新型基因转移系统介导p21基因抑制人脑胶质瘤的体外实验研究

为克服目前肿瘤基因治疗中载体系统效率低下,缺乏靶向性的不足,研究构建了受体介导的靶向性非病毒型基因转移系统,并利用该系统将p21基因导入体外培养的人脑胶质瘤细胞,观察p21对肿瘤细胞生长的抑制效应.方法:根据受体配体特异性结合理论,鉴于在多种肿瘤细胞表面存在表皮生长因子受体(EGF-R)的过量表达,分别设计合成了针对EGF-R的16肽GE7配体寡肽以及流感病毒血凝素功能域20肽HA20做为内吞小体释放寡肽,将两者分别与多聚阳离子鱼精蛋白(protamine)共价连接,籍静电效应与DNA形成复合体颗粒,构成受体介导的靶向性载体系统.以此载体系统携带β-gal报道基因转染体外培养的人脑胶质瘤细胞U251,x-     

新型基因转移系统在人肝癌基因治疗中的应用

目前基因治疗的主要问题之一是基因导入系统效率低下,缺乏靶向性.病毒类载体,如临床常用的逆转录病毒和腺病毒,体内导入率极低且无靶向性;脂质体等非病毒类载体系统,具有同样的局限性.人们曾经尝试用针对某种受体的配体及多聚阳离子多肽与DNA形成复合物,从而介导外源基因进入受体过度表达的细胞.但是,内吞小体和溶酶体融合后,内吞复合物会被溶酶体的酶系所降解.     

靶向于胰岛素样生长因子受体的基因导入系统

目的：建立一种新的以细胞表面受体为靶向的基因导入系统。方法：根据胰岛素样生长因子Ⅰ号及Ⅱ号受体（IGFIR,IGFⅡR）在人原发性肝癌中过量表达,设计并合成了针对IGFIR及IGFⅡR的14肽E5,同时合成了流感病毒血凝素功能域20肽HA20作为内吞小体释放寡肽（Endosome releasing loigopeptide,EROP),将它们分别与多聚阳离子多肽（Polycatiomic polypeptide,PCP)－多聚赖氨酸（Polylysine,PL)共价连接,通过静电效应与DNA形成受体介导的靶向性非病毒性基因导入系统（E5－PCP,PCP－HA20）。结果：体内、外实验证实此基因导入系统能高效且 相对靶向地将外源基因导入人肝癌细胞并表达。应用此系统将细胞周期素依赖的蛋白激酶抑制物基因p15,p16,p21导入肝癌细胞,明显抑制肝癌细胞生长。体内将p21导入荷瘤裸鼠,显著抑制裸鼠皮下肿瘤的生长。结论：受体介导的非病毒型基因导入系统高效具有相对靶向性,有可能成为新的肝癌基因治疗途径。     

受体介导基因转移的体外研究

目前 ,基因治疗大多使用重组病毒载体 ,转移和表达外源基因的效率较高 ,但所载片断较短、具潜在感染和插入突变的危险性。非病毒载体主要有脂质体和受体介导途径 ,前者虽有较高效率 ,但不具特异靶向性。使用超分子复合物载体 ,通过受体介导内吞作用 ,对真核生物进行外源基因的转移 ,是近十年发展起来的一种非病毒载体基因转移技术 ,可以把目的基因转移到特异组织细胞内。本研究检测受体介导基因转移靶向性和表达效率。首先构建含β ｇａｌ基因的超分子复合物载体 ,零长度二步法使转铁蛋白和多聚赖氨酸发生共价交联 ,在适当的反应条件下 ,质…     

完整肽聚糖对裸鼠腹腔巨噬细胞分泌和杀瘤功能的影响

目的：探索靶向性非病毒载体系统在人脑腕质瘤基因治疗中的应用。方法：组了ＥＧＦ－Ｒ介导的ＧＥ７基因转移系统,分别与β－ａｌ报道基因和Ｐ２１基因构成载体复合物,体外转染Ｕ２５１细胞,通过Ｘ－ｇａｌ染色、Ｗｅｓｔｅｒｎ ｂｌｏｔ分析,原位木端标记Ｄ梯带检测等,观察了外源基因的导入及对肿瘤细胞的抑制作用。结果：Ｘｇａｌ染色表明,外源基因的导入率可达８０％。转染Ｐ２１基因后,细胞生长受到明显抑制第７天生长抑     

靶向性非病毒载体GE7系统治疗人脑胶质瘤的体内外研究

目的：研究EGFR介导的靶向性非病毒载体GE7系统治疗人脑胶质瘤的体内外效应。方法：组建GE7基因转移系统，分别与βgal报道基因、p21WAF1/CIP1基因组成复合物，体外转导U251MG细胞，分析GE7系统导入效率；MTS法观察细胞生长曲线；FACS分析细胞周期变化。建立裸鼠皮下荷瘤模型，分别经瘤内和尾静脉注射导入DNA载体复合物，观察肿瘤生长抑制率。结果：GE7系统体外导入率最高达70%，细胞生长曲线呈低平型，细胞周期阻滞于G1期，平均凋亡指数为25.2%；瘤内注射和尾静脉注射抑瘤率分别为56.5%和60.2%。结论：GE7系统具有较高效率和靶向性，经肿瘤局部和全身途径导入p21WAF1/CIP1基因，均可诱导凋亡，抑制肿瘤生长。     

HSV－tk基因治疗人脑胶质瘤SHG_（44）的实验研究

为了研究脑肿瘤的基因治疗，构建了含有ｐｇｋ启动子驱动的ＨＳＶ－ｔｋ基因的反转录病毒载体ｐＬＮＴＫ，并成功地转移至人脑胶质瘤ＳＨＧ４４细胞。经体外实验证实，ＳＨＧＬＮＴＫ对阿昔洛韦（Ａｃｙｃｌｏｖｉｒ，ＡＣＶ）的杀伤敏感性高于ＳＨＧ４４约１０００倍，３Ｈ－ＴｄＲ掺入法证实，ＡＣＶ能够明显抑制ＳＨＧＬＮＴＫ的ＤＮＡ合成。体内实验证实，ＡＣＶ可抑制ＳＨＧＬＮＴＫ细胞在裸小鼠体内的肿瘤形成，原位基因转移治疗裸小鼠ＳＨＧ４４肿瘤效果明显。提示其可作为脑肿瘤治疗的一种新办法。     

多聚赖氨酸-硅纳米颗粒的生物相容性研究

背景与目的：多聚赖氨酸-硅纳米颗粒是一种新型的非病毒纳米颗粒基因传递载体。本研究主要阐述其生物相容性,为其进一步的体内应用提供实验依据。方法：细胞转染和流式细胞仪分析在含血清培养基中多聚赖氨酸-硅纳米颗粒介导质粒DNA和反义寡核苷酸的转染能力,随后通过血浆蛋白反应滤过试验和红细胞聚集试验进一步评价其生物相容性。结果：在含血清培养基中,多聚赖氨酸-硅纳米颗粒介导质粒DNA和反义寡核苷酸传递的能力显著下降。多聚赖氨酸-硅纳米颗粒／DNA(／ODN)复合物可与小鼠血浆蛋白成分结合,并可引起红细胞聚集。结论：多聚赖氨酸-硅纳米颗粒可能与含血清培养基中的血浆蛋白相互作用,影响体外细胞转染效率。多聚赖氨酸-硅纳米颗粒可能与体内外周血血浆蛋白和外周血红细胞反应而影响其体内基因传递能力,其生物相容性还有待于进一步提高。     

逆转录病毒载体介导人GM—CSF基因在HL—60白血病细胞的高效转…

探讨将外源基因导入白血病细胞的条件及可行性,为造血系统恶性肿瘤的基因治疗奠定基础。方法采用脂质体（ｌｉｐｆｅｃｔｉｎ）包装技术将Ｎ２Ａ／ＣＭＶ／ｈＧＭ－ＣＳＦ导入包装细胞ＰＡ３１７,获得重组逆转录病毒,将重组病毒悬液感染人髓系白血病ＨＬ－６０细胞,经Ｇ４１８筛选出ＨＬ－６０转基因细胞群,应１用ＭＴＴ法测定ＧＭ－ＣＳＦ活性。结果ＰＣＲ及Ｓｏｕｔｈｅｒｎｂｌｏｔ检测结果证实,ＧＭ－ＣＳＦ基因成功地转移     

逆转录病毒载体介导人 GM-CSF 基因在 HL-60 白血病细胞的高效转移和表达

目的探讨将外源基因导入白血病细胞的条件及可行性，为造血系统恶性肿瘤的基因治疗奠定基础。方法采用脂质体（ｌｉｐｏｆｅｃｔｉｎ）包装技术将Ｎ２Ａ／ＣＭＶ／ｈＧＭ－ＣＳＦ导入包装细胞ＰＡ３１７，获得重组逆转录病毒，将重组病毒悬液感染人髓系白血病ＨＬ－６０细胞，经Ｇ４１８筛选出ＨＬ－６０转基因细胞群，应１用ＭＴＴ法测定ＧＭ－ＣＳＦ活性。结果ＰＣＲ及Ｓｏｕｔｈｅｒｎｂｌｏｔ检测结果证实，ＧＭ－ＣＳＦ基因成功地转移并整合到ＨＬ－６０细胞基因组中，而未转基因和转空载体Ｎ２Ａ的ＨＬ－６０细胞经ＰＣＲ检测未能扩增出特异性片段。经ＧＭ－ＣＳＦ依赖株ＴＦ－１检测，转基因细胞分泌的ＧＭ－ＣＳＦ生物学活性为６０～２００ｎｇ／ｍｌ（细胞１×１０６，培养２４小时），而未转基因及转空载体Ｎ２Ａ的ＨＬ－６０细胞培养上清无ＧＭ－ＣＳＦ活性。结论逆转录病毒载体介导的ｈＧＭ－ＣＳＦ基因能在体外培养的人ＨＬ－６０白血病细胞中获得高效的转移和表达。     

增殖病毒对肿瘤的治疗作用

目的：探讨ＨＥＲ２／ｎｅｕ原癌基因胞外区片段ＤＮＡ免疫诱导的特异性细胞免疫应答及其在体内的抗瘤效应。方法：克隆并构建人ＨＥＲ２／ｎｅｕ原癌基因及其胞外区片段表达载体,分别转染ＳＰ２／０细胞以制备杀伤靶细胞。将质粒ＤＮＡ免疫小鼠,观察其诱导的细胞免疫应答,同时分析免疫动物体内抗瘤效应。结果：体外获得稳定表达ＨＥＲ２／ｎｅｕ基因的ＳＰ２／０靶细胞。目的基因ＤＮＡ免疫后,免疫鼠脾细胞在体外可检测到特异性杀伤作用,体内肿瘤细胞接种后可发现肿瘤结节出现时间延迟,肿瘤生长缓慢。结论： ＨＥＲ２／ｎｅｕ原癌基因胞外区片段ＤＮＡ免疫可诱导出特异细胞免疫应答,并具有一定抗瘤效应。     

重组人白介素2-卡介苗对荷瘤小鼠腹腔巨噬细胞体外杀伤活性的影响 *

目的：研究ＨＥＲ２／ｎｅｕ原癌基因胞外区片段（ＥＣＤ）ＤＮＡ直接肌肉注射在小鼠体内表达,体液免疫状况,利用小鼠流产模型观察其体内效应。方法：克隆并构建了人及大鼠ＨＥＲ２／ｎｅｕ原癌基因胞外区片段真核表达载体ｐＣＤＮＡ３－Ｘ,采用肌肉直接注射的方法免疫小鼠,分析目的基因在小鼠体内的表达及其诱导的兔疫应答。结果：在质粒ＤＮＡ直接注射部位可检测到目的基因的表达,并诱导了针对ＨＥＲ２／ｎｅｕ的抗体反应;同时诱发小鼠流产,使免疫鼠产仔数减少,可观察到流产结节形成。结论：通过基因免疫,可诱导针对ＨＥＲ２／ｎｅｕ原癌基因产物的有效免疫应答。     

HER2/neu肽诱导BALB/c小鼠产生特异性CTL及其抑瘤作用的研究

目的：探讨来源于ＨＥＲ２／ｎｅｕ原癌蛋白的多肽诱导特异性细胞免疫应答及其在体内的抗癌效应。方法：利用合成的２个具有小鼠Ｈ－２Ｋ＾ｄ分子结合基序的ＨＥＲ２／ｎｅｕ肽作为肿瘤排斥抗原,在体外刺激小鼠淋巴细胞以及皮下免疫小鼠,观察淋巴细胞的增殖能力、ＣＴＬ的杀伤活性以及ＨＥＲ２／ｎｅｕ肽的抑瘤作用。结果：ＨＥＲ２／ｎｅｕ肽在体内和体外对ＢＡＬＢ／ｃ小鼠淋巴细胞的增殖都有显显的促进作用。ＨＥＲ２／ｎｅｕ肽免疫ＢＡＬＢ／ｃ小鼠后,可以从小鼠淋巴细胞中诱导出肽特异性ＣＴＬ,这些ＣＴＬ可以特异地杀伤ＨＥＲ２／ｎｅｕ阳性ＳＰ２／０细胞,而对ＨＥＲ２／ｎｅｕ阴性ＳＰ２／０细胞却无明显的杀伤活性。ＳＰ２／０ＨＥＲ２细胞在ＨＥＲ２／ｎｅｕ肽免疫小鼠体内的生长受到抑制。结论：研究结果表明ＨＥＲ２／ｎｅｕ肽可以起到一定的抑瘤保护作用,提     

Antibody and CD8+ T cell responses against HER2/neu required for tumor eradication after DNA immunization with a Flt-3 ligand fusion vaccine.

PURPOSE: HER2/neu is frequently overexpressed in breast cancer. In a mouse model, vaccination with HER2/neu DNA elicits antibodies that confer partial protection against tumor challenge. EXPERIMENTAL DESIGN: To enhance antitumor immunity, we fused cDNA encoding Flt-3 ligand (FL) to the rat HER2/neu extracellular domain (neu), generating a chimeric FLneu molecule. FLneu and neu DNA vaccines were compared for immunogenicity and their ability to protect mice from tumor challenge. RESULTS: The neu vaccine generated a HER2/neu-specific antibody response. In contrast, vaccination with FLneu induced CD8+ T cells specific for HER2/neu but a negligible anti-HER2/neu antibody response. The switch from an antibody-mediated to T cell-mediated response was due to different intracellular localization of neu and FLneu. Although the neu protein was secreted, the FLneu protein was retained inside the cell, co-localizing with the endoplasmic reticulum, facilitating processing and presentation to T cells. The neu and FLneu vaccines individually conferred only weak tumor immunity. However, efficient tumor rejection was seen when neu and FLneu were combined, inducing both strong anti-HER2/neu-specific antibody and T cell responses. Adoptive transfer of both immune CD8+ T cells and immune sera from immunized mice was required to confer tumor immunity in na?ve hosts. CONCLUSIONS: These results show that active induction of both humoral and cellular immunity to HER2/neu is required for efficient tumor protection, and that neither response alone is sufficient.     

Delayed tumor onset and reduced tumor growth progression after immunization with a Her-2/neu multi-peptide vaccine and IL-12 in c-<Emphasis Type="Italic">neu</Emphasis> transgenic mice

Passive immunotherapy with monoclonal antibodies is a routinely performed but cost intensive treatment against certain cancers. Induction of humoral anti-tumor responses by active peptide immunization has therefore become a favorable treatment concept. We have recently identified three peptides representing B-cell epitopes of the extracellular domain of Her-2/neu each of them inducing Her-2/neu specific immune responses with anti-tumor activity in vitro. The present study was performed to evaluate the in vivo protective capacity of a combined vaccination with these three peptides in FVB/N transgenic mice spontaneously developing c-neu overexpressing breast cancers. The three Her-2/neu peptides coupled to tetanus toxoid were administered with or without addition of recombinant IL-12. At the time all untreated mice had developed tumors about 40% of peptide-immunized mice and nearly 60% of mice immunized with the peptide vaccine co-applied with IL-12 remained tumor free. Moreover, co-administration of IL-12 had a significant impact on the retardation of tumor progression. The enhanced anti-tumor efficacy of the vaccine by IL-12 was associated with a Th1 biased immune response as demonstrated by an increased IFN-γ production in vitro and elevated Her-2-specific IgG levels. Our findings clearly demonstrate that this multi-peptide vaccine is effective in tumor prevention and support its use against minimal disease, drug-resistant tumors or even for prophylaxis against cancers overexpressing Her-2/neu. Stefan Wagner and Joanna Jasinska contributed equally to this work.     

异位hCGβ基因免疫诱导的特异性抗肿瘤免疫应答

目的　观察异位hCGβ基因免疫诱导的肿瘤特异性免疫应答及其抗肿瘤作用 ,为肿瘤的免疫生物治疗寻求新途径。方法　构建含hCGβ编码基因的质粒TR4 2 1 hCGβ ,对BALB/c小鼠实施基因免疫 ,并以空质粒为对照。采用ELISA法和3 H TdR掺入法分别检测免疫小鼠血清中特异性抗hCGβ IgG抗体及其对肿瘤细胞体外生长的抑制作用 ;特异抗原体外刺激免疫小鼠脾细胞后 ,用3 H TdR掺入法和3 H TdR释放法分别检测其特异性增殖活性和细胞毒活性 ;皮下接种SP2 /0 hCGβ细胞攻击免疫小鼠 ,以成瘤率及实体瘤重量评估体内抗瘤效果。结果　全部TR4 2 1 hCGβ质粒免疫小鼠均产生高水平的抗hCGβ IgG抗体 ,该抗体可抑制肿瘤细胞的体外生长 ,与对照血清相比 ,差异有显著差性 (P <0 .0 5 ) ;hCGβ蛋白、灭活SP2 /0 hCGβ细胞以及两者混合均能刺激TR4 2 1 hCGβ质粒免疫小鼠脾细胞的体外增殖 (SI值分别为 1.5 3、1.81和 2 .0 5 ) ,与空质粒免疫小鼠相比 ,差异有显著性 (P <0 .0 1) ;特异抗原刺激的TR4 2 1 hCGβ质粒免疫小鼠脾细胞对SP2 /0 hCGβ细胞的细胞毒活性明显高于SP2 /0细胞(P <0 .0 1) ;空质粒免疫小鼠接种SP2 /0 hCGβ细胞后成瘤率为 10 0 % ,瘤重达 3.37g ,而TR4 2 1 hCGβ质粒免疫小鼠的成瘤率为 16 .6 6 % ,瘤重为     

Coexpression of Flt3 ligand and GM-CSF genes modulates immune responses induced by HER2/neu DNA vaccine

DNA vaccine and dendritic cells (DCs)-based vaccine have emerged as promising strategies for cancer immunotherapy. Fms-like tyrosine kinase 3-ligand (Flt3L) and granulocyte-macrophage-colony-stimulating factor (GM-CSF) have been exploited for the expansion of DC. It was reported previously that combination of plasmid encoding GM-CSF with HER2/neu DNA vaccine induced predominantly CD4(+) T-cell-mediated antitumor immune response. In this study, we investigated the modulation of immune responses by murine Flt3L and GM-CSF, which acted as genetic adjuvants in the forms of bicistronic (pFLAG) and monocistronic (pFL and pGM) plasmids for HER2/neu DNA vaccine (pN-neu). Coexpression of Flt3L and GM-CSF significantly enhanced maturation and antigen-presentation abilities of splenic DC. Increased numbers of infiltrating DC at the immunization site, higher interferon-gamma production, and enhanced cytolytic activities by splenocytes were prominent in mice vaccinated with pN-neu in conjunction with pFLAG. Importantly, a potent CD8(+) T-cell-mediated antitumor immunity against bladder tumors naturally overexpressing HER2/neu was induced in the vaccinated mice. Collectively, our results indicate that murine Flt3L and GM-CSF genes coexpressed by a bicistronic plasmid modulate the class of immune responses and may be superior to those codelivered by two separate monocistronic plasmids as the genetic adjuvants for HER2/neu DNA vaccine.     

Production of antibodies by single DNA immunization: comparison of various immunization routes

DNA immunization is a recent vaccination method that induces humoral and cellular immune responses in a range of hosts. Different immunization routes induce a different degree of the immune response. In the present report, we demonstrate that multiple intramuscular immunizations of plasmid DNA encoding various leukocyte surface molecules induced a specific antibody response. In contrast, a single intramuscular immunization could not induce antibody production. To study the induction of antibody response after a single immunization of plasmid DNA, mice were single-dose intramuscularly, intraperitoneally, intravenously and intrasplenically immunized, simultaneously, with the same preparation of plasmid DNA encoding CD147 membrane protein. We observed that only the intrasplenic route induced specific antibody production. The induction of antibody by intrasplenic immunization was confirmed by using plasmid DNA encoding CD54 molecule. By this single-dose DNA intrasplenic immunization, the generated antibodies could be detected in mice up to 6 months. These results suggest that the injected DNA is expressing the relevant protein antigen in the spleen for several months after injection. Our results demonstrate that direct immunization of antigen-encoding DNA into the spleen is a more effective method for induction of antibody production. This finding may support future investigations of DNA vaccination strategies that specifically promote the uptake of plasmid by splenocytes. Intrasplenic immunization may also be helpful in the understanding of the early events of the immune response to DNA vaccine and be useful as an effective route for the induction of immune responses.     

Anti-tumor activity and immune responses induced by human cancer-associated mucin core peptide

Objective: To investigate the immune responses induced by apomucin which is a mixture of mucin core peptide, in mice for elucidating the role of mucin core peptide in the modulation of cancers. Methods: Apomucin was isolated from human pancreatic cancer cell line SW1990. The mice were immunized with this apomucin (10μg/time×6) plus DETOX. Results: When immunized, all mice developed delayed-type hypersensitivity (DTH) after challenged with apomucin or synthetic peptide MUC-2 or MUC-3, while the mice immunized with apomucin alone did not develop DTH. No antibodies were detected by ELISA after immunization. When the spleen cells of vaccinated mice were cocultured with this apomucin (10–50μg/ml) and rhIL-2(50U/ml)in vitro, the proliferated lymphocytes showed cytotoxicity against human cancer cells, including colon cancer, gastric cancer, pancreatic cancer and leukemia as measured by Cr-51 release assay. Antibodies against MUC-2 and MUC-3 could block the cytotoxicity. Conclusion: It was identified that a vaccine combined of apomucin and immune adjuvant DETOX can induce cellular immune response and anti-tumor cytotoxicity in mice. This study was supported by National Natural Science Foundation of China No. 39570792.     

Adenovirus vaccination against neu oncogene exerts long-term protection from tumorigenesis in BALB/neuT transgenic mice

The transforming rat HER2/neu oncogene (neu), when embedded in the genome of transgenic BALB/c (neuT) mice, provokes the development of an invasive carcinoma in each of their 10 mammary glands. We used the neuT mice model system to evaluate the immunization efficiency and the protective effect of intramuscular injection of adenovirus (Ad) and/or of DNA with electrostimulation (DNA+ES), both expressing the rat p185(neu) protein. A neu cDNA sequence, which exclusively contains codons preferred by highly expressed mammalian genes, was used in this study. This "optimized" cDNA displayed higher expression in cultured cells and greater cell-mediated response than the original gene when injected as DNA+ES. Ad expressing the optimized sequence (Ad5-neu.opt) induced a higher immune response, as measured by the frequency of IFN-gamma-secreting spleen cells and antibody titers. Different Ad/DNA combinations and immunization schedules confirmed the superiority of Ad5-neu.opt in inducing a strong Th1-skewed humoral and CD8(+) cell-mediated response. Two Ad5-neu.opt injections of 10(9) viral particles at week 10 and 12 were sufficient to induce the highest response, which persisted at detectable levels up to 33 weeks of age. Anti-Ad5 antibodies elicited by previous injections neutralized the effect of an additional Ad5-neu.opt immunization at week 19. A group, which received 3 injections of DNA+ES at week 23, 27 and 31, in addition to the 3 Ad injections at week 10, 12 and 19 showed an increased frequency of IFN-gamma(+), CD8(+) PBMC at week 25, which persisted at detectable levels till week 38. Ad5-neu.opt administration at 10 and 12 weeks of age had a significant impact on tumor progression. At 44 weeks, 40% of the mice were completely protected from tumors with a mean tumor of 3.8. In contrast, control mice developed 10 tumors and died by week 27. Vaccination blocked the tumor development at the atypical hyperplasia stage present at the time of treatment. Tumors developing at later times express reduced levels of rat p185(neu) protein.