腺病毒介导IL-2基因转染的瘤苗体内抗肿瘤作用

目的 :探讨重组腺病毒介导的 IL- 2基因转染的瘤苗的体内抗肿瘤作用及其免疫学机制。方法 :应用腺病毒介导的鼠 IL- 2基因转染 CT2 6小鼠结肠癌细胞 ,灭活后用作瘤苗治疗荷瘤小鼠 ,观察皮下肿瘤生长及其存活期。采用乳酸脱氢酶释放法检测荷瘤小鼠脾细胞 CTL、L AK、NK细胞的杀伤活性。结果 :鼠 IL- 2基因转染瘤苗治疗能显著抑制荷瘤小鼠皮下肿瘤生长并明显延长其存活期 (P<0 .0 1)。体内免疫功能检测表明 ,鼠 IL- 2基因转染疫苗治疗组小鼠脾细胞 CTL 活性、L AK活性和 NK活性显著高于对照组 (P<0 .0 1)。结论 :腺病毒介导鼠 IL- 2基因转染的瘤苗体内具有较强的抗肿瘤效应 ,其机制可能是提高了荷瘤小鼠特异性和非特异性抗肿瘤免疫反应     

IL—2基因转染的肿瘤细胞增强机体免疫功能的实验研究

目的：探讨小鼠接种腺病毒介导的IL－2基因转移的CT26小鼠结肠腺癌细胞后机体免疫功能的变化（包括CTL、LAK细胞、NK细胞、巨噬细胞的杀伤活性变化）。方法：体外将腺病毒介导的小鼠IL－2基因转染CT26细胞（CT26-mIL-2)，然后将CT26－mIL2细胞接种于小鼠皮下。采用4h乳酸脱氢酸释放法检测荷瘤小鼠CTL、LAK、NK细胞的杀伤活性，MTT法检测巨噬细胞的杀伤活性。结果：接种CT26－mIL－2后第14d，小鼠脾细胞NK活性和诱导后的LAK活性和CTL活性均显著高于对照组（P＜0．01）；小鼠腹腔巨噬细胞数量显著增加，杀伤活性显著增强（P＜0．01）。结论：IL－2基因转染的肿瘤细胞能诱导机体的特异性和非特异性免疫功能参与机体的抗肿瘤作用。     

重组腺病毒介导mIL-4基因转染的G422胶质母细胞瘤体外生物学特性及体内致瘤性的改变

以重组的复制缺陷性腺病毒为载体将鼠IL-4基因转染G422小鼠脑胶质母细胞瘤细胞,观察其体外生物学特性和体内致瘤性的变化。结果显示:基因转染的肿瘤细胞有目的基因mRNA的表达,分泌高水平的mIL-4,和野生型G422细胞相比,其体外的细胞增殖能力无显著差异,而接种皮下后肿瘤生长延缓、小鼠的存活期延长。     

联合细胞因子基因转染对恶性胶质瘤的治疗作用及其免疫学机理的研究

人脑恶性胶质瘤的发病率高,疗效差,病人平均生存期不到一年.本研究将具有多种免疫学功能的IL-2基因和具有共刺激作用的B7-1分子的基因共转染恶性胶质瘤细胞,通过对其疗效的观察及对机体免疫功能的检测,探讨恶性胶质瘤免疫基因治疗的新途径.方法:①以重组腺病毒作载体,将IL-2基因和B7-1基因体外联合转染G422小鼠胶质母细胞瘤细胞,2h后洗去病毒.取1×10~5细胞,分别作小鼠皮下和脑内注射,观察肿瘤生长情况和动物存活期,注射后     

CD自杀基因联合淋巴细胞趋化因子基因疗法的肿瘤治疗作用及免疫机理研究

本研究以腺病毒作为载体,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5一氟胞嘧啶(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,小鼠存活期明显长于单用AdLtn治疗组或单用AdCD/5-FC治疗组.经联合治疗后小鼠脾细胞的NK活性和对(37结肠腺癌细胞的CTL杀伤活性明显增强.瘤体细胞FACS分析结果表明,经联合基因治疗后,肿瘤组织CD4~ 、CD8~ 细胞浸润增加,结肠腺癌细胞表达H-2Kd和B7-1分子明显增加.提示经CD自杀基因和Ltn基因联合治疗后,肿瘤细胞免疫原性增加.本研究结果表明联合应用自杀基因和Ltn基因治疗可以提高机体对肿瘤细胞免疫的应答,增加机体的抗肿瘤作用,是肿瘤基因治疗中一条新的途径.     

IL—12协同B7—1转基因细胞增强C57BL／6小鼠的抗肿瘤免疫

目的应用逆转录病毒构建IL-12、B7-1表达载体,以研究基因修饰的肿瘤细胞的癌疫苗作用.方法将两种表达载体分别转染EL-4胸腺瘤细胞,使该细胞能表达分泌B7-1或IL-12,并研究了该基因导入细胞的抗肿瘤免疫效果.结果当接种了EL-4/IL-12转染细胞后,在C57BL/6同系鼠中其基因导入细胞的肿瘤原性比较EL-4/Wt和EL-4/Neo组明显减少(P＜0.01),在EL-4/IL-12组的肿瘤被排斥后,实验动物体内诱发了抗EL-4/Wt的全身性、保护性免疫,用51Cr释放测定法证明有一个较强的抗EL-4/Wt和一个较弱的抗同系Lewis肿瘤细胞的CTL活性,体内淋巴细胞消除分析的结果提示减少的肿瘤原性主要与CD4+、CD8+和NK细胞有关.用EL-4/IL-12基因转染的瘤细胞进行疫苗治疗比较用EL-4/Neo瘤细胞作疫苗治疗能更加有效地延缓已建立的EL-4/Wt肿瘤的生长(P＜0.005),EL-4/IL-12和EL-4/B7-1基因联合转染组比用单一的转基因细胞增强了治疗效果(P＜0.005).结论研究结果提示应用IL-12进行血液肿瘤的治疗是有效的,IL-12和B7-1联合使用在未来人类癌症的治疗中有一定的应用前景.     

FasL和B7-1联合基因修饰的肺癌细胞诱导抗肺癌主动免疫

目的:探讨FasL和B7-1联合基因转移是否具有协同的抗肿瘤效应.方法:采用重组腺病毒载体将FasL和B7-1基因导入人肺癌细胞A-549,G418阳性克隆筛选,流式细胞分析,RT-PCR显示FasL和B7-1的表达,并且通过Hoechst33342染色和DNA片段分析检测肺癌细胞的凋亡;将携带FasL和B7-1基因的肺癌细胞(命名为A-549/FB-11)接种于C57BL/6小鼠背部皮下,观测其致瘤性能;A-549/FB-11致敏的小鼠对野生型瘤细胞是否具有免疫保护作用;用A-549和A-549/FB-11细胞分别经腹腔免疫小鼠,得到腹腔浸润淋巴细胞及致敏脾细胞,MTT法检测其体外杀伤实验.结果:FasL和B7-1基因在肺癌细胞中获得高表达并可诱导肺癌细胞的凋亡,双基因转染的肺癌细胞的致瘤性明显下降; A-549/FB-11 诱导的CTL (cytotoxic T lymphocyte)对A-549的杀伤活性显著高于野生型A-549诱导的CTL对相同靶细胞的杀伤活性,P<0.05;A-549 /FB- 11诱导的CTL对A-549/FB-11的杀伤率显著高于对野生型A-549的杀伤率,P<0.05.结论:FasL促进肺癌细胞凋亡,B7-1促进抗肺癌CTL的增殖、活化,在效应阶段两基因发挥着重要的协同作用.     

B7-1基因转染小鼠EL-4淋巴瘤诱导带瘤宿主抗瘤效应的体内研究

本文研究了IL-2、IL-4、IL-6基因转染后B16黑色素瘤细胞表面MHCⅠ类抗原及ICAM-1的表达水平,并探讨了其在CTL诱导过程中的作用.结果表明,IL-2、IL-4、IL-6基因转染的B16黑色素瘤细胞表面MHCⅠ类抗原及ICAM-1表达均高于野生型B16黑色素瘤细胞及转染对照质粒的B16黑色素瘤细胞.体内接种后,小鼠脾脏CTL活性明显增强,CTL培养体系中IFN-γ、TNF-α的分泌水平也增高.在CTL诱导体系中加入抗ICAM-1单抗可以抑制CTL的活化,加入抗MHCⅠ类分子单抗后可使CTL的活化完全阻断.这提示细胞因子基因转染可能通过使肿瘤细胞表面MHCⅠ类抗原、ICAM-1的表达增加,从而增强瘤苗的免疫原性.     

IL-2基因转导自身肿瘤特异性细胞毒T细胞对小鼠实体瘤作用的实验研究

许多研究发现自身肿瘤特异性细胞毒T细胞(CTL)能定位于体内肿瘤所在部位．其杀瘤细胞活性及特异性远在LAK细胞之上，来源和增殖性方面较TIL为优，因此可作为较为理想的载体细胞导入各种治疗基因。将IL-2基因导入CTL中表达并回输．通过其聚集在肿瘤发生局部持续分泌一定量的IL-2，激活机体肿瘤免疫反应或直接杀伤肿瘤细胞，     

IL-12协同B7-1转基因细胞增强C57BL/6小鼠的抗肿瘤免疫

目的:应用逆转录病毒构建IL-12、B7-1表达载体,以研究基因修饰的肿瘤细胞的癌疫苗作用.方法:将两种表达载体分别转染EL-4胸腺瘤细胞,使该细胞能表达分泌B7-1或IL-12,并研究了该基因导入细胞的抗肿瘤免疫效果.结果当接种了EL-4/IL-12转染细胞后,在C57BL/6同系鼠中其基因导入细胞的肿瘤原性比较EL-4/Wt和EL-4/Neo组明显减少(P＜0.01),在EL-4/IL-12组的肿瘤被排斥后,实验动物体内诱发了抗EL-4/Wt的全身性、保护性免疫,用51Cr释放测定法证明有一个较强的抗EL-4/Wt和一个较弱的抗同系Lewis肿瘤细胞的CTL活性,体内淋巴细胞消除分析的结果提示减少的肿瘤原性主要与CD4+、CD8+和NK细胞有关.用EL-4/IL-12基因转染的瘤细胞进行疫苗治疗比较用EL-4/Neo瘤细胞作疫苗治疗能更加有效地延缓已建立的EL-4/Wt肿瘤的生长(P＜0.005),EL-4/IL-12和EL-4/B7-1基因联合转染组比用单一的转基因细胞增强了治疗效果(P＜0.005).结论:研究结果提示应用IL-12进行血液肿瘤的治疗是有效的,IL-12和B7-1联合使用在未来人类癌症的治疗中有一定的应用前景.     

联合IL-2基因和B7-1基因转染G422细胞体外生物学特性

目的　通过研究联合基因转染后胶质母细胞瘤G42 2细胞生物学特性的变化 ,探讨脑胶质瘤细胞因子基因治疗的新途径。方法　通过重组腺病毒作载体 ,将IL 2基因和B7 1基因转染至小鼠G42 2细胞 ,通过CTLL 2细胞增殖的MTT法检测IL 2的分泌水平 ,FACS检测B7 1和ICAM 1的表达 ,MTT法检测转基因后G42 2细胞的增殖活性 ,双层琼脂糖培养法检测其克隆形成能力。结果　IL 2基因转染 4小时后即可检测到IL 2的分泌 ,2 4小时达高峰 ( 4 83U/ml) ,一周时表达明显下降 ,B7 1高表达 ,ICAM 1表达增加 ,增殖能力和克隆形成能力无变化。结论　重组腺病毒作载体 ,IL 2基因及B7 1基因共转染G42 2细胞 ,能得到目的基因的高效表达 ,同时促进了粘附分子ICAM 1的表达 ,体外增殖活性不受影响。     

COMBINED IL-2/IL-3 GENE THERAPY FOR G422 MOUSE GLIOBLASTOMA BY INTRATUMORAL INJECTION OF RECOMBINANT ADENOVIRUSES

ＣＯＭＢＩＮＥＤＩＬ２／ＩＬ３ＧＥＮＥＴＨＥＲＡＰＹＦＯＲＧ４２２ＭＯＵＳＥＧＬＩＯＢＬＡＳＴＯＭＡＢＹＩＮＴＲＡＴＵＭＯＲＡＬＩＮＪＥＣＴＩＯＮＯＦＲＥＣＯＭＢＩＮＡＮＴＡＤＥＮＯＶＩＲＵＳＥＳ１ＨｏｎｇＢｏ２洪波ＣａｏＸｕｅｔａｏ３曹雪涛Ｙｕ...     

Efficient inducation of local and systemic antitumor immune response by liposome-mediated intratumoral co-transfer of interleukin-2 gene and interleukin-6 gene.

Interleukin 2 (IL-2) expressing plasmid and interleukin 6 (IL-6)-expressing plasmid were encapsulated in liposome and administrated intratumoraly into tumor-bearing mice 4 days after subcutaneous inoculation of B16F10 melanoma cells. The results showed that treatment of tumor-bearing mice with IL-2 gene or IL-6 gene transfer inhibited the growth of subcutaneous tumor and prolonged the survival of tumor-bearing mice significantly when compared with the treatment of PBS or control gene transfer mediated by liposome (P < 0.01). Combined transfer of IL-2 gene and IL-6 gene was found to elicit inhibitory effects on the growth of B16F10 tumor more significantly and prolonged the survival period of tumor-bearing mice more obviously. We investigated the local immunity in tumor microenvironment and found that IL-2 and IL-6 gene transfer could significantly increase the expression of lymphocyte function-associated antigen-1 on tumor infiltrating lymphocytes (TIL) and MHC-I molecule on tumor cells freshly isolated from the tumor mass. The NK and CTL activity of TIL increased markedly after the combined transfer of these two cytokine genes. We also observed the systemic antitumor immune response in the tumor-bearing mice and demonstrated that NK and CTL activity of splenocytes and the production of IL-2, tumor necrosis factor and interferon-gamma from splenocytes increased obviously in mice after the combined transfer of IL-2 and IL-6 gene. In conclusion, local and systemic antitumor immunity of the tumor-bearing host could be induced efficiently after the combined gene transfer. The enhanced specific and non-specific antitumor immunity might be responsible for the more potent antitumor effects of the combined gene therapy.     

ANTITUMOR EFFECTS OF HUMAN IL-15 GENE MODIFIED LUNG CANCER CELL LINE

ＡＮＴＩＴＵＭＯＲＥＦＦＥＣＴＳＯＦＨＵＭＡＮＩＬ－１５ＧＥＮＥＭＯＤＩＦＩＥＤＬＵＮＧＣＡＮＣＥＲＣＥＬＬＬＩＮＥＳｈｅｎＹｏｎｇｑｕａｎ１沈永泉ＣｕｉＬｉａｎｘｉａｎ２崔莲仙ＨｅＷｅｉ１何维ＸｕｅＬｉ１薛莉ＢａＤｅｎｉａｎ１巴德年１Ｉｎｓｔ...     

瘤内／瘤周注射巨噬细胞集落刺激因子或（和）干扰素—γ基因转染的 …

目的 观察巨噬细胞集落刺激因子（Ｍ－ＣＳＦ）和干扰素－γ（ＩＦＮ－γ）基因单独或联合转染的巨噬细胞对局部黑色素瘤的治疗效果及相关免疫机理。     

NK and CD8+ T cell-mediated eradication of poorly immunogenic B16-F10 melanoma by the combined action of IL-12 gene therapy and 4-1BB costimulation

In previous reports, systemic administration of a stimulatory monoclonal antibody directed against the 4-1BB receptor had no effect on survival or tumor burden in mice inoculated with the poorly immunogenic B16-F10 melanoma. We combined IL-12 gene transfer with 4-1BB costimulation to explore a previously noted cooperative anti-tumor effect against this model tumor. We hypothesize that the innate immune response mediated by IL-12-activated natural killer (NK) cells initiates the activation of the immune system, leading to the priming of T cells, whereas 4-1BB costimulation enhances the function of primed tumor-specific T cells. The effect of the combination therapy on the growth of subcutaneous (s.c.) tumors and pulmonary metastasis was examined. The combination therapy significantly retarded the growth of subcutaneously-inoculated tumors, and 50% of tumor-bearing mice survived with complete tumor regression. In contrast, neither IL-12 gene transfer nor anti-4-1BB antibody administration alone was as effective. Enhanced CTL activity against both B16-F10 tumor cells and TRP-2-pulsed EL4 syngeneic tumor cells was observed in tumor-bearing animals treated with the combination therapy 2 weeks after treatment and, in long-term survivors from this combination therapy, at >120 days. In a pulmonary metastatic model, only the combination therapy generated significant protection against metastasis. In vivo depletion of NK or CD8(+) but not CD4(+) subsets eliminated the protective immunity. Furthermore, NK cell depletion significantly reduced both tumor-specific CTL activity and the number of tumor-specific IFN-gamma-producing cells, suggesting that this synergistic effect requires the participation of both NK and CD8(+) T cells.     

Overexpression of interleukin-15 in vivo enhances antitumor activity against MHC class I-negative and -positive malignant melanoma through augmented NK activity and cytotoxic T-cell response

Interleukin (IL)-15, a pleiotropic cytokine, is involved in the development and maintenance of NK cells and memory CD8+ T cells. We examined the effects of in vivo overexpression of IL-15 on protection against 2 types of murine B16 melanoma lines, MHC class I-negative B16.44 and MHC class I-positive B16F10 cells, using IL-15 transgenic (Tg) mice that we have recently constructed. The tumor growth was severely retarded in IL-15 Tg mice after subcutaneous (s.c.) inoculation with B16.44 or B16F10 cells. IL-15 Tg mice showed an augmented NK cell activity against B16.44 cells, and in vivo depletion of NK cells by anti-asialoGM1 Ab treatment abrogated the antitumor activity in IL-15 Tg mice. On the other hand, IL-15 Tg mice inoculated with B16F10 cells developed a significant level of CTL response against B16F10 cells, and in vivo depletion of CD8+ T cells by anti-CD8 MAb treatment abrogated the antitumor activity. Thus, overexpression of IL-15 augmented antitumor activity against different tumors via augmentation of different antitumor mechanisms. These results suggest a possible therapeutic application of IL-15 for human neoplasms expressing a wide range of MHC class molecules.     

人LAK细胞的诱导及其生物特性的研究

LAK cell activity of peripheral blood from normal donors was induced by interleukin 2 (IL-2). Optimal induction of LAK cells was obtained by 3-4 day incubation of PBMC in the presence of 30-40 units/ml of IL-2. Several cell lines Raji, CEM, HeLa, 109 (esophageal carcinoma) and OC (ovarian carcinoma) which were NK cell resistant tumor cells could be lysed by LAK cells. PHA was unable to markedly induce the LAK cell activity. Although both LAK and NK cell activity was decreased by mitomycin C treatment, their degree of susceptibility to such treatment was quite different. NK cells were more sensitive to mitomycin C than LAK cells. In contrast to the significant inhibition of the NK cell-mediated lysis by the addition of anti-CD 2 monoclonal antibody during cytotoxicity assay LAK cell-mediated lysis was not affected. These results indicate that LAK cells are capable of lysing a variety of tumor cells that are resistant to NK cells and their biological characteristics are also different.