脂质体干扰素α对小鼠Lewis肺癌转移及脾细胞增殖的影响

Ｃ５７ＢＬ／６Ｎ小鼠尾静脉注射Ｌｅｗｉｓ肺癌细胞后，腹腔注射脂质体干扰素α（Ｌ－ＩＦＮ－α）和未包裹干扰素α（ＩＦＮ－α），连续１０ｄ；测小鼠肺湿重、肺转移结节数和ＣｏｎＡ诱导的脾细胞ＤＮＡ掺入量。结果ＩＦＮ－α５、２０万ｕ·ｋｇ（－１）组，小鼠肺湿重较对照组减小１４．１、１９．６％，肿瘤肺转移结数减少３２．７、５０．０％，ＣｏｎＡ刺激的脾细胞ＤＮＡ掺入量增加２４．６、４６．８％。１／１０剂量的Ｌ－ＩＦＮ－α可产生相同的药理活性。小鼠环磷酸胺５０ｍｇ·ｋｇ（－１）ｉｐ３ｄ后，尾静脉注射瘤细胞，实验处理同上。结果Ｌ－ＩＦＮ－α和ＩＦＮ－α对环磷酰胺处理小鼠的Ｌｅｗｉｓ肺癌细胞肺转移的抑制作用更为明显。本文结果表明，ＩＦＮ－α可抑制小鼠Ｌｅｗｉｓ肺癌转移，促进ＣｏｎＡ刺激的脾细胞增殖，并与剂量明显相关。脂质体包裹ＩＦＮ－α可使其生物活性提高约１０倍。对免疫功能受抑小鼠作用更为显著。     

瘤内/瘤周注射巨噬细胞集落刺激因子或(和)干扰素γ基因转染的巨噬细胞对肿瘤的治疗作用

目的观察巨噬细胞集落刺激因子（ＭＣＳＦ）和干扰素γ（ＩＦＮγ）基因单独或联合转染的巨噬细胞（ｍａｃｒｏｐｈａｇｅ，Ｍ）对局部黑色素瘤的治疗效果及相关免疫机理。方法荷瘤小鼠经基因转染的巨噬细胞治疗后，观察其长期存活期并检测其体内抗肿瘤免疫功能。用４小时５１Ｃｒ释放法检测荷瘤小鼠脾细胞自然杀伤细胞（ＮＫ）、细胞毒Ｔ淋巴细胞（ＣＴＬ）活性，间接ＭＴＴ法检测荷瘤小鼠腹腔巨噬细胞的杀伤活性，常规方法检测脾细胞诱导上清中肿瘤坏死因子（ＴＮＦ）、白细胞介素２（ＩＬ２）、γ干扰素（ＩＦＮγ）活性，局部肿瘤经治疗后行常规病理分析。结果经ＩＦＮγ基因及联合基因转染的巨噬细胞治疗后的荷瘤小鼠有２５％能长期存活，体外诱导的ＣＴＬ和小鼠腹腔巨噬细胞杀伤活性显著增强，脾细胞经诱导产生的细胞因子有不同程度的增加，与对照组相比差别显著。常规病理显示，ＭＣＳＦ和ＩＦＮγ联合基因转染的巨噬细胞治疗的小鼠肿瘤局部有大量的淋巴细胞浸润。结论ＭＣＳＦ和ＩＦＮγ基因转染的巨噬细胞瘤内瘤周注射对肿瘤有较好的治疗效果，其机制除了与巨噬细胞在局部直接接触杀伤瘤细胞有关外，宿主抗肿瘤免疫功能的增强亦起了重要的作用     

腺病毒介导的人GM-CSF基因转染瘤苗增强机体免疫功能的实验研究

目的 研究腺病毒介导的人ＧＭ－ＣＳＦ基因转染瘤苗体内抗肿瘤免疫作用极其机理。方法 应用ＧＭ－ＣＳＦ基因转染、的瘤苗对小鼠肝癌模型进行免疫基因治疗,观察该疗法对荷瘤小鼠脾细胞ＮＫ、ＬＡＫ、ＣＴＬ活性的影响、脾淋巴细胞围化反应的影响以及荷瘤习的生存期。结果 经ＧＭ－ＣＳＦ基因转染瘤苗治疗后,插细胞ＣＴＬ活性显著升高,而ＮＫ、ＬＡＫ细胞活性未见明显增强、脾淋巴细胞转化反应显著增强、生存期显著延长。结论     

吐温80及温热对荷瘤鼠肿瘤坏死因子的影响

ＢＡＬＢ／Ｃ小鼠经腹接种Ｂ１６黑色素瘤细胞建立荷瘤鼠模型，经静脉注射吐温８０及腹部４１℃温热，于处理后１周和２周测定肿瘤坏死因子（ＳｅｒｕｍＴｕｍｏｒＮｅｅｒｏｓｉｓＦａｅｔｏｒ，ＳＴＮＦ）活性及血清唾液酸含量，并观察作用后荷瘤鼠死亡率，结果显示，荷瘤鼠ＳＴＮＦ活性及唾液酸含量持续保持相对高水平（正常ＢＡＬＢ／Ｃ鼠ＳＴＮＦ活性检测不出；唾液酸含量〈５０ｍｇ／ｍｌ）。单独以吐温８０处理和吐温８０合并     

牛膝多糖抗肿瘤作用及免疫机制实验研究

作者研究了牛膝多糖（ＡＢＰ）对Ｓ＿（１８０）荷瘤小鼠的抑瘤作用和脾细胞诱生ＴＮＦ和ＬＡＫ细胞活性的影响。结果证实ＡＢＰ２５～１００ｍｇ·Ｋｇ￣（－１）·ｄ￣（－１）×７的抑瘤率为３１％～４０％。环磷酰胺１２．５ｍｇ／ｋｇ单次的抑瘤率为１７％，与ＡＢＰ１００ｍｇ·ｋｇ￣（－１）·ｄ￣（－１）合用的抑瘤率为５８％，有明显协同作用。ＡＢＰ１～２μｇ／ｍｌ对小鼠肉瘤Ｓ＿（１８０）细胞和人白血病Ｋ＿（５６２）细胞的增殖均有明显抑制作用，ＡＢＰ５０及１００ｍｇ／ｋｇ腹腔注射能明显提高Ｓ＿（１８０）荷瘤小鼠ＬＡＫ细胞活性和ＴＮＦ－β生成。诱生ＴＮＦ的达峰时间是２次腹腔注射后的第８天。为探讨其抗肿瘤机理，对Ｓ＿（１８０）细胞膜成份进行了分析，结果显示ＡＢＰ与细胞接触２４小时，引起细胞膜唾液酸含量升高，膜磷脂含量降低，这些变化差异均有显著性意义（Ｐ＜０．０５或Ｐ＜０．０１）；但膜胆固醇含量、膜流动性（Ｃ／Ｐ比值）不受影响。提示ＡＢＰ的抗瘤机理与其增强宿主免疫功能及改变细胞膜生化特性有关。     

肺癌生物学特性与灌注化疗疗效和生存关系的初步探讨

本文对１７例Ⅲ期肺癌，测定了癌细胞ＤＮＡ含量，ＤＮＡ指数（ＤＩ），细胞增殖核抗原（ＰＣＮＡ）。１７例肺癌均给支气管动脉灌注化疗。小细胞肺癌（ＳＣＬＣ）和非小细胞肺癌（ＮＳＣＬＣ）分别缩小４８．８±３１．０％，３６．８±１７．１％（Ｘ±ＳＥ）。化疗后７例开胸手术６例切除肿瘤。ＳＣＬＣ和ＮＳＣＬＣ的中位生存期分别为３个月和８．５个月。在ＮＳＣＬＣ中，ＰＣＮＡ与肿瘤缩小范围呈负相关（ｒ＝－０．４７）；ＤＮＡ超四倍体百分比和ＤＩ存活时间呈正相关（（ｒ＝０．５１，ｒ＝０．５６）。ＳＣＬＣ则相关性差。结果表明，ＰＣＮＡ、ＤＡＮ和ＤＩ是ＮＳＣＬＣ判定疗效和预后的有用指标。     

粘附性淋巴因子活化的杀伤细胞抗自体白血病细胞作用的研究

建立了一套培养和处理粘附ＬＡＫ细胞（Ａ－ＬＡＫ）的系统，将１２例缓解期急性髓系白血病（ＡＭＬ）患者（ＲＰＳ）的Ａ－ＬＡＫ细胞与常规制备ＬＡＫ细胞（ＲＴ－ＬＡＫ）进行了对照研究，结果显示ＲＰＳ－Ａ－ＬＡＫ细胞于培养第１０天时其扩增指数（２０．１±１３．９）较ＲＴ－ＬＡＫ细胞的扩增指数（７．５±２．１）明显提高（Ｐ＜０．０５）。形态学研究显示Ａ－ＬＡＫ细胞主要由大颗粒淋巴细胞组成，免疫表型分析显示其主要由ＣＤ１６＋的ＮＫ细胞组成，ＲＴ－ＬＡＫ细胞主要由ＣＤ３＋的Ｔ细胞组成。其杀伤活性与ＣＤ１６＋ＮＫ细胞之间有很好的相关性（ｒ＝０．８２，Ｐ＜０．０５）。这提示ＣＤ１６＋ＮＫ细胞是Ａ－ＬＡＫ细胞的主要组成细胞，代表了杀伤功能最强的亚群。     

腺病毒介导的胞嘧啶脱氨酶自杀基因联合IL-2基因疗法的肿瘤治疗作用及免疫机理分析

目的以腺病毒作为载体，将大肠杆菌胞嘧啶脱氨酶（ＣＤ）基因与小鼠ＩＬ－２基因联合转移，研究其体内抗肿瘤作用及免疫机理。方法小鼠皮下接种黑色素瘤Ｂ１６Ｆ１０细胞后３天，肿瘤局部注射表达ＩＬ－２的重组腺病毒ＡｄＩＬ－２和表达ＣＤ的重组腺病毒ＡｄＣＤ，然后连续１０天给予５－氟胞嘧啶（５－Ｆｃ）３００ｍｇ／ｋｇ进行治疗。结果联合治疗组荷瘤小鼠皮下肿瘤结节的生长明显受到抑制，小鼠存活期明显长于ＡｄＩＬ－２、ＡｄＣＤ／５－Ｆｃ、ＡｄｌａｃＺ／５－Ｆｃ或ＰＢＳ组。经联合治疗后，小鼠脾细胞的ＮＫ活性和ＣＴＬ杀伤活性明显增强；肿瘤瘤体内ＣＤ４、ＣＤ８细胞浸润增加；肿瘤细胞表达Ｈ－２Ｋｂ和Ｂ７－１分子明显增加。结论联合应用自杀基因和ＩＬ－２基因治疗，一方面可以明显抑制荷瘤小鼠肿瘤生长，另一方面可以提高机体对肿瘤细胞免疫应答，增加机体的抗肿瘤作用，是肿瘤基因治疗中一条行之有效的途径。     

AK细胞及环磷酰胺治疗肝癌的实验研究

为研究１ 种新的过继免疫化疗治疗肝癌的方法,采用肝癌细胞株Ｈ２２ 接种于近交系Ｂａｌｂ／ｃ 小鼠皮下,制成肿瘤模型。使用ＩＬ２ 、肿瘤活化的杀伤细胞（ＡＫ细胞）及环磷酰胺（Ｃｙ） 进行治疗,检测小鼠脾淋巴细胞ＮＫ、ＬＡＫ及ＣＴＬ 活性,用流式细胞仪检测Ｌ３Ｔ４ 亚群及Ｌｙｔ２ 亚群含量。结果表明在过继免疫化疗组小鼠ＬＡＫ 及ＣＴＬ活性明显高于其它治疗组（Ｐ＜ ０．０１）,荷瘤小鼠肿瘤结节生长较ＩＬ２ 组及Ｃｙ 组明显缓慢（Ｐ＜ ０．０１）,生存期也明显高于其它各治疗组（Ｐ＜０ ．０１）。该研究表明过继免疫化疗有较强的抗肿瘤作用。     

ＫＩＦ４Ａ在非小细胞肺癌中的表达及其与预后的相关性

目的　研究ＫＩＦ４Ａ蛋白在非小细胞肺癌（ＮＳＣＬＣ）组织中的表达及其与预后的相关性。方法　选取术后经病理证实的１１５例ＮＳＣＬＣ和１９例肺良性病变（５例肺结核、４例支气管扩张、６例肺大疱、４例炎性假瘤）的标本,运用免疫组化ＥｎＶｉｓｉｏｎ法检测ＫＩＦ４Ａ蛋白的表达,采用χ２检验分析ＫＩＦ４Ａ蛋白在ＮＳＣＬＣ组织和良性组织中的表达差异及ＮＳＣＬＣ中ＫＩＦ４Ａ蛋白表达与临床病理特征的相关性。采用Ｋａｐｌａｎ-Ｍｅｉｅｒ生存分析和Ｃｏｘ回归法分析ＫＩＦ４Ａ蛋白在ＮＳＣＬＣ组织中表达和预后的关系。结果　ＫＩＦ４Ａ在ＮＳＣＬＣ组织中阳性表达率为６０％,显著高于对照组的３１.６％,与Ｔ分期、淋巴结转移明显相关。生存分析提示,ＫＩＦ４Ａ阳性表达的ＮＳＣＬＣ患者预后较差（Ｐ＝０.００３）;Ｃｏｘ回归分析结果表明,ＫＩＦ４Ａ蛋白也是影响ＮＳＣＬＣ预后的独立危险因素（相对危险度１.９４４,Ｐ＝０.０３２）。结论　ＫＩＦ４Ａ蛋白在ＮＳＣＬＣ组织中的阳性表达率高于肺良性病变组织;ＫＩＦ４Ａ蛋白的高表达与ＮＳＣＬＣ患者预后相关,可以作为判断预后的参考指标。     

Polyporus sp.MO5多糖的抗肿瘤作用

目的研究真菌Polyporus sp.M05多糖PSM-a体内对荷瘤小鼠S180的抑瘤作用及其机制.方法 MTT法检测PSM-a体外对S180细胞株的增殖抑制作用.建立S180小鼠模型,灌胃治疗,每日检测肿瘤体积并计算瘤体比和抑瘤率.实验结束后处死小鼠,MTT比色法测定小鼠自然杀伤(NK)细胞及淋巴因子活化杀伤细胞(LAK)的杀伤活性.HE染色检测肿瘤组织细胞坏死情况.结果 PSM-a在体外能抑制S180细胞生长;在体内能显著降低荷瘤小鼠的瘤重和瘤体比,250μg/ml的PSM-a对肿瘤抑制率高达80%以上;PSM-a能促进NK细胞及LAK细胞的杀伤活性;病理切片显示PSM-a作用后引起肿瘤组织坏死.结论 PSM-a对S180荷瘤小鼠肿瘤的生长有明显的抑制作用,其机制可能与提高机体免疫细胞对肿瘤细胞的杀伤活性有关.     

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     

转移因子对肿瘤细胞的抑制作用

用匀浆透析法制备正常转移因子（Ｎ—ＴＦ）和Ｓ１８０瘤细胞转移因子（Ｓ１８０－ＳＴＦ）。并观察其对Ｓ１８０细胞的杀伤及抑制作用。结果显示Ｎ—ＴＦ和Ｓ１８０－ＳＴＦ均不能杀伤Ｓ１８０细胞，但可抑制Ｓ１８０细胞ＤＮＡ合成，其抑制强度与转移因子剂量成正比，而与特异性无关。Ｓ１８０－ＳＴＦ还能明显抑制Ｓ１８０细胞移植瘤的生长。     

Human interleukin 4 regulates the phenotype of lymphocytes generated during mixed lymphocyte culture and inhibits the IL-2-induced development of LAK function in normal and leukaemic cells

This study examined the immunoregulatory role of recombinant interleukin 4 (IL-4), also known as B-cell stimulating factor 1, on the generation of cytotoxic effector cells from normal and leukaemic human blood mononuclear cells. When tested on cells from normal individuals, the addition of IL-4 to mixed lymphocyte cultures led to a dose-dependent proliferation of T-helper cells (CD3, 4 positive) with a concomitant decrease in phenotypic and functional cytotoxic T cells and natural killer (NK) cells. IL-4 also inhibited the interleukin-2 (IL-2)-induced generation of lymphokine-activated killer (LAK) activity when added at the beginning of mixed lymphocyte culture. When tested on mature leukaemic NK cells, IL-4 also inhibited the ability of IL-2 to induce LAK function using a short-term culture system. These results show that IL-4 acts on both normal and leukaemic cells and suggests that it acts at more than one level during the development of LAK function.     

Effect of flavone acetic acid (NSC 347 512) on splenic cytotoxic effector cells and their role in tumour necrosis

Flavone acetic acid (FAA), an antitumour agent currently undergoing clinical trial, has immune-modulatory effects on various cytotoxic cells in mice. Natural killer (NK) cell activity in the spleen was augmented 4 h after FAA treatment, and when spleen cells were cultured with interleukin-2 to induce the production of lymphokine-activated-killer (LAK) cells, higher levels of LAK cell activity were generated by spleen cells from FAA-treated animals than by spleen cells from untreated, control mice. The response to FAA by spleen cells from mice bearing the Colon 38 tumour was compared to that of non-tumour bearers. Activity against NK-sensitive YAC-1 tumour targets was augmented to a similar degree, and no activity against NK-resistant P815 targets was detected. FAA was shown to induce haemorrhagic necrosis in the P815 tumour grown as a subcutaneous solid tumour. Furthermore, haemorrhagic necrosis was induced by FAA on Colon 38 tumours growing in mice which had been depleted of NK activity by treatment with anti-asialo GM-1 antibody. Thus, although NK activity could be involved in the long-term host response to the tumour, it does not appear to be a major determinant of FAA-induced haemorrhagic tumour necrosis.     

Heterogeneity of human lymphokine (IL-2)-activated killer (LAK) precursors and regulation of their LAK induction by blood monocytes

Highly purified lymphocytes (greater than 99%) and monocytes (greater than 90%) were isolated by CCE from peripheral blood of healthy donors. Blood lymphocytes were separated by this CCE into 9 subpopulations. The NK activities of these lymphocyte fractions against NK-sensitive K-562 cells and their LAK activities against NK cell-resistant target (Daudi) cells were assayed promptly or after incubation of the fractions for 4 days with or without an optimal concentration of IL-2. NK and LAK activities were measured by 4-hr 51Cr-release assay. On the basis of their NK and LAK activities, these lymphocyte fractions were classified into 3 subpopulations of LAK precursors: one lacking both NK and LAK activities (Fr.2), one with moderate NK activity but low LAK activity (Fr.5), and one possessing both NK and LAK activities (Fr.8). Addition of autologous fresh monocytes to the lymphocyte cultures resulted in a significant increase in induction of LAK activity in Fr.2 and Fr.5. This up-regulation of lymphocytes in Fr. 2 and Fr.5 by monocytes was confirmed in parallel experiments by measuring the blastogenic response of the lymphocytes to IL-2. Deletion of lymphocytes in Fr. 8 of CD16+ (Leu-11+) NK cells resulted in 74% reduction in LAK induction, whereas depletion of mixtures of monocytes and lymphocytes in Fr. 2 of cells reacting with CD3+ (OKT3+) antibody resulted in a 66% reduction in LAK induction. This up-regulation of LAK cell induction from LAK precursors by monocytes was confirmed using 4 lines of human lung cancer cells as targets for LAK activity. These results clearly indicate that human monocytes may cause up-regulation of the expression of IL-2-induced LAK activity in T cells and in a subpopulation of NK cells.     

Phenotypical analysis of effector cells on nonspecific cancer cell therapy

In the present study, we examined the contributions of lymphocyte subpopulations in lymphokine activated killer (LAK) activity. LAK cells prepared from peripheral blood mononuclear cells (PBMC) of healthy donors showed highly cytotoxic activities against target tumor cells. When CD16 and CD56 positive cells in LAK cells were depleted by magnetic cell sorting, their cytotoxic activities were dramatically decreased. In contrast, little change was observed by the depletion of CD3 positive cells, suggesting that CD16 and/or CD56 positive populations, but not CD3 positive populations, including natural killer (NK) cells are the major cell types involved in LAK activity. Indeed, NK-enriched LAK cells prepared by culturing PBMC with IL-2 and OK-432 showed a more potent LAK activity than conventional LAK cells and CD3-activated T cells. These results suggest that selective expansion and activation of CD16 and CD56 positive cells in LAK cells is a useful strategies to improve their anti-tumor potential in nonspecific immunotherapy, and possibly in combination therapy with other target immunotherapies as well.     

Synergistic Suppressive Effect of Double Transfection of Tumor Necrosis Factor-α and Interleukin 12 Genes on Tumorigenicity of Meth-A Cells

Tumor necrosis factor-α(TNF-α) and interleukin 12 (IL-12), both potent antitumor cytokines, are known to be involved in the host's antitumor immune surveillance in tumor bearers, via different mechanisms. The former enhances the activities of dendritic cells, natural killer/lymphocyteactivated killer (NK/LAK) and cytotoxic T lymphocyte (CTL), while the latter induces Th1-type cellular immunity and enhances the activities of natural killer T (NKT), NK/LAK and CTL. In the present study, in the expectation of synergistic actions of these cytokines in stimulating the host's immune responses, we investigated the feasibility of a cancer vaccine involving double transfection with both genes in a murine model. The expression of major histocompatibility complex (MHC) class I, class II and B7.1 on the surface of the double transfectants was enhanced as revealed by FACS analysis. A significant decrease in tumorigenicity was observed in mice inoculated with the double transfectants. Cytotoxicity assay revealed that the activities of NK/LAK and CTL from spleens of mice bearing the double transfectants were enhanced. The induction of tumor-specific immunity was confirmed by rechallenge with parental Meth-A cells in mice that had rejected the double transfectants. Thus, double transfection of TNF-α and IL-12 genes was considered to bring about synergistic suppressive effects on the tumorigenicity of transfectants through the activation of killer cells by produced cytokines and the enhancement of expression of MHC class I, II and B7.1 molecules.     

Synergistic suppressive effect of double transfection of tumor necrosis factor-alpha and interleukin 12 genes on tumorigenicity of Meth-A cells.

Tumor necrosis factor-alpha (TNF-alpha) and interleukin 12 (IL-12), both potent antitumor cytokines, are known to be involved in the host's antitumor immune surveillance in tumor bearers, via different mechanisms. The former enhances the activities of dendritic cells, natural killer / lymphocyte-activated killer (NK / LAK) and cytotoxic T lymphocyte (CTL), while the latter induces Th1-type cellular immunity and enhances the activities of natural killer T (NKT), NK / LAK and CTL. In the present study, in the expectation of synergistic actions of these cytokines in stimulating the host's immune responses, we investigated the feasibility of a cancer vaccine involving double transfection with both genes in a murine model. The expression of major histocompatibility complex (MHC) class I, class II and B7.1 on the surface of the double transfectants was enhanced as revealed by FACS analysis. A significant decrease in tumorigenicity was observed in mice inoculated with the double transfectants. Cytotoxicity assay revealed that the activities of NK / LAK and CTL from spleens of mice bearing the double transfectants were enhanced. The induction of tumor-specific immunity was confirmed by rechallenge with parental Meth-A cells in mice that had rejected the double transfectants. Thus, double transfection of TNF-alpha and IL-12 genes was considered to bring about synergistic suppressive effects on the tumorigenicity of transfectants through the activation of killer cells by produced cytokines and the enhancement of expression of MHC class I, II and B7.1 molecules.