用单倍体相合小鼠FBL-3红白血病细胞株移植建立CB6F1小鼠红白血病模型

本研究探讨建立F1代单倍体相合小鼠FBL-3红白血病模型的可行性,并观察FBL-3细胞在小鼠体内的生物学特性。将FBL-3H2-d小鼠红白血病细胞经尾静脉分别接种给小鼠C57BL/6和CB6F1H-2b/d(C57BL/6×BALB/c),观察两种小鼠的生存时间和染色体变化,并对濒死小鼠的肝、脾、肺和肾进行病理检查,部分进行电子显微镜检查。分析骨髓和脾脏细胞染色体核型及MHC分子表达情况。结果表明:静脉接种103-107个白血病细胞的情况下,C57BL/6小鼠和CB6F1小鼠发病率分别为100%和92.5%;接种的细胞数量与存活时间呈线性关系;接种相同数量FBL-3细胞的CB6F1小鼠平均生存时间较C57BL/6小鼠延长。白血病细胞主要侵及肝、脾、骨髓、肺和肾组织,糖原染色阳性、氯醋酸染色部分阳性,过氧化物酶、碱性磷酸酶、丁酸染色均为阴性。电子显微镜观察到细胞内病毒样颗粒。脾脏和骨髓细胞染色体多数为非二倍体,且H-2b表达率升高,H-2d表达率降低。结论:经尾静脉接种FBL-3细胞可以建立CB6F1小鼠红白血病模型。     

肿瘤坏死因子α基因转导的肿瘤浸润淋巴细胞体外生物学活性及不同转输方式的比较

背景利用肿瘤过继免疫和基因转移技术将表达肿瘤坏死因子α的载体导入运载细胞回输到体内,使肿瘤坏死因子α在肿瘤局部高浓度表达,既可增加肿瘤坏死因子α直接杀伤肿瘤细胞的能力,又能减少对其他组织的毒副作用.目的探讨肿瘤坏死因子α基因转导的肿瘤浸润淋巴细胞体外生物学活性及不同转输方式对荷瘤裸鼠肿瘤细胞的抑制作用.设计以实验动物为观察对象的随机对照实验.单位中国医科大学肿瘤研究所.材料实验于2000-01/2001-12在中国医科大学肿瘤研究所和中国医科大学实验动物部完成.TJ8510细胞(人脑恶性胶质瘤细胞系细胞)天津医科大学总医院神经病学研究所提供;实验动物先天性无胸腺BALB/C裸鼠36只.方法在肿瘤坏死因子α反转录病毒转移系统的建立及运载细胞肿瘤浸润淋巴细胞制备的基础上,利用构建的单克隆病毒细胞株PLC-2和PLJC-5将标记基因NeoR和目的基因肿瘤坏死因子α分别导入到肿瘤浸润淋巴细胞中,然后对其进行细胞增殖、肿瘤坏死因子表达、体外抗瘤活性的检测.将36只裸鼠接种肿瘤后随机分为6组,局部转输对照组、局部转输肿瘤浸润淋巴细胞组、局部转输肿瘤坏死因子肿瘤浸润淋巴细胞组、静脉转输对照组、静脉转输肿瘤浸润淋巴细胞组、静脉转输肿瘤坏死因子肿瘤浸润淋巴细胞组,对荷瘤裸鼠的治疗作用进行观察.主要观察指标①基因转导前后肿瘤浸润淋巴细胞的增殖情况和肿瘤浸润淋巴细胞的肿瘤坏死因子α的表达.②体外抗瘤活性的测定.③动物实验结果.结果①肿瘤浸润淋巴细胞、NeoR-肿瘤浸润淋巴细胞及肿瘤坏死因子肿瘤浸润淋巴细胞3组细胞的体外增殖活性差异无显著性意义(P＞0.05).②肿瘤浸润淋巴细胞与NeoR-肿瘤浸润淋巴细胞的肿瘤坏死因子α分泌量差异无显著性意义(P＞0.05);而肿瘤坏死因子肿瘤浸润淋巴细胞的肿瘤坏死因子α分泌量与NeoR-肿瘤浸润淋巴细胞、肿瘤浸润淋巴细胞相比差异有显著性意义(P＜0.01),而且肿瘤坏死因子肿瘤浸润淋巴细胞在体外培养30 d,可持续表达高水平的肿瘤坏死因子α.③肿瘤浸润淋巴细胞与NeoR肿瘤浸润淋巴细胞对TJ8510细胞的杀伤活性差异无显著性意义(P＞0.05),而肿瘤坏死因子肿瘤浸润淋巴细胞与前两者相比杀伤活性有明显地提高(P＜0.01).④动物实验结果转输肿瘤坏死因子肿瘤浸润淋巴细胞40d后,肿瘤局部转输肿瘤坏死因子肿瘤浸润淋巴细胞组肿瘤体积小于局部转输对照组[(307±42),(2 048±278)mm3,P＜0.01],静脉转输肿瘤坏死因子肿瘤浸润淋巴细胞组肿瘤体积小于静脉转输对照组[(954±195),(1 989±305)mm3,P＜0.05].结论肿瘤坏死因子α基因转导的肿瘤浸润淋巴细胞能有效地表达肿瘤坏死因子,其在体外和荷瘤裸鼠体内的抗肿瘤效果明显高于肿瘤浸润淋巴细胞.静脉转输人脑胶质瘤肿瘤浸润淋巴细胞对人脑胶质瘤裸鼠皮下实体瘤生长无明显抑制效应,但静脉转输肿瘤坏死因子肿瘤浸润淋巴细胞却能明显抑制肿瘤的生长,局部转输肿瘤坏死因子肿瘤浸润淋巴细胞较静脉转输肿瘤坏死因子肿瘤浸润淋巴细胞对肿瘤生长的抑制效果更为明显,提示过继免疫基因治疗胶质瘤以局部转输方式为佳.     

肿瘤坏死因子α基因转导的肿瘤浸润淋巴细胞体外生物学活性及不同转输方式的比较

背景：利用肿瘤过继免疫和基因转移技术将表达肿瘤坏死因子α的载体导入运载细胞回输到体内，使肿瘤坏死因子α在肿瘤局部高浓度表达，既可增加肿瘤坏死因子α直接杀伤肿瘤细胞的能力，又能减少对其他组织的毒副作用。目的：探讨肿瘤坏死因子α基因转导的肿瘤浸润淋巴细胞体外生物学活性及不同转输方式对荷瘤裸鼠肿瘤细胞的抑制作用。设计：以实验动物为观察对象的随机对照实验。单位：中国医科大学肿瘤研究所。材料：实验于2000-01／2001—12在中国医科大学肿瘤研究所和中国医科大学实验动物部完成。TJ8510细胞（人脑恶性胶质瘤细胞系细胞）：天津医科大学总医院神经病学研究所提供；实验动物：先天性无胸腺BALB／C裸鼠36只。方法：在肿瘤坏死因子α反转录病毒转移系统的建立及运载细胞肿瘤浸润淋巴细胞制备的基础上，利用构建的单克隆病毒细胞株PLC-2和PLJC-5将标记基因Neo^R和目的基因肿瘤坏死因子α分别导入到肿瘤浸润淋巴细胞中，然后对其进行细胞增殖、肿瘤坏死因子表达、体外抗瘤活性的检测。将36只裸鼠接种肿瘤后随机分为6组，局部转输对照组、局部转输肿瘤浸润淋巴细胞组、局部转输肿瘤坏死因子肿瘤浸润淋巴细胞组、静脉转输对照组、静脉转输肿瘤浸润淋巴细胞组、静脉转输肿瘤坏死因子肿瘤浸润淋巴细胞组，对荷瘤裸鼠的治疗作用进行观察。主要观察指标：①基因转导前后肿瘤浸润淋巴细胞的增殖情况和肿瘤浸润淋巴细胞的肿瘤坏死因子α的表达。②体外抗瘤活性的测定。③动物实验结果。结果：①肿瘤浸润淋巴细胞、Neo^R-肿瘤浸润淋巴细胞及肿瘤坏死因子肿瘤浸润淋巴细胞3组细胞的体外增殖活性差异无显著性意义（P〉0．05）。②肿瘤浸润淋巴细胞与Neo^R-肿瘤浸润淋巴细胞的肿瘤坏死因子α分泌量差异无显著性意义（P〉0．05）；而肿瘤坏死因子肿瘤浸润淋巴细胞的肿瘤坏死因子α分泌量与Neo^R-肿瘤浸润淋巴细胞、肿瘤浸润淋巴细胞相比差异有显著性意义（P〈0．01），而且肿瘤坏死因子肿瘤浸润淋巴细胞在体外培养30d，可持续表达高水平的肿瘤坏死因子α。③肿瘤浸润淋巴细胞与Neo^R肿瘤浸润淋巴细胞对淋巴细胞对TJ8510细胞的杀伤活性差异无显著性意义（P〉0．05），而肿瘤坏死因子肿瘤浸润淋巴细胞与前两者相比杀伤活性有明显地提高（P〈0．01）。④动物实验结果：转输肿瘤坏死因子肿瘤浸润淋巴细胞40d后，肿瘤局部转输肿瘤坏死因子肿瘤浸润淋巴细胞组肿瘤体积小于局部转输对照组[（307&;#177;42），（2048&;#177;278）mm^3，P〈0．01]，静脉转输肿瘤坏死因子肿瘤浸润淋巴细胞组肿瘤体积小于静脉转输对照组[（954&;#177;195），（1989&;#177;305）mm^3，P〈0．05]。结论：肿瘤坏死因子α基因转导的肿瘤浸润淋巴细胞能有效地表达肿瘤坏死因子，其在体外和荷瘤裸鼠体内的抗肿瘤效果明显高于肿瘤浸润淋巴细胞。静脉转输人脑胶质瘤肿瘤浸润淋巴细胞对人脑胶质瘤裸鼠皮下实体瘤生长无明显抑制效应，但静脉转输肿瘤坏死因子肿瘤浸润淋巴细胞却能明显抑制肿瘤的生长，局部转输肿瘤坏死因子肿瘤浸润淋巴细胞较静脉转输肿瘤坏死因子肿瘤浸润淋巴细胞对肿瘤生长的抑制效果更为明显，提示过继免疫基因治疗胶质瘤以局部转输方式为件。     

TIMP-2基因对白血病细胞系SHI-1细胞在裸鼠体内生长的调节作用

目的 研究高表达组织型基质金属蛋白酶抑制剂-2(TIMP-2)基因对人急性单核细胞白血病细胞系SHI-1细胞在裸鼠体内浸润能力的影响.方法 ①经尾静脉将转染TIMP-2基因的SHI-1(SHI-1-TIMP-2)细胞与转染空载体MSCV的SHI-1(SHI-1-MSCV)细胞1×107分别接种于经切脾、环磷酰胺腹腔注射和全身亚致死量照射等预处理的6周龄裸鼠体内.接种后第30天各组处死8只裸鼠,其余8只观察生存期,通过病理学检测及CD45抗体免疫组织化学染色检测SHI-1细胞在裸鼠体内各脏器中浸润情况,并埘中枢神经系统白血病(CNSL)进行分级.②将5×106个SHI-1-TIMP-2和SHI-1-MSCV细胞分别接种于未经预处理的6周龄裸鼠前肢腋侧皮卜,各组10只.分别丁第23及30大各处死5只,测量肿瘤体积,并行TIMP-2及vWF抗体免疫组化染色,观察TIMP-2蛋白表达及微血管密度.结果 经尾静脉接种SHI-1-TIMP-2细胞的裸鼠生存期较接种SHI-1-MSCV细胞者明显缩短,体内各脏器中成瘤增加,苏木精-伊红染色及CD45抗体免疫组化染色显示脏器中白血病细胞浸润增加,CNSL发生程度更严重.接种于裸鼠皮下时,尽管SHI-1-TIMP-2细胞成瘤时间提前,其在第23及30天时所形成的瘤块体积较SH1-1-MSCV细胞组显著减少,并出现中央坏死区,免疫组化染色示瘤块TIMP-2蛋白表达增加,而微血管密度减少.结论 TIMP-2基因具有多样性,在肿瘤细胞浸润及其转移中似有双相调节作用.     

抗血管内皮生长因子发夹状核酶基因抑制白血病细胞裸鼠体内生长和肿瘤内血管生成

目的探讨抗血管内皮生长因子(VEGF)发夹状核酶基因对白血病细胞裸鼠体内生长和肿瘤内血管生成的影响。方法采用脂质体介导的方法将抗 VEGF 发夹状核酶基因真核表达载体pcDNA-RZ 转染白血病细胞系 K562,G418抗性筛选获得阳性克隆;抽提基因组 DNA,用 PCR 方法验证核酶基因已转入 K562细胞;荧光定量 PCR 和免疫印迹反应检测白血病细胞中 VEGF mRNA 和蛋白表达量的改变;将白血病细胞皮下接种 BALB/c 裸鼠,观察转染细胞在裸鼠体内成瘤及生长情况;组织形态学和免疫组织化学法检测裸鼠移植瘤微血管密度(MVD)。结果抗 VEGF 发夹状核酶基因真核表达载体 pcDNA-RZ 转入白血病细胞系 K562(K562/RZ),G418筛选两周获得阳性克隆,PCR 检测证实核酶基因整合入白血病细胞基因组 DNA;与 K562及 K562/PC 细胞(转染空质粒的 K562细胞)相比,K562/RZ 细胞 VEGF mRNA 和蛋白的表达量明显降低。接种 K562、K562/PC 和 K562/RZ 细胞组小鼠肿瘤终体积分别为(3.21±0.89)cm~3,(3.42±1.01)cm~3,(1.71±0.94)cm~3;肿瘤重量分别为(4.43±0.87)g,(3.96±0.94)g,(2.24±0.56)g;瘤体内 MVD 分别为4.70±1.25,4.67±1.31和1.80±1.55。以上各组之间的差异均有统计学意义(P<0.01)。结论转导抗 VEGF 发夹状核酶基因能减少白血病细胞中 VEGF 的合成,细胞裸鼠致瘤能力明显减弱,瘤体内血管形成能力降低。     

K562/NOD-SCID小鼠白血病模型的建立

本研究探讨人CML急性变的白血病细胞在NOD—SCID小鼠体内建立白血病模型的方法并研究其生长特性。首先将K562细胞接种于全身受照射后的裸鼠，待皮下成瘤后取出局部瘤块，选取无坏死的瘤组织制成瘤细胞悬液，再腹腔接种于全身受照射后的NOD—SCID小鼠。结果表明：成功建立全身播散的白血病模型。4周时外周血涂片可见白血病细胞，晚期浸润肝、脾、骨髓等造血器官，白细胞上升到接种前的8—10倍，血涂片中白血病细胞达20％-30％。腹腔局部出现瘤块，多位于腹腔内或大网膜，较少累及其他器官。结论：腹腔接种K562瘤细胞于全身照射后的NOD—SCID小鼠能建成全身播散的白血病模型，该模型较好地反映白血病在体内的演变过程，是进行新药疗效试验、生物导向治疗及基因治疗的理想工具。     

卡介苗抑制裸鼠白血病移植瘤生长及抗肿瘤的实验研究

本研究通过建立人白血病突变株细胞异种移植模型探讨卡介苗(bacillus calmette-guerin vaccine,BCG)的抗白血病作用。对8-10周龄的BALB/c裸鼠皮下接种1×107/ml人急性髓系白0细胞,于4-6天可形成皮下浸润的白血病裸鼠模型,随后将其分为2组:对照组和实验组。对照组于肿瘤内接种生理盐水,实验组又分为T1组(BCG组)、T2组(灭活的BCG组)。观察各组裸鼠带瘤生存情况,以及通过对肿瘤组织及多个脏器组织行石蜡切片HE染色,在光学显微镜下观察肿瘤内的形态变化。结果表明,HL-60细胞植入裸鼠皮下后各组裸鼠体表接种部位形成淡绿色的肿块,同时有局部转移及肝脏脾脏转移。在BCG组、灭活的BCG组均出现不同程度的肿瘤组织坏死现象。结论 :用白血病HL-60细胞建立了白血病移植裸鼠模型,有全身肿瘤转移的现象,在接种BCG后均未出现明显的结核感染情况。BCG自身对肿瘤组织有明显的破坏作用。     

survivin基因RNA干涉对宫颈癌裸鼠移植瘤生长及凋亡和顺铂化疗敏感性的影响

目的观察survivin基因RNAi对宫颈癌裸鼠移植瘤生长、凋亡和化疗敏感性的影响。方法随机选择雌性BALB/C-nu/nu裸小鼠24只,细胞接种法建立4组人宫颈癌裸鼠皮下移植瘤模型,每天观察裸鼠一般状况及肿瘤生长情况,通过绘制肿瘤生长曲线并计算肿瘤生长抑制率,观察survivin基因RNAi对人宫颈癌裸鼠皮下移植瘤生长的影响;通过免疫组化SP法检测各组移植瘤组织中survivin蛋白表达情况,TUNEL染色观察survivin基因RNAi对人宫颈癌裸鼠皮下移植瘤凋亡的影响;当肿瘤体积达0.2cm3时给予顺铂化疗以观察survivin基因RNAi对人宫颈癌裸鼠皮下移植瘤化疗敏感性的影响。结果成功建立4组人宫颈癌裸鼠皮下移植瘤模型,接种HeLa-s2组裸鼠肿瘤体积在每个检测点均明显小于接种HeLa组;观察结束时,接种HeLa-s2组裸鼠瘤重明显小于接种HeLa组,分别为：（0.369±0.043）g和（1.150±0.136）g（P〈0.05）;接种HeLa-s2组裸鼠肿瘤生长抑制率为67.9%。免疫组化结果显示：接种HeLa-s2组裸鼠survivin蛋白表达显著下降;TUNEL染色结果显示：接种HeLa-s2组裸鼠细胞凋亡明显增多,凋亡指数（AI）值达（22.73±1.37）%。顺铂化疗后不同检测点接种HeLa-s2组裸鼠肿瘤体积明显小于接种HeLa组,肿瘤生长明显受抑;观察结束后,接种HeLa-s2组裸鼠瘤重明显小于接种HeLa组,分别为：（0.323±0.058）g和（1.347±0.173）g（P〈0.05）;接种HeLa-s2组裸鼠肿瘤细胞凋亡明显增多,与接种HeLa组AI比较,接种HeLa-S2组AI明显升高,分别为：（37.38±1.01）%和（5.19±0.61）%（P〈0.05）。结论 survivin基因RNAi可通过下调移植瘤组织survivin蛋白表达抑制移植瘤生长并促进其凋亡,并通过增加顺铂化疗诱导的细胞凋亡,增强顺铂化疗对移植瘤的生长抑制,进而提高移植瘤对顺铂化疗的敏感性。     

人肿瘤坏死因子α基因对绒癌耐药细胞Bcl-2表达影响的体内研究

目的研究转导人肿瘤坏死因子α(human tumor necrosis factor alpha,hTNF-α)基因对裸鼠绒癌耐药细胞移植瘤组织中Bcl-2表达的影响。方法以JEG-3、JEG-3/VP2和JEG-3/VP2hTNF三种绒癌细胞系接种18只裸鼠,采用RT-PCR、苏木精-伊红染色、免疫组织化学的方法,观察移植瘤细胞的凋亡和Bcl-2表达的情况。结果在接种JEG-3/VP2/hTNF-α的移植瘤组织中,存在hTNF-α基因的表达;JEG-3/VP2/hTNF-α组的Bcl-2阳性表达的细胞较少,但凋亡细胞数较多,与JEG-3组相近;而JEG-3/VP2组则相反。结论转导hTNF-α基因可能使绒癌耐药瘤细胞产生Bcl-2的量减少,进而促使了绒癌耐药瘤细胞的凋亡。     

人急性单核细胞白血病细胞系SHI-1在裸鼠体内的高致瘤性及其机制的初步研究

本研究确定人急性单核细胞白血病细胞系SHI-1在裸鼠体内的高致瘤性,并对其成瘤机制进行初步探讨。将SHI-1细胞接种至裸鼠皮下,观察肿瘤生长情况;取肿瘤组织行病理检测、R显带核型分析;RT-PCR检测MLL-AF6融合基因和VEGF基因的转录;明胶酶谱法检测培养上清中基质金属蛋白酶-9（MMP-9）和MMP-2的表达;体外穿膜实验观察迁移能力。结果表明：注射SHI-1细胞的16只裸小鼠均于皮下出现肿块;瘤体由白血病细胞组成;注射的裸鼠中有MLL/AF6融合基因和VEGF基因的转录;在无血清培养上清中MMP-9和MMP-2的表达明显高于对照细胞;SHI-1细胞有较强的迁移能力,MMP-2的阻断抗体可显著抑制其体外迁移能力。结论：SHI-1在裸鼠体内有极高的成瘤率,其机制可能与p53基因的异常、高水平VEGF基因的转录、金属蛋白酶的高表达和较强的体内浸润能力有关。     

白细胞介素6基因转染的白血病细胞克隆株的建立及其生物学特性的观察

作者将人白细胞介素６（ＩＬ－６）基因转染至ＦＢＬ－３红白血病细胞，建立了高分泌ＩＬ６的ＦＢＬ－３细胞克隆株，并观察了其生物学特性。采用磷酸钙ＤＮＡ共沉淀法将ＩＬ－６表达载体ＢＣＭＧＮｅｏ－ＩＬ－６转移至ＦＢＬ－３细胞中，通过Ｇ４ｌ８抗性筛选、有限稀释法和上清中ＩＬ－６活性的测定，从多株阳性克隆中筛选到一株高分泌ＩＬ－６（２２５．６Ｕ／ｍｌ）的克隆株。体外观察表明，ＩＬ－６基因转染的ＦＢＬ－３细胞体外生长能力、集落形成能力均减弱，且其生长抑制程度与分泌ＩＬ－６水平呈正相关。给小鼠皮下接种后肿瘤结节形成率降低，生长速度减慢，荷瘤小鼠存活期延长。以上结果表明，ＩＬ－６基因转染的ＦＢＬ－３细胞致瘤性下降，这为将该细胞制备成新型瘤苗治疗白血病打下了基础。     

白细胞介素2基因和白细胞介素3基因共转染白血病瘤苗的抗白…

目的：研究白细胞介素２（ＩＬ－２）和白细胞介素３（ＩＬ－３）基因共转染的白血病细胞瘤苗对白血病的小鼠的治疗效果。方法：用ＩＬ－２重组腺病毒（Ａｄ－ＩＬ－２）和（或）ＩＬ－３重组腺病毒转染红白轿病细胞株ＦＢＬ－３，＾６０Ｃｏ射后制备成瘤苗对实验性白血病小鼠进行治疗，观察肿瘤生长，小鼠生存期及治疗后小鼠腹腔巨噬细胞，ＮＫ细胞，诱导杀伤性Ｔ淋巴细胞（ＣＴＬ）杀伤活性，并与低剂量环磷酰胺合用，观察其抗肿瘤     

IL—2基因修饰增强白血病抗原冲击的树突状细胞诱导的抗肿瘤？…

目的 研究腺病毒介导的白细胞介素（ＩＬ）２基因修饰能否使抗原冲击的树突状细胞（ＤＣ）在体内诱导出更强的抗肿瘤免疫反应。方法 用小鼠粒－巨噬细胞击落刺激因子（ｍＧＭ－ＣＳＦ）和ｍＩＬ－４扩增上鼠骨髓ＤＣ在ＦＢＬ－３红白血病细胞冻融抗原冲击的同时转染ＩＬ－２腺病毒。观察其体外分泌ＩＬ－２的水平,刺激Ｔ细胞增殖的能力,其体内皮下免疫后小鼠引流淋巴结细胞数和组分的变化以及主艉地产生细胞毒性Ｔ淋巴细胞（ＣＴ     

GM—CSF基因修饰,抗原预激的树突状细胞体内诱导白血病细胞分化？…

目的：探讨树突状细胞在肿瘤治疗过程中的作用及机制。方法：以小鼠ＦＢＬ－３红白血病细胞皮下接种Ｃ５７ＢＬ／６小鼠建立荷瘤模型，采用流式细胞仪分析和透射电镜等技术，观察了粒－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）基因修饰的抗原预激的树突状细胞体内治疗作用。结果：采用ＧＭ－ＣＳＦ基因修饰的抗原预激的树突状细胞治疗，可以明显抑制白血病细胞生长；白血病细胞表达ＣＤ３４水平增加，同时ＭＨＣ－Ⅱ，Ｂ７－１，Ｂ７－     

白细胞介素6基因转移的白血病细胞体内诱导小鼠免疫功能的实验研究

作者观察了人白细胞介素６（ＩＬ－６）基因转移的、能高分泌ＩＬ－６的ＦＢＬ－３红白血病细胞对机体抗肿瘤免疫功能的影响。结果发现，高分泌ＩＬ－６的红白血病细胞接种到小鼠体内后，能显著提高小鼠脾脏的细胞毒性Ｔ淋巴细胞、ＮＫ活性及ＩＬ－２诱导的ＬＡＫ活性，小鼠脾细胞诱生的ＩＬ－２、肿瘤坏死因子和粒－巨噬细胞系集落刺激因子水平亦增高，腹腔巨噬细胞杀伤活性明显增强。结果表明，高分泌ＩＬ－６的ＦＢＬ－３红白血病细胞体内接种后能显著地增强机体的抗肿瘤免疫功能。研究结果为白血病的生物治疗以及ＩＬ－６基因转移的新型瘤苗的应用研究提供了实验依据。     

Augmentation of the anti-tumor therapeutic efficacy of long-term cultured T lymphocytes by in vivo administration of purified interleukin 2

Spleen cells from C57BL/6 mice immunized in vivo with a syngeneic Friend virus-induced leukemia, FBL-3, were specifically activated by culture for 7 d with FBL-3, then nonspecifically induced to proliferate in vitro for 12 d by addition of supernatants from concanavalin A- stimulated lymphocytes containing interleukin 2 (IL-2). Such long-term cultured T lymphocytes have previously been shown to specifically lyse FBL-3 and to mediate specific adoptive therapy of advanced disseminated FBL-3 when used as an adjunct to cyclophosphamide (CY) in adoptive chemoimmunotherapy. Because the cultured cells are dependent upon IL-2 for proliferation and survival in vitro, their efficacy in vivo is potentially limited by the availability of endogenous IL-2. Thus, the aim of the current study was to determine whether exogenously administered purified IL-2 could augment the in vivo efficacy of long- term cultured T lymphocytes. Purified IL-2 alone or as an adjunct to CY as ineffective in tumor therapy. However, IL-2 was extremely effective in augmenting the efficacy of IL-2-dependent long-term cultured T lymphocytes in adoptive chemoimmunotherapy. The mechanism by which IL-2 functions in vivo is presumably by promoting in vivo growth and/or survival of adoptively transferred cells. This assumption was supported by the findings that IL-2 did not enhance the modest therapeutic efficacy of irradiated long-term cultured cells that were incapable of proliferating in the host and was ineffective in augmenting the in vivo efficacy of noncultured immune cells that are not immediately dependent upon exogenous IL-2 for survival.     

Antigen-driven long term-cultured T cells proliferate in vivo, distribute widely, mediate specific tumor therapy, and persist long- term as functional memory T cells

Mice bearing disseminated syngeneic FBL-3 leukemia were treated with cyclophosphamide plus long term-cultured T cells immune to FBL-3. The cultured T cells for therapy had been induced to grow in vitro for 62 d by intermittent stimulation with irradiated FBL-3. At the time of therapy, such antigen-driven long term-cultured T cells were greatly expanded in number, proliferated in vitro in response to FBL-3, and were specifically cytotoxic. Following adoptive transfer, donor T cells persisting in the host were identified and counted using donor and host mice congenic for the T cell marker Thy-1. The results show that antigen-driven long term-cultured T cells proliferated rapidly in vivo, distributed widely in host lymphoid organs, and were effective in tumor therapy. Moreover, the already rapid in vivo growth rate of donor T cells could be augmented by administration of exogenous IL-2. When cured mice were examined 120 d after therapy, donor L3T4+ T cells and donor Lyt-2+ T cells could be found in large numbers in host ascites, spleen, and mesenteric and axillary lymph nodes. The persisting donor T cells proliferated in vitro, and became specifically cytotoxic in response to FBL-3, demonstrating that antigen-driven long term-cultured T cells can persist long term in vivo and provide immunologic memory.     

转染血管内皮细胞生长因子受体基因的K562细胞在裸鼠体内生长的实验研究

目的 观察人白血病细胞转染可溶性内皮细胞生长因子受体基因（sFlt-1)后,对其在裸鼠体内生长的影响。方法 ①将内皮细胞生长因子受体－1（Flt-1)的配体结合区基因片断与免疫球蛋白重链的恒定区基因片断重组成Flt-Ig融合基因,插入到pcDNA3质粒;②采用电穿方法转染K562细胞,经过筛选、克隆,用RT－PCR检测Flt-1 mRNA的表达;③将表达Flt-Ig基因的细胞和转染对照载体的细胞分别给裸鼠移植,动态观察肿瘤的生长。结果 Flt-Ig融合基因转染K562细胞后,获得5株Flt-Ig mRNA表达阳性的细胞株,取一株扩增后移植给6只裸鼠,移植后第14,21和28天实验组肿瘤的体积约为对照组的1／2,明显小于对照组。结论 转染Flt-Ig融合基因的K562细胞,在裸鼠体内生长受到明显抑制,这可能与转染后的肿瘤细胞表达可溶性Flt-Ig蛋白,中和了肿瘤细胞分泌的内皮细胞生长因子,抑制肿瘤血管新生有关。     

Retroviral immunotoxin gene therapy of leukemia in mice using leukemia-specific T cells transduced with an interleukin-3/Bax fusion protein gene

In past studies, we showed that T cells transduced with retroviral diphtheria immunotoxin (IT) target genes could serve as vehicles for delivering IT to tumors in vivo. We took advantage of the observation that antigen-specific T cells are able to penetrate tumors to design an approach delivering combined cellular and humoral therapy directly to the tumor site. To improve tumor specificity, we selected interleukin (IL)-3 as a ligand because its receptor is selectively overexpressed on myeloid leukemia progenitors. Because Bcl-2 family proteins show structural similarity to diphtheria toxin (DT), we constructed a unique retroviral IT using Bax, a proapoptotic member of the Bcl-2 family, in place of DT. Bax was chosen because several studies showed that its transduction induces lethal apoptosis in different cancers. The retroviral construct for gene therapy included IL-3 positioned downstream of its 80 amino acid leader, and permitted cotranslational protein synthesis of hybrid IL-3/human Bax fusion protein. Other vectors were constructed with IL-3 fused to DT or Pseudomonas exotoxin. Retroviral vectors were used to transiently transduce C8, a CD4(+) T cell clone that specifically recognized FBL-3, a lethal myeloid leukemia. Supernatants collected from transduced cells showed proapoptotic activity and selectively inhibited FBL-3 cells in vitro. Intraperitoneal injection of transduced but not nontransduced C8 into mice with subcutaneous tumors or systemic cancer significantly inhibited tumor growth. These results indicate that retroviral IT made with IL-3 and various toxic proteins may be useful in patients with acute myelogenous leukemia (AML). Furthermore, the Bax construct may be particularly useful as a nonimmunogenic substitute for bacterial toxins in retIT.     

Therapy of disseminated murine leukemia with cyclophosphamide and immune Lyt-1+,2- T cells. Tumor eradication does not require participation of cytotoxic T cells

The ability of noncytolytic Lyt-1+,2- T cells immune to FBL-3 leukemia to effect eradication of disseminated FBL-3 was studied. Adult thymectomized, irradiated, and T-depleted bone marrow-reconstituted (ATXBM) B6 hosts were cured of disseminated FBL-3 by treatment with 180 mg/kg cyclophosphamide (CY) and adoptively transferred Lyt-1+,2- T cells obtained from congenic B6/Thy-1.1 donors immune to FBL-3. Analysis of the T cell compartment of ATXBM hosts treated and rendered tumor-free by this therapy revealed that the only T cells present in the mice were donor-derived Lyt-1+,2- T cells. In vitro stimulation of these T cells with FBL-3 tumor cells, which express class I but no class II major histocompatibility complex antigens, induced lymphokine secretion, but did not result in the generation of cytotoxic T lymphocytes (CTL). Thus, in a setting in which mice lack Lyt-2+ T cells, and in which no CTL of either host or donor origin could be detected, immune Lyt-1+,2- T cells, in conjunction with CY, mediated eradication of a disseminated leukemia. The results suggest that delayed-type hypersensitivity responses induced by immune T cells represent a potentially useful effector mechanism for in vivo elimination of disseminated tumor cells.