T细胞及相关免疫分子在淋巴细胞趋化因子增强肿瘤自杀基因疗法中的作用

本室以往的研究表明,用腺病毒作为载体将大肠杆菌胞嘧啶脱氨酶(CD)基因与小鼠淋巴细胞趋化因子(Itn)基因联合体内转染,具有显著的抗肿瘤效应.本文对其免疫机理进行了进一步研究,发现CT26结肠腺癌细胞体外经过CD/Ltn基因转染并给予前体药物5-FC后,CT26结肠腺癌细胞表达CD80和CD54分子明显增加,提示经CD自杀基因和Ltn基因联转移后,肿瘤细胞免疫原性增加.荷瘤小鼠体内经联合治疗后,小鼠脾细胞分泌IL-2和IFN-γ明显增加.体内用抗CD4、CD8抗体阻断实验研究结果的表明,联合应用自杀基因和Ltn基因治疗主要通过诱导CD8~ T细胞发挥抗肿瘤作用.     

联合自杀基因与IL-2基因转移疗法的抗肿瘤效果及其抗肿瘤免疫应答的诱导作用

单用自杀基因疗法或单用细胞因子基因疗法抗肿瘤效果不理想,本研究中我们观察了大肠杆菌胞嘧啶脱胺酶(CD)基因与白细胞介素2(IL-2)基因联合转移对荷瘤小鼠的治疗效果及其对抗肿瘤免疫的诱导作用。复制荷瘤小鼠模型后在荷瘤部位注射表达CD基因的重组腺病毒(AdCD)及表达小鼠IL-2基因的重组腺病毒(AdIL2),并连续10天、每天1次腹腔注射5氟胞嘧啶(5FC)对荷瘤小鼠进行治疗。结果表明,AdCD/5FC/AdIL2联合基因治疗能显著抑制荷瘤小鼠皮下肿瘤的生长,并明显延长其生存期(P<0.01)。联合基因治疗组小鼠肿瘤细胞发生明显的坏死,瘤内及瘤周有大量的炎性细胞浸润,瘤内CD4~ 和CD8~ T细胞明显增加,脾细胞NK和CIL杀伤活性明显高于单用AdCD/5FC、对照病毒AdLacZ/5FC或PBS组。实验结果表明,联合应用自杀基因与细胞因子基因治疗可更有效诱导机体的抗肿瘤免疫反应,从而更显著地抑制荷瘤小鼠肿瘤的生长。     

IL-2基因与自杀基因联合转移对小鼠红白血病的治疗作用

用腺病毒作为载体,将大肠杆菌胞嘧啶脱氨酶(CD)基因体外转染小鼠红白血病FBL-3细胞,结果CD基因转移后该细胞对5-氟胞嘧啶(5-FC)的敏感性提高了近1000倍,FBL-3细胞有明显的凋亡发生,且观察到明显的旁观者效应.小鼠体内接种FBL-3红白血病细胞后3天,肿瘤局部注射小鼠白细胞介素2(IL-2)基因的表达载体(Ad-IL-2)和Ad-CD腺病毒载体,然后连续10天给予5-FC 300mg/kg行治疗.结果FBL-3皮下肿瘤的生长明显受到抑制,部分小鼠肿瘤消失.联合治疗小鼠的存活期明显大于单用Ad-IL-2治疗组和单用自杀基因CD与5-FC治疗组.体内免疫功能检测表明,经小鼠自杀基因与IL-2联合基因治疗后小鼠脾细胞的CTL杀伤     

大肠杆菌CD自杀基因转染诱导肿细胞凋亡及旁观者效应的研究

以重组腺病毒AdCD为载体将大肠杆菌胞嘧啶脱氨酶(CD)基因体外传染小鼠红白血病细胞FBL3,结果显示,转染了CD基因的FBL3细胞对5-氟胞嘧啶(5-FC)的敏感性显著提高,进一步研究发现,AdCD/5-FC系统可以诱导肿瘤细胞发生凋亡;将经AdCD/5-FC处理过的FBL3细胞上清倍比稀释后,加入到野生型FBL3细胞中,发现当上清仅占6.25％时,即对野生型FBL3细胞发挥明显的杀伤作用,提示旁观者效应在AdCD介导的细胞毒作用中起着重要的作用。本实验还观察了CD基因体内转染后的杀伤效果,荷瘤小鼠局部注射AdCD并连续10天给予5-FC(300mg/kg)治疗后,小鼠皮下肿瘤结节的生长受到明显抑制。     

腺病毒介导的胞嘧啶脱氨酶自杀基因联合IL-2基因疗法的肿瘤治疗作用及免疫机理分析

目的以腺病毒作为载体，将大肠杆菌胞嘧啶脱氨酶（ＣＤ）基因与小鼠ＩＬ－２基因联合转移，研究其体内抗肿瘤作用及免疫机理。方法小鼠皮下接种黑色素瘤Ｂ１６Ｆ１０细胞后３天，肿瘤局部注射表达ＩＬ－２的重组腺病毒ＡｄＩＬ－２和表达ＣＤ的重组腺病毒ＡｄＣＤ，然后连续１０天给予５－氟胞嘧啶（５－Ｆｃ）３００ｍｇ／ｋｇ进行治疗。结果联合治疗组荷瘤小鼠皮下肿瘤结节的生长明显受到抑制，小鼠存活期明显长于ＡｄＩＬ－２、ＡｄＣＤ／５－Ｆｃ、ＡｄｌａｃＺ／５－Ｆｃ或ＰＢＳ组。经联合治疗后，小鼠脾细胞的ＮＫ活性和ＣＴＬ杀伤活性明显增强；肿瘤瘤体内ＣＤ４、ＣＤ８细胞浸润增加；肿瘤细胞表达Ｈ－２Ｋｂ和Ｂ７－１分子明显增加。结论联合应用自杀基因和ＩＬ－２基因治疗，一方面可以明显抑制荷瘤小鼠肿瘤生长，另一方面可以提高机体对肿瘤细胞免疫应答，增加机体的抗肿瘤作用，是肿瘤基因治疗中一条行之有效的途径。     

自杀基因与GM-CSF基因联合治疗对小鼠黑色素瘤的治疗作用及其机理研究

将小鼠粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因转移至肿瘤细胞内可以大大增加肿瘤细胞的免疫原性,有效诱导肿瘤细胞特异性的抗肿瘤免疫,但是该作用不够强大,对已建立的肿瘤作用较弱.而自杀基因治疗虽然可以消除肿瘤负荷,但是不能有效地诱导抗肿瘤免疫应答.本研究以腺病毒作用载体,将小鼠GM-CSF基因与大肠杆菌胞嘧啶脱氨酶基因(CD)联合转移,在体内可以观察到显著的抗肿瘤作用.小鼠体内接种黑色素瘤B16F10细胞后3天,肿瘤局部注射表达GM-CSF的腺病毒载体Ad-GM-CSF和表达CD基因     

p16基因重组质粒的构建及其对人肺癌细胞的抑制作用

表达大肠杆菌胞嘧啶脱胺酶(CD)基因的重组腺病毒AdCD体外转染小鼠黑色素瘤细胞B16F10,结果显示转染了CD基因的B16F10细胞对5-氟胞嘧啶(5FC)的敏感性显著提高.将经AdCD/5FC系统处理的B16F10细胞上清倍比稀释后.加至野生型B16F10细胞中,发现当上清仅占6.25%时即可对野生型B16F10细胞发挥明显的杀伤作用,提示AdCD/5FC介导的旁观者效应可能是通过5FC经CD酶代谢产生的毒性产物扩散而实现的.本实验还观察了CD基因体内转染后的杀伤效果,荷瘤小鼠经注射AdCD并连续10天给予5FC治疗后,与PBS、对照病毒AdLacZ/5FC治疗小鼠比较,小鼠肿瘤生长明显受到抑制,小鼠存活期明显延长.     

CD自杀基因联合淋巴细胞趋化因子基因疗法的肿瘤治疗？…

本研究以腺病毒作为载体，将大肠杆菌胸嘧啶脱氨酶（ＣＤ）基因与小鼠淋巴细胞趋化因子（Ｌｔｎ）基因体内联合转染，观察了其抗肿瘤效应并分析了免疫机理。小鼠皮下接种结肠腺癌ＣＴ２６细胞后３天，肿瘤局部注射表达Ｌｔｎ的重组腺病毒ＡｄＬｔｎ和表达ＣＤ的重组腺病毒ＡｄＣＤ，然后连续１０天给予５－氟胸嘧啶（５－ＦＣ）３００ｍｇ／ｋｇ进行治疗，结果表明，联合治疗组荷瘤小鼠皮下肿瘤结节的生长受到明显抑制，小鼠存活期明     

腺病毒介导CD/5FC“自杀基因”疗法对人肝癌裸鼠和小鼠结肠腺癌的治疗作用研究

“自杀基因”疗法应用于肿瘤治疗时,通常是在肿瘤局部直接转染“自杀基因”,以期达到在肿瘤局部发挥抗肿瘤作用而避免全身毒性.在本实验中,我们观察了腺病毒介导的“自杀基因”疗法(CD/5FC系统)对人肝细胞癌SMMC-7721及小鼠结肠腺癌CT26的生长抑制作用,证实了腺病毒介导的CD/5FC系统在体外能杀伤人肝癌细胞SMMC-7721,并且能明显地抑制裸鼠SMMC-7721肿瘤模型的生长,对小鼠的结肠腺癌CT26也有一定的治疗作用.同时观察了以AFP启动子驱动的CD基因合并5FC的使用对AFP阳性肝癌细胞的特异性杀伤作用,证实了以肝癌特异启动子驱动的CD基因能在高表达AFP的肝癌细胞HepG2中特异表达,合并5FC的使用能在体外特异地杀伤HepG2细胞.这一组织特异性的“自杀基因”系统能特异地抑制高水平分泌AFP的人肝细胞癌裸鼠模型7721AFP( )的生长,上述研究结果表明,腺病毒介导的CD/5FC系统是一个有效的治疗方案,组织特异性的表达调控更能体现该疗法的实用价值.     

腺病毒介导的CD/5FC自杀基因疗法对小鼠结肠腺癌的治疗作用及相关免疫机理的研究

大肠杆菌胞嘧啶脱氨酶CD可以将前体药物5FC代谢为毒性产物5FU,具有抗肿瘤作用.本课题我们观察了腺病毒介导的CD/5FC自杀基因疗法对小鼠结肠癌生长的治疗作用并探讨其作用机理.结果表明,首先,CD/5FC自杀基因疗法小鼠结肠癌CT26体内外生长均具有显著的抑制作用,4O%的荷瘤小鼠经过治疗后长期存活;其次,我们还研究了自杀基因疗法治疗肿瘤时可能涉及的免疫机理.结果发现在以CD/5FC系统治疗结肠腺癌小鼠过程中能在一定程度上诱导出机体的抗肿瘤免疫反应,包括脾脏CT6L活性的增高、肿瘤局部浸润免疫细胞表达,DEC-205、B7-2、I-A~(b,d)分子增加等.本实验结果提示虽然CD/5FC系统可以诱导机体产生一定的抗肿瘤免疫反应,但其程度不够显著.     

腺病毒介导的胞嘧啶脱氨酶自杀基因联合IL—2基因疗法的肿瘤治 …

以腺病毒作为载体,将大肠杆菌胞嘧啶脱氨酶基因与小鼠ＩＬ－２基因联合转移,研究其体内抗肿瘤作用及免疫机理。方法小鼠皮下接种黑色素瘤Ｂ１６Ｆ１０细胞后３天,肿瘤局部 注射表达ＩＬ－２的重要腺病毒ＡｄＩＬ－２和表达的ＣＤ的重组腺病毒ＡｄＣＤ,然后连续０天给予５－氟胞嘧啶３００ｍｇ／ｋｇ进行治疗。     

Enhanced antitumoral effect of adenovirus-mediated cytosine deaminase gene therapy by induction of antigen-presenting cells through stem cell factor/granulocyte-macrophage colony-stimulating factor gene transfer

Suicide gene therapy has been studied intensively for the treatment of cancer. A limited antitumoral effect was obtained by intratumoral injection of adenovirus harboring Escherichia coli cytosine deaminase gene (AdCD) in tumor-bearing mice followed by continuous administration of 5-fluorocytosine (5FC). To address the drawbacks of the limited potential for the induction of antitumoral immunity by CD suicide gene therapy, we hypothesized that antigen-presenting cells (APCs) might contribute to the efficient induction of an antitumoral immune response in tumor-bearing mice undergoing suicide gene therapy. We preinjected the mice with murine stem cell factor (SCF)-encoding adenovirus (AdSCF) and murine granulocyte-macrophage colony-stimulating factor (GM-CSF)-encoding adenovirus (AdGM-CSF); after 7 days, the mice were inoculated with CT26 colon adenocarcinoma. AdCD was injected intratumorally into tumor-bearing mice followed by 5FC administration. The results showed that AdSCF/AdGM-CSF treatment could increase the number, surface molecule expression, and function of APCs efficiently. A more significant growth inhibition of established tumors and a prolongation of the survival period were observed in tumor-bearing mice after AdSCF/AdGM-CSF pretreatment in combination with AdCD/5FC therapy when compared with mice treated with AdSCF or AdGM-CSF in combination with AdCD/5FC, or AdCD/5FC alone (P < .01). Cytotoxic T-lymphocyte activity was induced efficiently after the combined therapy, and mRNA of tumor necrosis factor-alpha, interleukin-4, interferon-gamma, and interleukin-2 was present in the tumor mass after combined therapy, suggesting that a more potent antitumoral response was induced by enhanced APCs. Our results demonstrated that AdSCF/AdGM-CSF pretreatment could activate APCs, and that these APCs could present the tumor antigens released from AdCD/5FC-killed tumor cells and activate the antitumoral response of the host, thus increasing the therapeutic efficiency of suicide gene therapy.     

氟胞嘧啶/胞嘧啶脱氨酶自杀基因疗法结合热休克蛋白-多肽复合物治疗小鼠胃癌

目的；研究氟胞嘧啶／胞嘧啶脱氨酶（5－FC／CD）基因疗法与热休克蛋白－多肽复合物（FSP70－PC）免疫疗法联合抗肿瘤效果。方法；将携带CD基因的重组腺病毒注射到小鼠MFC瘤体内，腹腔注射5－FC，同时皮下接种HSP70－PC。结果；经联合治疗后，70％荷瘤小鼠肿瘤体答缩小，消退，小鼠存活期延长，细胞毒T淋巴细胞（CTL）杀伤活性增高，CD4^ 及CD^8 T细胞浸润明显。结论：5－FC／CD基因疗法结合HSP－PC免疫疗法抗小鼠MFC瘤作用显著，具有临床应用前景。     

Dose of adenoviral vectors expressing interleukin-2 plays an important role in combined gene therapy with cytosine deaminase/5-fluorocytosine: preclinical consideration.

Using a syngeneic murine model, we investigated the therapeutic efficacy of combined gene therapy using adenoviral vectors expressing murine interleukin-2 (AdmIL-2) and Escherichia coli cytosine deaminase (AdCD). In a subcutaneous tumor model, tumor-bearing mice were treated with an intratumoral injection of adenoviral vectors and received an intraperitoneal administration of 5-fluorocytosine (5-FC). Only the mice treated with AdCD (2 x 10(8) pfu) and an intermediate dose of AdmIL-2 (1 x 10(6) pfu) survived significantly longer than mice treated with AdCD alone (P < 0.01). Moreover, 40% of these treated mice obtained complete remission from tumor-bearing status. The cytotoxicity of splenocytes obtained from the treated mice was related to the survival period. Tumor-specific cytotoxic T lymphocyte assay showed that the cell-mediated cytotoxic response was specific for parental tumor cells. In a hepatic metastasis model, mice treated with an intravenous administration of both AdCD (2 x 10(8) pfu) and an intermediate dose of AdmIL-2 (1 x 10(6) pfu) demonstrated the most significant reduction of metastatic foci and the longest survival following a 5-FC administration. These results suggest that gene therapy combined with AdmIL-2 and AdCD may be a promising strategy for clinical application and, in addition, that translation of combined gene therapy from murine models into the clinical setting will require careful attention to the variables of cytokine expression levels in the design of clinical trials and in the evaluation of treatment efficacy.     

Dose of Adenoviral Vectors Expressing Interleukin-2 Plays an Important Role in Combined Gene Therapy with Cytosine Deaminase/5–Fluorocytosine: Preclinical Consideration

Using a syngeneic murine model, we investigated the therapeutic efficacy of combined gene therapy using adenoviral vectors expressing murine interleukin-2 (AdmIL-2) and Escherichia coli cytosine deaminase (AdCD). In a subcutaneous tumor model, tumor-bearing mice were treated with an intratumoral injection of adenoviral vectors and received an intraperitoneal administration of 5–fluorocytosine (5–FC). Only the mice treated with AdCD (2×10⁸ pfu) and an intermediate dose of AdmIL-2 (1×10⁶ pfu) survived significantly longer than mice treated with AdCD alone ( P <0.01). Moreover, 40% of these treated mice obtained complete remission from tumor-bearing status. The cytotoxicity of splenocytes obtained from the treated mice was related to the survival period. Tumor-specific cytotoxic T lymphocyte assay showed that the cell-mediated cytotoxic response was specific for parental tumor cells. In a hepatic metastasis model, mice treated with an intravenous administration of both AdCD (2×l0⁸ pfu) and an intermediate dose of AdmIL-2 (1×10⁶ pfu) demonstrated the most significant reduction of metastatic foci and the longest survival following a 5–FC administration. These results suggest that gene therapy combined with AdmIL-2 and AdCD may be a promising strategy for clinical application and, in addition, that translation of combined gene therapy from murine models into the clinical setting will require careful attention to the variables of cytokine expression levels in the design of clinical trials and in the evaluation of treatment efficacy.     

自杀基因疗法联合化疗对癌胚抗原表达型肺肿瘤的杀伤作用

目的：探讨阳离子脂质体介导E.coli cd/HSV-1tk融合自杀基因联合长春瑞滨对癌胚抗原阳性肺癌的体内杀伤作用。方法：裸鼠皮下接种肺癌细胞SPCA－1后第5、9、13天,腹腔注射阳离子脂质体／pE-CEAcd-tk复合物,于第6－15天连续腹腔注射5－氟胞嘧碇＋丙氧鸟苷前体药物进行自杀基因治疗。于第5、13天给予长春瑞滨进行联合治疗。结果：阳离子脂质体介导融合自杀基因联合长春瑞滨使小鼠皮下的肿瘤结节的生长受到显著抑制,其平均瘤体体积显著小于单独自杀基因或化疗治疗组的平均瘤体体积（P＜0．01）。结论：阳离子脂质体介导E.coli cd/HSV-1tk融合基因联合长春瑞滨化疗在体内在有效抑制肺瘤的生长,为癌胚抗原阳性肺癌的治疗提供了1条新的途径。