小鼠对IFN-α1转染的Friend白血病细胞的局部和全身反应

3C18系Frienol白血病细胞(FLC)对小鼠IFN-α/β具有完全的抵抗性,经IFN-αl基因转染后此抵抗性仍存在,且能分泌高剂量的IFN,接种小鼠后能出现持续数周的干扰素血症,故作者选用此细胞建立模型,观察接种IFN-αl基因转染FLC后小鼠的局部和全身反应,以进一步了解宿主抑制此肿瘤生长的机制。 应用逆转录病毒将IFN-αl基因转入FLC,选出克隆IFN-αlCl-11FLC,可向培养液中分泌IFN-αl256～512U/ml,对照转染的FLC(TC-2)不分泌IFN。具有免疫活性的DBA2 /bg小鼠皮下注射TC-2 FLC后可生成快速生长的肿瘤,接种后20～35天后肿瘤呈现大面积坏死,小鼠存活时间和存活数量较对照组显著增多。IFN-αl C1-11 FLC接种缺失NK细胞活性的bg/bg小鼠后呈现相似的致瘤性下降现象,用单抗去除DBA/2小鼠体内的CD4~ CD8~ 细胞或粒细胞并不影响实验组小鼠的肿瘤生长曲线、肿瘤发生率及存活时间;经一系列处理后获得完全性免疫抑制的SIA裸鼠用于实验,实验组小鼠肿瘤与对照组TC-2肿瘤生长曲线仅稍有差异,并不显著,二者在存活时间、存活数量上并无差异。光镜检查发现在第7、14天,接种TC-2细胞小鼠肿瘤在皮肤表面破溃,在肿瘤边缘仅见少量巨噬细胞和中性粒细胞;在接利IFN-αl C1-11细胞小鼠,肿瘤仍在网状胶原层,有广泛的细胞坏死和凋亡,肿     

EL_4瘤细胞产生的IL-2可诱导NK细胞和T细胞介导免疫

全身应用rIL-2治疗癌症患者,尽管具有明显的抗肿瘤作用,但由于严重的毒副作用,其疗效受到一定限制。为探讨一种避免全身毒副反应的IL-2免疫治疗方法,以逆转录病毒(PLJ)为载体,将IL-2cDNA转染小鼠胸腺瘤EL_4细胞,用IL-2依赖细胞株CTLL2筛选一株高分泌IL-2(500IU/ml/24h/5 ×10~5细胞)细胞株(EL_(4P)IL-2),另外以同样方法构建不分泌IL-2的EL_(4P)细胞株为对照株,体外大量扩增这两种细胞株。将多种剂量的EL_(4P)IL-2或EL_(4P)细胞分别皮下接种于C57BL/ 6小鼠,观察诱发肿瘤的情况,结果发现,小鼠对EL_(4P)IL-     

经组成性活化Akt1修饰的鼠胚胎肝前体细胞体内形成肿瘤的观察

目的:观察组成性活化Akt1(myr-Akt1)导入的p53-/-鼠胚胎肝前体细胞(fetal hepatic progenitor cells, FHPCs)体外培养的特点和小鼠体内肿瘤模型的建立.方法:利用磁性细胞分选系统(magnetic cell-sorting system,MACS)分离上皮细胞钙黏蛋白(E-cadherin)阳性的p53-/-小鼠FHPCs,经细胞体外培养和反转录病毒导入myr-Akt1后,接种到C57BL/6小鼠的脾脏.应用RT-PCR和免疫组织化学法分析肝细胞分化和生长相关基因mRNA和蛋白的表达.结果:导入myr-Akt1后可明显提高E-cadherin阳性p53-/-小鼠FHPCs的体外生长和克隆形成能力,并可导致p16Ink4a和p19Arf mRNA表达水平下降;将该细胞接种到小鼠脾脏后可形成肿瘤,肿瘤细胞内GSK3β呈磷酸化,β-catenin呈一定水平表达.结论:成功建立了经Akt1修饰的p53-/-鼠FHPCs体内肿瘤模型,为探讨肝癌发生的分子机制和对相关抗癌药物的研究提供了平台.     

树突状细胞融合及体外致敏的瘤苗诱导抗肿瘤免疫应答

观察骨髓来源的树突状细胞在保护小鼠免受骨髓瘤攻击及对荷瘤小鼠的治疗作用.本文用mGM-CSF和L929细胞上清从BALB/c小鼠的骨髓中培养树突状细胞,将树突状细胞与小鼠NS-1骨髓瘤细胞融合(FD),并用瘤细胞制备的抗原体外冲击致敏树突状细胞(DC~ ).FD每鼠1×10~5,DC~ 每鼠2×10~5分别在第0,7天经尾静脉注射,第14天每只小鼠皮下接种NS-1骨髓瘤5×10~6,观察两种瘤苗保护小鼠免受肿瘤攻击的预防作用.FD(每鼠1×10~5)和DC~ (每鼠2×10~5)分别间隔1周治疗荷瘤7d和14d的小鼠2次,观察小     

癌得清注射液的抗癌作用

 本文用金黄色葡萄球菌(Staphylococcusaureus)、乙型溶血性链球菌(StreP－tococcushemolyticus)、卡介苗(BCG)、伤杆菌(Salmonellatyphi)等多种细菌抗原免疫SD大鼠,并从其脾脏、胸腺等淋巴组织通过透析的方法获得透析外液,再加上构才己等中药透析的小分子(MW≤10KD)物质,制备成混合制剂(癌得清)。然后以DBA/2小鼠荷L5178Y、P815肿瘤为模型,分别在肿瘤细胞接种后及接种前后腹腔注射上述制剂,观察其抑瘤作用和荷瘤小鼠脾细胞NK、LAK活性及产生IL－2活性的影响。结果表明,该制剂可明显抑制L5178Y、P815肿瘤生长并能显著增强荷瘤小鼠脾脏NK、LAK活性,增强脾细胞产生IL－2的能力。     

rBCG-IL-2对肿瘤生长抑制作用和免疫刺激作用的研究

 目的　将表达IL-2重组BCG用于动物实验,观察其对肿瘤生长和机体免疫功能的影响。方法　采用基因工程技术和电转化技术,构建重组卡介苗即rBCG-IL-2和rBCG,并用瘤株重组BCG接种荷瘤小鼠,几周后观察肿瘤生长情况和检测巨噬细胞的杀伤活性。结果　rBCG-IL-2能表达分泌IL-2,其表达量为9.87ng/ml±4.56ng/ml,rBCG-IL-2与rBCG抑制肿瘤生长的作用明显优于对照组,并能提高小鼠巨噬细胞杀瘤率。结论　rBCG-IL-2能有效抑制肿瘤生长,较早刺激机体的免疫功能,提高巨噬细胞的杀瘤活性。     

IL-3基因转染的黑色素瘤细胞株的建立及其体外生长性质的观察

为了探讨不同细胞因子基因修饰的肿瘤细胞制备的瘤苗对膀胱癌的治疗作用,作者构建了表达不同细胞因子的逆转录病毒载体N_2/IL-2、N_2/CMV/GM-CSF、DC/TK/IL-1α、N_2/CMV/IL-1β、DC/SV/R/IFN-γ,将其分别转染包装细胞GP env AM12,获得含病毒的细胞上清,再将其分别感染小鼠膀胱癌细胞MBT-2,经筛选获得不同的高分泌株。将这些高分泌株分别接种C3H小鼠后发现分泌IFN、IL-1α、IL-1β或GM-CSF的MBT-2细胞在体内的生长较野生型MBT-2细胞缓慢。而分泌IL-2的MBT细胞在体内的生长受到完全的抑制。在裸鼠体内,仅分泌IL-2的MBT-2细胞的生长受到抑制。免疫组化分析表明,在这些细胞接种处出现CD4~ 及CD8~ T细胞的浸润,在     

癌得清注射液的抗癌作用

本文用金黄色葡萄球菌（Staphylococcusaureus）、乙型溶血性链球菌（StreP－tococcushemolyticus）、卡介苗（BCG）、伤杆菌（Salmonellatyphi）等多种细菌抗原免疫SD大鼠，并从其脾脏、胸腺等淋巴组织通过透析的方法获得透析外液，再加上构才己等中药透析的小分子（MW≤10KD）物质，制备成混合制剂（癌得清）。然后以DBA／2小鼠荷L5178Y、P815肿瘤为模型，分别在肿瘤细胞接种后及接种前后腹腔注射上述制剂，观察其抑瘤作用和荷瘤小鼠脾细胞NK、LAK活性及产生IL－2活性的影响。结果表明，该制剂可明显抑制L5178Y、P815肿瘤生长并能显著增强荷瘤小鼠脾脏NK、LAK活性，增强脾细胞产生IL－2的能力。     

鲁山冬凌草甲素对小鼠淋巴细胞白血病L1210细胞动力学的影响

采用[~3H]TdR标记的放射自显影术和有丝分裂阻断法研究鲁山冬凌草甲素对小鼠淋巴细胞白血病L1210细胞动力学的影响。实验用DBA/2小鼠,(?)号兼用,体重18—22克,IP接种L1210细胞10~6/鼠,所有实验均采用d6带瘤小鼠,Go期细胞杀伤作用取d9小鼠。[~3H]dR和核4乳胶均由中     

博莱霉素连续给药和间断给药的抗肿瘤作用及血浆动力学

Sikic 等发现连续皮下注射博莱霉素的Lewis 肺癌小鼠比间断皮下注射的动物生存期更长;肿癌生长速度更慢。本文研究连续腹腔注射(C.i.p)或间断腹腔注射(I.i.p)博莱霉素对白血病小鼠脾集落生长的影响,结果发现C.i.p 在提高博莱霉素抗白血病活性上与合理的血浆动力学有关。实验中给DBA/2小鼠iv 10~6P_(388)腹水癌细胞,Ⅰ.i.p 组动物于接种癌细胞的1～6天,每日ip 博莱霉素(2～10单位/公斤/日),C.i.p 组于第1天腹腔内埋藏     

白细胞介素2基因转染肿瘤细胞体外生物学特性的研究

应用逆转录病毒载体PLXSN将人的IL-2基因和抗新霉素基因(Neo~R)插入人肺腺癌细胞,转染以后,经G418筛选,获得阳性细胞.将转染与未转染细胞作比较,观察了两者在体外的生长特性.测量了阳性细胞上清液中IL-2含量.PCR证实了外源性基因的插入及持续稳定表达.另外在电镜下,可见转染细胞表面绒毛变短变少,此变化是否会影响细胞的浸润性及转移性尚待进一步研究.     

Reinforced Cytotoxicity of Lymphokine-activated Killer Cells toward Glioma Cells by Transfection of the Killer Cells with the γ-Interferon Gene

Lymphokine-activated killer (LAK) cells generated from peripheral blood lymphocytes incubated with recombinant interleukin-2 were transfected with the human γ-interferon (HuIFN-γ) gene by means of liposomes having a positive charge on their surface. The cells secreted significant amounts of HuIFN-γ (reaching more than 5 U/ml) into the culture medium. The HuIFN-γ produced by the cells induced intercellular adhesion molecule-1 (ICAM-1) and enhanced the expression of Fas antigen on the surface of human glioma cells. Also, LAK cells transfected with HuIFN-γ gene exhibited reinforcement of cytotoxicity toward human glioma cell lines (U251-MG and SK-MG-1). Furthermore, the reinforcement was significantly quenched by anti-ICAM-1 and/or anti-TNF-α monoclonal antibody.     

Induction by human interferon and murine h-2l(d) gene of the surface expression of endogenous hla class-I free heavy-chains in hct human colon-carcinoma cells, deficient in Beta-2-microglobulin

The colon cancer cell line HCT fails to express HLA class I antigens on the cell surface as a consequence of two independent mutations occurring in both copies of the beta2-microglobulin (beta2-mu) gene. Restoration of HLA class I antigen expression in HCT cells transfected with the wild-type human beta2-mu gene is accompanied by the expression of beta2-mu-free HLA class I heavy chains on the cell surface. HCT cells expressing a transfected H-2L(d) gene (HCT-L(d) transfectants) exhibited high levels of H-2L(d) heavy chains on the cell surface, following treatment with human interferon a (IFN-alpha). IFN-treated HCT-L(d) transfectants also expressed high levels of endogenous beta2-g-free HLA class I heavy chains on the cell surface. Incubation of HCT-L(d) transfectants at 26-degrees-C for 24 hours is associated with a striking increase in the surface expression of H-2L(d) heavy chains (comparable to that observed at 37-degrees-C in the presence of IFN), without a concomitant increase in the surface expression of beta2-mu-free HLA class I heavy chains, suggesting that IFN and low temperature act by different mechanisms. These results also indicate that human IFN and H-2L(d) heavy chain can act synergistically in inducing the surface expression of endogenous beta2-mu-free HLA class I heavy chains by HCT cells. The combination of 26-degrees-C incubation and IFN treatment results in the surface expression of beta2-mu-free HLA class I heavy chains in HCT-neo transfectants, suggesting that IFN plays a crucial role in allowing the surface expression of endogenous HLA class I heavy chains in beta2-mu-negative HCT cells.     

Retroviral vector-mediated lymphokine gene transfer into human renal cancer cells.

Effective vaccination against cancer, either for prophylaxis or therapy, has been an elusive goal for years. Cytokine gene therapy offers a novel approach to generate immunogenic tumor cell vaccines. To examine the feasibility of cytokine gene transfer into human renal cancer (RC) cells, we introduced the cDNAs for human interleukin-2 (IL-2) or interferon-gamma (IFN-gamma) into various RC cell lines with retroviral vectors. Using the NIH3T3 amplification assay, no replication competent retroviral particles were detectable in cell culture supernatants taken from gene-modified RC cell lines. Efficient expression of both lymphokines was achieved. Depending on the cell line and the vector construct used, lymphokine gene-modified human RC cell lines released 4 to 29 units/10(6) cells of IL-2, or up to 10 units/10(6) cells of IFN-gamma within 48 h. Fluorescence-activated cell sorter analysis of SK-RC-29 cells releasing IFN-gamma showed increased expression of major histocompatibility complex class I antigen, beta 2-microglobulin, and ICAM-1, as well as induction of major histocompatibility complex class II antigen expression [human leukocyte antigen(HLA)-DR, -DP], but no changes in these cell surface markers were observed with SK-RC-29 cells releasing IL-2. Following in vitro gamma-irradiation with 5,000 or 10,000 rad, growth of lymphokine gene-modified RC cells was abrogated, but their capability to release lymphokine and express lymphokine-induced antigenic determinants, such as HLA-DR, was retained. Tumor formation by the human RC cell line SK-RC-29 in BALB/c nude mice was not affected by IFN-gamma secretion, but was inhibited by in vivo release of IL-2 from s.c. injected tumor cells. These studies demonstrate the feasibility of retroviral mediated lymphokine-gene transfer into human RC cells and suggest a means for generating autologous or HLA-matched allogeneic tumor cell vaccines for the treatment of patients with renal cell carcinoma.     

Human lung carcinoma cells engineered to release IL2, IL7, GM-CSF and TNF alpha

A human lung adenocarcinoma cell line (LC89) was transduced with the IL2, IL7, GM-CSF and TNF alpha genes by retroviral vector mediated infection. This induced the constitutive and stable release of all cytokines. No difference or modulation was found in the parental and gene transduced LC89 cells with regard to cytokine receptor expression, in vitro cell growth and proliferation, nor in cell surface expression of different adhesion molecules. Following injection into immunosuppressed nu/nu mice, IL2 gene transduced LC89 cells lost their tumorigenic potential. LC89 cells engineered to release IL7 and TNF alpha grew in nu/nu mice, but in 40% of the animals tumor regression was observed. GM-CSF gene transduced LC89 cells showed a tumorigenic capacity identical to that of the parental clone. The levels of TGF beta(1) released by IL2, IL7 and GM-CSF gene transduced LC89 cells were markedly reduced compared to those of the parental and TNF alpha gene transduced cells. The results of this study support the concept that human lung cancer cells engineered with different cytokine genes maintain their intrinsic morphologic and proliferative features, while their tumorigenic and immunosuppressive capacities can be profoundly down-modulated. Both these effects are optimally achieved following insertion of the IL2 gene, suggesting that vaccination protocols with IL2 gene transduced tumor cells may be considered for the management of human lung cancer.     

Antagonistic effects of c-myc and Epstein-Barr virus latent genes on the phenotype of human B cells

Epstein-Barr virus (EBV) immortalized cells and Burkitt lymphoma cells have a completely different growth pattern and phenotype. EBV immortalized cells express a set of 11 viral genes to accommodate B cell activation and proliferation, whereas EBV-positive Burkitt lymphoma cells highly express the c-myc oncogene that is activated through translocation into 1 of the immunoglobulin loci and EBNA1 as the only viral protein. We have developed a primary human B cell line conditionally immortalized by Epstein-Barr virus in which the viral gene program responsible for the induction of proliferation can be switched on and off by the addition or withdrawal of estrogen (cell line EREB2-5). Starting from this cell line we have generated 2 daughter cell lines that proliferate in a c-myc dependent fashion, 1 with a highly active exogenous c-myc gene (cell line A1) and 1 with a regulatable c-myc gene that can be switched on by withdrawal and switched off by addition of tetracycline (cell line P493-6). The comparison of the 3 cell lines has allowed us to dissect the contribution of c-myc and EBV genes to the regulation of the growth pattern and expression of cell surface molecules. We show that MYC and EBNA2 (and their respective target genes) have opposing effects on the expression of several surface markers involved in B cell activation. We show that MYC contributes to the phenotype of Burkitt lymphoma cells by upregulating CD10 and CD38 and downregulating activation markers. The phenotype of the cells is determined on one hand by the absence of the viral gene products EBNA2 and LMP1 that mediate the phenotype of activated lymphoblasts and to a lesser extent by an active contribution of the c-myc gene.     

A Murine Monoclonal Antibody that Recognizes an Extracellular Domain of the Human c-erbB-2 Protooncogene Product

A marine IgM monoclonal antibody, designated SV2–61, was generated against human c-erbB-2 gene-transfected NIH-3T3 (SV11) cells. SV2–61 defined a 185-kDa molecule present on the surface of SV11 cells, another line of c-erbB-2 gene-transfected NIH-3T3 (A4–15) cells, and MKN-7 human gastric cancer cell line carrying an amplified human c-erbB-2 gene. The SV2–61-defined antigen was found to show protein kinase activity in vitro . The SV2–61 was reactive with human c-erbB-2 gene-transfected NIH-3T3 cell lines but not with transfectants carrying c-erbB-2 gene mutants which lack a coding region for the extracellular domain. It was reactive with a portion of human epithelial cell lines hut not with native NIH-3T3, TGF-a-coding gene-, activated c-raf gene- or Ha-ras gene-transfected NIH-3T3 cells, or non-epithelial human cells. These results indicate that the SV2–61 is an antibody which recognizes an extracellular domain of the c-erbB-2 gene product, 185-kDa protein.     

A murine monoclonal antibody that recognizes an extracellular domain of the human c-erbB-2 protooncogene product

A murine IgM monoclonal antibody, designated SV2-61, was generated against human c-erbB-2 gene-transfected NIH-3T3 (SV11) cells. SV2-61 defined a 185-kDa molecule present on the surface of SV11 cells, another line of c-erbB-2 gene-transfected NIH-3T3 (A4-15) cells, and MKN-7 human gastric cancer cell line carrying an amplified human c-erbB-2 gene. The SV2-61-defined antigen was found to show protein kinase activity in vitro. The SV2-61 was reactive with human c-erbB-2 gene-transfected NIH-3T3 cell lines but not with transfectants carrying c-erbB-2 gene mutants which lack a coding region for the extracellular domain. It was reactive with a portion of human epithelial cell lines but not with native NIH-3T3, TGF-alpha-coding gene-, activated c-raf gene- or Ha-ras gene-transfected NIH-3T3 cells, or non-epithelial human cells. These results indicate that the SV2-61 is an antibody which recognizes an extracellular domain of the c-erbB-2 gene product, 185-kDa protein.     

Single-chain antibody-mediated gene delivery into ErbB2-positive human breast cancer cells

Targeted gene transfer by nonviral vectors can be achieved through incorporation of specific ligand(s) into the vectors. In this study, the effects of incorporation of an anti-ErbB2 single-chain antibody fragment (ScFv) into nonviral vectors for targeted gene delivery were investigated. The ML39 ScFv, selected from a human ScFv phage display library and affinity matured in vitro (K(d)=1 x 10(-9) M), was used as ligand specific for the extracellular domain of the tumor surface protein, ErbB2. Two approaches were taken: (a) development of a vector that is composed of a bifunctional fusion protein capable of binding DNA with the ErbB2-specific ML39 ScFv at its N-terminus and a truncated form of human protamine at its C-terminus, and (b) formulation and evaluation of delivery vectors consisting of three independent components including ML39 ScFv, protamine, and cationic lipids. We demonstrate that fusion proteins comprised of the ML39 ScFv and a truncated form of protamine, denoted as ScFv-P-S, can selectively deliver exogenous DNA into ErbB2(+) cells, with an 8- to 10-fold increase in expression levels of the luciferase reporter gene in ErbB2(+) cells as compared to ErbB2(-) cells. In addition, vectors formulated by appropriately mixing DNA, ScFv, protamine, and lipids in vitro could even more efficiently deliver the reporter gene into ErbB2(+) cells with approximately 5-fold increase in gene expression in ErbB2(+) cell as compared to ErbB2(-) cells. Expression and refolding of the ScFv fusion proteins, in addition to determination of optimal conditions for vector development using these approaches, are discussed.     

Transfection-induced Tumor Necrosis Factor-α Increases the Susceptibility of Human Glioma Cells to Lysis by Lymphokine-activated Killer Cells: Continuous Expression of Intercellular Adhesion Molecule-1 on the Glioma Cells

To develop more effective adoptive immunotherapy, we transfected the human tumor necrosis factor-α (TNF-α) gene into human glioma cells (U251-SP), which were used as target cells. TNF-α is known to increase both the expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of glioma cells and the susceptibility of glioma cells to lymphokine-activated killer (LAK) cell cytolysis. We compared the expression of ICAM-1 induced by TNF-α generated by the TNF-α gene-transfected cells with that induced by exogenously added TNF-α. When the TNF-α gene was transfected into U251-SP cells, the expression of ICAM-1 was detected on the cell surface from 3 days after the transfection and continued until at least 9 days. In contrast, it was expressed only transiently in the case of exogenously added TNF-α. Also, the cytolytic activity of LAK cells induced by transfection-induced TNF-α was significantly stronger than that induced by exogenously added TNF-α. The increased susceptibility was quenched by anti-ICAM-1 monoclonal antibody. These data indicated that continuous expression of ICAM-1 induced by TNF-α gene transfection of glioma cells resulted in higher cytolytic activity of LAK cells.