目的研究如何提高胶质瘤细胞的免疫原性,探讨胶质瘤免疫治疗的新途径.方法用重组腺病毒作载体,将共刺激分子B7-1基因和具有多重免疫功能的IL-2基因转入G422小鼠胶质母细胞瘤细胞中,取1×105个细胞(体积0.1 ml),在小鼠皮下接种,分野生型对照组、lacZ组、IL-2组、B7-1组和B7-1+IL-2组.接种后不同时间测量肿瘤大小.两周后检测荷瘤小鼠脾细胞的NK、LAK和CTL的活性及细胞因子的分泌.结果B7-1+IL-2组肿瘤生长明显受抑制(P＜0.05).B7-1组的NK、LAK、CTL的杀伤活性及脾细胞分泌细胞因子的水平都很低,与对照组相差不显著.IL-2组的CTL的杀伤活性较高,与对照组相差显著(P＜0.05),而NK和LAK活性及细胞因子水平与对照组相差不显著.B7-1+IL-2组的NK、LAK、CTL和脾细胞分泌细胞因子的活性与对照组相比,相差都非常显著(P＜0.01).结论单纯B7-1基因转染G422细胞不能增强其免疫原性;IL-2基因转染后,对机体的免疫反应有一定的增强作用,但效果不够理想;B7-1基因和IL-2基因联合转染G422细胞则可以明显增强其免疫原性,并有效激活荷瘤小鼠的特异性抗肿瘤免疫反应.

我们用腺病毒作载体,将B7-1基因和IL-2基因联合转染脑胶质瘤细胞后小鼠皮下接种,观察了其对机体免疫功能的影响.

Objective To seek a new way of gene therapy for brain glioma by studying on IL-2 gene and B7-1 gene cotransfected G422 murine glioblastoma cells.Methods IL-2 gene and B7-1 gene were co-transfected into G422 murine glioblastoma cells mediated by recombinant adenovirus in vitro.The proliferation of G422 cells was tested by MTT method and the level of IL-2 secreted by gene modified G-422 cells was tested by MTT method of the proliferation of CTLL-2.The expression of B7-1 and ICAM-1 on gene transfected G422 cells were assayed by FACS.Their colon-forming ability was tested with bilayer agrose culture after gene transfection.Results 4h after IL-2 gene transfection,IL-2 could be detected in the supernatant of gene modified G422 cells,and the level of IL-2 reached the highest level from 24 to 72 hours,but decreased markedly one week later.B7-1 was highly expressed and the expression of ICAM-1 was enhanced.Conclusion IL-2 gene and B7-1 gene could be co-transfected into G422 cells and could be expressed effectively mediated by recombinat adenovirus,which results in enhanced expression of ICAM-1 of the cells,whereas the proliferation of gene modified G422 cells is not changed.

The evidemce that Kupffer cells (KC) are capable of controlling metastatic growth in the liver is largely circumstantial.However,the ability of KC to function as antigen-presenting cells was not much studied. Thus,the aim of this study was to characterize the effect on the antigen-presenting function of Kupffer cells by intrasplenic transplantation of GM-CSF gene-modified fetal liver cells (FILC-GM)in combination with IL-2 and 5-Fu. Two weeks after the tumor-bearing mice was treated,strong increase in KC numbers and percentage of phagocytosis of 99mTc within the liver tissue were observed. At the same time, co-stimulator antigen CD40 expression on KC were determined by fluorescence-activated cell sorter( FACS) and found that its expression level was upregulated. In additioni, OVA-specific autogenetic T cells mixed lymphocyte reaction induced by KC have increased. Moreover, we observed that blocking CD40/CI40L interaction with anti-CD40L mAbs caused significantly inhibition of T cell proliferation response and inhibition of IL-2 and IL-12 production.These results indicated that intrasplenic transplantation of GM-CSF gene therapy in combination with immuno-chemotherapy could efficientiy activate the KC immunologic function, particularly its antigen-presenting function.

Purpose: Tumor suppressor gene therapy and cytokine gene therapy have limited antitumor effects when used alone. Thus, in the present study, we investigated the antitumor potentials of the combined transfer of the p16 tumor suppressor gene and the murine granulocyte-macrophage colony-stimulating factor (GM-CSF) gene. Methods: The adenovirus-harboring p16 gene (Adp16) and adenovirus-harboring: GM-CSF (AdGMCSF) gene were utilized for the treatment of established tumors in vivo. The mice were inoculated s.c. with Renca renal carcinoma cells and 3 days later received an intratumoral injection of Adp16 in combination with AdGMCSF. Results: The results demonstrated that tumor-bearing mice treated with Adp16 and AdGMCSF showed more potent inhibition of tumor growth and a prolonged survival period than mice treated with Adp16, AdGMCSF, adenovirus-expressing beta -galactosidase or PBS (P < 0.01). Treatments of the mice with Adp16 alone or AdGMCSF alone also showed obvious antitumor effects as compared with those mice treated with PBS (P < 0.05). After combined p16 and AdGMCSF gene therapy, the expression of H-2K(d) and Fas molecules on freshly isolated tumor cells increased markedly, and more CD4+ T cells and CD8+ T cells infiltrated in the tumor sites. The cytotoxicity of natural killer cells and specific cytotoxic T lymphocytes increased more significantly after the combined therapy. Conclusions: Our results demonstrated that combination p16 gene and GM-CSF gene therapy could inhibit the growth of established tumors in mice more significantly through efficient induction of antitumor immunity.

目的：了解 IL-2基因修饰的细胞毒 T淋巴细胞（ cytotoxic T lymphocytes,CTL）的增殖和杀伤活性，探索细胞因子基因疗法及被动免疫治疗脑胶质瘤的新途径。方法：用昆明种小鼠的脾细胞体外诱导 CTL，用腺病毒载体转染 IL-2基因，观察其体外增殖活性和杀伤活性，再用 G422小鼠胶质母细胞瘤细胞建立肺转移瘤模型， 36 h后经过继回输， 2周后计数肺的肿瘤结节，观察 IL-2基因转染的 CTL对实验性肺转移瘤的治疗作用。结果：重组腺病毒载体在 MOI(multiplicity of infection)为 100时，转染率达 96.8％， IL-2基因修饰的 CTL增殖活性、 IL-2的分泌（ 248 u）和体外杀伤活性 (36.4％ )明显增强，对实验性肺转移瘤的治疗作用都显著增强，肺转移结节数 (28)显著减少（ P<0.01）。结论： IL－ 2基因转染的 CTL过继回输，可直接杀伤和诱导激活机体特异性抗肿瘤免疫反应，使体内抗肿瘤效果显著增强，有效抑制实验性肺转移瘤的生长，为胶质瘤的过继免疫治疗提供了新的思路和实验依据。

Objective To develop a genetically modified fetal liver cells (FLC) based transplantation system that can release therapeutic levels of hematopoietic growth factors into the system circulation which can facilitate treatment of patient receiving cytokine therapy following chemotherapy. Method Examine adeno virus mediated gene transfer to isolated murine FLC and evaluate the biocharacterization of intrasplenic transplantation of gene modified murine FLC. Results Substantial transfection rate of 80%～85% were achieved at a ratio of 50 for 2 hr of exposure. Gene modified FLC (FLC GM) labeled with 111 In were injected into the allogenic mice, spleen, the %ID/g of liver was 20%～25% at 24 hr and 50%～55% at 48 hr after transplantation. In addition, serum concentration of GM CSF in mice with intrasplenic transplantation reached its maximum at 48 hr [(356 ±58 ) pg/ml]. Conclusion Intrasplenic transplantation of FLC GM can be predominantly localized in liver and spleen, and engraft rapidly and maintain normal function, which represent a critical step toward successfully accomplishing liver directed gene therapy.

Objective: To investigate the effects of macrophages transfected with IL 2 gene. Methods: IL 2 gene was transfected into murine peritoneal macrophages by adenovirus. Levels of TNF, IL 1 and NO in the supernatant of macrophages as well as cytotoxicity of macrophages to target cells L1210 and Renca cells were assayed. Results: The productions of TNF,IL 1 and NO in the supernatant of IL 2 gene modified macrophages increased, cytotoxicity of the macrophages to L1210 cells and Renca cells also increased significantly. Conclusion: Transfection of IL 2 gene to macrophages can enhance their cytotoxicity, thus provide experimental basis for immunotherapy of tumor with IL 2 gene modified macrophages.

目的:研究巨噬细胞游走抑制因子(MIF)基因修饰的FBL3红白血病细胞的生物学性质变化,为探讨其体内诱导抗肿瘤免疫反应的作用奠定基础.方法:以腺病毒介导,将MIF基因导入小鼠FBL3红白血病细胞,观察其体外生物学性质变化.将MIF基因修饰的FBL3细胞(MIF-FBL3)接种小鼠体内,观察其致瘤性和局部病理学变化,同时检测细胞因子mRNA表达.结果:腺病毒能有效地将MIF基因导入FBL3细胞,MIF的分泌可持续10 d.MIF-FBL3细胞形态、增殖能力及表面MHC-Ⅰ,MHC-Ⅱ,B7-1,B7-2,ICAM-1等分子的表达与野生型FBL3细胞相比无明显变化,但在小鼠体内的致瘤性降低,小鼠存活期延长,肿瘤局部可检测出mTNF-α mRNA和mLtn mRNA的表达,并有明显的单核和淋巴细胞浸润.结论:MIF-FBL3细胞体外生物学性质无明显改变,但可能通过分泌MIF在体内激活抗肿瘤免疫功能.

Objective To investigate the antitumor effects of p16 and GM CSF combined gene therapy for renal cell carcinoma (renca) and elucidate the immune mechanisms of its antitumor response. Methods The model of renal tumor bearing mice by injected subcutaneously was developed. The tumor bearing mice were grouped as 5 groups at random 3 days after injection of Renca cells. Different groups of the mice were subjected to different treatments. The antitumor immune responses and the survival time of the mice were tested. Histopathologic analysis of tumor tissue was performed. Results The mice receiving combined therapy of AdP16+AdGM CSF were able to survive much longer than all the other mice receiving control therapies ( P 0.01). Two of 10 mice after AdP16/AdGM CSF combined therapy were found to be tumor free and lived more than 90 days, while the control animals died within 52 days. Enhancement of the NK cytotoxicity (33%) were observed more obviously in tumor bearing mice after combined therapy when compared with that of the other 4 groups ( P 0.01). Conclusion p16 and GM CSF combined gene therapy can enhance the antitumor immune responses of mice and inhibit the growth of pre established renal cell carcinoma, also prolong the survival time of tumor bearing mice.\;

目的:观察体外肿瘤抗原冲击致敏的白细胞介素2(IL-2)基因修饰的巨噬细胞对肾癌小鼠的治疗效果并探讨其相关的免疫机理.方法:通过重组腺病毒的介导,将IL-2基因转入新鲜分离的小鼠腹腔巨噬细胞,经肿瘤抗原冲击致敏后回输治疗原位肾癌小鼠,采用4 h 51 Cr释放法检测脾脏NK和CTL活性.结果:IL-2基因修饰的巨噬细胞经肿瘤抗原冲击后体内回输可使肾癌小鼠肺转移结节明显减少,存活期明显延长,40%肾癌小鼠达到长期存活.治疗后荷瘤小鼠脾脏NK和CTL活性显著提高.结论:IL-2基因修饰的巨噬细胞经肿瘤抗原冲击后自体回输是治疗肾癌的有效方法.

目的：观察白细胞介素２ （ＩＬ２） 基因修饰的巨噬细胞ＩＬ２ 动态分泌水平及对其表面分子的表达和抗原提呈能力变化的影响。方法：通过重组腺病毒介导，将ＩＬ２ 基因转染至腹腔巨噬细胞，ＭＴＴ法检测其ＩＬ２ 动态分泌水平，ＦＡＣＳ检测巨噬细胞表型，混合淋巴细胞反应法（ＭＬＲ） 测定巨噬细胞抗原提呈能力。结果：ＩＬ２ 基因修饰４ ｈ 后巨噬细胞即可表达较高水平的ＩＬ２ ，１８ ～４８ ｈ 之间的ＩＬ２ 分泌处于高峰。ＩＬ２ 基因修饰的巨噬细胞ＭＨＣＩＩ、Ｂ７１、Ｂ７２ 和ＣＤ４０ 表达增加，抗原提呈能力提高。结论：ＩＬ２ 基因修饰的巨噬细胞能有效表达ＩＬ２ ，与其功能密切相关的ＭＨＣＩＩ、Ｂ７ 、ＣＤ４０ 表达增加，其抗原提呈能力增强，本实验为巨噬细胞免疫治疗肿瘤提供了实验依据。

动物试验证实，经脾脏移植的肝细胞５０％能够选择性移入肝脏，并在其中较长期存活和有效表达外源基因。我们通过脾内途径移植腺病毒载体ＧＭ ＣＳＦ基因修饰的胎肝细胞，观察其对化疗造血损伤的恢复影响。材料和方法１动物与主要试剂ＢＡＬＢ／ｃ小鼠，４～６周龄，Ｃ５...

In the present study, FBL-3 murine erythroleukemia cells were transacted with human OSM(hOSM) gene by recombinant adenovirus, then the immunological properties of hOSM gene-transfected FBL-3 cells(FBL-3-OSM+) were investigated. 4 hours after transfection with hOSM gene, hOSM could be detected in the supernatant of FBL-3-OSM+ cells and hOSM secretion peaked at 24 h. The proliferation of FBL-3-OSM+ cells was inhibited markedly. The clonal formation of FBL-3-OSM+ cells was suppressed more obviously in comparison with wild-type FBL-3 cells when analysed in clonal argar culture. Flow cytometry analysis showed that FBL-3-OSM+ cells expressed higher levels of Fas protein, B7 and ICAM-1 molecuIes.FBL-3-OSM+ cells also expressed higher level of MHC class I molecules(H-2Kb) but remained unchanged in expression of MHC class II molecules (la). CD14, which is a specific marker of monocyte/macrophage and not expressed on the wild-type FBL-3 cells, was also detected on the surface of FBL-3-OSM+ cells. The results suggested that OSM gene transfer could increase the immunogenicity of FBL-3 cells and promote their differentiation into macrophage-like cells. The data outline a promising approach to OSM gene therapy of leukemia mediated by recombinant adenovirus.