重组p53腺病毒联合中药治疗小鼠腹水瘤的疗效

目的： 〖HT5"SS〗探讨p53腺病毒（简称Adp53）注射液联合中药得力生及消癌平治疗小鼠腹水瘤的协同作用。〖HT5W〗方法：〖HT5"SS〗建立昆明小鼠H22肝癌腹水瘤模型,建模48 h后分组分别给予Adp53、中药得力生和消癌平或两药联用。用药1周后检测体重、腹围、腹水量、腹水瘤细胞计数,流式细胞仪测定瘤细胞凋亡百分率以及细胞周期情况。〖HT5W〗结果：〖HT5"SS〗 Adp53、得力生、消癌平单药治疗组,除体重以外的上述指标与对照组比较均有统计学差异（P＜0.05）,Adp53+得力生和Adp53＋消癌平组的腹围增长率、腹水量、瘤细胞计数、晚期细胞凋亡率均明显少于各单药治疗组（P＜0.05）。 〖HT5W〗结论： 〖HT5"SS〗腹腔注射Adp53、得力生和消癌平均可抑制H22细胞生长并减少腹水形成,Adp53与中药得力生、消癌平联合腹腔内给药对治疗腹水瘤有协同作用。     

腺病毒介导p53基因联合胸腺肽治疗腹腔转移癌的实验研究

 目的 评价p53腺病毒注射液与胸腺肽α1（Tα1）联合腹腔给药对腹膜转移癌模型的协同抑制作用。方法 建立H22腹水瘤的昆明小鼠模型，48 h后以p53腺病毒注射液、Tα1等腹腔注射，治疗周期为1周，治疗后测量腹围、体重，计量腹水，计数瘤细胞数，以流式细胞分析检测瘤细胞凋亡、死亡比例及细胞周期。结果 p53腺病毒注射液腹腔局部给药1周，出现瘤细胞G0／G1期阻滞；其和Tα1联用后，未生成腹水的小鼠增多，生成腹水的小鼠的腹水量和瘤细胞数均较单用时明显减少，死亡细胞数明显增高。结论 p53腺病毒注射液可阻滞腹水瘤细胞增生，联合Tα1腹腔给药，对瘤细胞具有协同杀伤作用，抑制腹膜转移癌的发生。     

消癌平治疗小鼠恶性腹水的实验研究

目的:观察消癌平注射液治疗小鼠肝癌H22细胞腹水瘤效果,并初步探讨其作用机制。方法:建立小鼠H22腹水瘤模型,随机区组分为生理盐水组和消癌平组,每组小鼠各20只。建模后48 h开始给药,消癌平组连续腹腔给药(0.2 mL/d)6 d,生理盐水组给予同体积生理盐水。末次给药24 h后处死各组小鼠半数。计算各组小鼠平均腹水量,腹水抑制率、腹水中瘤细胞数、肿瘤细胞生存率;并检测腹水肿瘤细胞凋亡、细胞周期及Bcl-2蛋白表达。剩余小鼠继续观察一般状态及生存期。结果:和生理盐水组相比,消癌平组小鼠精神好、动作灵敏、反应快;消癌平组平均腹水体积(7.5±1.3)mL低于生理盐水组平均腹水体积(10.3±1.7)mL,P<0.05;消癌平组腹水中肿瘤细胞数(2.0±1.1)×107明显低于生理盐水组腹水中肿瘤细胞数(6.6±2.8)×107,P<0.01;消癌平组腹水肿瘤细胞生存率(34%)明显低于生理盐水组肿瘤细胞生存率(58%),P<0.001;消癌平组小鼠生存期(16.0±1.5)d高于生理盐水组小鼠生存期(14.2±1.1)d,P<0.05。消癌平组小鼠腹水H22细胞凋亡增加,P<0.05;H22细胞G2/M期百分...     

Ad-hTERTp-HSV-TK/GCV系统对肝癌腹水抑制效应的实验研究

目的:探讨由人端粒酶逆转录酶启动子驱动单纯疱疹病毒胸腺激酶基因的重组腺病毒,联合更昔洛韦(GCV)对肝细胞癌腹水生成的影响.方法:将H22细胞注入小鼠腹腔建立肝癌腹水瘤模型,并通过观察各组小鼠体质量、腹围、腹水量、生存时间、脏器及细胞凋亡率等指标的差异.揭示Ad-hTERTp-HSV-TK/GCV系统对肝癌腹水的治疗作用.结果:经过Ad-hTERTp-HSV-TK/GCV治疗的小鼠,显示出明显的腹水抑制作用,其腹水量显著低于各对照组(P<0.01),生存时问延长(P<0.01),细胞凋亡率提高(P<0.01).并且无明显毒副反应.而单独使用Ad-hTERTp-HSV-TK和GCV对腹水生成无明显作用.结论:Ad-hTERTp-HSV-TK/GCV系统对肝癌细胞有较强的靶向杀伤作用,治疗肝癌腹水效果显著,对其临床应用具有理论意义.     

得力生注射液联合健择对肝癌HepG2细胞株增殖与凋亡的影响

目的:探讨得力生注射液(DLS)、健择(GEM)单药及联合作用对人肝癌HepG2细胞株的增殖抑制和诱导凋亡作用。方法:MTT法观察不同浓度得力生注射液、健择单药及联合应用对HepG2细胞株生长的抑制作用;采用流式细胞仪检测上述药物单独或联合应用对HepG2细胞株凋亡率的影响以及得力生注射液对细胞周期的影响。结果:得力生注射液、健择单药及联合作用对HepG2细胞株的抑制作用均呈明显的剂量与时间依赖关系。均可诱导细胞凋亡。联用时有协同作用。得力生注射液主要使HepG2细胞株阻滞于G1期,与对照组比较,实验组细胞凋亡率增高(P<0.05)。结论:得力生注射液、健择能够抑制人肝癌HepG2细胞增殖并诱导凋亡。两药联合有协同作用。     

重组人血管内皮抑制素对肝癌H22腹水瘤小鼠腹水的抑制作用

目的探讨血管内皮抑制素腹腔内给药对H22小鼠腹水瘤生长及腹水的抑制作用。方法取110只昆明小鼠腹腔内接种H22细胞（2x106细胞／只）,建立小鼠腹水瘤模型,接种后随机分为5组：模型对照组（0．9％NS）;低剂量血管内皮抑制素组（4mg／kg）;中剂量血管内皮抑制素组（8mg／kg）;高剂量血管内皮抑制素组（12mg／kg）;阳性对照组（顺铂0．6mg／kg）。接种24h后连续10d给予各药物腹腔内注射,停药24h后处死各组小鼠,测量腹水量并取腹水行相关检测;解剖小鼠,观察腹腔脏器及肺脏的转移情况;通过Evan蓝的吸光度值反映小鼠的腹膜渗透性;观察各组小鼠的生存时间。结果与模型对照组相比,中、高剂量血管内皮抑制素组可显著抑制荷H22腹水瘤小鼠腹水的生成,并减少腹腔内及肺脏转移,延长生存时间。结论血管内皮抑制素腹腔内用药治疗荷H22腹水瘤小鼠腹水具有显著的抑制作用。     

得力生注射液联合TP方案化疗治疗中晚期非小细胞肺癌的临床观察

目的:观察得力生注射液与TP方案化疗联合应用治疗非小细胞肺癌的近期疗效及毒副反应。方法:将60例非小细胞肺癌病人随机分为治疗组和对照组,治疗组用得力生注射液 TP方案治疗,对照组用TP方案治疗。结果:治疗组有效率为60%,对照组有效率为56%,两组对比差异无显著意义(P>0.05);治疗组生活质量增强者占66%,对照组增加者占40%,治疗组较对照组生活质量有明显的提高(P<0.05)。消化道反应治疗组Ⅰ、Ⅱ、Ⅲ度占80%(24/30),对照组占50%(15/30),差异有显著意义(P<0.01)。结论:得力生注射液对非小细胞肺癌有效,对改善临床症状、减轻化疗副反应和提高生活质量方面均有较好作用。     

TP方案联合得力生注射液治疗中晚期非小细胞肺癌62例的临床观察

目的: 观察得力生注射液与TP方案联合应用治疗非小细胞肺癌的近期疗效及不良反应.方法: 将62例非小细胞肺癌病人随机分为治疗组和对照组,治疗组用得力生注射液TP方案治疗,对照组用TP方案治疗.结果: 治疗组有效率为60%,对照组有效率为56%,两组对比差异无显著性(P>0.05);治疗组生活质量提高者占66%,对照组提高者占40%,治疗组较对照组生活质量有明显提高(P<0.05).I、Ⅱ、Ⅲ度消化道反应发生率对照组80%(24/30),治疗组50%(16/32),差异有显著意义(P<0.01).结论: 得力生注射液对非小细胞肺痛有效,在改善临床症状、减轻化疗副反应和提高生活质量方面均有较好疗效.     

得力生注射液联合肝动脉化疗栓塞治疗原发性肝癌的临床研究

目的评价得力生注射液联合经肝动脉化疗栓塞术(transcatheterarterialchemoembolization,TACE)治疗原发性肝癌(hepatocelluarcarcinoma,HCC)患者的临床效果及不良反应。方法选择无手术指征的HCC患者62例,随机分为试验组(得力生联合TACE)32例和对照组(单纯TACE)30例,选用FAP肝动脉化疗方案,试验组加用得力生注射液50ml/d静滴,15d~20d。治疗2个周期后观测患者的近期疗效、细胞免疫功能、生活质量及不良反应。结果试验组有效率为56.3%(18/32),对照组为43.3%(13/30),两组之间差异无统计学意义,(P>0.05)。试验组较对照组生活质量(KPS评分)明显提高,(P<0.05)。试验组细胞免疫功能提高,其中NK细胞升高明显,差异有统计学意义,(P<0.05)。试验组化疗毒副反应减轻,与对照组比较无统计学意义,(P>0.05)。结论得力生注射液联合TACE治疗能提高HCC患生存质量,增强患者的细胞免疫功能。     

得力生腹腔内注射治疗恶性腹水疗效观察

目的:观察得力生注射液腹腔内注射治疗恶性腹水的近期疗效及毒副作用。方法:58例确诊的消化道肿瘤并发癌性腹水患者,随机分为二组:治疗组29例,腹腔内注射得力生;对照组29例,腹腔内注射顺铂,观察患者的近期疗效、生活质量及毒副反应。结果:得力生注射液腹腔内注射有效率为37.9%;而对照组腹腔内注射顺铂的有效率为27.6%,二组比较差异无显著性,但治疗组生活质量(KPS评分)改善者明显高于对照组,且毒副反应发生率明显低于对照组。结论:得力生注射液对恶性腹水有明显的治疗效果,并且具有显著提高患者生存质量的作用。     

Administration of wild-type p53 adenoviral vector synergistically enhances the cytotoxicity of anti-cancer drugs in human lung cancer cells irrespective of the status of p53 gene

Recombinant adenovirus mediated p53 gene transfer combined with anti-cancer drugs has clinical potential for gene therapy of lung cancer. We constructed a recombinant adenoviral vector expressing wild-type p53 cDNA (Ad-p53), and assessed the efficacy of a combined treatment with Ad-p53 and six anti-cancer drugs (cisplatin, 5-fluorouracil, doxorubicin, docetaxel, irinotecan, and etoposide) for human lung cancer cell lines, H1299 (with deleted p53), RERF-LC-OK (with mutant p53), and A549 (with wild-type p53). The infection of the Ad-p53 vector into H1299 cells, RERF-LC-OK cells, or A549 cells increased the sensitivity to all six drugs regardless of the cellular p53 status, and a synergism was observed by the isobolic method in combination studies (D<1). We conclude that our strategy using adenoviral mediated p53 gene transfer to cancer cells can enhance the cytotoxic effect of anti-cancer drugs, which leading to an improvement of lung cancer chemotherapy.     

Late resistance to adenoviral p53-mediated apoptosis caused by decreased expression of Coxsackie-adenovirus receptors in human lung cancer cells

Adenovirus-mediated wild-type p53 gene transfer induces apoptosis in a variety of human cancer cells. Although clinical trials have demonstrated that a replication-deficient recombinant adenovirus expressing the wild-type p53 gene (Ad-p53) is effective in suppressing growth of non-small cell lung cancer (NSCLC), we often experienced late resistance to this treatment. To elucidate the mechanism of late resistance to Ad-p53 in human lung cancer cells, we generated 5 different resistant variants from p53-susceptible H1299 NSCLC cells by repeated infections with Ad-p53. We first examined the transduction efficiency of adenoviral vector by Ad-LacZ transduction followed by X-gal staining in parental and 5 resistant H1299 cell lines. Their sensitivity to viral infection decreased in correlation with the magnitude of resistance, and Ad-p53-mediated tumor suppression could be restored by dose escalation of Ad-p53 in the resistant variants. The expression of Coxsackie and adenovirus receptor (CAR) and alphaV integrins, which are cellular receptors for attachment and internalization of the virus, respectively, was next investigated in these cell lines. Flow cytometry revealed that alphaVbeta3 and alphaVbeta5 integrin expression was consistent, while p53-resistant cell lines showed that diminished CAR expression correlated with the magnitude of the resistance. Our results demonstrated that decreased CAR expression could be one of the mechanisms of late resistance to Ad-p53, which may have a significant impact on the outcome of adenovirus-based cancer gene therapy.     

Phosphorylation of Thr18 and Ser20 of p53 in Ad-p53-induced apoptosis

The p53 protein plays a critical role in inducing cell cycle arrest or apoptosis. Because p53 is inactivated in human gliomas, restoring p53 function is a major focus of glioma therapy. The most clinically tested strategy for replacing p53 has been adenoviral-mediated p53 gene therapy (Ad-p53). In addition to their therapeutic implications, investigations into Ad-p53 provide model systems for understanding p53's ability to induce cell cycle arrest versus apoptosis, particularly because wild-type p53 cells are resistant to Ad-p53-induced apoptosis. Here we use Ad-p53 constructs to test the hypothesis that simultaneous phosphorylation of p53 at threonine 18 (Thr18) and serine 20 (Ser20) is causally associated with p53-mediated apoptosis. Studies using phosphorylation-specific antibodies demonstrated that p53-induced apoptosis correlates with phosphorylation of p53 at Thr18 and Ser20 but not with carboxy-terminal phosphorylation (Ser392). To prove a causal relationship between apoptosis and Thr18 and Ser20 phosphorylation of p53, the effects of an adenoviral p53 construct that was not phosphorylated (Ad-p53) was compared with a Thr18/Ser20 phosphomimetic construct (Ad-p53-18D20D) in wild-type p53 gliomas. Whereas treatment with Ad-p53 resulted only in cell cycle arrest, treatment with Ad-p53-18D20D induced dramatic apoptosis. Microarray and Western blot analyses showed that only Ad-p53-18D20D was capable of inducing expression of apoptosis-inducing proteins. Chromatin immunoprecipitation assays indicated that the protein product of Ad-p53-18D20D, but not Ad-p53, was capable of binding to apoptosis-related genes. We thus conclude that phosphorylation of Thr18 and Ser20 is sufficient for inducing p53-mediated apoptosis in glioma cells. These results have implications for p53 gene therapy and inform other strategies that aim to restore p53 function.     

p73 beta-expressing recombinant adenovirus: a potential anticancer agent

Tumor suppressor p53-based gene therapy strategy is ineffective in certain conditions. p73, a p53 homologue, could be a potential alternative gene therapy agent as it has been found to be an important determinant of chemosensitivity in cancer cells. Previously, we have reported the generation of a replication-deficient adenovirus expressing p73 beta (Ad-p73). In this study, we evaluated the therapeutic potential of Ad-p73 against a panel of cancer cells (n=12) of different tissue origin. Ad-p73 infected all the cell lines tested very efficiently resulting in several-fold increase in p73 beta levels, which is also functional as it activated the known target gene p21(WAF1/CIP1). Infection with Ad-p73 resulted in potent cytotoxicity in all the cell lines tested. The mechanism of p73-induced cytotoxicity in these cell lines is found to be due to a combination of cell cycle arrest and induction of apoptosis. In addition, exogenous overexpression of p73 by Ad-p73 infection increased the chemosensitivity of cancer cells by many fold to commonly used drug adriamycin. Moreover, Ad-p73 is more efficient than Ad-p53 in enhancing the chemosensitivity of mutant p53 harboring cells. Furthermore, Ad-p73 infection did not induce apoptosis in human normal lung fibroblasts (HEL 299) and human immortalized keratinocytes (HaCaT). These results suggest that Ad-p73 is a potent cytotoxic agent specifically against cancer cells and could be developed as a cancer gene therapy agent either alone or in combination with chemotherapeutic agents.     

Ad-p53与Ad-p16联合对人肺腺癌GLC-82细胞作用的体外实验研究 *

探讨腺病毒介导的p53与p16基因的联合对人肺腺病GLC-82疗效提高的可能性。方法：将分别携有p53基因、p16基因重组腺病毒(Ad-p53与Ad-p16)联合使用,通过细胞生长和存活实验、克隆形成实验、流式细胞分析、TUNEL检测、RT-PCR分析、免疫组织化学实验,观察其对人肺腺癌GLC-82细胞的作用。结果：在总感染强度相同的前提下,Ad-p53与Ad-p16联合导入GLC-82细胞,对细胞生长存活及克隆形成能力的抑制、以及所造成的凋亡效果比施用单种基因的效果强,表明p53基因与p16基因在体外疗效上互为协同。结论： Ad-p53与Ad-p16联合,可以提高对人肺腺癌GLC-82细胞的疗效。     

Proapoptotic and antitumor activities of adenovirus-mediated p202 gene transfer.

Purpose and Experimental Design: p202, a mouse IFN-inducible protein, is a member of the 200-amino acid repeat family. Enforced p202 expression in stable cancer cell lines resulted in growth inhibition in vitro and tumor suppression in vivo. However, to study the immediate effect of p202 and test the potential efficacy of p202 treatment, an efficient gene delivery system for p202 is required. For these purposes, an adenoviral vector expressing the p202 gene (Ad-p202) was generated. We examined the effects of Ad-p202 infection on human breast cancer cells. Furthermore, we tested the efficacy of Ad-p202 treatment on breast and pancreatic cancer xenograft models. RESULTS: We found that Ad-p202 infection induces growth inhibition and sensitizes the otherwise resistant cells to tumor necrosis factor alpha-induced apoptosis. In addition, we demonstrated for the first time that Ad-p202 infection induces apoptosis and that activation of caspases is required for the full apoptotic effect. More importantly, we showed the efficacy of Ad-p202 treatment on breast cancer xenograft models, and this antitumor effect correlated well with enhanced apoptosis in Ad-p202-treated tumors. CONCLUSIONS: We conclude that Ad-p202 is a potent growth-inhibitory, proapoptotic, and tumor-suppressing agent. Ad-p202 may be further developed into an efficient therapeutic agent for human cancer gene therapy.     

Ad—p53与Ad—p16联合对人肺腺癌GLC—82细胞作用的研究

目的：探讨腺病毒介导的ｐ５３与ｐ１６基因联合对人肺腺癌ＧＬＣ－８２疗效提高的可能性。方法：将分别携有ｐ５３基因、ｐ１６基因重组腺病毒（Ａｄ－ｐ５３与 Ａｄ－ｐ１６）联合使用,通过细胞生长和存活实验、克隆形成实验、流式细胞分析、ＴＵＮＥＬ检测、ＲＴ－ＰＣＲ分析、免疫组织化学实验,观察其对人肺腺癌ＧＬＣ－８２细胞的作用。结果：在总感染强度相同的前提下,Ａｄ－ｐ５３与Ａｄ－ｐ１６联合导入ＧＬＣ－８２细胞,对细胞生长存活及克隆形成能力的抑制、以及所造成的凋亡效果比施用单种基因的效果强,表明ｐ５３基因与ｐ１６基因在体外疗效上互为协同。结论：Ａｄ－ｐ５３与Ａｄ－ｐ１６联合,可以提高对人肺腺癌ＧＬＣ－８２细胞的疗效。