氧化低密度脂蛋白刺激人肾小球系膜细胞产生单核细胞趋化蛋白-1由核因子-κB活化调控

目的 研究核因子ＫＢ（ＮＦ－ＫＢ）在氧化低密度脂蛋白（Ｏｘ- ＬＤＬ）诱导的体外培养的人肾小球系膜细胞表达单核／巨噬细胞趋化蛋白－１（ＭＣＰ－１）中的作用。方法 采用凝胶迁移率变动分析检测ＮＦ－ＫＢ的ＤＮＡ结合活性变化，以免疫组织化学观测细胞内ｐ６５的核转位，用细胞ＥＬＩＳＡ法检测细胞内 ＭＣＰ－１及ＩＫＢα蛋白含量变化。结果 不同浓度（１０、２５、５０、１００μｇ／ｍｌ）Ｏｘ－ＬＤＬ刺激肾小球系膜细胞均可引起细胞ＮＦ-ＫＢ的ＤＮＡ结合活性增强、ＩＫＢα蛋白表达下降以及ＭＣＰ－１蛋白表达增强，以５０μｇ／ｍｌ刺激１ｈ ＮＦ－ＫＢ活化及ＩＫＢα表达减弱最明显，作用２４ｈＭＣＰ-１表达水平最高。ＮＦ－ＫＢ俯活化的同时伴有ｐ６５核转位。上述效应可被ＮＦ－ＫＢ特异性抑制剂吡咯二硫氨基甲酸酯（ＰＤＴＣ）所抑制。结论Ｏｘ－ＬＤＬ刺激人肾小球系膜细胞产生ＭＣＰ－１是由ＮＦ－ＫＢ调控，ＮＦ－ＫＢ参与了脂质肾损害的发病过程。     

沙培林对脾切除后实验性胃癌大鼠免疫杀伤细胞的活性作用

通过观察沙培林（ＯＫ－４３２）对脾切除后胃癌大鼠免疫杀伤的细胞作用，发现ＯＫ－４３２小时剂长期治疗后，外周血淋巴细胞（ＰＢＬ）的ＮＫ和ＡＤＣＣ细胞活性分别显著高于生理盐水非治疗组，ＯＫ－４３２治疗同时保留脾脏，ＰＢＬ和ＮＫ和ＡＤＣＣ活性更辊显著高地高于切脾免疫治疗组。在ＯＫ－４３２腹腔注射大鼠中，模拟切脾组的ＰＢＬ的ＮＫ和ＡＤＣＣ的细胞活性均高于切脾组，提示在腹腔注射ＯＫ－４３２治疗中，保留脾脏有     

胞嘧啶脱氨酶基因对胃癌细胞的杀伤作用

目的探讨胞嘧啶脱氨酶基因对胃癌细胞的杀伤作用。方法应用胞嘧啶脱氨酶（ＣＤ）基因,通过逆转录病毒载体转入人胃癌细胞Ｍ８５中,经０．４ｍｇ／ｍｌ的Ｇ４１８筛选阳性克隆扩增后进行前药５ＦＣ敏感试验。结果转ＣＤ自杀基因的Ｍ８５细胞较亲代Ｍ８５细胞对前药的敏感性显著增高（Ｐ＜０．０５）。体外实验中还观察到,前药在对转自杀基因的Ｍ８５细胞产生杀伤作用同时,还对与其共培养的亲代Ｍ８５细胞产生明显的杀伤作用,即“旁观者效应”。进一步将转基因Ｍ８５细胞和未转基因Ｍ８５细胞接种至裸鼠皮下,发现两者成瘤性无明显差异;前药５ＦＣ对未转基因肿瘤的生长无影响,而对转基因肿瘤的生长明显抑制。结论本研究为ＣＤ基因治疗胃癌向临床过渡提供了依据。     

卡介苗激活杀伤细胞抗膀胱肿瘤作用研究

为了进一步探讨ＢＣＧ抗膀胱肿瘤的作用机理，采取１５例膀胱肿瘤患者外周血单个核细胞（ＰＢＭＣ），置于含ＢＣＧ或ＩＬ２的培养基中培养，计算扩增倍数，检测培养细胞抗自体及异体膀胱癌细胞活性。结果：卡介苗激活杀伤细胞（ＢＡＫ）与淋巴因子激活杀伤细胞（ＬＡＫ）分别于培养第７和第３天达增殖高峰，对自体瘤杀伤率分别为３６．２％和３１．４％，差异有显著性（Ｐ＜０．０５）；对异体瘤杀伤率分别为２５．２％和２８．３％，差异无显著性。结果表明：ＢＡＫ细胞抗自体瘤活性高于ＬＡＫ细胞，死ＢＣＧ对ＰＢＭＣ无激活作用，ＢＡＫ细胞抗肿瘤效应可能是ＢＣＧ抗膀胱肿瘤重要作用机理之一。     

胃癌患者脾细胞免疫功能的研究

目的 探讨胃癌患者脾脏的细胞免疫功能，方法 检测进展胃癌患者脾静脉血淋巴细胞（ＳＶＬ），和脾细胞（ＳＣ）的自然杀伤细胞（ＮＫ），ＬＡＫ细胞的杀伤活性及Ｔ细胞亚群（ＴＣ）与胃良性病变的ＳＶＬ及外伤性脾破裂ＳＣ的对照比较。结果 胃癌患者脾脏ＮＫ，ＬＡＫ细胞杀伤活性显著降低（Ｐ〈０．０２，Ｐ〈０．０１），ＣＤ４减少，ＣＤ８增加，ＣＤ４／ＣＤ８比值显著降低（Ｐ〈０．０２～０．０１）。结论 进展期胃癌患者脾     

BCG激活淋巴细胞对膀胱肿瘤细胞的抗肿瘤作用研究

利用ＢＣＧ激活的外周血单核细胞（ＰＢＭＮＣ）作为肿瘤杀伤细胞———ＢＡＫ细胞，观察其对移行上皮肿瘤细胞系ＢＴＡ和１１例原代培养细胞的细胞毒作用。结果表明，单独ＢＣＧ对培养肿瘤细胞没有细胞毒作用；ＮＫ细胞仅表现较低的细胞毒作用，在效靶比４０∶１对ＢＴＡ的特异性杀伤率为１７．６３％，对原代细胞为１２．４９％；ＢＡＫ细胞分别为３１．２６％和１９．１６％（Ｐ＜０．０５）；ＬＡＫ细胞分别为３５．５３％和２４．２１％；对ＢＴＡ细胞的细胞毒作用ＬＡＫ细胞＞ＢＡＫ细胞（Ｐ＜０．０５），对原代细胞的细胞毒作用ＬＡＫ细胞和ＢＡＫ细胞之间无显著性差异（Ｐ＞０．０５）。认为ＢＡＫ细胞是不同于ＬＡＫ细胞的一类肿瘤杀伤细胞，在ＢＣＧ抗肿瘤的过程中起着重要作用。     

抗白介素2受体α链单抗及AK细胞治疗肝癌的实验研究

探讨一种新的过继免疫治疗肝癌方法,方法将肝癌细胞株接种于近交系ＢＡＬＢ／ｃ小鼠皮下,制成荷瘤小鼠模型,用ＩＬ－２／ＡＫ细胞／抗白介素２受体α链单抗治疗,以发现其抗肿瘤作用。结果荷瘤小鼠脾淋巴细胞ＮＫ活性无明显变化,ＬＡＫ和ＣＴＬ活性及淋巴细胞刺激指数在联合治疗组均高于其他各组;肝癌皮下结节在联合治疗组明显受到抑制成荷瘤小鼠模型 肿瘤小鼠生存期明显延长。结论ＩＬ－２,αＩＬ－２αＭＡｂ及ＡＫ细胞联合     

TK基因/9-丙氧鸟苷联合mGM-CSF基因治疗胃癌

目的 观察自杀基因ＴＫ对小鼠胃癌的杀伤作用,并联合ｍＧＭ－ＣＳＦ基因,观察免疫反应在增强ＴＫ杀伤性中的作用。方法 应用逆转录病毒方法转导自杀基因ＴＫ,应用脂质体方法转导细胞因子基因ｍＧＭ－ＣＳＦ。观察ＴＫ基因体外对小鼠胃癌６１５小鼠前胃癌（ＭＦＣ）细胞的杀伤性和旁观者效应;体内实验中将病毒上清注入瘤体内,并结合应用其前药９－丙氧鸟苷（ＧＣＶ）和ｍＧＭ－ＣＳＦ基因,分别观察肿瘤的生长情况。通过病理切     

胃泌素受体拮抗剂抑制胃癌细胞生长的可行性研究

目的　探讨应用胃泌素受体拮抗剂治疗胃癌的可行性。方法　在体外条件下观察胃泌素及其受体拮抗剂丙谷胺对胃癌细胞株ＭＫＮ４５ 的细胞活力、细胞内ｃＡＭＰ浓度及细胞凋亡的影响;在体内条件下观察胃泌素受体拮抗剂丙谷胺对胃癌细胞株ＭＫＮ４５ 移植瘤的面积、重量及胃癌细胞ＤＮＡ 的影响。结果　在体外条件下,胃泌素能促进胃癌细胞株ＭＫＮ４５ 的增殖,细胞内ｃＡＭＰ浓度增高,细胞凋亡百分率降低,而丙谷胺能完全阻断胃泌素的作用。在体内条件下,丙谷胺能抑制胃癌细胞株ＭＫＮ４５ 移植瘤的生长,肿瘤面积、重量及胃癌细胞ＤＩ、ＤＮＡ 含量、ＳＰＦ均低于对照组。结论　胃泌素受体拮抗剂能够抑制胃泌素受体阳性的胃癌细胞生长,有望为胃癌的治疗提供一种新手段。     

MHC基因胸腺转移诱导异基因小鼠皮肤移植免疫耐受

目的通过胸腺内注射质粒ＰＸＮ（Ｎ２Ｂ１９Ｈ２Ｋｂ）,表达外源性主要组织相容性抗原复合物（ＭＨＣ）抗原,以小鼠皮肤移植为模型,诱导移植免疫耐受,为免疫耐受提供理论和实验依据。方法应用逆转录病毒载体介导的基因转移技术,通过ＢＡＬＡ／Ｃ胸腺内注射质粒ＰＸＮ（Ｎ２Ｂ１９Ｈ２Ｋｂ）,将外源性ＭＨＣ基因转移到胸腺细胞,１４天后行异基因小鼠皮肤移植。应用聚合酶链反应（ＰＣＲ）,反转录聚合酶链反应（ＲＴＰＣＲ）,单克隆抗体免疫荧光染色流式细胞仪检测胸腺细胞ＤＮＡ,ｍＲＮＡ和ＭＨＣ蛋白质表达。结果外源性ＭＨＣ基因已整合到靶细胞染色体ＤＮＡ并有效地转录,胸腺细胞表面有外源性ＭＨＣ分子表达,转染效率为５％,胸腺内注射质粒ＰＸＮ（Ｎ２Ｂ１９Ｈ２Ｋｂ）能明显延长异基因小鼠的皮肤移植时间,平均２７天,对照为１０天（Ｐ＜０００１）。受体鼠脾细胞对ＣｏｎＡ的增殖反应在正常范围。结论供体鼠ＭＨＣ基因转移至受体鼠胸腺诱导了对供体皮肤移植的免疫耐受。     

Cisplatin augments cytotoxic T-lymphocyte-mediated antitumor immunity in poorly immunogenic murine lung cancer

OBJECTIVE: Many tumors are poorly immunogenic and resistant to cytotoxic T-lymphocyte-mediated cell lysis. Because cisplatin has been demonstrated to increase tumor cell Fas receptor expression, we hypothesized that cisplatin will enhance cytotoxic T-lymphocyte tumor cell killing and augment the antitumor effect of an active immunotherapy strategy in a poorly immunogenic murine lung cancer model. METHODS: Lewis lung carcinoma cells were exposed to cisplatin in vitro, and Fas receptor expression and apoptosis in response to an agonistic anti-Fas antibody were quantified using flow cytometry. Wild-type and Fas ligand-deficient mice bearing Lewis lung carcinoma flank tumors were then treated with intraperitoneal cisplatin as well as an intratumoral injection of an adenovirus gene transfer vector encoding CD40 ligand. End points included tumor size, animal survival, and Fas expression (determined using immunofluorescence). Cytotoxicity assays were performed using splenocytes from adenovirus gene transfer vector encoding CD40 ligand-treated animals as effectors and cisplatin-treated Lewis lung carcinoma cells as targets. RESULTS: Cisplatin induced heightened expression of Fas receptor on Lewis lung carcinoma cells in vitro and in vivo and enhanced apoptosis in cells exposed to an agonistic anti-Fas antibody. In vivo, the combination of 1 dose of intraperitoneal cisplatin and intratumoral adenovirus gene transfer vector encoding CD40 ligand inhibited tumor growth and prolonged survival compared with adenovirus gene transfer vector encoding CD40 ligand alone, resulting in a higher cure rate. This effect was lost in Fas ligand-deficient mice. Splenocytes from adenovirus gene transfer vector encoding CD40 ligand-treated wild-type mice lysed cisplatin-treated Lewis lung carcinoma cells more efficiently than untreated Lewis lung carcinoma cells, an effect lost in splenocytes from Fas ligand-deficient mice. CONCLUSION: Cisplatin augments the antitumor effect of a cytotoxic T-lymphocyte-mediated immunotherapy strategy, resulting in a higher cure rate than seen with immunotherapy alone. This effect is associated with the enhanced ability of cytotoxic T lymphocytes to lyse tumor cells that have been exposed to cisplatin through Fas/Fas ligand interactions.     

胃癌乳斑微转移在胃癌干细胞及前驱细胞研究中的意义

目的：探讨胃癌乳斑微转移模型在胃癌干细胞及前驱细胞研究中的意义。方法：胃癌细胞系MFC,1×10~4细胞数量注射到小鼠腹腔内,72 h后取大网膜,收集乳斑微转移灶内的胃癌细胞团;以不同细胞数量进行NOD/SCID小鼠皮下注射,观察细胞的致瘤能力,移植瘤HE染色,免疫组化分析CEA、CK20表达;免疫细胞化学分析乳斑微转移灶内的胃癌细胞团干细胞相关标志物的表达。结果：皮下注射16周后,微转移灶内的胃癌细胞团组中,皮下注射数量为5×10~2个细胞有1个部位产生肿瘤组织：注射数量为1×10~3个细胞时,5个部位均能产生肿瘤组织。对照组皮下注射为5×10~4个细胞时,仅1个部位出现肿瘤,而注射部位全部出现肿瘤组织,需要5×10~6个以上的细胞数量。两组皮下肿瘤组织分别经HE染色和免疫组化分析CEA、CK20表达,与对照组比较无统计学差异。微转移灶内细胞团高表达CD133和CD44,而CD324的表达则明显下降。结论：胃癌乳斑微转移模型内存在丰富的胃癌于细胞及前驱细胞;大网膜乳斑微环境具有富集胃癌干细胞及前驱细胞的作用。CD133~＋、CD44~＋和CD324~-可能是胃癌干细胞的表面标志物。     

放射性^32磷—玻璃微球治疗裸鼠人肝癌移植瘤的研究

目的探讨３２磷玻璃微球（３２ＰＧＭＳ）局部注射对裸鼠人肝癌移植瘤的治疗作用和超微结构影响。方法建立裸鼠人肝癌移植瘤模型后用３２ＰＧＭＳ进行治疗,取治疗后瘤组织用透射电镜观察。结果实验组瘤细胞大量坏死（３５％～７０％）,残留的瘤细胞在分化上出现了一些差异,部分瘤细胞出现了高分化肝癌的形态特征;肿瘤间质见淋巴细胞浸润和纤维结缔组织增生,但无强烈的急性放射性炎反应;瘤体周围肌纤维和皮肤均无改变。对照组肿瘤示低分化肝癌,其中有少量死亡瘤细胞（４％）。结论３２ＰＧＭＳ抗肝癌作用明显,并有一定的免疫调节和促分化功能,对治疗区周围组织亦无损伤。     

CD基因联合GM-CSF基因对胰腺癌治疗作用的实验研究

目的：观察自杀基因CD联合GM—CSF基因的抗胰腺癌作用。方法：应用逆转录病毒方法转导CD和GM—CSF基因。皮下接种胰腺癌细胞TD2，肿瘤局部注射重组表达的pVITR02-CD-GM—CSF，然后连续10d给予5-氟胞嘧啶（5-Fc）300mg／kg治疗，观察肿瘤的生长情况。结果：CD／5-Fc联合GM—CSF治疗后，小鼠肿瘤体积显著缩小，存活期明显延长，与对照组以及GM—CSF组和CD／5-Fc组比较差异有统计学意义（P〈0．05，P〈0．01），且肿瘤瘤体内CD8^＋细胞浸润增加，MHC—Ⅰ和B7—1表达明显增加。结论：CD基因联合细胞因子基因GM—CSF可增强CD的抗肿瘤作用，其疗效要好于GM—CSF或CD基因单独治疗。     

携带4-1BBL基因的胃癌细胞总RNA转染树突状细胞疫苗的研究

目的 观察携带4-1BBL基因的胃癌细胞总RNA转染树突状细胞疫苗,在小鼠体内诱导的抗肿瘤效应以及对小鼠免疫力的影响.方法 用脂质体法把pMKITneo/4-1 BBL质粒导入小鼠胃癌细胞MFC内,再提取此细胞的总RNA并转染至树突状细胞内制备MFC/4-1BBL/DC疫苗;把MFC细胞注射于20只615小鼠皮下建立荷瘤小鼠胃癌模型,并随机分为对照组、DC组、MFC/DC组和MFC/4-1BBL/DC组,每组5只;在MFC细胞接种后第7、14天给予相应DC疫苗治疗,于肿瘤细胞接种后第21天,处死实验动物,测量瘤重、测定肿瘤细胞的凋亡率及外周血的CD4+、CD8+T细胞和NK细胞的含量.结果 MFC/4-1BBL/DC组小鼠平均瘤重(2.06±0.39)g显著低于对照组(3.82±0.57)g、DC组(3.63±0.51)g、MFC/DC组(2.67±0.32)g(P＜0.05);且MFC/4-1BBL/DC组肿瘤细胞凋亡率(28.58±2.84)%明显高于MFC/DC组(25.03±1.88)%、DC组(20.66±2.71)%及对照组(19.09±2.73)%(P＜0.05);MFC/4-1BBI/DC组小鼠外周血CID4+T、NK细胞数明显高于对照组和其他治疗组(P＜0.05).结论 携带4-1BBL基因的胃癌细胞总RNA转染树突状细胞疫苗能够提高荷瘤机体的免疫能力、抑制肿瘤细胞的生长、促进肿瘤细胞的凋亡.

Abstract：

Objective To observe the induced anti-tumor effects and immune state in vivo by the transfection of dendritic cell vaccine into the total RNA of gastric cancer cells carrying 4-1BBL gene.Methods The liposome-mediated pMKITneo/4-1BBL gene was inserted into the MFC gastric cancer cells,and the cell total RNA was extracted and transfected into dendritic cells to make the MFC/4-1BBL/DC vaccine.After MFC cells were injected into the 615 mouse to establish tumor-bearing mouse model,and those 20 mice were randomly divided into control group,DC group,MFC/DC group,MFC/4-1BBL/DC group.The dendritic cell vaccine was subcutaneously injected on the day 7 and 14 after inoculation of tumor cells,and on the day 21 animals were killed and tumor weight was measured,tumor cell apoptosis rate was assayed and the contents of CD4 +,CD8 + T cells and NK cells in peripheral blood were analyzed.Results The mean tumor weight in MFC/4-1BBL/DC group (2.06 ±0.39) g was significantly less than that in control group (3.82 ±0.57) g,DC group (3.63 ±0.51 ) g,MFC/DC group (2.67 ±0.32) g (P ＜0.05).Moreover the apoptosis rate in MFC/4-1BBL/DC group (28.58 ± 2.84 ) % was significantly higher than that in MFC/DC group (25.03 ± 1.88)%,DC group (20.66 ±2.71 )% and control group ( 19.09 ±2.73 )% (P ＜0.05).The percentage of CD4 + T,NK cells in MFC/4-1BBL/DC group was significantly higher than in other groups ( P＜0.05).Conclusion The transfection of dendritic cell vaccine into the total RNA of gastric cancer cells carrying 4-1BBL gene can increase immunity of the bearing tumor mice,inhabit tumor growth and promote tumor cell apoptosis.     

腺病毒介导p16基因转染对胃癌细胞作用的研究

目的　观察以腺病毒为载体介导 p16基因转染对胃癌细胞 (MGC80 3)生长的影响。方法　重组腺病毒Ad p16感染胃癌细胞 ,观察胃癌细胞生长情况 ;用Ad p16感染胃癌细胞接种于大鼠体内 ,Ad lacZ为对照组 ,每组大鼠 10只 ,观察大鼠接种的阳性率 ;用重组腺病毒Ad p16作用于胃癌大鼠模型 ,Ad lacZ为对照组 ,每组大鼠 10只 ,观察大鼠肿瘤的生长情况。结果　p16可以抑制胃癌细胞生长 ,促进胃癌细胞的凋亡 ,经Ad p16感染的胃癌细胞肿瘤发生率明显下降 (由 90 %降至 2 0 % )。腺病毒介导 p16基因作用胃癌大鼠模型 2周后肿瘤的重量 ( 2 .2 1± 0 .2 6 )g明显低于对照组 ( 5 .6 4± 0 .5 3)g ,差异有显著性 (P <0 .0 0 1)。应用免疫组织化学检查可见肿块有明显的淋巴细胞侵润。结论　腺病毒介导 p16基因对胃癌细胞有明显的抑制作用。     

Potent antitumor effects of CD154 transduced tumor cells in experimental bladder cancer

PURPOSE: Current intravesical immunotherapy for bladder cancer with bacillus Calmette-Guerin instillations is standard treatment for patients with high risk superficial tumors but relapses are common. We evaluated the tumor vaccine concept in murine bladder cancer by comparing tumor cell transduction with genes coding for the immunostimulatory molecules CD154, interleukin (IL)-12 and CD80 to design a novel vaccination strategy. MATERIALS AND METHODS: Adenoviral vectors were used to transduce murine bladder cancer MB-49 cells with genes coding for CD154, IL-12 and CD80. Parental or transduced MB-49 cells were injected subcutaneously into syngeneic mice. The effects of transgene expression on tumorigenicity and the generation of protective immunological memory against challenge with parental tumor were studied. RESULTS: All 76 animals injected with parental MB-49 cells had tumors within 8 to 12 days. Tumor cell expression of CD154 combined with IL-12 completely inhibited tumor outgrowth with all 21 mice tumor-free and CD154 transduction alone was almost as effective with 33 of 35 tumor-free. IL-12 production by tumor cells delayed tumor outgrowth and 4 of 10 mice remained tumor-free. Over expression of CD80 had no effect on tumorigenicity. CD154 expressing tumors were rapidly infiltrated with large numbers of CD4+ and CD8+ T cells. Mice vaccinated 4 times with adenoviral CD154 transduced MB-49 cells were completely protected against challenge with parental tumor. Co-injection of CD154 modified cells with parental MB-49 cells retarded tumor growth. CONCLUSIONS: Our experimental results suggest that the potent antitumor effects of CD154 gene transduction should be considered for immunostimulatory gene therapy for bladder cancer.     

散发性大肠癌肿瘤相关基因位点杂合性丢失分析

目的　分析大肠癌组织中抑癌基因及肿瘤相关基因位点的变化与预后的关系。方法　７９例大肠癌组织经组织微解剖分离癌组织和正常组织,分别进行１１ 个染色体上１４ 个位点的杂合性丢失（ｌｏｓｓｏｆｈｅｔｅｒｏｚｙｇｏｓｉｔｙ,ＬＯＨ）分析。结果　肿瘤组织中ＬＯＨ呈现一复杂的征象,５ｑ１２ 及ＲＢ基因位点的ＬＯＨ 与较好的预后有关。结论　不同的抑癌基因及肿瘤相关基因与大肠癌发生有关。５ｑ１２ 位点的ＬＯＨ 与较好预后相关的意义有待今后进一步研究。