. J Med Chem. 2003; 46(25): 5533-5545.
[9] Tamiya T, Ono Y, Wei MX, et al. Escherichia coli gpt gene sensitizes rat glioma cells to killing by 6-thioxanthine or 6-thioguanine. Cancer Gene T her. 1996; 3(3): 155-162.
[10] Parker WB, King SA, Allan PW, et al. In vivo gene therapy of cancer w ith E. coli purine nucleoside phosphorylase. Hum Gene Ther. 1997; 8(14):
1637-1644.
[11] Qian C, Bilbao R, Bruna O, Prieto J. Induction of sensitivity to ganciclov ir in human hepatocellular carcinoma cells by adenovirus-mediated gene tra nsfer of herpes simplex virus thymidine kinase. Hepatology.1995, 22(1): 11 8-123.
[12] Gerolami R, Cardoso J, Lewin M, et al. Evaluation of HSV-tk gene thera py in a rat model of chemically induced hepatocellular carcinoma by intrat umoral and intrahepatic artery routes. Cancer Res.2000, 60(4): 993-1001.
[13] Kwon HC, Kim JH, Kim KC, et al. In vivo antitumor effect of herpes si mplex virus thymidine kinase gene therapy in rat hepatocellular carcinoma: feasibility of adenovirus-mediated intra-arterial gene delivery. Mol Cells.20 01, 11(2): 170-178.
[14] Van Dillen IJ, Mulder NH, Vaalburg W, et al. Influence of the bystander effect on HSV-TK/GCV gene therapy. A review. Curr Gene Ther. 2002, 3: 307-322.
[15] Lu SY, Sui YF, Li ZS, et al. Construction of a regulable gene therapy v ector targeting for hepatocellular carcinoma. World J Gastroenterol.2003, 9 (4): 688-691.
[16] Cao G, Kuriyama S, Gao J, et al. Gene therapy for hepatocellular carcino ma based on tumour-selective suicide gene expression using the alpha-fetopr otein (AFP) enhancer and a housekeeping gene promoter. Eur J Cancer.200 1, 37(1): 140-147.
[17] He P, Tang ZY, Ye SL, et al. The targeted expression of interleukin-2 in human hepatocellular carcinoma cells. J Exp Clin Cancer Res.2000, 19(2):1 83-187.
[18] Lai CM, Lai YK, Rakoczy PE. Adenovirus and adeno-associated virus vec tors. DNA Cell Biol. 2002; 21(12): 895-913.
[19] Guo W, Gosselin MA, Lee RJ. Characterization of a novel diolein-based LPDⅡ vector for gene delivery. J Control Release. 2002, 83(1): 121-132.
[20] Prabha S, Zhou WZ, Panyam J, et al. Size-dependency of nanoparticle-me
diated gene transfection: studies with fractionated nanoparticles. Int J Pharm. 2002, 244(1-2): 105-115.
[21] 杜仕国，施冬梅，韩其文。纳米颗粒的液相合成技术。粉末冶金技术。2000，18(1)：46-50。
[22] Moghimi SM, Hunter AC, Murray JC. Long-Circulating and Target-Specific Nanoparticles: Theory to Practice. Pharmacol Rev. 2001, 53(2): 283-318.
[23] Mulle RH. Colloidal carriers for controlled drug deliverly and targeting. Wissenschaftliche Verlagsgesellschaft[M]. Stuttgraft. 1991.
[24] Prabha S, Zhou WZ, Panyam J, et al. Size-dependency of nanoparticle-me diated gene tansfection: studies with fractionated nanoparticles. Int J Pharm. 2002, 244(1-2): 105-115.
[25] Bellocq NC, Pun SH, Jensen GS, et al. Transferrin-containing, cyclodextrin polymer-based particles for tumor-targeted gene delivery. Bioconjug Che m. 2003; 14(6): 1122-1132.
[26] Chen Y, Xue Z, Zheng D, et al. Sodium chloride modified silica nanopart icles as a non-viral vector with a high efficiency of DNA transfer into cells. Curr Gene Ther. 2003, 3(3): 273-279.
[1] Watanabe,K., Saito,A. and Tamaoki,T. Cell-specific enhancer activity in a far upstream region of the human alpha-fetoprotein gene. J. Biol. Chem. 1987; 262 (10): 4812-4818.
[2] Qian C, Bilbao R, Bruna O, Prieto J. Induction of sensitivity to ganciclovir in human hepatocellular carcinoma cells by adenovirus-mediated gene transfer of herpes simplex virus thymidine kinase. Hepatology. 1995; 22(1): 118-123.
[3] Gerolami R, Cardoso J, Lewin M, et al. Evaluation of HSV-tk gene therapy in a rat
model of chemically induced hepatocellular carcinoma by intratumoral and intrahepatic artery routes. Cancer Res.2000; 60(4): 993-1001.
[4] Kwon HC, Kim JH, Kim KC, et al. In vivo antitumor effect of herpes simplex virus thymidine kinase gene therapy in rat hepatocellular carcinoma: feasibility of adenovirus-mediated intra-arterial gene delivery. Mol Cells.2001; 11(2): 170-178.
[5] Matono S, Tanaka T, Sueyoshi S, et al. Bystander effect in suicide gene therapy is directly proportional to the degree of gap junctional intercellular communication in esophageal cancer. Int J Oncol. 2003; 23(5): 1309-1315.
[6] Van Dillen IJ, Mulder NH, Vaalburg W, et al. Influence of the bystander effect on HSV-TK/GCV gene therapy. A review. Curr Gene Ther. 2002; 3: 307-322.
[7] Ishikawa H, Nakata K, Mawatari F, et al. Retrovirus-mediated gene therapy for hepatocellular carcinoma with reversely oriented therapeutic gene expression regulated by alpha-fetoprotein enhancer/promoter. Biochem Biophys Res Commun. 2001; 4: 1034-1040.
[8] Qiao J, Doubrovin M, Sauter BV, et al. Tumor-specific transcriptional targeting of suicide gene therapy. Gene Ther. 2002; 3:168-175.
[9] Kitazono M, Chuma Y, Aikou T, et al. Adenovirus HSV-TK construct with thyroid-specific promoter:erthancerment of activity and specificity with histone deacetylase inhibitors and agents modulating the camp pathway. Int J Cancer.2002; 3: 453-459.
[10] Chen XP, Zhao H, Zhao XP. Alternation of AFP-mRNA level detected in blood circulation during liver resection for HCC and its significance. World J Gastroenterol. 2002; 8:818-821.
[11] Lu SY, Sui YF, Li ZS, et al. Construction of a regulable gene therapy vector targeting for hepatocellular carcinoma. World J Gastroenterol.2003; 9(4): 688-691.
[12] Nakaya H, Ishizu A, Ikeda H,et al. In vitro model of suicide gene therapy for alpha-fetoprotein-producing gastric cancer. Anticancer Res. 2003; 23(5A): 3795-3800.
[13] Cao G, Kuriyama S, Gao J, et al. Gene therapy for hepatocellular carcinoma based on tumour-selective suicide gene expression using the alpha-fetoprotein (AFP) enhancer and a housekeeping gene promoter. Eur J Cancer.2001; 37(1): 140-147.
[14] He P, Tang ZY, Ye SL, et al. The targeted expression of interleukin-2 in human hepatocellular carcinoma cells. J Exp Clin Cancer Res.2000; 19(2): 183-187.
[15] Ido A, Nakata K, Kato Y, et al. Gene therapy for hepatoma cells using a retrovirus vector carrying herpes simplex virus thymidine kinase gene under the control of human α-fetoprotein gene promoter. Cancer Res. 1995; 55: 3105-3109.
[16] Ido A, Uto H, Moriuchi A, et al. Gene therapy targeting for hepatocellular carcinoma: selective and enhanced suicide gene expression regulated by a hupoxia-inducible enhancer linked to a human α-fetoprotein promoter. Cancer Research. 2001; 61: 3016-3021.
[17] Yakikawa H, Mafune K, Hamada H,et al. An advanced strategy of enhanced specific gene expression for hepatocellular carcinoma. Int J Oncol. 2003; 22(5): 1051-1056.
[18] Kazutada Watanabe, Akira Saito, Taiki Tamaoki. Cell-specific enhancer activity in a far upstream region of the human α-fetoprotein gene. J Biol Chem. 1987; 262(10): 4812-4818.
[19] Cui J, Yang D. Construction of c-fms antisense eukaryotic expressing vector beating AFP enhancer and its clinical significance. Zhonghua Gan Zang Bing Za Zhi. 2001; 9(5): 297-299.
[1] Andreas Zimmer. Antisense oligonucleotide delivery with polyhexylcyanoacrylate nanoparticles as carries. METHODS: A companion to methods in enzymology.
1999, 18: 286-295.
[2] 基因表达技术。李育阳主编。科学出版社，2001。
[3] 基因治疗。顾健人，曹雪涛主编。科学出版社，2001。
[4] Wu Q, Moyana T, Xiang J. Cancer gene therapy by adenovirus-mediated gene transfer. Curr Gene Ther 2001; 1(1): 101-122.
[5] Rainov NG, Ren H. Clinical trials with retrovirus mediated gene therapy--what have we learned? J Neurooncol. 2003; 65(3): 227-236.
[6] Watanabe T. Adenovirus-mediated CTLA4Ig gene therapy in cardiac xenotransplantation. Hokkaido Igaku Zasshi. 2004; 79(1): 47-53.
[7] Buning H, Nicklin SA, Perabo L,et al. AAV-based gene transfer. Curr Opin Mol Ther. 2003; 5(4): 367-375.
[8] Goins WF, Wolfe D, Krisky DM, et al. Delivery using herpes simplex virus: an overview. Methods Mol Biol. 2004; 246: 257-299.
[9] Marti WR, Schutz A, Oertli D, et al. Recombinant vaccinia viruses as efficient vectors of biologically active, human B7 costimulation molecules. Langenbecks Arch Chir Suppl Kongressbd. 1998; 115(Suppl Ⅰ): 131-136.
[10] Lai CM, Lai YK, Rakoczy PE. Adenovirus and adeno-associated virus vectors. DNA Cell Biol. 2002; 21(12): 895-913.
[11] Fox JL. Gene therapy safety issues come to fore. Nat Biotechnol. 1999; 17(12): 1153.
[12] Wu X, Li Y, Crise B, Burgess SM. Transcription start regions in the human genome are favored targets for MLV integration. Science. 2003; 300(5626): 1749-1751.
[13] Sun X, Zhang HW, Zhang ZR. Transfection efficiency of pORF lacZ plasmid lipopolyplex to hepatocytes and hepatoma cells. World J Gastroenterol. 2004; 10(4): 531-534.
[14] Kwoh DY, Coffin CC, Lollo CP, et al. Stabilization of poly-L-lysine/DNA polyplexes for in vivo gene delivery to the liver. Biochim Biophys Acta. 1999, 1444(2): 171-190.
[15] Golan R, Pietrasanta LI, Hsieh W, et al. DNA toroids: stages in condensation.
Biochemistry. 1999, 38(42): 14069-14076.
[16] J unghans M, Kreuter J, Zimmer A. Antisense delivery using prtamine-oligonucleotide particles. Nucleic Acids Res. 2000, 28: E45.
[17] Jeong JH, Park TG. Ploy(L-lysine)-g-poly(D,L-lactic-co-glycolic acid) micelles for low cytotoxic biodegradable gene delivery carriers. J Control Release. 2002, 82(1): 159-166.
[18] Jeong JH, Song SH, Lira DW. DNA transfection using linear poly(ethylenimine) prepared by controlled acid hydrolysis of poly(2-ethyl-2-oxazoline). J Control Release. 2001, 73(2-3): 391-399.
[19] Lee JH, Lim YB, Choi JS, et al. Polyplexes assembled with internally quaternized PAMAM-OH dendrimer and plasmid DNA have a neutral surface and gene delivery potency. Bioconjug Chem. 2003; 14(6): 1214-1221.
[20] Maruyama K, Iwasaki F, Takizawa T, et al. Novel receptor-mediated gene delivery system comprising plasmid/protamine/sugar-containing polyanion ternary complex. Biomaterials. 2004; 25(16): 3267-3273.
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