新城疫病毒HN基因重组体对人胃癌细胞BGC-823凋亡作用机制研究

目的本实验研究旨在探讨含新城疫病毒HN基因的重组质粒对人胃癌细胞BGC-823凋亡的联合作用机制。方法将含有HN基因的重组质粒pIRVP3IL-18HN经脂质体介导转染人胃癌细胞BGC-823，通过MTT方法检测细胞活力；电镜下观察细胞形态；罗丹明123和DCFA染色测定线粒体跨膜电位（△ψ）和活性氧（ROS）水平变化；底物染色反应检测Caspase-3活性。结果重组质粒pIRVP3IL-18HN转染人胃癌细胞BGC-823后，MTT法结果显示致肿瘤细胞死亡率升高，细胞活力下降；电镜观察肿瘤细胞呈现明显凋亡状态；与阴性空白质粒对照显示，线粒体△ψ下降，ROS水平升高；Caspase-3活性被激活。结论含新城疫病毒HN基因的重组质粒可以诱导人胃癌细胞BGC-823进入特定的凋亡程序，从而导致人胃癌细胞BGC-823的凋亡。     

重组人血管内皮抑素联合顺铂体外干预人胃癌细胞生长的研究

目的研究重组人血管内皮抑制素（恩度）联合顺铂对人胃癌细胞株BGC823和SGC7901增殖及凋亡的影响,并探讨其抑制增殖及诱导凋亡的机制。方法采用四甲基偶氮唑盐（MTT）法检测不同浓度顺铂单药（单药组）及其与恩度联合应用（联合组）对BGC823和SGC7901细胞的增殖抑制率;采用流式细胞术分析凋亡细胞比例;采用Westernblot法分析增殖相关蛋白Ki-67及凋亡相关蛋白Bcl-2表达的变化。结果联合组对BGC823和SGC7901细胞的抑制率明显高于单药组;流式细胞术结果表明,正常对照组、单药组和联合组细胞的早期凋亡率依次增加;与单药组比较,联合组Ki-67、Bcl-2基因（蛋白）表达率显著降低。结论与顺铂单独应用相比,恩度联合顺铂对BGC823和SGC7901细胞增殖的抑制作用和诱导凋亡能力均明显增强。     

腺病毒介导p53基因对人胃癌细胞热增敏的作用

目的 评价腺病毒介导p53基因对人胃癌细胞热增敏的作用。方法 以重组腺病毒介导p53基因悬液（Adp53）感染4种不同p53状况的人胃癌细胞，用免疫组化法和Western blot法检测p53蛋白在胃癌细胞中的表达；用经胞存活分数来反映经胞增殖状况；用TUNEL法来检测细胞凋亡，感染Adp53的W和M胃癌细胞经42℃2h或43℃0.5h加温后24h，用流式细胞计检测细胞周期分布和凋亡；胃癌细胞的种植肿瘤内注射Adp53悬液后48h，行43℃0.5h加温，以肿瘤相对体积增长曲线观察肿瘤抑制情况。结果 高效靶比（100MOI）Adp53产生细胞高转染率和p53基因在4种胃癌细胞中均高表达，并产生G2/M期阻滞和凋亡及细胞增殖抑制，Adp53基因的作用不依赖胃癌细胞内在的p53状态。如果以凋亡作为热效应，Adp53对2种加温方式的热增敏比，对W细胞为1.6-3.3，而对M细胞为1.8-2.1,Adp53对W细胞肿瘤43℃0.5h加温的热增敏比为1.7，而对M细胞肿瘤为1.6。结论 腺病毒介导p53基因提高了人胃癌细胞的热敏感性，这种作用不依赖于细胞内在p53状况，本实验为p53基因治疗与热疗结合提供了可靠的实验依据。     

重组人p53腺病毒联合苦参碱对肺癌A549细胞生长抑制作用及其机制探讨

目的观察重组人p53腺病毒(rAd-p53)联合苦参碱对肺癌A549细胞生长的协同抑制作用,探讨协同抑制作用的机制。方法通过MTT及流式细胞技术观察rAd-p53和苦参碱单药与联合用药对肺癌A549细胞生长抑制率及凋亡率,用Western blot检测p53、bcl-2、bax蛋白的表达情况。结果 (1)rAd-p53、苦参碱对肺癌A549细胞均具有生长抑制作用,生长抑制率呈现时间和剂量依赖性(P<0.05)。(2)rAd-p53联合苦参碱对肺癌A549细胞生长有协同抑制作用,与单药相比,抑制率及细胞凋亡率明显增强(P<0.05);不同的联合加药顺序对细胞生长抑制没有明显差异(P>0.05)。(3)单药rAd-p53组、联合用药组的肿瘤细胞有明显的p53蛋白表达,bax蛋白在联合用药组中表达量最高,bcl-2联合用药组中表达量最低。结论重组人p53腺病毒联合苦参碱对肺癌A549细胞生长具有协同抑制作用,能抑制细胞增殖和促进细胞凋亡,且呈现时间和剂量依赖性;协同抑制作用可能通过上调促凋亡蛋白bax的表达,下调抑凋亡蛋白bcl-2的表达实现的。     

米非司酮增加宫颈癌Caski细胞对顺铂敏感性的研究

目的：探讨米非司酮作用于人宫颈鳞癌Caski细胞后对顺铂敏感性的影响和机制。为临床应用米非司酮治疗宫颈鳞癌提供实验依据。方法：体外培养人宫颈鳞癌Caski细胞，分别或联合应用不同浓度的米非司酮、顺铂处理Caski细胞，采用四甲基偶氮唑蓝比色法测定米非司酮对Caski细胞增殖活性的作用及其对顺铂敏感性的影响；流式细胞术观察各组细胞凋亡率，并分析细胞周期的变化；异硫氰酸荧光素（FITC）荧光标记流式细胞术（FCM）法测定米非司酮对Caski细胞HPV—E6，p53，Bcl-2，Bax蛋白的表达变化。结果：四甲基偶氮唑蓝比色法结果显示，1．25、2．5mg／L的米非司酮对Caski细胞无显著的抑制作用，其与顺铂合用时能增强顺铂对Caski细胞增殖抑制作用。流式细胞术结果显示，米非司酮（1．25mg／L）对Caski细胞无明显的诱导凋亡作用，但能促进顺铂（1．0、2．0、4．0mg／L）诱导其凋亡。FITC荧光标记FCM法结果显示，米非司酮作用于Caski细胞后，HPV—E6、Bcl-2蛋白表达下调，p53、Bax蛋白表达上调，呈浓度依赖方式。结论：米非司酮能增强顺铂对Caski细胞的增殖抑制和诱导凋亡并对Caski细胞有增殖抑制和化疗增敏作用，与下调HPV16-E6、Bcl-2蛋白表达，上调p53、Bax蛋白表达有关。     

阿霉素联合顺铂对人胃癌组织增殖和凋亡影响作用的研究

目的研究阿霉素联合顺铂对人胃癌组织增殖和凋亡的影响作用,初步探讨阿霉素联合顺铂治疗胃癌的可能机制。方法常规培养人胃癌细胞株SGC-7907,用不同浓度的阿霉素、顺铂分别单独及联合用药作用于细胞。采用MTT法检测细胞增殖活力;流式细胞术检测细胞周期分布、凋亡的变化;Western blot检测相关蛋白的表达。结果阿霉素和顺铂能抑制SGC-7907细胞的增殖,两种药物联合应用时抑制率远远高于单药组(P0.01)。流式细胞测定仪显示单用阿霉素、单用顺铂以及联合用药在24 h内对SGC-7907细胞细胞凋亡率分别为(11.89±1.25)%、(19.51±1.33)%、(30.02±2.49)%,联合用药组明显高于单独用药组(P0.01)。Western blot检测显示,联合用药组Bax增加和Bcl-2减小更为明显,caspase-3蛋白的表达显著增强,与单药组相比差异显著(P0.01)。结论阿霉素与顺铂联合用药效果明显,可抑制胃癌细胞增殖,促进胃癌细胞凋亡,其作用可能与激活Bax、抑制Bcl-2和活化caspase-3有关。     

组蛋白去乙酰化酶抑制剂MS-275对卵巢癌SKOV3细胞增殖凋亡的影响

目的探讨MS-275对卵巢癌SKOV3细胞增殖凋亡的影响及其作用机制。方法 MTT法检测MS-275和顺铂单药及联合用药对SKOV3细胞增殖的影响,并计算半抑制浓度IC50;流式细胞术检测MS-275和顺铂单药及联合用药对SKOV3细胞凋亡的影响;Western-Blot法检测各组BCL2、survivin及p21蛋白的表达。结果 MS-275和顺铂两单药均可抑制SKOV3的增殖,各浓度组具有统计学差异(P0.05),联合用药时可显著降低顺铂半抑制浓度(P0.05);同时两单药组可促进SKOV3细胞凋亡(P0.05),联合用药时凋亡作用更显著(P0.05);MS-275和顺铂可显著抑制SKOV3细胞BCL-2和survivin蛋白的表达,增强p21的表达(P0.05)。结论 MS-275抗SKOV3细胞增殖,促进其凋亡及增强顺铂敏感性的机制可能与抑制HDAC活性,抑制BCL-2和survivin蛋白表达,促进p21蛋白表达有关。     

重组人P53腺病毒对逆转非小细胞肺癌细胞株A549/顺铂耐药的研究

目的 探讨重组人P53腺病毒(Ad-P53)对已产生顺铂耐药的人肺腺癌细胞的耐药逆转作用.方法 将重组腺病毒载体所携带的野生型p53基因导入人肺腺癌耐药细胞株A549/顺铂,并联合应用化疗药物顺铂,通过Western blot法分析外源野生型p53基因在细胞内的表达,MTT法观察其对细胞生长的影响.结果 Western blot法证实外源p53基因能在A549/顺铂细胞中高效表达.MIT法观察到Ad-P53对肺癌细胞的抑制作用呈时间依赖性和剂量依赖性效应(P均<0.05).100MOI Ad-PS3与0.5 mg/L顺铂联合应用后72 h,对A549/顺铂细胞的生长抑制率达(41.53±0.59)%,显著高于单用Ad-P53组[(15.69±0.96)%]和顺铂组[(7.40±1.13)%,P<0.001].结论 Ad-P53腺病毒可以逆转肺腺癌细胞对顺铂的耐药性.     

姜黄素联合顺铂对人前列腺癌PC-3细胞株的影响

目的观察姜黄素联合顺铂(DDP)对人前列腺癌PC-3细胞株抗肿瘤活性作用的影响,并探讨其作用机制。方法采用MTT法检测姜黄素单独或联合顺铂对人前列腺癌PC-3细胞株的抑制率;采用流式细胞术检测不同浓度姜黄素和顺铂单药、联合处理后对人前列腺癌PC-3细胞株凋亡率变化;RT-PCR检测姜黄素单独或联合顺铂对人前列腺癌PC-3细胞株作用后P53的mRNA蛋白表达。结果不同浓度的姜黄素组随着药物浓度增加、作用时间延长,细胞抑制率上升,差异有统计学意义(P<0.05);联合用药组明显高于单一用药组(P<0.05),两药联合具有协同作用;姜黄素和顺铂均促进人前列腺癌PC-3细胞株凋亡,联合用药可显著增加凋亡率;RT-PCR结果显示联合用药组能使P53 mRNA蛋白表达水平较单药组上升。结论姜黄素对人前列腺癌PC-3细胞株具有生长抑制作用,且具有剂量和时间依赖性,其与顺铂联合具有协同抑制作用,其协同作用可能与上调P53 mRNA表达有关。     

LHRH-PE40诱导分化胃癌细胞凋亡的研究

目的研究NF-κB mRNA和Caspase-3mRNA及其蛋白在诱导分化BGC-823细胞中的表达,探讨LHRH-PE40与胃癌细胞凋亡的关系。方法应用RT-PCR、Western blot法检测BGC-823细胞中NF-κB P65mR-NA、Caspase-3mRNA表达及其蛋白的表达情况。结果 LHRH-PE40诱导BGC-823细胞凋亡时,NF-κB mRNA和Caspase-3mRNA表达水平有所增加,NF-κB P65和Caspase-3P20活性片段也增加。结论 LHRH-PE40可以诱导胃癌细胞内NF-κB的活化;引起Caspase-3级联反应,促进细胞凋亡。     

野生型p53基因转染对人胃癌细胞的作用

目的评价外源性p53基因对人胃癌细胞生物学行为的影响。方法以复制缺陷型重组腺病毒为载体,将人野生型p53基因导入不同p53状态的3种人胃癌细胞系,用免疫组化法检测p53基因在胃癌细胞中的表达;用流式细胞计数、细胞DNA片段化分析检测细胞周期分布和凋亡。结果在高MOI病毒剂量下,外源p53基因在3种胃癌细胞的胞核中均高效表达,并可使细胞产生明显的G2／M阻滞和凋亡,而且这种作用不依赖细胞内在的p53基因状态。结论无论体外还是体内实验,野生型p53基因转染胃癌细胞后均有明显的生物效应。该实验为进一步开展p53基因治疗的临床研究提供了理论依据。     

Adenovirus-mediated transfer of p53 and p16(INK4a) results in pancreatic cancer regression in vitro and in vivo

Pancreatic cancer has a very poor prognosis. Current chemotherapy and radiotherapy regimens are only moderately successful. The tumour suppressor genes p53 and p16(INK4a)encode cell cycle regulatory proteins that are important candidates for gene replacement therapy. Over 80% of pancreatic adenocarcinoma cases lack detectable p16 protein while over 60% contain mutated p53 protein. We used replication-deficient recombinant adenoviruses to reintroduce wild-type p16 and p53 into pancreatic cancer cells in vitro and into subcutaneous pancreatic tumours in an animal model to determine the effect on tumour growth. Significant growth inhibition was observed in all five human pancreatic cell lines with these viruses (P < 0.002) compared with similar control viruses expressing either luciferase or beta-galactosidase. G1 arrest was observed in all cell lines 72 h after infection with Adp16. Infection with Adp53 caused significant levels of apoptosis (P < 0.004). Apoptosis was also observed to a lesser degree (P < 0.03) with the Adp16 vector. Subcutaneous pancreatic tumours, generated in nu-nu mice demonstrated significant growth suppression following injection of Adp53, Adp16 and a combination of both Adp53 and Adp16 (P < 0.0001). These results show that transfer of wild-type p53 and p16 produces significant growth suppression of pancreatic cancer in vitro and in vivo.     

RNA干扰敲除Smac基因的表达促进人肺癌细胞的生长及增强顺铂耐药性

目的 探讨RNA干扰敲除Smac基因的表达对肺癌细胞的生长及顺铂(cDDP)耐药性的影响.方法 通过转染含有以人Smac基因为靶基因的慢病毒载体系统,即含有小分子干扰RNA序列的pGC-FU载体,分别在A549和95D细胞中实施Smac基因敲除.通过转染含有Smac全长编码序列的pGC-FU载体来实现Smac的高表达.细胞生长、细胞周期及凋亡采用四甲基偶氮唑盐(MTT)法、克隆形成实验及流式细胞仪测定.药物耐药用10 μg/ml顺铂检测.结果 Smac下调表达促进A549和95D细胞中肺癌细胞的生长及增强顺铂耐药性;Smac高表达抑制A549细胞生长并且增强其对顺铂的敏感性.结论 Smac抑制肺癌细胞生长并且增强其对顺铂的敏感性.     

Tumor suppression and therapy sensitization of localized and metastatic breast cancer by adenovirus p53

We have examined the effects of a replication-defective adenovirus encoding p53 (RPR/INGN 201 [Ad5CMV-p53]; Adp53), alone or in combination with the breast cancer therapeutic doxorubicin (Adriamycin), to suppress growth and induce apoptosis in breast cancer cells in vitro. We have also examined the in vivo effect of intratumoral administration of Adp53, alone or in combination with doxorubicin, to suppress the growth of established subcutaneous MDA-MB-435 breast cancer tumors. Finally, using the MDA-MB-435 orthotopic model of metastatic breast cancer, we have examined the effect of systemic administration of Adp53, alone or in combination with doxorubicin, to reduce the incidence of metastases. We find that whereas in vitro treatment of cells with Adp53 reduces [(3)H]thymidine incorporation by about 90% at 48 hr, cell viability at 6 days is reduced by only some 50% relative to controls. Although apoptosis is detectable in Adp53-treated cultures, these results suggest that a large fraction of Adp53-treated cells merely undergo reversible cell cycle arrest. Combined treatment with Adp53 and doxorubicin results in a greater than additive loss of viability in vitro and increased apoptosis. In vivo, locally administered Adp53 suppresses growth of established subcutaneous tumors in nude mice and suppression is enhanced by doxorubicin. In the metastatic breast cancer model, systemic administration of Adp53 plus doxorubicin leads to a significant reduction in the incidence of metastases relative to Adp53 or doxorubicin alone. Taken together, these data indicate an additive to synergistic effect of Adp53 and doxorubicin for the treatment of primary and metastatic breast cancer.     

Histone deacetylase inhibitor FK228 enhances adenovirus-mediated p53 family gene therapy in cancer models

Therapeutic replacement of the wild-type p53 gene has been pursued as a potential gene therapy strategy in a variety of cancer types; however, some cancer models are resistant to p53 in vivo and in vitro. Therefore, to improve p53 gene therapy, it is important to overcome the resistance to p53-mediated apoptosis. Histone deacetylase inhibitors are a novel class of chemotherapeutic agents that are able to reverse the malignant phenotype of transformed cells. A natural histone deacetylase inhibitor, FK228, is reported to enhance adenovirus infection due in part to the up-regulation of coxsackievirus adenovirus receptor expression. In this study, preclinical experiments were done to establish a mechanistic rationale for the combination of adenovirus-mediated p53 family gene transfer and FK228 pretreatment in future clinical trials. Pretreatment with FK228 enhanced apoptosis in human cancer cells through enhanced transduction of Ad-p53. FK228 also induced hyperacetylation of the p53 protein and specifically enhanced p53-mediated Noxa expression. Additionally, the combination of FK228 and Ad-p53 induced Bax translocation to the mitochondria. The double knockdown of Bax and Noxa expression by small interfering RNA antagonized the synergistic effect of Ad-p53 and FK228 on apoptosis induction. In human cancer xenograft models, FK228 significantly increased the therapeutic effectiveness of p53 as well as p63 gene therapy. These results provide a strong rationale for combining p53 gene therapy and FK228 pretreatment in cancer therapy.     

全反式维A酸诱导肺鳞癌细胞凋亡与Rb基因表达的关系

目的研究全反式维A酸(ATRA)诱导肺鳞癌细胞株A2凋亡作用并初步探讨其作用机制,寻找肺癌治疗的新途径。方法体外培养A2细胞,随机分为两组,实验组加ATRA使其终浓度为5μmol/L,对照组加入二甲亚砜使其终浓度为0.1%,继续培养48 h后用流式细胞仪技术分别检测Rb、p53基因表达率,同时用DNA凋亡分析法检测肿瘤细胞凋亡发生率,研究三者之间的相关关系。结果A2细胞实验组中细胞凋亡发生率显著增高(P<0.01),Rb和p53基因表达率显著增强(P<0.01)。A2细胞中凋亡发生率与Rb基因表达率之间正相关(P<0.05),与p53基因表达率之间亦呈正相关关系(P<0.05)。结论ATRA可能通过上调Rb和p53基因表达途径使A2细胞阻滞在G0/G1期,进而诱导肺癌细胞A2凋亡。     

腺病毒介导的NDRG2基因对前列腺癌细胞株DU145的影响

目的:探讨腺病毒介导的N-myc下游调节基因2(NDRG2)基因对前列腺癌细胞株DU145增殖抑制及诱导其凋亡的作用.方法:以携带人NDRG2基因的腺病毒载体转染体外培养的前列腺癌细胞株DU145.采用westernblot检测目的基因的表达,通过细胞生长实验、平板克隆实验检测NDRG2对DU145细胞增殖能力的影响.流式细胞仪检测转染前后细胞凋亡的情况:光镜观察细胞形态学的改变.结果:DU145细胞经腺病毒转染后westernblot检测有NDRG2蛋白(40 kD)特异表达.MTT比色及平板克隆实验结果显示NDRG2对DU145细胞生长有明显抑制作用(P<0.05).流式细胞检测结果显示Ad-NDRG2组凋亡率明显高于对照组及Ad-LacZ组,差异有显著性(P<0.05).与对照组及Ad-LacZ组比较,光镜下观察可见Ad-NDRG2转染的细胞生长状态明显变差,细胞变圆,边缘模糊.结论:通过腺病毒载体使细胞表达NDRG2基因,可以明显抑制DU145细胞的生长和增殖,并可诱导细胞凋亡.     

Late expression of p53 from a replicating adenovirus improves tumor cell killing and is more tumor cell specific than expression of the adenoviral death protein

Gene transfer of p53 induces cell death in most cancer cells, and replication-defective adenoviral vectors expressing p53 are being evaluated in clinical trials. However, low transduction efficiency limits the efficacy of replication-defective vector systems for cancer therapy. The use of replication-competent vectors for gene delivery may have several advantages, holding the potential to multiply and spread the therapeutic agent after infection of only a few cells. However, expression of a transgene may adversely affect viral replication. We have constructed a replicating adenoviral vector (Adp53rc) that expresses high levels of p53 at a late time point in the viral life cycle and also contains a deletion of the adenoviral death protein (ADP). Adp53rc-infected cancer cells demonstrated high levels of p53 expression in parallel with the late expression pattern of the adenoviral fiber protein. p53 expression late in the viral life cycle did not impair effective virus propagation. Survival of several lung cancer cell lines was significantly diminished after infection with Adp53rc, compared with an identical p53-negative control virus. p53 expression also improved virus release and spread. Interestingly, p53 was more cytotoxic than the ADP in cancer cells but less cytotoxic than the ADP in normal cells. In conclusion, late expression of p53 from a replicating virus improves tumor cell killing and viral spread without impairing viral replication. In addition, in combination with a deletion of the ADP, specificity of tumor cell killing is improved.