局部注射小鼠白细胞介素12逆转录病毒包装细胞对大鼠肝癌的治疗

目的:研究低剂量环磷酸胺(Cy)联合MHC Ⅰ类限制性肿瘤抗原多肽Mutl致敏、白细胞介素2(IL-2)基因修饰的树突状细胞(DCs)对转移性肺癌小鼠的治疗作用及其免疫学机理.方法:制备小鼠骨髓来源的DCs,用转移性Lewis肺癌特异性多肽Mutl预激经IL-2基因修饰的DCs联合低剂量Cy治疗转移性肺癌小鼠.通过FACS分析其脾细胞内T淋巴细胞比例的变化,~51Cr释放法检测CTL和NK细胞杀伤活性.结果:肿瘤抗原多肽致敏、IL-2基因修饰的DCs与小剂量Cy联合后,能比单用DCs更有效地治疗转移性肺癌,小鼠脾细胞中CD8~ T细胞和NK1.1~ 细胞明显比例升高,联合治疗组诱导出的CTL杀伤活性最高.结论:以肿瘤抗原多肽冲击致敏的IL-2基因修饰的DCs联合小剂量Cy能更有效地促进荷瘤宿主免疫应答,具有显著地体内抑制肺癌转移的效果.     

低剂量环磷酰胺增强MHCⅠ类限制性肿瘤抗原多肽冲击致敏的树突状细胞的抗肿瘤转移效果

目的:研究低剂量环磷酸胺(Cy)联合MHC Ⅰ类限制性肿瘤抗原多肽Mutl致敏、白细胞介素2(IL-2)基因修饰的树突状细胞(DCs)对转移性肺癌小鼠的治疗作用及其免疫学机理.方法:制备小鼠骨髓来源的DCs,用转移性Lewis肺癌特异性多肽Mutl预激经IL-2基因修饰的DCs联合低剂量Cy治疗转移性肺癌小鼠.通过FACS分析其脾细胞内T淋巴细胞比例的变化,~51Cr释放法检测CTL和NK细胞杀伤活性.结果:肿瘤抗原多肽致敏、IL-2基因修饰的DCs与小剂量Cy联合后,能比单用DCs更有效地治疗转移性肺癌,小鼠脾细胞中CD8~+T细胞和NK1.1~+细胞明显比例升高,联合治疗组诱导出的CTL杀伤活性最高.结论:以肿瘤抗原多肽冲击致敏的IL-2基因修饰的DCs联合小剂量Cy能更有效地促进荷瘤宿主免疫应答,具有显著地体内抑制肺癌转移的效果.     

白介素-12基因转染致敏树突状细胞体外诱导特异抗前列腺癌效应的实验研究

背景与目的前列腺癌是泌尿系统疾病中发病率和死亡率较高的恶性肿瘤,临床积极寻求最佳治疗方案。白细胞介素-12(IL-12)具有促T细胞增殖分化和抗肿瘤免疫作用,负载肿瘤抗原的树突状细胞(DC)疫苗可诱导机体产生特异性的抗肿瘤免疫效应,联合两者抗肿瘤效应,探讨逆转录病毒介导白细胞介素-12(IL-12)基因转染DC体外诱导特异免疫杀伤前列腺癌细胞的效能及其机制。方法构建IL-12基因逆转录病毒重组质粒,转染PA317细胞后获得相应病毒,转染前列腺癌(PCa)患者外周血DC成DC-IL-12细胞。以HSP70-PC-3m肿瘤混合肽负载DC-IL-12细胞得致敏的DC。MTT法检测T淋巴细胞(1×105个/ml)增殖分化能力,细胞毒实验检测DC-IL-12诱导的细胞毒T淋巴细胞(CTL)及其上清液对PC-3m细胞株杀伤作用。结果DC经IL-12修饰后48h分泌高水平IL-12(26.35±3.12)ng/L及IFN-r(778±28)ng/L,均显著高于未转染组(P<0.05)。DC-IL-12细胞诱导的CTL及其上清液对前列腺癌PC-3m细胞均有显著杀伤作用,杀伤率显著高于未转染组,分别为(76.43±6.20)%vs(42.12±2.80)%和(66.38±7.56)%vs(14.23±5.42)%(P<0.05),对膀胱癌细胞EJ无明显杀伤作用。结论IL-12基因修饰增强PC-3m细胞抗原致敏DC体外诱导同源T淋巴细胞产生特异性的抗前列腺癌免疫效能,其机制与IL-12基因转染DC后促进分泌IL-12、IFN-r及增强T淋巴细胞活化状态密切相关。     

突变Ki-ras基因修饰的肺癌DC疫苗的体外抗癌活性

目的 探讨以Ki-ras基因12位密码子点突变为靶点，构建DC-Ad-Ki-ras(V12)肺癌疫苗及其抗肺癌免疫治疗的可能性。方法 应用重组腺病毒的方法将人Ki-ras基因12位密码子点突变(由甘氨酸转变为缬氨酸)的cDNA导入DC，构建DC-Ad—Ki-ras(V12)肺癌疫苗，以流式细胞术、PCR、MLR和CTL等方法检测其免疫活性。结果 (1)DC-Ad-Ki-ras(V12)肺癌疫苗小仅能表达人Ki—ras(V12)基因，且能明显刺激T细胞增殖及提高CTL的杀伤作用。(2)突变Ki-ras基因修饰的DC免疫小鼠后，可产生针对表达Ki—ras(V12)基因的Lewis肺癌的特异件杀伤作用，但对B16黑色素瘤细胞则无明显杀伤作用。结论 以人突变Ki—ras基因修饰的肺癌DC疫苗可在体外特异性诱导抗可表达Ki-ras(V12)基因的Lewis肺癌的活性。     

肿瘤抗原多肽致敏白介素-18基因修饰的树突状细胞对转移性肺癌治疗作用的研究

IL-18是新近发现的细胞因子,可诱生IFN-2的产生、增强NK活性和Th1型免疫反应具有抗肿瘤作用.本研究的目的是研究转染IL-18基因的DC以肿瘤抗原肽冲击致敏后对转移性肺癌的治疗作用.首先从C57BL/6小鼠骨髓培养DC,按MOI=100进行体外转染AdIL-18及AdLacZ基因,X-gal染色提示转染效率大于95%;转染smIL-18后24h、48h,ELISA测定上清中IL-18因子水平分别为(45±8)ng/ml和(54±7)ng/ml;以RT-PCR方法可检测到含插入信号肽的IL-18mRNA的表达,对照DC上清中则未检出;测定DC-IL-18培养上清的活性发现IL-18基因转染之DC上清能显著刺激ConA活化的小鼠脾脏T细胞产生IFN-γ,且与IL-12有协同作用,对照组DC上清则无此作用.FACS检测msIL-18基因修饰前后DC表型发现,提示msIL-18基因修饰后24h,荧光强度无明显变化,但la,B7-2,     

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.     

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-23基因修饰的树突细胞疫苗联合β-榄香烯对小鼠胰腺癌生长的影响

背景与目的:树突细胞(dendriticcells,DC)疫苗是目前最具应用潜能的治疗性疫苗,功能性细胞因子能显著增强DC的活性及其诱导的宿主抗肿瘤作用,利用细胞因子基因修饰DC是当今肿瘤免疫治疗中最活跃的领域。本研究探讨β-榄香烯联合白细胞介素-23(interleukin-23,IL-23)修饰的DC疫苗对胰腺癌小鼠的协同抗肿瘤作用。方法:克隆并构建IL-23基因真核双表达载体,转染DC并负载肿瘤抗原后制备成疫苗。将IL-23基因转染的DC疫苗、空质粒转染的DC疫苗、未转染的DC疫苗及对照生理盐水分别注射小鼠,体外观察各组小鼠脾脏T淋巴细胞IFN-γ及IL-4的分泌。体内观察β-榄香烯联合DC疫苗对荷胰腺癌小鼠肿瘤生长的抑制作用及对小鼠存活期的影响。结果:基因测序证实IL-23基因克隆及双表达载体构建成功,转染后DC共刺激分子MHC-Ⅰ和MHC-Ⅱ的表达增强。接种IL-23转染DC疫苗后小鼠的免疫防御能力显著增强,有效地延缓并防御接种肿瘤的发生。DC介导的免疫应答促进了IFN-γ生成型Th1细胞的产生,未转染DC疫苗组和空质粒转染DC疫苗组IL-4分泌量与IL-23转染的DC疫苗组比较差异有显著性(P<0.05);IL-23转染DC疫苗组IFN-γ的分泌与其他各组比较差异有显著性(P<0.01)。β-榄香烯联合IL-23转染DC疫苗组肿瘤生长受到显著抑制,该组小鼠存活期与DC组、NS对照组及β-榄香烯组比较差异有极显著性(P<0.01)。结论:IL-23修饰DC疫苗可强化宿主针对特异肿瘤的Th1及CTL的免疫应答,使宿主不仅产生防御性免疫反应而且增强自动免疫能力。β-榄香烯有一定的协同抗癌作用。     

基因腺病毒感染树突状细胞诱导CTL对胶质瘤细胞的杀伤作用

目的:研究EPHA2基因修饰的树突状细胞(dendritic cell,DC)疫苗诱导细胞毒性T淋巴细胞(cytotoxic T lympho-cyte,CTL)对U251胶质瘤细胞的杀伤效应,为胶质瘤的免疫治疗提供新的方法。方法:将重组EPHA2腺病毒rAd-EPHA2感染HLA-A2阳性的人外周血来源的DC,制备EPHA2基因修饰的DC疫苗,Western blotting和FACS方法检测感染后DC的EPHA表达。以DC疫苗体外刺激HLA-A2阳性的单个核细胞,酶联免疫斑点实验(enzyme-linked immunospot assay,ELISPOT)和标准51Cr释放实验分别检测DC疫苗所诱导的CTL活性和对HLA-A2阳性的U251细胞的杀伤作用(另设rAd-Lac Z感染的DC组和PBS组作为对照)。结果:成功制备了EPHA2基因修饰的DC疫苗,其可有效表达EPHA2蛋白。与感染rAd-LacZ的DC和PBS组相比,感染rAd-EPHA2的DC疫苗能有效激发CTL活性[(187±21)vs(12±4)、(18±5)个,P<0.01];所诱导的CTL对胶质瘤U251细胞有明显的杀伤效应[(45.7±6.8)%vs(7,1±4.5)%,P<0.01],对自身淋巴细胞没有杀伤效应。结论:EPHA2基因修饰的DC疫苗能有效激发CTL活性,并对胶质瘤U251细胞有明显的杀伤活性。     

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.     

Immunotherapy of spontaneous metastatic lung cancer with tumor antigen-pulsed,interleukin-12 gene-modified dendritic cells

Interleukin-12 (IL-12), with the ability of inducing production of interferon-gamma and enhancing of NK activity and Th1 response, has potent antitumor role and has been used in treatment of tumors[1-7]. Dendritic cells (DC) are the uniquely potent APCs involved in the initiation of immune responses. As adjuvants for Ag delivery, DC pick up Ags in the periphery and carry them to T cells area in lymphoid organs to prime the immune responses. With the development of the methods for propaga…     

Route of administration influences the antitumor effects of bone marrow-derived dendritic cells engineered to produce interleukin-12 in a metastatic mouse prostate cancer model

Gene-modified dendritic cells (DC) provide unique therapeutic strategies for prostate cancer; however, the comparative evaluation of specific delivery options using appropriate preclinical models has not been described. In this study, bone marrow-derived DC were genetically engineered to express high levels of interleukin-12 (IL-12) with or without the costimulatory molecule B7-1, by ex vivo infection with recombinant adenoviral vectors. We used an orthotopic metastatic mouse prostate cancer preclinical model (178-2 BMA) to compare two therapeutic protocols for DC delivery, in situ and subcutaneous. DC were generated from bone marrow of syngeneic 129/Sv mice by culturing in the presence of GM-CSF and IL-4. In vitro DC/IL-12 or DC/IL-12/B7 produced high levels of biologically active IL-12. In situ delivery of DC/IL-12 or DC/IL-12/B7 induced a significant suppression of primary tumor growth compared to DC/beta gal controls (P=.0328 and P=.0019, respectively), as well as reduced numbers of spontaneous lung metastatic nodules (P=.1404 and P=.0335, respectively). In survival experiments, in situ DC/IL-12 injection demonstrated a small but statistically significant advantage (P=.0041). Subcutaneous, tumor lysate pulsed DC/IL-12 significantly decreased tumor size (P=.0152) and increased survival (P=0.0433) compared to HBSS controls but the decrease in the number of spontaneous lung metastases did not achieve statistical significance. Both in situ and subcutaneous treatments enhanced cytolytic activities of natural killer (NK) cells and cytotoxic T lymphocytes (CTL). In this preclinical model, gene-modified DC-based intratumoral immunotherapy was shown to be an effective therapeutic strategy for locally advanced prostate cancer based on tumor growth suppression, inhibition of metastasis and survival improvement.     

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。     

Antitumor Activity of Lentivirus-mediated Interleukin -12 Gene Modified Dendritic Cells in Human Lung Cancer in Vitro

Objectives: Dendritic cell (DC)-based tumor immunotherapy needs an immunogenic tumor associatedantigen (TAA) and an effective approach for its presentation to lymphocytes. In this study we explored whethertransduction of DCs with lentiviruses (LVs) expressing the human interleukin-12 gene could stimulate antigenspecificcytotoxic T cells (CTLs) against human lung cancer cells in vitro. Methods: Peripheral blood monocytederivedDCs were transduced with a lentiviral vector encoding human IL-12 gene (LV-12). The anticipated targetof the human IL-12 gene was detected by RT-PCR. The concentration of IL-12 in the culture supernatant of DCswas measured by ELISA.Transduction efficiencies and CD83 phenotypes of DCs were assessed by flow cytometry.DCs were pulsed with tumor antigen of lung cancer cells (DC+Ag) and transduced with LV-12 (DC-LV-12+Ag).Stimulation of T lymphocyte proliferation by DCs and activation of cytotoxic T-lymphocytes (CTL) stimulatedby LV-12 transduced DCs pulsed with tumor antigen against A549 lung cancer cells were assessed with methylthiazolyltetrazolium (MTT). Results: A recombinant lentivirus expressing the IL-12 gene was successfullyconstructed. DC transduced with LV-12 produced higher levels of IL-12 and expressed higher levels of CD83than non-transduced. The DC modified by interleukin -12 gene and pulsed with tumor antigen demonstratedgood stimulation of lymphocyte proliferation, induction of antigen-specific cytotoxic T lymphocytes and antitumoreffects. Conclusions: Dendritic cells transduced with a lentivirus-mediated interleukin-12 gene have anenhanced ability to kill lung cancer cells through promoting T lymphocyte proliferation and cytotoxicity.     

Adenovirus-mediated CD40 ligand gene-engineered dendritic cells elicit enhanced CD8(+) cytotoxic T-cell activation and antitumor immunity

CD40L, the ligand for CD40 on dendritic cells (DCs), plays an important role in their activation and is essential for induction of antigen-specific T-cell responses. In the present study, we investigated the efficacy of antitumor immunity induced by vaccination with DCs engineered to express CD40L and pulsed with Mut1 tumor peptide. Our data show that transfection of DCs with recombinant adenovirus AdV-CD40L resulted in activation of DCs with up-regulated expression of proinflammatory cytokines (IL-1beta and IL-12), chemokines (RANTES, IP-10, and MIP-1alpha), and immunologically important cell surface molecules (CD54, CD80, and CD86). Our data also demonstrate that DCs transfected with AdV-CD40L (DC(CD40L)) are able to stimulate enhanced allogeneic T-cell proliferation and Mut1-specific CD8(+) cytotoxic T-cell responses in vitro. Vaccination of mice with Mut1 peptide-pulsed control virus-transfected DC (DC(pLpA)) could only protect mice from challenge of a low dose (0.5 x 10(5) cells per mouse, 8/8 mice), but not a high dose (3 x 10(5) cells per mouse, 0/8 mice) of 3LL tumor cells. However, vaccination of Mut1 peptide-pulsed AdV-CD40L-transfected DC(CD40L) induced an augmented antitumor immunity in vivo by complete protection of mice (8/8) from challenge of both low and high doses of 3LL tumor cells. Thus, DCs engineered to express CD40L by adenovirus-mediated CD40 ligand gene transfer may offer a new strategy in production of DC cancer vaccines.