CD3单克隆抗体活化的杀伤细胞对人胃癌细胞株MNK45体外杀伤及…

用人的外周血单个核细胞（ＰＢＭＣ）与抗ＣＤ３单克隆抗体和基因重组的人ＩＬ－２，共同培养制备ＣＤ３ＡＫ细胞，ＰＢＭＣ与ｒＩＬ－２共同培养制备ＬＡＫ细胞，用单纯ＰＢＭＣ作为对照细胞。对这三种细胞的增殖及在体外和裸鼠体内的抗人胃癌细胞株ＭＮＫ４５作用进行了实验研究。体外试验结果表明，ＣＤ３ＡＫ对ＭＮＫ４５有明显杀伤作用，而ＬＡＫ细胞杀伤率较低，二者差异有非常显著性意义（Ｐ＜０．０１）；用ＭＴＴ法和台盼蓝     

小鼠H-2K b基因转染人肝癌细胞后激活免疫排斥反应的研究

目的：增强人肝癌细胞的免疫原性以激活宿主免疫细胞识别杀伤相应的肝癌细胞。方法：以脂质体介导小鼠Ｈ－２Ｋｂ基因转染肝癌细胞株,并检测Ｈ－２Ｋｂ抗原表达情况,然后用Ｈ－２Ｋｂ基因转染后的肝癌细胞体外激活效应细胞杀伤活性,再进行裸鼠动物实验。结果：Ｈ－２Ｋｂ基因转染人肝癌细胞株ＨｅｐＧ２后,Ｓｏｕｔｈｅｍ印迹杂交结果显示,Ｈ－２Ｋｂ基因整合于肿瘤细胞染色体中,ＲＮＡ点杂交结果显示,Ｈ－２ＫｂＤＮＡ已转录成ｍＲＮＡ。免疫组化及流式细胞仪检测显示,Ｈ－２Ｋｂ抗原已在肝癌细胞胞膜上表达。转染后肝癌细胞在体外能强烈诱发效应细胞毒性,这种细胞毒性现象在裸鼠实验中得到进一步验证。结论：异源ＭＨＧ－Ｉ类基因Ｈ－２Ｋｂ转染肝癌细胞后可增强其免疫原性并激活免疫效应细胞杀伤活性。     

CD_3单克隆抗体活化的杀伤细胞对人胃癌细胞株MNK_（45）体外杀伤及裸鼠体

用人的外周血单个核细胞（ＰＢＭＣ）与抗ＣＤ３单克隆抗体和基因重组的人ＩＬ－２，共同培养制备ＣＤ３ＡＫ细胞（ＣＤ３ＭｃＡｂＡｃｔｉｖａｔｅｄＫｉｌｌｅｒＣｅｌｌｓ），ＰＢＭＣ与ｒＩＬ－２共同培养制备ＬＡＫ细胞，用单纯ＰＢＭＣ作为对照细胞。对这三种细胞的增殖及在体外和裸鼠体内的抗人胃癌细胞株ＭＮＫ４５作用进行了实验研究。体外试验结果表明，ＣＤ３ＡＫ对ＭＮＫ４５有明显杀伤作用，而ＬＡＫ细胞杀伤率较低，二者差异有非常显著性意义（Ｐ＜０．０１）；用的ＭＴＴ法和台盼蓝活细胞计数法测定这三种细胞的增殖能力和细胞数量的增加，表明ＣＤ３ＡＫ的增殖能力和扩增数量均明显高于ＬＡＫ细胞（Ｐ＜０．０１）。裸鼠体内试验结果显示，对照组及ＬＡＫ组全部长出瘤结节，而ＣＤ３ＡＫ组无一例长出皮下结节，且生存期明显长于前者。     

双特异抗体介导的CD3AK对人小细胞肺癌细胞株的细胞毒作用

目的：研究在双特异性抗体的介导下ＣＤ３ＡＫ细胞（ＣＤ３单克隆栓激活的杀伤细胞）对人小细胞肺癌细胞株ＬＴＥＰ－ｓｍｌ的细胞毒作用。方法：用＾３１Ｃｒ－Ｎａ２ＣｒＯ４释放试验检测单抗（２Ｄ６、ＵＣＨＴ１）和双特异性抗体（ＷＳＴ－Ｈ７）与ＣＤ３ＡＫ共同对人小细胞肺癌细胞株的杀伤活性。持异性抗体体外能明显增强ＣＤ３ＡＫ细胞对人小细胞肺细胞株ＬＴＥＰ－ｓｍｌ的杀伤活性结论：与单纯ＣＤ３ＡＫ相比,加入双特异性     

IL—2／LAK疗法对肿瘤患者免疫功能的影响

我们观察了２０例肿瘤患者应用ＩＬ－２／ＬＡＫ疗法前后其免疫指标的改善，包括Ｔ淋巴细胞亚群，ＬＡＫ细胞活性，白介素Ⅱ分泌细胞及血清白介素Ⅱ受体。通过治疗显示：ＣＤ４／ＣＤ８比值，ＬＡＫ活性，白介素Ⅱ膜受体及白介素Ⅱ分泌细胞的水平均显著升高，而可溶性白介素Ⅱ受体减少，提示ＩＬ－２／ＬＡＫ可以改善机体免疫，功能。     

人类LAK细胞体外实验性扩增及生物学活性分析

由癌症患者外周血中分离出少量ＬＡＫ前体细胞。在ＬＡＫ细胞培养体系中加入重组人类白细胞介素２，植物血球凝集素及辅助细胞，明显提高了ＬＡＫ细胞的体外增殖能力并延长了ＬＡＫ细胞的存活时间。表型分析发现，ＬＡＫ－Ｌ细胞的ＩＬ－２受体（ＩＬ－２－Ｒ－）表达水平明显高于ＬＡＫ－Ｓ细胞，ＬＡＫ－Ｌ细胞的体外大量增殖具有以ＯｋＴ＾＋ ８表型为主的单亚群增殖特性。体外杀伤实验及裸小鼠体内成瘤抑制实验结果表明，用此法     

3H—TdR法检测LAK细胞活性的建立及人肺腺癌细胞株（GLC—82）对LAK细胞的…

用３Ｈ－ＴｄＲ释放法检测了个旧人肺腺癌细胞株（ＧＬＣ－８２），小鼠肥大细胞瘤（Ｐ８１５）及人红白血病细胞株（Ｋ５６２）对自然杀伤细胞（ＮＫ细胞），洒巴因子激活的杀伤细胞（ＬＡＫ细胞）的敏感性。结果表明：用３Ｈ－ＴｄＲ释放法检测ＮＫ、ＬＡＫ细胞活性具有灵敏、准确、重复性好的优点，适用于国内一般实验室开展；ＧＬＣ－８２细胞对ＮＫ细胞不敏感，而对ＬＡＫ细胞敏感，提示人肺腺癌用ＬＡＫ细胞治疗可能有效，ＧＬ     

人参皂甙与IL-2协同诱导PBMC对恶性脑胶质瘤细胞杀伤活性研究

目的：探索提高恶性脑胶质瘤治疗效果的新途径。方法：用人参皂甙（ｇｉｎｓｅｎｏｓｉｄｅ,ＧＳ）与白细胞介素２（ＩＬ－２）协同诱导人外周血单个核细胞（ＰＢＭＣ）制成ＧＳ－ＬＡＫ细胞,用ＭＴＴ法测定其对恶性脑胶质瘤细胞（ＢＴ３２５）的杀伤活性,并与ＬＡＫ细胞相比较。结果：效应细胞ＧＳ－ＬＡＫ在增殖数量和杀伤恶性脑胶质瘤细胞活性等方面均优于ＬＡＫ细胞,且ＩＬ－２用量减少。结论：采用ＧＳ－ＬＡＫ细胞,可提高效应细胞的数量和活性,为恶性脑胶质瘤的免疫治疗打下了理论基础。     

IL-4基因及B7-1基因共转染新型瘤苗的制备及其治疗作用的初步研究

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，并探讨了基估ＣＴＬ诱导过程中的作用。结果表明，ＩＬ－２，ＩＬ－４，ＩＬ－６基因转染Ｂ１６黑素瘤细胞表面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。     

IL－2／LAK疗法对肿瘤患者免疫功能的影响

我们观察了２０例肿瘤患者应用ＩＬ－２／ＬＡＫ疗法前后其免疫指标的改善，包括Ｔ淋巴细胞亚群、ＬＡＫ细胞活性、白介素Ⅱ膜受体、白介素Ⅱ分泌细胞及血清白介素Ⅱ受体。通过治疗显示：ＣＤ４／ＣＤ８比值、ＬＡＫ活性、白介素Ⅱ膜受体及白介素Ⅱ分泌细胞的水平均显著升高，而可溶性白介素Ⅱ受体减少，提示ＩＬ－２／ＬＡＫ可以改善机体免疫功能。     

白细胞介素2基因治疗耐三苯氧胺乳腺癌的实验研究

为探讨耐三苯氧胺（Ｔａｍｏｘｉｆｅｎ，ＴＡＭ）乳腺癌细胞因子基因治疗的可行性及效果，在体外将逆转录病毒载体介导的白细胞介素２（ＩＬ－２）基因导入耐ＴＡＭ大鼠乳腺癌细胞株Ｙ２，并进行了动物体内抗肿瘤实验研究。结果显示：同亲代Ｙ２细胞比、基因修饰的ＹＩＬ－２细胞形态和细胞生长特性发生改变。ＰＣＲ显示ＩＬ－２基因成功地整合到ＹＩＬ－２细胞内。ＹＩＬ－２细胞（２．０×１０６个／只）在大鼠右腋皮下失去致瘤性，并阻断等量混合的Ｙ２细胞的致瘤性，但阻断作用随亲代细胞数量的增多而减弱。同样数量无ＩＬ－２基因转移的Ｙ２细胞致瘤率为１００％。结果表明ＹＩＬ－２细胞具有抗肿瘤活性。     

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.     

Suppression of the tumorigenic growth of Burkitt's lymphoma cells in immunodeficient mice by cytokine gene transfer using EBV-derived episomal expression vectors

Epstein-Barr virus (EBV)-based expression vectors were tested for cytokine gene transfer-mediated induction of an immune response against human lymphoma cells. These vectors express the EBV latent gene EBNA 1 and carry the EBV latent origin of replication (ori P) for episomal replication in transfected cells. In addition, 3 human immunoglobulin light chain enhancer elements augment expression in B-cells. The suitability of these vectors for expression of cytokine genes in human lymphoma cells in vitro has been demonstrated. In order to extend these experiments in vivo, highly tumorigenic Burkitt's lymphoma (BL) cells were transfected with different cytokine genes of human and murine origin cloned into the EBNA 1/ori P vectors. Tumorigenicity of the transfectants was measured after inoculation into nude mice. No effect on tumorigenicity was observed after hIL 6 transfection and an inconsistent effect after hTNFalpha transfection. In contrast, complete suppression of tumor outgrowth occurred in hIL 10 transfectants. This tumor suppressive effect, however, was restricted to the IL 10 transfectants themselves and not directed against non-transfected cells. By comparison, mIL 4 transfected BL cells also were non-tumorigenic. However, co-inoculation of mIL 4 transfected and non transfected cells resulted in suppression of the tumorigenicity of the non-transfected cells. Thus, highly tumorigenic BL cells in nude mice are sensitive to immune effector mechanisms triggered by cytokine expression. In this experimental model, EBNA 1/ori P expression vectors are a suitable tool for cytokine gene transfer mediated induction of an anti-lymphoma immune response of the host.     

LRRC4基因表达能降低胶质母细胞瘤细胞系U251的生长和成瘤潜能

背景与目的：LRRC4是作者最近克隆的一个新基因,该基因在原发性脑肿瘤活检标本中明显表达下调。本研究旨在研究LRRC4基因是否具有抑制脑肿瘤生长的能力。方法：LRRC4基因的全长编码区被亚克隆至表达载体pcDNA3．1中,应用脂质体转染的方法将重组的质粒载体导入胶质母细胞瘤细胞系U251,经G418筛选,建立稳定表达LRRC4基因的U251的细胞系。采用细胞增殖实验、软琼脂实验、肿瘤形成实验来考察LRRC4基因表达对于细胞生长和肿瘤形成的影响。结果：经过脂质体转染和筛选,建立了稳定表达LRRC4全长编码区的U251细胞系,用于进一步实验。比较未转染组和转染空白载体组,Northern blot实验证实转染了LRRC4基因的细胞LRRC4 mRNA的表达增强。细胞增殖一定时间后,转染LRRC4基因的细胞较未转染细胞的生长速度明显减慢,克隆形成率明显降低。将这些细胞注射入无胸腺裸鼠体内,40天后处死裸鼠,测量肿瘤大小,结果显示,转染LRRC4基因的细胞形成的肿瘤明显小于对照组。结论：LRRC4基因可转染于人脑胶质母细胞瘤细胞系U251。LRRC4在U251细胞的表达有抑制瘤细胞增殖和抑制裸鼠移植瘤的形成和生长的作用。     

分泌型人白细胞介素18重组质粒的构建及其真核表达

目的：构建带IL—12 P40信号肽序列的分泌型IL—18质粒，使其在真核细胞中稳定表达。方法：根据IL—12 P40的信号肽cDNA序列及人成熟IL—18序列设计4条特异性引物，用RT—PCR法自人树突状细胞中分别扩增IL—12 P40信号肽序列(片段A)及成熟IL—18基因(片段B)，用重叠延伸PCR法拼接和扩增融合基因(片段C)，连接制备pGEM—T克隆载体，亚克隆至pcDNA3．1^ 。转染NCI—H460人肺癌细胞株，经RT—PCR和Western blot检测IL—18的表达。结果：表达质粒的酶切图谱符合预期的结果，测序结果与预期的cDNA序列完全一致，转染细胞株表达了IL—18。结论：成功地构建了含IL—12 P40信号肽序列的IL—18表达质粒，并在人肺癌细胞株中得到了表达。     

Vaccination with allogeneic GM-CSF gene-modified lung cancer cells: antitumor activity comparing with that induced by autologous vaccine

OBJECTIVE: The aim of this study was to investigate the whole allogeneic (differing tissue-type) tumor cells as vaccine in the mouse lung cancer model. The immunogenic and antitumor activity of allogeneic vaccine was compared with that of autologous cancer cell vaccine. METHODS: C57/BL mice inoculated with Lewis lung cancer (LLC) cells were used as the animal model to test the effects of allogeneic vaccination. LA795 and LLC lung cancer cell lines, which were transfected with the mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) gene, were administered as allogeneic and autologous tumor vaccine, respectively. The irradiated tumor cells were administered as subcutaneous vaccines before the tumor challenge. The immunity of cancer vaccine was tested by mouse interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) lactate dehydrogenase (LDH) assays. The serum level of IFN-gamma and interleukin (IL)-4 was tested using the enzyme-linked immunosorbent assay method. RESULTS: Prophylactic vaccination with allogeneic LA795 cells protected against the LLC tumor challenge in C57/BL. The tumor growth was inhibited and the survival was accordingly prolonged. The cytotoxicity of the spleen cells or the purified CD(8)(+) T-cells against LLC cells in the mice immunized with either the autologous or allogeneic cancer cell vaccine was significantly increased, relative to that of the control, untreated group (p<0.05). ELISPOT IFN-gamma assays showed that spleen cells from mice immunized with LA795 cells could be activated after coculture with irradiated LLC cells. In addition, the serum level of Th1-king cytokine IFN-gamma significantly increased after vaccination; however, no statistically difference was found in Th2-kind cytokine IL-4. CONCLUSIONS: The allogeneic cancer vaccine could induce immune responses and protection against lung cancer, which had no significant difference with that of autologous vaccine.     

逆转录病毒介导的IL6/IL2融合基因转导对小鼠B16细胞粘附及转移特性的影响

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,将其转染小鼠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.实验结果表明,融合基因转导的细胞所分泌产生的融合蛋白具有IL-2和IL-6两种细胞因子活性.对肿瘤细胞体外粘附及转移能力的测定结果表明,经融合基因修饰的B16细胞对细胞外基质的主要成分Laminin及重组基底膜Matrigel的粘附得到抑制,同时伴有实验性转移能力降低.这一结果提示,融合基因转导可明显降低肿瘤细胞的转移能力,其机制可能与基因修饰细胞相关生物学特性的改变及其对机体的免疫刺激活性有关.     

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...     

白细胞介素-18基因转染的肺癌细胞与树突状细胞融合体的抗肿瘤作用

目的研究白细胞介素-18(IL-18)基因转染的肺癌细胞与树突状细胞(DC)融合体的抗肿瘤作用。方法(1)从人外周血单核细胞诱导培养DC,与IL-18基因转染的NCI-H460肺癌细胞融合,经免疫磁珠筛选。(2)设转染组(GT组)、空载体转染组(PT组)、未转染组(NT组)和对照组(BC组),分别以IL-18转染的融合细胞、pcDNA3.1~ 转染的融合细胞、未转染的融合细胞活化的T细胞为效应细胞,BC组不含效应细胞,用乳酸脱氢酶(LDH)法测定效应细胞对NCI-H460细胞的杀伤作用。(3)荷瘤裸鼠皮下注射上述3种效应细胞,以BC组为空白对照,比较4组裸鼠肿瘤体积和重量。结果3组效应细胞在体外对NCI-H460细胞的杀伤率分别为53.1%,30.1%和31.5%;3组裸鼠肿瘤体积及肿瘤重量明显低于BC组,以GT组最低。结论IL-18基因转染的融合细胞可有效诱导抗肿瘤免疫。     

联合B7-1分子、IL-2及LAK细胞治疗肝癌的实验研究

目的：研究共刺激信号在肿瘤免疫中的作用。方法：构建含人B7-1基因的真核表达载体PRCCMV-B7-1，脂质体介导DNA转移法将人B7-1基因转入人肝癌细胞株SMMC7721，建立新的细胞株SMMC-B7-1，PCR及逆转录PCR（RT-PCR），流式细胞仪检测，四唑卤（MTT）比色试验体外检测白细胞介素-2（IL-2）激活的LAK细胞（LAK-C），对两种肝癌细胞的杀伤活性，结果：PCR，RT-PCR法证实SMMC-B7-1细胞稳定表达B7基因，LAK细胞对SMMC-B7-1细胞的杀伤活性明显高于SMMC7721，结果有统计学意义。结论：B7基因可以体外转染人肝癌细胞，并表达有活性的B7分子，明显增强IL-2激活的LAK细胞的肿瘤杀伤作用。为进一步开展临床应用奠定基础。