抗肝癌免疫纳米颗粒的制备及其对肝癌细胞增殖的影响

目的:制备抗肝癌单链抗体二聚体高分子免疫纳米颗粒,观察其对肝癌细胞增殖的影响.方法:采用离子交联的方法,以壳聚糖水溶性衍生物多糖为基材,将已获得的抗肝癌单链抗体二聚体BDM制备成免疫纳米颗粒,检测纳米颗粒的表征、包封率及载药量,并通过MTT法观察免疫纳米颗粒对肝癌细胞株增殖的影响.结果:制备粒径为100-200nm的抗肝癌单链抗体二聚体高分子纳米颗粒,最佳包封率为53%,栽药量为75μg/mg抗体二聚体,抗肝癌单链抗体二聚体高分子纳米颗粒显示较好的抗肿瘤作用,其对肝癌细胞的抑制率为34%左右,且有浓度依赖性.结论:成功制备了抗肝癌单链抗体二聚体高分子纳米颗粒,初步应用具有抑瘤性,为下一步开展体内肝癌的放射免疫诊断和靶向治疗奠定了基础.     

特异性抗肝癌单链抗体二聚体在毕赤酵母中的表达、纯化及生物学活性鉴定

目的:实现特异性抗肝癌单链抗体二聚体在毕赤酵母中高效表达、纯化并鉴定其生物学活性.方法:构建酵母表达载体pGAPZαA-BDM,转化感受态大肠杆菌DH5α,选择测序正确的阳性克隆进行扩增后,电转化酵母细胞,表达抗体二聚体,对表达抗体进行纯化、浓度检测,并鉴定其对肝癌细胞的结合活性,免疫组织化学检测二聚体对肝癌组织抗原的特异性.结果:测序显示成功构建酵母表达载体pGAPZαA-BDM,表达96 h抗体收获量最大,二聚体表达量为30 mg/L菌液,为大肠杆菌的300倍.抗肝癌单链抗体二聚体BDM与三种肝癌细胞结合,而与正常肝细胞不结合,结合效价为1:128.免疫组织化学显示二聚体与肝癌组织结合的阳性率比肝硬化、胃癌、正常肝组织高,差异有统计学意义.结论:成功制备了高表达量、高特异性、较好的活性及稳定性的抗肝癌单链抗体二聚体BDM,为制备免疫纳米颗粒及开展肝癌的放射免疫诊断和靶向治疗奠定了基础.     

^99mTc—MIBI显像预测局部晚期非小细胞肺癌同步放化疗疗效临床研究

目的探讨^99mTc—MIBI显像预测局部晚期非小细胞肺癌同步放化疗疗效的价值。方法42例晚期非小细胞癌患者分为同步放化疗有效组和无效组,于同步放化疗前行^99mTc—MIBI肺显像,并对所摄取比值行半定量分析。采用t检验及秩和检验分析同步放化疗两组早期相肿瘤／正常肺组织摄取比值（ER）和延迟相肿瘤／正常肺摄取比值（DR）、滞留指数（RI）之间的差别。结果^99mTc—MIBI显像结果中,同步放化疗有效组的ER、DR平均值及RI中位值均显著高于同步放化疗无效组（P均〈0．05）。结论^99mTc—MIBI显像在预测局部晚期非小细胞肺癌同步放化疗疗效方面具有重要的临床价值。     

^99mTc-甲氧基异丁基异腈双时相核素显像对甲状腺结节良、恶性鉴别的价值探讨

^99m锝（^99mTc）-甲氧基异丁基异腈（MIBI）作为一种心肌显像剂目前在临床上广泛应用,自1987年Muller首次报道其在分化型甲状腺癌的肺转移灶浓聚后,^99mTc-MIBI显像被广泛用于探查各种肿瘤。但近年来有报道认为其在鉴别甲状腺良、恶性结节方面价值有限。本研究旨在评价^99mTc-MIBI对甲状腺结节的诊断价值。     

核素99mTc标记的红细胞扫描对子宫内膜异位症的诊断价值

对20例子宫内膜异位症患者进行^99mTc标记的红细胞扫描（^99mTc—RBC）、B超检查及血清CA-125检测。结果^99mTc—RBC扫描显示的异位病灶大小明显小于B超显影，且CA-125〉200V／ml者比〈200U／ml者显像更清晰，时间更早。认为^99mTc—RBC扫描在早期诊断子宫内膜异位症方面有一定的临床应用价值。     

099mTc—MIBI硝酸异山梨酯介入显像对冠脉支架置入术后心肌血流灌注的评估

目的：探讨99mTc锝-甲氧基异丁基异腈（99mTc—methoxyisobutyl isonitrile,Tc—MIBI）评估冠状动脉支架置入术（支架置入术）后损伤区心肌细胞血流灌注状况的价值。方法：40例行支架置入术患者,分别在置入术前、后进行99mTc—MIBI心肌静态显像及其硝酸异山梨酯（ISDN）介入显像,对比分析损伤区心肌细胞血流灌注改善程度。结果：40例患者冠状动脉共有360个节段。术前Tc—MIBIISDN介入显像异常10g个节段（29．2％）,显著少于Tc—MIBI静态显像异常的178个节段（49．4％,P〈0．01）。支架置入术后ISDN介入显像异常91个（25．3％）节段也显著少于静态显像异常的112个节段（31．1％,P〈0．05）。结论：99mTc—MIBI—ISDN介入显像较99mTc—MIBI心肌静态显像更能反映支架置入术后心肌血流灌注情况。     

99Tcm标记抗人膀胱癌单抗放射免疫显像

目的探讨99Tcm-BDI-1在荷人膀胱癌裸鼠体内的分布及对肿瘤的导向定位性能。方法用亲和层析法得到单克隆抗体BDI-1,用99Tcm标记BDI-1,并以99Tcm-免疫球蛋白作为对照,进行荷人膀胱癌裸鼠的放射免疫显像。用感兴趣区技术获得全身和肿瘤放射性计数及T/NT。显像24h后处死裸鼠测定体内放射性分布及每克组织,百分注射剂量率(%ID/g)、肿瘤与正常组织的放射性比值(T/NT)。结果99Tcm-BDI-1的标记率为(67.3±7.1)%和99Tcm-球蛋白的标记率为(66.4±6.9)%,放化纯度均>90%。荷人膀胱癌裸鼠放射免疫显像结果显示,静脉注射99Tcm-BDI-1后肿瘤显影清楚,而99Tcm-球蛋白注射后肿瘤无明显显影。全身和肿瘤的放射性计数均随时间的延长而减低,注射99Tcm-BDI-1的裸鼠的T/NT随时间增高,而注射99Tcm-球蛋白的裸鼠的T/NT随时间降低。99Tcm-BDI-1注射24h后肿瘤%ID/g为22.7,除肾脏以外,肿瘤与全身其他正常组织有较高的比值(T/NT>5),而99Tcm-球蛋白注射24h后肿瘤%ID/g为0.33,除肾脏以外,肿瘤与全身其他各正常组织的比值均较小(T/NT<5.12)。结论99Tcm-BDI-1显示了良好的免疫活性和良好的肿瘤导向定位的特性,有望成有膀胱癌早期诊断的有效方法。     

^99mTc—PMT全身显像搜寻肝细胞癌肝外转移灶

16例肝细胞性肝癌伴有肝外转移病灶的患者，用^99mTc－吡哆－5－甲基色氨酸（^99mTc-PMT）作全身显像，以观察肝外病灶的放射性浓聚。其中骨转移9例，肺转移6例，纵隔软组织转移1例。静脉注射555MBq(15mCi)^99mTc-PMT后，先按常规方法作肝为像，并在1，2，或3h后追加全身显像或避开肝胆道加摄局部处。本组16例中8例转移病灶表现为异常放射性集积，病灶分别位于胸骨柄，胸椎，股骨颈，肺及纵隔等相应部位。检测阳性率为50．0％。由于^99mTc-PMT肝显像的组织特异性高，对疑及肝细胞癌肝外转移的患者，可用本方法初筛搜寻病灶。     

99mTc标寡核苷酸用于家兔粥样斑块分子显像研究

目的:建立锝-99m(99m Tc)标记寡核苷酸的方法,并用于家兔动脉粥样硬化斑块分子显像.方法:以高胆固醇饲料喂饲新西兰大耳白兔约2个月,复制成动脉粥样硬化家兔模型.寡核苷酸进行99m Tc标记后注人家兔,进行生物学分布以及体内、体外显像研究.结果:寡核苷酸99m Tc标记率为(77.8±6.8)%(n=7).生物学分布实验表明,与SON相比,c-mycASON在粥样斑块中的浓聚程度明显要高.c-mycASON体内显像可见腹主动脉部位散在性显像剂浓聚,体外主动脉显像提示放射性浓聚灶与斑块所在部位吻合.未偶联组未见阳性显像结果.结论:99m Te标记反义寡核苷酸有望成为一种新的显像剂,在分子水平上用于动脉粥样硬化的早期、特异和无创性的诊断.     

^99mTc-Gd-DTPA-BMA的制备及其生物学分布研究

目的以99m锝（99mTc）标记钆双胺（Gd-DTPA-BMA）制备99mTc-Gd-DTPA-BMA并对其基本生物学分布特征进行研究,旨在为SPECT-MRI双模式显像剂的应用提供依据。方法以氯化亚锡为还原剂,采用一步法以99mTc分别标记Gd-DTPA-BMA、二乙三胺五乙酸（DTPA）,纸层析法测定两标记物标记率、不同pH值及室温下观察稳定性;将99mTc-Gd-DTPA-BMA用生理盐水稀释后注射至小鼠尾静脉,记录注药后1~240 min每克组织百分注射剂量率（%ID/g）;取家兔3只耳缘静脉分别弹丸注射99mTc-Gd-DTPA-BMA和99mTc-DTPA,采用SPECT仪以肾动态显像模式行双时相采集,记录分肾摄取百分比（Uptake%）、达高峰时间（Tmax）、半排时间（T1/2）。结果 99mTc-Gd-DTPA-BMA标记率和稳定性均较高,通过静脉注入小鼠体内后主要分布于肾脏;99mTc-Gd-DTPA-BMA和99mTc-DTPA均经家兔肾脏排泄,其他脏器摄取率较低,但前者Tmax及T1/2显著大于后者（P〈0.05）。结论 99mTc标记Gd-DTPA-BMA具有标记率和稳定性较高等优点,所得99mTc-Gd-DTPA-BMA静脉注入体内后主要分布于肾脏并经肾脏排泄;此为SPECT-MRI双模式显像剂的制备及应用提供了依据。     

抗肝癌单链免疫毒素的构建、表达及导向研究

目的 制备高效、低毒的抗肝癌单链免疫毒素。 方法 将人突变型肿瘤坏死因子α(mTNF α)与人源化抗肝癌单链抗体hscFv25基因连接,构建pGEx4T-1/hscFv25-mTNF α融合蛋白原核表达载体,在大肠杆菌中表达并纯化目的蛋白,经western blot鉴定之后,进行荷肝癌(SMMC-7721)裸鼠体内初步抑瘤实验,并对治疗后裸鼠肿瘤组织进行抗TNF α的免疫组织化学染色。 结果 原核表达载体pGEX4T-1/hscFv25 mTNF α融合蛋白的表达量占细菌总蛋白的12％,hscFv25-mTNFα治疗组对荷肝癌裸鼠的有效率为5/5,其中2/5为完全缓解,3/5为部分缓解,疗效明显高于mTNFα对照组(F=8.70,P<0.05),治疗组裸鼠的肝、肺等组织中未见转移性病灶,经hscFv25-mTNFα治疗后的肿瘤组织,呈TNFα弥漫阳性反应,阳性颗粒主要位于瘤细胞的胞质中。 结论 hscFv25-mTNFα是高效、低毒的抗肝癌单链免疫毒素。     

中药复方“松友饮”联用干扰素α影响姑息性肝切除术后残癌生长转移的实验观察

目的研究中药＂松友饮＂联用干扰素（IFN）α对高转移人肝细胞癌（HCC）裸鼠原位移植瘤姑息性肝切除模型残癌生长、转移和生存期的影响。方法以高转移人肝癌MHCC97H细胞建立裸鼠原位移植瘤姑息性肝切除模型,72只成模裸鼠随机分成对照组、＂松友饮＂组、IFNα组及＂松友饮＂＋IFNα组（每组18只）,姑息性肝切除术后24 h开始每天1次灌胃给药及皮下注射,连续5周测量裸鼠体质量。最后一次处理结束48 h后每组随机处死6只裸鼠,计算肿瘤体积及转移情况;剩余裸鼠用于观察生存期。结果对照组、＂松友饮＂组、IFNα组及＂松友饮＂＋IFNα组肿瘤体积分别为（1205.2±581.3）、（724.9±337.6）、（507.6±367.0）、（245.3±181.2）mm3（F=6.410,P〈0.05）;肺转移结节数（n）分别为13.8±3.5、12.1±3.8、7.4±2.8、4.5±1.9（F=22.930,P〈0.05）;生存期分别为（62.1±2.0）、（76.2±3.2）、（81.1±2.5）、（88.3±2.5）d（Chi-Square=45.704,P〈0.05）。联合用药组同单药组比较,疗效差异有统计学意义。结论连续5周联用＂松友饮＂增强IFNα抑制姑息性肝切除术后残癌生长、转移并延长荷瘤裸鼠生存期,对HCC患者有潜在临床应用价值。     

肝癌细胞不同制悬方法建立裸鼠移植瘤模型的比较

目的比较肝癌细胞不同制悬方法建立裸鼠皮下瘤及原位瘤模型,优选建模方法。方法将肝癌细胞分别采用PBS缓冲液(PBS组)、无血清DMEM溶液(DMEM组)和含10%血清的DMEM溶液(血清DMEM组)3种液体制悬后注入裸鼠皮下建立肝癌裸鼠皮下瘤模型,比较皮下瘤的体积、瘤重及成瘤率;将肝癌细胞分别采用PBS缓冲液(PBS组)、无血清DMEM溶液(DMEM组)和含10%血清的DMEM溶液(血清DMEM组)3种液体制悬后注入裸鼠肝脏建立肝癌裸鼠原位瘤模型,比较原位瘤的成瘤率。结果3组裸鼠皮下瘤模型的皮下瘤体积及瘤重相比,差异无统计学意义(P0.05),但是PBS组及DMEM组的成瘤率(90%和100%)明显高于血清DMEM组(40%),差异有统计学意义(P0.05);裸鼠原位瘤模型中PBS组的成瘤率(90%)明显高于DMEM组(40%)及血清DMEM组(20%),差异有统计学意义(P0.05)。结论采用PBS缓冲液或无血清DMEM溶液对肝癌细胞稀释制悬可以有效建立裸鼠皮下瘤模型,采用PBS缓冲液对肝癌细胞稀释制悬可以有效建立裸鼠原位瘤模型。     

Construction of non-covalent single-chain Fv dimers for hepatocellular carcinoma and their biological functions

AIM: To create new diabodies with improved binding activity to antigen of the variable light - variable heavy (VH-VL) oriented single-chain Fv dimers genes (scFv).METHODS: The linker between VH and VL genes was shortened to 3-5 amino acid residues and cloned into the vector pCANTAB5E. The recombinant plasmids were transformed into TG1 cells and sequenced. The positive transformed cells were infected by M13K07 helper phage to form human recombinant phage antibodies. Expressed products were identified by SDS-PAGE, Western blotting, size exclusion gel chromatography (SEC), ELISA and immunohistochemistry.RESULTS: Three scFv (scFv-3, scFv-4, scFv-5) were constructed successfully with binding ability to hepatocellular carcinoma 3.5-6 fold greater than their parental scFv. The single-chain Fv dimer (scFv-5, termed BDM3) with the best binding ability was successfully expressed in Yeast pichlia, as shown by. SDS-PAGE and Western blotting. SEC results suggested the molecular weight of the expressed products was about 61 kDa. Expressed products showed significantly stronger binding to hepatocellular carcinoma cells than scFv, still having 50% binding activity even after 16 h incubation as 37°C. The purified dimers were bound specifically to the tumor antigen of HCC.CONCLUSION: we have generated scFv dimers by shortening a series of linkers to 3-5 amino acid residues in VH-linker-VL orientation, resulting in highly stable and affinity-improved dimeric molecules. These will become an attractive targeting moiety in immunotherapeutic and diagnostic applications for HCC.     

Direct technetium-99m labeling of anti-hepatoma monoclonal antibody fragment:a radioimmunoconjugate for hepatocellular carcinoma imaging

AIM:To directly radiolabel an anti-hepatoma mAb fragment HAb18 F(ab')(2) with (99m)Tc by stannousreduced method, and assess the stability, biodistribution and radioimmun oimaging (R II).METHODS:Immunoreactive fraction was determined according to Lindmo's method. Ellman's reagent was used to determine the number of thiols in the reduced F(ab') (2). Labeling efficiency and homogeneity were measured by paper chromatography, sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. Challenge assay involved the incubation of aliquots of labeled antibody in ethylenediaminetetraacetate (EDTA) and L-cysteine (L-cys) solutions with different molar ratio at 37° for 1h, respectively. Investigations in vivo utilized nude mice bearing human hepatocellular carcinoma (HHCC) xenografts with gamma camera imaging and tissue biodistribution studies at regular intervals.RESULTS:The labeling procedure was finished within 1.5h compared with the pretinning method which would take at least 21h. In vitro studies demonstrated that the radiolabeled mAb fragment was homogeneous and retained its immunoreactivity. Challenge studies indicated that (99m)Tc-labeled HAb18 F(ab') (2) in EDTA is more stable than in L-cys. Imaging and biodistribution showed a significant tumor uptake at 24h post injection of (99m)Tc-labeled HAb18 F(ab') (2). The blood, kidney, liver and tumor uptakes at 24h were 0.56 ± 0.09, 56.45 ± 11.36,1.43 ± 0.27 and 6.57 ± 3.01 (%ID/g) respectively.CONCLUSION:(99m)Tc-HAb18 F(ab') (2) conjugate prepared by this direct method appears to be an effective way to detect hepatoma in nude mice model.     

肝细胞癌患者PBMCs中T-bet、GATA3和Foxp3 mRNA水平及其意义

目的探讨肝细胞癌患者外周血单个核细胞(PBMC)中T-bet、GATA3和Foxp3 mRNA水平变化及意义。方法在肝细胞癌患者20例和健康对照人群10例,采用RT-PCR法检测PBMC中T-bet、GATA3和FoxP3mRNA水平。结果 HCC患者和健康对照T-bet水平分别为0.554±0.030和0.514±0.071(P=0.391);HCC患者和健康对照GATA3水平分别为0.956±0.030和0.535±0.028(P<0.01);HCC患者和健康对照FoxP3水平分别为0.976±0.073和0.772±0.083(P<0.01);HCC患者和健康对照T-bet/GATA3比值分别为0.697±0.078和0.963±0.133(P<0.01)。结论肝细胞癌患者PBMC中GATA3和FoxP3 mRNA水平上调,可能参与了HCC的发生和发展过程。     

肿瘤相关糖蛋白-72嵌合锚定T细胞对肝癌的抑癌效应

目的制备肿瘤相关糖蛋白-72(TAG-72)嵌合锚定T细胞,体内外实验检测其对肝癌的抑癌活性。方法分离健康人外周血单个核细胞(PBMC),采用脂质体lipofectamineTM2000介导的细胞转染,将已制备的重组真核表达载体抗TAG-72 scFv-CD3ζ-pcDNA3.0导入PBMC,获得肿瘤相关糖蛋白-72(TAG-72)嵌合锚定T细胞,以此为实验组治疗细胞;用SDS-PAGE检测TAG-72嵌合锚定T细胞中抗TAG-72 scFv-CD3ζ嵌合分子的表达,并用Western blotting证实。用转染空载体pcDNA3.0的PBMC作为对照组治疗细胞。将实验组及对照组的治疗细胞分别经尾静脉注射治疗荷TAG-72+肝癌细胞HepG2、荷TAG-72-肝癌细胞BEL7402裸鼠动物模型,观察对裸鼠的抑癌效应。结果 SDS-PAGE检测TAG-72嵌合锚定T细胞嵌合分子抗TAG-72 scFv-CD3ζ的表达,显示大小为47 kDa的融合蛋白片段,与抗TAG-72 scFv-CD3ζ的理论值相符;Western blotting证实该片段CD3呈阳性表达。TAG-72嵌合锚定T细胞与肝癌细胞HepG2共培养后可对后者产生G1期阻滞。用TAG-72嵌合锚定T细胞经尾静脉注射治疗荷瘤裸鼠,发现实验组细胞对荷HepG2细胞裸鼠存活时间、体质量、肿瘤包块大小等方面疗效优于荷BEL7402细胞裸鼠的疗效,两者比较差异有统计学意义(P0.05)。结论 TAG-72嵌合锚定T细胞可特异性抑制荷TAG-72+肝癌细胞裸鼠的肝癌细胞生长;该治疗策略将为肝癌的临床诊治提供一种有希望的途径。     

经沉默免疫负调控基因技术处理的树突状细胞联同细胞毒性T淋巴细胞对HepG2细胞移植瘤的抑制作用

目的观察经沉默免疫负调控基因(iAPA)技术处理的树突状细胞(DC)联同细胞毒性T淋巴细胞(CTL)(iAPA-DC/CTL)对HepG2细胞移植瘤的抑制作用。方法利用人肝癌细胞系HepG2建立裸鼠皮下移植瘤模型,将12只裸鼠随机分为2组:生理盐水对照组(C组)和iAPA-DC/CTL组(DC组),每组6只,行iAPA-DC/CTL治疗4次(1周/次)后处死。实验期间观察各组裸鼠的肿瘤生长,测量肿瘤长短径并描绘肿瘤生长曲线,称量瘤重并计算抑瘤率,病理检测。两组间均数比较采用成组t检验。结果造模成功率为92.31%。C组和DC组肿瘤体积分别为:(697.69±143.99)、(485.64±188.75)mm3,DC组生长相对缓慢(t=2.28,P0.05);C组和DC组肿瘤重量分别为:(0.32±0.07)、(0.22±0.08)g,DC组肿瘤重量小于对照组(t=2.31,P0.05),抑瘤率为30.39%。肿瘤免疫组化染色后T淋巴细胞计数分别为:C组未见、DC组(39.74±5.11)个/高倍视野,DC组肿瘤内T细胞数多于对照组(t=19.05,P0.05)。结论 iAPA-DC/CTL能够有效抑制HepG2细胞裸鼠皮下移植瘤的生长。     

B3(Fv)-PE38KDEL, a single-chain immunotoxin that causes complete regression of a human carcinoma in mice.

The genes encoding the heavy- and light-chain Fv regions of the monoclonal murine antibody B3, which recognizes a carbohydrate antigen on the surface of many human carcinomas, were cloned by PCR techniques and used to generate single-chain immunotoxins containing Pseudomonas exotoxin (PE). The light and heavy chains were connected by a flexible linker to form a single-chain antigen-binding protein, B3(Fv), which was in turn fused to truncated forms of PE lacking the cell-binding domain. The single-chain Fv and two different B3(Fv) immunotoxins, B3(Fv)-PE40 and B3(Fv)-PE38KDEL, were expressed in Escherichia coli and the single-chain immunotoxins were purified to near homogeneity. Both recombinant immunotoxins were shown to be cytotoxic specifically to carcinoma cell lines that express the B3 antigen on their surface; B3(Fv)-PE38KDEL was significantly more active. Furthermore, intravenous administration of B3(Fv)-PE38KDEL caused complete regression of human epidermoid carcinomas growing subcutaneously in immunodeficient mice.     

二硫代氨基甲酸吡咯烷联合苦参碱对人肝癌裸鼠移植瘤生长的抑制作用

目的 探讨二硫代氨基甲酸吡咯烷(PDTC)抑制核因子-κ B(NF-κ B)活化后对苦参碱抑制人肝癌裸鼠移植瘤生长的影响.方法 建立人肝癌细胞HepG2裸鼠皮下移植瘤模型,随机分为对照组(灭菌等渗盐水)、苦参碱组(35 mg/kg)、PDTC组(120 mg/kg)和PDTC(120 mg/kg)+苦参碱(35 mg/kg)联合组,腹腔注射用药.绘制肿瘤生长曲线,测定肿瘤生长抑制率;TdT介导的dUTP缺口末端标记法检测肿瘤细胞凋亡情况;电泳迁移率变动分析法检测细胞核内NF-κB的活化水平;免疫组织化学法检测肿瘤组织bcl-2和bax蛋白表达水平;RT-PCR法检测肿瘤细胞NF-κB、bcl-2和bax的mRNA表达水平.多组间比较用SNK-q检验,单独效应比较采用LSD法,相关分析采用Pearson法进行分析.结果 PDTC增强了苦参碱对肿瘤增殖的抑制作用(P＜0.05);苦参碱在诱导肿瘤细胞凋亡的同时激活NF-κB;PDTC能显著抑制苦参碱诱导的NF-κB活化,NF-κB活性的灰度值由93.64±2.95降至65.78±5.65(F=124.754,P＜0.01),同时促进苦参碱诱导肿瘤细胞凋亡,细胞凋亡指数由55.9%±2.8%升高至74.3%±4.8%(P＜0.05).NF-κB的mRNA表达水平与bcl-2的mRNA表达水平呈正相关(r=0.983,P＜0.01).结论苦参碱诱导皮下移植瘤细胞凋亡的同时激活NF-κB;PDTC可通过抑制NF-κB的活化而下调bcl-2的表达,改变bcl-2与bax的比值,增强苦参碱诱导肿瘤细胞凋亡的作用.

Abstract：

Objective To investigate the relationship between activation of nuclear factor-κ-gene binding (NF-κ B) and apoptosis induced by matrine(MT) in transplanted tumor of human hepatocellular carcinoma in nude mouse. Methods Tumors were established by injection of hepatocellular carcinoma cell line HepG2 into the back of nude mice. The mice were divided randomly into four groups: Control group, MT group (35 mg/kg), PDTC group (120 mg/kg) and Combination group: PDTC+MT group (120 mg/kg+35 mg/kg), the reagents were injected peritoneally. The tumor growth curve of nude mice bearing transplanted tumor were observed and the inhibition ratios were evaluated. Apoptosis of carcinoma cells was analyzed by TUNEL. The DNA-bingding activity of NF-κ B was determined by electrophoretic mobility shift assay (EMSA). Expression of bcl-2 and bax in carcinoma tissue were detected by immunohistochemical method.NF-κ B mRNA, bcl-2 mRNA and bax mRNA in carcinoma tissue were detected by RT-PCR. Results Pyrrolidine dithiocarbamate (PDTC) could enhance the inhibition of matrine on carcinoma proliferation (P＜0.05). The apoptosis and activation of NF-κB in carcinoma cells could be induced by matrine. PDTC significantly suppressed NF-κ B activation induced by matrine in carcinoma cells from 93.64±2.95 to 65.78±5.65 (F=124.754, P＜0.01). Meanwhile, PDTC increased the apoptosis induced by matrine from 55.9%±2.8%to 74.3%±4.8% (P＜0.05).A positive correlation observed between the expressions of NF-κ B and of bcl2 (Pearson correlation coefficient=0.983,P＜0.01). Conclusions Matrine could induce apoptosis and activation of NF-κ B in transplanted tumor. PDTC could increase apoptosis in hepatocellular carcinoma cells might be due to the suppression of NF-κ B activation and the enhancement of bcl-2 expression.