叶酸修饰的G5-PAMAM-D介导HSV-TK/GCV自杀基因系统治疗前列腺癌的体外实验研究*

目的:探讨叶酸修饰的第五代聚酰胺- 胺型树枝状聚合物（G5-PAMAM-D-fol ）作为基因载体进行前列腺癌自杀基因治疗的可行性。方法:以G5-PAMAM-D-fol 为基因载体将含有自杀基因HSV-TK 的重组质粒pcDNA 3-tk转染至前列腺癌细胞系PC- 3 和LNCaP ,24h 后以RT-PCR 法检测HSV-TK 基因在两种前列腺癌细胞系中的mRNA 表达;再次转染后24h,对转染的两种细胞分别给予不同浓度的前体药物更昔洛韦（GCV ）,48h 后采用四甲基偶氮唑盐比色法（MTT 法）检测细胞抑制率。结果:RT-PCR结果证明G5-PAMAM-D-fol 可将重组质粒pcDNA 3-tk转染2 种前列腺癌细胞并转录HSV-TK 基因。MTT 实验反映出A 组（G5-PAMAM-D-fol/pcDNA 3-tk）对PC- 3 和LNCaP 细胞有明显的杀伤作用,并且比B 组（G5-PAMAM-D/pcDNA3-tk）的细胞抑制率更高。C 组（G5-PAMAM-D-fol ）和D 组（G5-PAMAM-D）随着GCV 浓度的增加细胞生长并未受到明显的抑制,而且前者并未表现出比后者更明显的细胞杀伤作用。结论:G5-PAMAM-D-fol 能够在体外将自杀基因重组质粒成功转入前列腺癌细胞并表达HSV-TK 基因,其靶向性强且细胞毒性低。      

肿瘤HSV-tk/GCV自杀基因治疗系统的增效策略

HSV-tk/GCV系统是目前应用最广泛的自杀基因治疗系统之一,其重要性日益上升.然而,许多缺陷严重限制了其应用广度和治疗效率.为满足更多领域的研究需要并实现临床治疗,科研人员投入了大量精力改善HSV-tk/GCV系统,增强其治疗效率.增效策略主要包括加强亲和性、提高表达水平、双自杀基因系统联合、诱发免疫系统协同作用、激活细胞周期、与其他肿瘤治疗方法相结合等.本文在阐明HSV-tk/GCV系统的自杀机理的基础上,分析了多种增效策略及效果,列举了应用领域并对未来发展作探讨.     

HSV-TK/GCV体系对前列腺癌PC-3m细胞杀伤作用的实验研究

目的：探讨HSV－TK／GCV体系对前列腺癌PC-3m细胞的杀伤作用。方法：应用逆转录病毒载体将HSV－TK基因转染PC－3m细胞，经RT－PCR鉴定后，MTT、流式细胞仪、电镜等方法检测丙氧鸟苷（GCV）对转染后PC－3m细胞的杀伤作用，同时以未转染PC－3m细胞为对照。结果：GCV对正常PC－3m细胞毒性较低，而对转染后PC－3m细胞有较强的细胞毒作用，但旁观者效应不明显。结论：HSV－TK／GCV体系对前列腺癌细胞具有明显杀伤作用，有必要进一步研究。     

腺相关病毒载体及其介导的自杀基因疗法

自杀基因疗法是肿瘤治疗的方法之一。腺相关病毒（AAV）载体介导的自杀基因疗法尤其备受关注。通过对AAV载体的选择改建，调控外源基因的有效表达，提高基因转染的效率，充分发挥自杀基因的杀伤作用及旁观者效应来达到治疗肿瘤的目的。本文就AAV载体及其介导的自杀基因疗法方面的进展作一综述。     

非病毒载体系统在基因治疗中的应用

非病毒载体系统具有低毒、低免疫原性和相对靶向性等优点,是新兴发展起来的基因转移系统。本文综述了目前基因治疗中所用的几种非病毒载体系统及其研究进展。     

非病毒载体系统在基因治疗中的应用

非病毒载体系统具有低毒、低免疫原性和相对靶向性等优点,是新兴发展起来的基因转移系统.本文综述了目前基因治疗中所用的几种非病毒载体系统及其研究进展.     

HSV-tk／GCV基因治疗在卵巢癌中的研究进展

卵巢癌自杀基因治疗多采用HSV-tk／GCV系统。该系统的旁观者效应可在一定程度上弥补基因转染率低的缺陷，通过缝隙连接胞间通信(GJIC)增强剂可提高该系统的旁观者效应，通过选择构建高效能载体系统、应用靶向性调控机制或与其他方法联合治疗可增强疗效。该系统治疗卵巢癌具有广阔的发展前景。     

自杀基因在肝癌治疗中的应用现状

目前，在自杀基因治疗肝癌的研究中，通过联合基因治疗，受体介导定位、改变酶特性，寻找新的自杀基因系统，以及选择不同用药策略和途径等，方法将自杀基因高效导入靶细胞， 并使之表达，从而增强特异性杀伤肝脏肿瘤细胞作用。临床应用前景广阔。     

两种基于PNP/ Mep-dR 系统的新型融合自杀基因载体的构建及鉴定

 目的 构建两个新型融合自杀基因表达载体并检测其表达。方法 利用DNA重组技术制备两个融合基因PNP-TK和PNP-CD，将二者分别插入真核表达载体pcDNA3．0中，构建两个融合基因表达载体pcDNA3．0／PNP-TK和pcDNA3．0／PNP-CD；经酶切、PCR及测序鉴定各个重组体。结果 成功构建了两个新型融合基因表达载体并在肝癌细胞株HepG2中检测到二者的表达。结论 两个基于PNP／Mep-dR系统的融合自杀基因表达载体是肿瘤基因治疗中杀瘤谱广、杀伤效应强大的理想载体。     

基因治疗中非病毒载体的基因导入方法

随着基因治疗研究的深入,人们发现,基因治疗的关键是选择恰当的载体及导入方法,使目的基因获得靶向性导入,稳定有效的表达,可控性增强。目前用于基因治疗的载体有两种:病毒载体和非病毒载体。虽然基因治疗的临床实验研究多采用病毒载体,但是不同的病毒载体由于各自存在许多不足     

壳聚糖纳米粒介导的HSV-tk基因对前列腺癌的体外杀伤作用

目的：考察壳聚糖纳米粒作为自杀基因载体进行前列腺自杀基因治疗的可行性，为前列腺癌的基因治疗寻找新的基因载体。方法：将壳聚糖纳米粒包裹的含报告基因的质粒pEGFP—C1分别转染前列腺癌细胞PC-3和22Rvl，并观察EGFP的表达情况。随后将含HSV-tk自杀基因的真核高效表达质粒pcDNA3-tk经壳聚糖纳米粒包裹后转染上述2种前列腺癌细胞，转染48h后，以RT-PCR检测HSV-tk基因的表达情况。最后对转染的2种细胞给予不同浓度的前体药更昔洛韦（ganci—clovir，GCV）作用24h后，采用MTT比色法测定药物对细胞增殖的影响，并进行统计学分析。结果：壳聚糖纳米粒能够将质粒pEGFP-C1转入2种前列腺癌细胞，并表达EGFP，在PC-3细胞中壳聚糖纳米介导的转染效率高于脂质体。采用壳聚糖纳米粒转染pcDNA3-tk于2种前列腺癌细胞后，RT-PCR证明在癌细胞中有HSV-tk基因的表达。给予前体药物GCV后，实验组细胞生长抑制率与只给壳聚糖或裸质粒的对照组相比明显增高，说明HSV-tk自杀基因纳米粒在细胞中表达，从而使HSV-tk／GCV自杀基因系统发挥杀伤细胞的作用。结论：壳聚糖纳米粒能将质粒基因pcDNA3-tk转入前列腺癌细胞并表达胸苷激酶发挥作用，可做为前列腺癌自杀基因治疗的基因载体。     

Double‐targeted and double‐enhanced suicide gene therapy mediated by generation 5 polyamidoamine dendrimers for prostate cancer

Herpes simplex virus (HSV)‐thymidine kinase (TK)/ganciclovir (GCV) system is one of the most widely used and efficient suicide gene therapy for prostate cancer, but the lack of favorable gene vector and target limits its application. In this study, we established a novel system using nonviral gene vector G5‐PAMAM‐D to express HSV‐TK and connexin43 (Cx43) gene driven by prostate‐specific membrane antigen (PSMA) promoter, and evaluated the anti‐tumor effect of this system. G5‐PAMAM‐D delivered PSMAe/p‐TK‐Cx43 showed expression of TK and Cx43 only in LNCaP cells, but not in PC‐3 and other cells. The transfection efficiency of this system was comparable to lipofectamine 2000 by propidium iodide staining assay. With gemcitabine, folate‐G5‐PAMAM‐D delivered PSMAe/p‐TK‐Cx43 (folate‐G5‐PAMAM‐D/PSMAe/p‐TK‐Cx43) significantly decreased prostate cancer LNCaP cell proliferation and promoted apoptosis in vitro. With gemcitabine, the systemic deliver of folate‐G5‐PAMAM‐D/PSMAe/p‐TK‐Cx43 significantly inhibited tumor growth in the LNCaP xenograft animal model. Our study demonstrates that this double‐targeted and double‐enhanced system is effective in inducing cell growth inhibition and apoptosis in vitro and suppressing tumor growth in vivo. In conclusion, Cx43 and gemcitabine combined with HSV‐TK/GCV gene therapy using nonviral vector G5‐PAMAM‐D hold great potential as a novel approach for the gene therapy of prostate cancer. © 2011 Wiley Periodicals, Inc.     

HSV-tk/GCV gene therapy mediated by EBV-LMP1 for EBV-associated cancer

Background

To investigate the feasibility of gene therapy in treating Epstein-Barr virus (EBV)-associated cancer by employing the suicide gene, herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV), which uses the signaling pathway through the HIV-long terminal repeat (LTR) gene which is expressed from a nuclear factor-κB (NF-κB)-binding motif-containing promoter that is regulated by EBV-latent membrane protein 1 (LMP1) via NF-κB.

Methods

First, we constructed the plasmid pVLTR-tk, which was regulated by EBV-LMP1 via NF-κB, and then investigated the cytotoxic effect of the pVLTR-tk/GCV on cancer cells, using MTT assays, clonogenic assays, flow cytometry, and animal experiments.

Results

The activation of TK was increased after transfection of the pVLTR-tk into the EBV-LMP1 positive cells. After GCV treatment, the clonogenicity and survival of the cells substantially declined, and a bystander effect was also observed. The LMP1 positive cells exhibited remarkable apoptosis following pVLTR-tk/GCV treatment, and the pVLTR-tk/GCV restrained tumor growth in vivo for EBV-LMP1 positive cancers.

Conclusion

The pVLTR-tk/GCV suicide gene system may be used as a new gene targeting strategy for EBV-associated cancer.     

The latest advances of experimental research on targeted gene therapy for prostate cancer

The absence of ef ective therapies for castration-resistant prostate cancer (CRPC) establishes the need to de-velop novel therapeutic modality, such as targeted gene therapy, which is ideal for the treatment of CRPC. But its application has been limited due to lack of favorable gene vector and the reduction of“bystander ef ect”. Consequently, scientists al over the world focus their main experimental research on the fol owing four aspects:targeted gene, vector, transfer means and comprehensive therapy. In this paper, we reviewed the latest advances of experimental research on targeted gene therapy for prostate cancer .     

Combination effect of oncolytic adenovirotherapy and TRAIL gene therapy in syngeneic murine breast cancer models

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy and oncolytic adenovirotherapy have been investigated extensively in xenografic human tumor models established in immunocompromised nude mice. However, the effects of these therapies on syngeneic murine tumors in immunocompetent settings were not well documented. We hypothesized that TRAIL gene therapy used with an oncolytic adenovirus would overcome the weaknesses of the two therapies used individually. In this study, we evaluated the antitumor effects of an oncolytic adenovirus, Delta24, in both human and murine breast cancer cell lines. We also analyzed the effects of TRAIL gene therapy combined with oncolytic virotherapy in these cancer cells. Our results showed that Delta24 can replicate and help the E1-deleted adenovector replicate in murine cancer cells. We also found that these two therapies combined had greater antitumor activity than either one alone in both human and murine breast cancer cells lines and in the syngeneic breast cancer models established in immunocompetent mice. Moreover, Delta24 virotherapy alone and combined with TRAIL gene therapy dramatically reduced the spontaneous liver metastasis that originated in the subcutaneous 4T1 tumor established in Balb/c mice. These findings provide important considerations in the development and preclinical assessments of oncolytic virotherapy.     

Transcription-targeted gene therapy for androgen-independent prostate cancer

The Escherichia coli enzyme (purine nucleoside phosphorylase, PNP) gene is delivered directly into PC3 tumors by one injection of replication-deficient human type-5 adenovirus (Ad5). Expressed PNP converts the systemically administered prodrug, 6MPDR, to a toxic purine, 6MP, causing cell death. We sought to increase the specificity of recombinant Ad vectors by controlling PNP expression with the promoter region from the androgen-dependent, prostate-specific rat probasin (Pb) gene. To increase its activity, the promoter was combined with the SV40 enhancer (SVPb). Cell lines were transfected with plasmids containing both a reporter gene, under SVPb control, and a reference gene cassette to allow normalization of expression levels. Plasmids expressed approximately 20-fold more reporter in prostate cancer than in other cells, but surprisingly, the SVPb element was both androgen-independent and retained substantial prostate specificity. Killing by Ad5-SVPb-PNP vector of cell lines cultured with 6MPDR for 6 days was 5- to 10-fold greater in prostate cancer than in liver or lung cells. In vivo, a single intratumoral injection of Ad5-SVPb-PNP (4 x 10(8) pfu), followed by 6MPDR administration twice daily for 6 days, significantly suppressed the growth of human prostate tumors in nude mice and increased their survival compared to control animals. Thus, the androgen-independent, prostate-targeting Ad5 vector reduces human prostate cancer growth significantly in vitro and in vivo. This first example of an androgen-independent vector points the way toward treatment of emerging androgen-independent prostate cancer in conjunction with hormone ablation therapy at a time when the tumor burden is low.     

Mouse macrophage metalloelastase gene delivery by HVJ-cationic liposomes in experimental antiangiogenic gene therapy for murine CT-26 colon cancer

We previously demonstrated that gene replacement of mouse macrophage metalloelastase (MME) into murine melanoma cells that grow rapidly and are MME deficient suppresses the primary tumor growth in vivo by halting angiogenesis. The aim of the present study was to evaluate the effectiveness of gene therapy against cancer using a cDNA-encoding MME gene. In a subcutaneous tumor model of CT-26 mouse colon cancer cells that are MME deficient, syngeneic mice repetitively treated with direct injections into the tumors of MME- hemagglutinating virus of Japan (HVJ), a type of HVJ-cationic liposome encapsulating a plasmid expressing MME, developed smaller tumors (210 +/- 47.2 mm(3) versus 925 +/- 156 mm(3) mean +/- SEM; p = 0.0004) with fewer microvessels (10.25 +/- 1.03 vs. 17.25 +/- 2.14; p = 0.03) than control mice. TUNEL staining revealed a significant increase of apoptotic cells in the MME-HVJ liposomes-treated tumors compared with control tumors. MME was effectively expressed in the s.c. tumors treated with MME-HVJ liposomes, inducing angiostatin generation in those tumors, as demonstrated by Western blot analysis. In conclusion, our study demonstrated that repeated in vivo transduction of the MME gene directly into the tumors using HVJ-cationic liposomes suppressed the tumor growth by an antiangiogenic mechanism, providing, then, a feasible strategy for gene therapy of cancer.     

AFP启动子与胸苷激酶基因治疗肝癌的实验研究

目的:探讨AFP启动子与胸苷激酶疗法对肝癌的作用.方法:采用含tk基因与潮霉素磷酸转移酶hy基因融合基因的真核表达载体及仅含潮霉素磷酸转移酶hy的空载体,以脂质体为介导,将这两种质粒分别转染肝癌细胞系HEPG2.潮霉素 B药物敏感实验筛选HEPG2,不同浓度丙氧鸟苷分别作用于 HEPG2,HEPG2-AFP-hytk 及 HEPG2-hy.结果:潮霉素B药物敏感实验筛选HEPG2,12天时HEPG2全部死亡的最低浓度为60μg/ml,30μmol/L丙氧鸟苷可最大限度杀死HEPG2-AFP-hytk.杀伤效应随时间而增强,作用24小时后开始出现毒性作用,至96小时死亡率>80%.仅含有18%的HEPG2-AFP-hytk 就可使周围20% 的HEPG2细胞死亡,总死亡率为38%.结论:60μg /ml潮霉素是筛选 HEPG2-AFP-hytk及 HEPG2-hy 阳性克隆的最佳剂量.30μmol/L 丙氧鸟苷是作用于HEPG2-AFP-hytk的最佳剂量.HSV-tk/GCV系统大于18%HEPG2-AFP-hytk开始具有旁观者效应,可提高疗效.     

HSV—tk／GCV系统对神经胶质瘤的自杀基因治疗研究

目的在ｉｎｖｉｔｒｏ和ｉｎｖｉｖｏ水平建立致死神经胶质瘤的ＨＳＶ－ｔｋ／ＧＣＶ自杀基因系统,并验证该系统的有效性。方法用携带单纯疱疹病毒胸苷激酶（ＨＳＶ－ｔｋ）基因的重组逆转录病毒,感染大鼠神经胶质瘤细胞Ｃ６,筛选稳定表达ｔｋ的克隆细胞Ｃ６／ｔｋ;经生长抑制试验,比较Ｃ６／ｔｋ细胞对三种核苷类似物ＧＣＶ、ＢＶｄＵ和ＡＣＶ的敏感性;Ｃ６／ｔｋ细胞在裸鼠皮下成瘤,用ＧＣＶ进行治疗并观察疗效。结果ｔｋ基因整合入Ｃ６细胞并在Ｃ６／ｔｋ细胞中稳定表达;生长抑制试验表明,ＧＣＶ是最为有效的原药,Ｃ６／ｔｋ细胞对ＧＣＶ高度敏感,ＩＣ５０＜０．２μｍｏｌ／Ｌ,而野生型Ｃ６细胞和转染空载病毒载体的Ｃ６／０细胞对ＧＣＶ的ＩＣ５０≥１００μｍｏｌ／Ｌ,相差５００多倍。裸鼠ｉｎｖｉｖｏ实验得到相应结果,治疗组肿瘤受到明显抑制和杀伤。结论在ｉｎｖｉｔｒｏ和ｉｎｖｉｖｏ水平,表达ｔｋ基因的肿瘤细胞均被ＧＣＶ有效杀伤,这表明ＨＳＶ－ｔｋ／ＧＣＶ自杀基因系统有可能成为基因治疗脑瘤的有效方法。