逆转录病毒介导的IL6／IL2融合基因转导对小鼠B16细胞…

为探讨细胞因子之间对肿瘤细胞修饰的协同效果及融合基因用于肿瘤细胞转染的可行性，我们采用ＰＣＲ及柔性接头连接的方法。构建了含人ＩＬ６／ＩＬ２融合基因的逆转录病毒载体，将其转梁小鼠Ｂ１６黑色素瘤细胞，对基因转导后细胞的粘附及实验性肺转移能力进行了测定。     

IL—6基因转导对人乳腺癌细胞系MCF—7细胞体内外生长特性的影响

为探讨ＩＬ－６基因转导对人乳腺癌细胞体内外生长特性的影响，我们构建了含人ＩＬ－６ｃＤＮＡ的逆转录病毒载体ｐＬＩＬ６ＳＮ。采用脂质体介导的方法将ｐＬＩＬ６ＳＮ导入ＭＣＦ－７细胞中，对基因转导细胞的体外生长、粘附特性及裸鼠体内的实验性转移能力进行了测定。实验结果表明，经ＩＬ－６基因修饰的ＭＣＦ－７细胞虽显示了明显的体外生长抑制，裸鼠体内实验性转移能力却获得增强。文中对其可能的影响机制进行了探讨     

IL-2基因修饰的细胞毒 T淋巴细胞抗肿瘤效应的研究

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样伤活性,再用G422小鼠胶质母细胞瘤细胞建立肺转移瘤模型,36h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（multipliciy of infection)为100时,转染率达96．8％,IL－2基因修饰CTL增殖活性、IL－2的分泌（248u)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。     

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.     

白细胞介素-2基因修饰肿瘤细胞引发凋亡的研究

用重组有人白细胞介素-2(IL-2)含信号肽cDNA的缺陷型逆转录病毒将人IL-2基因整合于鼠肝癌细胞株H22染色体后，PCR、RT-PCR及生物学等方法测定确证有人IL-2基因存在、表达及活性IL-2的分泌α透射电镜观察了IL．2基因修饰前后H22细胞韵超微结构，发现IL-2基因修饰H22细胞中有26%的细胞其DNA含量低于肿瘤细胞的近4倍体呈亚2倍体，     

转导B7-1基因消除IFN-γ对黑色素瘤转移潜力的增强作用

γ干扰素(IFN-γ)可通过增加MHCⅠ类分子或其它机制增强多种肿瘤的转移能力,而将T细胞共刺激分子B7基因导入肿瘤细胞能增强机体免疫系统对肿瘤的攻击。本文以Lipofectamine转染法将小鼠B7-1(mB7-1)基因导入B16黑色素瘤低转移株,导入空载体(只含neo基因)的B16细胞作对照。亲本B16细胞和基因转导细胞(B16-B7-1和B16-neo)经100U/ml IFN-γ预处理36小时后进行实验性肺转移试验,同时流式细胞分析细胞表面MHCⅠ类和Ⅱ类分子的表达。结果发现,IFN-γ预处理明显增强B16和B16-neo细胞的肺转移能力,而经IFN-γ预处理的B16-B7-1细胞转移能力并不增强,其实验性肺转移结节数与未经处理的对照组无差别。流式细胞分析显示IFN-γ预处理使B16、B16-neo和B16-B7-1三种细胞的MHCⅠ类(H-2K~b和H-2D~b)分子均明显增高,但IFN-γ增加B16-B7-1细胞MHCⅡ类分子I-A~b表达程度显著高于其它两种细胞。结果表明,转导B7-1基因可降低IFN-γ诱导的B16细胞转移能力,MHCⅡ类分子表达增高可能在其中起一定作用。     

IL-12协同B7-1诱导机体抗肿瘤免疫的实验研究

目的研究白细胞介素-12(Interleukin-12,IL-12)协同共刺激分子B7-1,联合诱导机体抗肿瘤免疫对大鼠卵巢上皮癌的治疗作用并探讨其机理.方法用携带有小鼠IL-12和B7-1的逆转录病毒表达载体感染大鼠卵巢癌细胞株NuTu-19,建立高表达细胞株NuTu-19/IL-12、NuTu-19/B7-1及双基因共表达细胞株NuTu-19/IL-12-B7-1,并以转染空载体pLXSN的细胞NuTu-19/Neo为对照.用经丝裂霉素C处理的各种基因修饰的肿瘤细胞免疫动物,观察卵巢癌腹腔转移模型动物生存期,及其诱导细胞毒性T淋巴细胞(cytotoxic lymphocyte,CTL)杀伤活性的作用.结果经各种基因修饰的肿瘤细胞免疫后,大鼠脾淋巴细胞增殖能力有不同程度的提高,CTL杀伤同源肿瘤细胞的活性明显增强,IL-12和B7-1基因联合修饰的肿瘤细胞免疫动物对模型动物生存期的延长具有显著意义(P＜0.05).结论IL-12和B7-1基因联合修饰的肿瘤细胞可以刺激机体CTL增殖、成熟,增强CTL对肿瘤细胞的识别和杀伤活性等,两者联合具有明显的协同效应.联合免疫基因治疗作为一种新的卵巢癌治疗方法,值得深入研究.     

IL-24对B16细胞生长抑制作用的体内外实验研究

目的:构建白细胞介素24(Interleukin24,IL24)真核表达质粒,研究其体内外表达对肿瘤细胞的生长抑制作用。方法:采用重组DNA技术构建IL24真核表达质粒pEGFPIL24。用脂质体法将重组质粒及空载体体外转染B16细胞,再经激光扫描共聚焦显微镜(LSM)观察其表达,用MTT法检测B16细胞的体外增殖能力,用流式细胞仪检测细胞周期。小鼠实体瘤模型研究基因转染对肿瘤的体内生长抑制作用。结果:pEGFPIL24转染B16细胞后,LSM可观察到IL24在细胞中的表达。转染IL24基因的B16细胞体外增殖能力明显受到抑制,细胞被阻滞在G2/M期。与对照组相比,IL24基因治疗组肿瘤在体内的生长受到明显抑制,P<0.05。结论:IL24基因转染的肿瘤细胞体外生长受抑。于荷瘤小鼠的瘤内注射pEGFPIL24可抑制肿瘤生长,提示IL24具有明显的体内外抗肿瘤作用。     

细胞因子基因转导的人肿瘤细胞生物学行为研究

对细胞因子基因修饰的人肝癌和胃癌细胞用电子显微术进行了细胞形态学观察、细胞生长曲线及克隆形成率测定、以及细胞周期的测定,结果发现:1.与未经修饰原肿瘤及空白载体修饰肿瘤细胞相比,扫描电镜观察到细胞因子基因修饰肿瘤细胞的表面形态及细胞形态有很大不同,如绒毛增多变细长、细胞变细长等,透射电镜没有发现细胞器等超微结构有明显改变;2.细胞因子基因修饰肿瘤细胞株的克隆形成率下降,增殖速度也变慢,但流式细胞仪测定的细胞周期却无明显变化;3.在细胞因子基因修饰肿瘤细胞培养过程中经常发现有原因不明的死亡现象,细胞变圆并浮起,将这类细胞收集抽提的DNA经琼脂糖凝胶电泳后发现DNA发生了弥散性降解,对以上现象的初步解释是:细胞因子基因地肿瘤细胞基因组中的整合及表达可能:1)对肿瘤细胞有一定的诱导分化作用,如IFN-γ;2)使肿瘤细胞发生坏死,如TNF-α;或促进细胞程序化死亡的发生,如IL-2。     

IL-18基因修饰增强肿瘤细胞裂解物致敏的树突状细胞的抗肿瘤效应

目的:观察经肿瘤细胞裂解物致敏的白细胞介素18(IL-18)基因修饰的树突状细胞(DC)体内诱导的抗肿瘤免疫应答反应.方法:体外培养的小鼠骨髓树突状细胞经IL-18重组腺病毒感染后(IL18-DC),再经Hepal-6肝癌细胞裂解物冲击致敏后通过皮下注射用于荷瘤小鼠的治疗.用ELISA检测细胞因子,4 h51Cr释放法检测NK细胞活性及CTL杀伤活性.结果:致敏IL18-DC组体内诱导NK细胞活性与未致敏IL18-DC组无明显差别(P＞0.05),但明显高于致敏DC组和DC组(均P＜0.01);致敏IL18-DC组体内诱导特异性CTL杀伤活性明显高于IL18-DC组、致敏DC组和DC组(均P＜0.01);致敏IL18-DC组免疫治疗作用明显优于未致敏IL18-DC组、致敏DC组和DC组(均P＜0.01).结论:肿瘤细胞裂解物致敏的IL-18基因修饰的DC疫苗进行体内免疫治疗,能诱导出显著的抗肿瘤免疫反应,为DC介导的肿瘤基因治疗开辟了新的途径.     

EFFECTS OF IL-6/IL-2 FUSION GENE TRANSFECTION ON TUMOUR CELL BIOLOGICAL CHARACTERISTICS IN VITRO AND IN VIVO

ＥＦＦＥＣＴＳＯＦＩＬ６／ＩＬ２ＦＵＳＩＯＮＧＥＮＥＴＲＡＮＳＦＥＣＴＩＯＮＯＮＴＵＭＯＵＲＣＥＬＬＢＩＯＬＯＧＩＣＡＬＣＨＡＲＡＣＴＥＲＩＳＴＩＣＳＩＮＶＩＴＲＯＡＮＤＩＮＶＩＶＯＹｕＸｉａｏｄａｎ于晓ＴａｎｇＰｅｉｘｉａｎ唐佩弦ＺｈａｏＣｈ...     

A study on anti-tumor immunity induced by gene-modified melanoma B16 cells

T cell-mediated cell immunity is the main anti-tumor immunity in which the effector T cells need specific antigen and costimulatory signals. One of the vaccines applied in tumor immunotherapy is the gene-modified tumor cell vaccine. One potential method to increase cell epitope density is to link the antigen with the major histcompatibility complex subunit beta2m. Our previous research indicated that the strategy of epitope fusion gene OVA-linker-beta2m can promote the formation of specific compounds on the tumor cell surface in vitro. In this study, we constructed two coexpression vectors pGL3-CD80-OVA-linker-beta2m and pGL3-IL21-OVA-linker-beta2m, in order to explore the cooperative action of CD80 or interleukin-21 (IL21) with the epitope fusion gene in anti-tumor immunity. Results showed that gene-modified B16 cells (B16/OVA, B16/CD80-OVA and B16/IL21-OVA) grew slower than B16 cells in vitro and in vivo, especially the B16/IL21-OVA subline, which illustrated that such gene modification decreased oncogenicity of malignant tumor cells. On the other hand, gene-modified tumor cell subline immunization can induce effective long-term anti-tumor immunity defending tumor cell attacks. IL21 played a more cooperative role with the OVA-linker-beta2m than CD80 in this study. This strategy might lay foundations for the research of a new type of tumor vaccine.     

Reduced Invasive and Metastatic Potentials of KAI1-transfected Melanoma Cells

KAI1 is a metastasis suppressor gene for human prostate cancer. To reveal the effect of KAI1 on the in vivo metastasis of tumors other than prostatic cancer, we transfected a human KAI1 cDNA into highly metastatic B16-BL6 murine melanoma cells and established stable transfectant clones with different expression levels of KAI1 message. The following results were obtained with the use of those transfectants. (1) Cell aggregation assay revealed a significantly enhanced Ca²⁺-independent aggregation of B16-BL6 cells by KAI1 cDNA transfection compared with mock transfectants ( P <0.01). (2) The in vivo phagokinetic activity and invasive ability of KAI1 transfectants were clearly decreased as compared with those of mock transfectants ( P <0.01). There was no significant effect of KAI1 expression on the in vitro or in vivo proliferation of B16-BL6 cells. (3) Lung colony formation of intravenously injected KAI1 transfectants in nude mice was significantly reduced as compared with mock transfectants or parental B16-BL6 cells ( P <0.01). These data suggest that KAI1 expression gives rise to the suppression of invasive and metastatic potentials of B16-BL6 cells.     

Impaired Cytotoxic Activity of Interleukin 2 (IL 2)-cultured Large Granular Lymphocytes (LGL) in Childhood Acute Lymphoblastic Leukemia. Quantitative Measurement of IL 2 Receptor Expression of IL 2-cultured LGL

Large granular lymphocytes (LGL) of the natural killer (NK) cell lineage were highly purified and their interleukin 2 (IL 2) receptors (IL 2R) were investigated in childhood acute lymphoblastic leukemia (ALL). Not only NK activity but also 3-day recombinant IL 2-cultured lymphokine activated killer (LAK) activity were decreased in ALL, despite normal percentages of LGL, CD16 (Leul1)⁺ and CD56 (NKH1)⁺ cells. The cytotoxic activity of IL 2-cultured LGL, but not IL 2-cultured T cells, was significantly decreased in ALL, indicating a selective defect of LGL among the killer cells. IL 2R (CD25) numbers/cell of 3-day IL 2-cultured LGL were not decreased in ALL by flow cytometric analysis. Scatchard plot analysis demonstrated that high affinity IL 2R numbers/cell of 3-day IL 2-cultured LGL were normal but the binding affinity level of the receptors in these cells in ALL was one-third of that in the similarly-cultured control cells, suggesting inadequate IL 2-IL 2R interaction is responsible, at least in part, for their reduced cytotoxic activity.     

Antitumor Effects of Interleukin-2 Gene-modified Fibroblasts in an Orthotopic Colon Cancer Model

We transduced the interleukin-2 (IL-2) gene into murine fibroblasts BALBCL7 or murine colon cancer CT26 using a retroviral vector. BALBCL7 transduced with IL-2 gene secreted 748 pg/ml of IL-2, whereas IL-2 gene-modified CT26 secreted 1,167 pg/ml of IL-2 (48 h incubation, 1×10⁶/ml). Then, we inoculated gene-modified BALBCL7 and/or CT26 cells into BALB/c female mice, and observed the tumor growth. The tumor growth was inhibited in mice inoculated with parental CT26 plus IL-2 gene-modified BALBCL7, compared with that in mice given parental CT26 alone ( P < 0.01). Moreover, we investigated the cytotoxic activity of spleen cells derived from mice treated with gene-modified cells, and performed phenotypic analysis of the effector cells. The killer cells derived from mice inoculated with IL-2 gene-modified BALBCL7 plus parental CT26 showed higher cytotoxic activity than those from mice inoculated with CT26 alone. The cytotoxic activity was almost completely blocked by anti-CD8 antibody (Ab), and partially blocked by anti-asialo GM1 Ab. Next, we inoculated CT26 tumor tissue into murine cecum orthotopically, and treated the animals with gene-modified BALBCL7 plus parental CT26. The tumor size in the cecum was significantly decreased, compared with parental CT26 alone ( P < 0.01).     

Chemotherapy and immunotherapy of tumours induced by gene-modified HPV16-transformed cells

HPV16 E6/E7 transformed mouse kidney cells designated MK16/1/IIIABC (MK16) were modified by the insertion of a suicide gene, viz. the thymidine-kinase gene of herpes simplex virus (HSV TK). Tumour induction by these cells, designated N2A, was suppressed by ganciclovir (GCV). The growth of already established tumours was partially inhibited by GCV. This effect was markedly potentiated by a single dose of cyclophosphamide (Cy). Ganciclovir- or GCV+ Cy-cured mice were not protected against challenge with MK16 cells. N2A tumour growth was suppressed by simultaneous administration of MK16-derived, non-oncogenic B9 and 181 cells, which express either mouse GM-CSF or mouse IL2, respectively, in addition to HSV TK. The animals treated were protected against challenge with MK16 cells. Animals with already established N2A tumours were treated with GCV and/or repeated doses of B9 or 181 cells. Ganciclovir treatment alone and immunotherapy alone resulted in partial suppression of tumour growth but not in tumour cure. On the other hand, combined chemo- and immunotherapy resulted in tumour rejection by nearly all animals. Similar results were obtained if the immunotherapy with homologous gene-modified cells was substituted by treatment with anti-CD4 antibody. The animals cured of tumours with GCV combined with cell-based vaccine therapy but not those cured by GCV and anti-CD4 antibody treatment were found resistant to challenge with MK16 cells. The present results suggest that combined specific and non-specific chemo- and immunotherapy of tumours induced by appropriately gene-modified cells might provide a special advantage in the treatment of established tumours.     

Vaccination of dendritic cells loaded with interleukin-12-secreting cancer cells augments in vivo antitumor immunity: characteristics of syngeneic and allogeneic antigen-presenting cell cancer hybrid cells.

Cancer immunotherapy by fusion of antigen-presenting cells and tumor cells has been shown to induce potent antitumor immunity. In this study, we characterized syngeneic and allogeneic, murine macrophage/dendritic cell (DC)-cancer fusion cells for the antitumor effects. The results showed the superiority of allogeneic cells as fusion partners in both types of antigen-presenting cells in an in vivo immunotherapy model. A potent induction of tumor-specific CTLs was observed in these immunized conditions. In addition, the immunization with DC-cancer fusion cells was better than that with macrophage-cancer fusion cells. Both syngeneic and allogeneic DC-cancer fusion cells induced higher levels of IFN-gamma production than macrophage-cancer fusion cells. Interestingly, allogeneic DC-cancer fusion cells were superior in that they efficiently induced Th1-type cytokines but not the Th2-type cytokines interleukin (IL)-10 and IL-4, whereas syngeneic DC-cancer fusion cells were powerful inducers of both Th1 and Th2 cytokines. These results suggest that allogeneic DCs are suitable as fusion cells in cancer immunotherapy. To further enhance the antitumor immunity in the clinical setting, we prepared DCs fused with IL-12 gene-transferred cancer cells and thus generated IL-12-secreting DC-cancer fusion cells. Immunization with these gene-modified DC-cancer fusion cells was able to elicit a markedly enhanced antitumor effect in the in vivo therapeutic model. This novel IL-12-producing fusion cell vaccine might be one promising intervention for future cancer immunotherapy.     

IL-2基因修饰鼠肝癌细胞引起的凋亡研究

用重组人白细胞介素-2(IL-2)含信号肽cDNA的缺陷型逆转录病毒将人IL-2基因整合于鼠肝癌细胞株H22染色体后，PCR及RT—PCR方法确证有人IL-2基因存在及表达，流式细胞仪检测表明IL-2基因修饰后的H22细胞中DNA低含量细胞明显多于对照组。用透射电镜分别观察了IL-2基因修饰H22细胞和未经修饰H22细胞的超微结构，发现IL-2基因修饰H22细胞其染色体有固缩等凋亡现象发生，说明人IL-2基因在鼠肝癌细胞中的表达具有促使肿瘤细胞发生凋亡的作用，这可能也是IL-2基因修饰后其致瘤性下降的重要机制之一。     

Interleukin-1B (IL1B) and interleukin-6 (IL6) gene polymorphisms are associated with risk of chronic lymphocytic leukaemia

Common polymorphisms in genes encoding for cytokines implicated in the inflammatory response and Th1/Th2 balance might play a role in the development and prognosis of chronic lymphocytic leukaemia (CLL). To test the hypothesis, we investigated 13 single nucleotide polymorphisms (SNPs) in nine of such genes in a population-based case-control study, conducted in the Italian region of Sardinia in 1999-2003. Forty incident CLL cases and 113 population controls were available for study. The following SNPs were selected: IL1A-889C > T, IL1RN 9589A > T, IL1B-31C > T, IL1B-511C > T, IL2-384T > G, IL6-174G > C, IL6-597G > A, IL10-1082A > G, IL10-3575T > A, TNF-308G > A, LTA- 91A > C, LTA 252A > G and CARD15 nt1007. After adjusting by age and gender, individuals homozygous for the IL1B-511T allele run a lower risk of CLL (OR = 0.1, 95% CI 0.0, 0.8, p = 0.032), while risk showed a 4.5-fold increase associated with the genotype homozygous for the IL6-174C allele (OR = 4.5; 95% CI 1.1, 19.3, p = 0.041). Individuals homozygous for the IL6-174C allele and carrying the homozygous IL1B-511C allele showed an 11-fold increase in CLL risk (OR = 11.4, 95% CI 1.9, 69.4, p = 0.008). None of the other interleukin SNPs evaluated showed any association with CLL risk. Large multicentre pooled studies are warranted, achieving the statistical power required to confirm whether IL6 and IL1B gene polymorphisms might play a role in CLL development and prognosis, as well as the null associations herein reported.     

Simultaneous targeting of IL2 and IL12 to Hodgkin's lymphoma cells enhances activation of resting NK cells and tumor cell lysis

Hodgkin's disease (HD) is characterized by the accumulation of functionally anergic T cells in the vicinity of the malignant Hodgkin/Reed-Sternberg (H/RS) cells. To revert cellular anergy against H/RS cells, we generated an anti-CD30-antibody-interleukin-(IL)-2 and an anti-CD30-antibody-IL12 fusion protein that target IL2 and IL12, respectively, specifically to CD30+ H/RS cells. Both antibody-cytokine fusion proteins act cooperatively in the activation of resting NK cells, the induction of IFN-g gamma secretion and enhanced target cell lysis. The cooperative activity of the targeted cytokines suggests that the application of both antibody-cytokine fusion proteins may be particularly suitable for the specific immunotherapy of Hodgkin's lymphoma.