p27kip1基因转移对食管癌细胞生存素表达和端粒酶活性的影响

背景：p27kip1是新近发现的一种抑癌基因，早期研究表明p27kip1基因转移能显著抑制食管癌细胞和人食管癌裸鼠移植瘤的生长，表明该基因疗法可能是食管癌治疗的新途径，但其抑癌机制尚未完全阐明。目的：研究p27kip1基因转移对食管癌细胞生存素（survivin）表达和端粒酶活性的影响。从而阐明p27kip1的抑癌机制，为p27kip1基因治疗食管癌提供理论依据。方法：将携带p27kip1基因的重组腺病毒（Ad—p27kip1）和LacZ重组腺病毒（Ad—LacZ）分别转染食管癌细胞系Eca9706，观察细胞形态变化。以免疫细胞化学染色和蛋白质印迹法检测p27kip1和生存素的表达．以端粒重复序列扩增程序（TRAP）聚合酶链反应（PCR）-酶联免疫吸附测定（ELISA）检测端粒酶活性。结果：经Ad—p27kip1转染后，Eta9706细胞变圆，呈葡萄串样聚集以致脱落。细胞p27kip1表达明显增强，生存素表达降低，端粒酶活性显著受抑制。结论：p27kip1基因抑制食管癌细胞生长的作用机制可能与下调生存素表达和抑制端粒酶活性有关。     

p27kip1基因转染联合化疗对胃癌细胞生长的影响

背景:p27kip1是一种抑癌基因,早期研究显示p27kip1基因转染能明显抑制胃癌、食管癌细胞生长,表明该基因疗法可能是治疗胃癌、食管癌的新途径,深入阐明p27kip1的抑癌机制,可为p27kip1基因治疗胃癌、食管癌提供可靠的理论基础.目的:研究p27kip1基因转染联合化疗对胃癌细胞生长的影响.方法:将p27kip1重组腺病毒(Adp27kip1)转染胃癌细胞SGC-7901,免疫细胞化学法检测p27kip1的表达.加入5-氟尿嘧啶(5-FU)和顺铂(DDP)后,甲基噻唑基四唑(MTT)法测定细胞生长抑制率,3H-胸腺嘧啶核苷(3H-TdR)掺入试验测定DNA的合成.结果:Adp27kip1转染胃癌细胞后,p27kip1表达明显增强.加入5-FU和DDP后,细胞生长抑制率显著增高,3H-TdR掺入量显著降低(P＜0.01).结论:p27kip1基因转染联合化疗能明显抑制胃癌细胞的生长.     

突变型p27对大肠癌裸鼠模型肿瘤生长及转移的作用

目的研究外源性突变型p27(p27mt)基因对大肠癌体内抑制作用。方法以腺病毒为载体,采用细菌内同源重组方法制备Ad-p27mt,应用细胞移植法把人大肠癌SW480细胞接种于BALB/c裸鼠皮下,构建荷大肠癌裸鼠模型,将已成功构建的Ad-p27mt及Lac-Z采用瘤体内直接注射法注射到瘤体内,绘制肿瘤生长曲线,计算肿瘤生长抑制率,进行细胞凋亡及MMP-9检测。结果Ad-p27mt基因治疗组肿瘤生长明显受到抑制,有更强促凋亡作用,MMP-9表达明显降低,与Lac-Z、对照组比较,有显著性差异。结论Ad-p27mt基因对大肠癌肿瘤生长有明显抑制作用并能减少其转移。     

RNA干扰沉默survivin基因对结直肠癌裸鼠移植瘤的影响

目的利用RNA干扰(RNAi)技术抑制survivin基因的体内表达,探讨survivin沉默对结直肠癌裸鼠移植瘤生长的抑制作用。方法将survivin-siRNA重组腺病毒载体pBAsi-survivin导入裸鼠皮下的移植瘤内,观察不同时间点对肿瘤生长的抑制作用以及肿瘤的抑制率。同时用RT-PCR方法检测干预组(成功转染质粒组),对照组(空载体对照组)和空白对照组(生理盐水)中survivin mRNA表达情况。Western印迹检测survivin蛋白的表达。结果与对照组相比,survivin-siRNA重组载体导入裸鼠皮下移植瘤后,肿瘤体积增长缓慢,随着时间的延长,肿瘤生长抑制作用明显,survivin mRNA表达明显降低,survivin蛋白表达下降。结论 survivin-siRNA重组载体能显著抑制结直肠癌裸鼠移植瘤的增殖,Survivin mRNA和蛋白表达明显降低,不同时间点肿瘤抑制率不同,且随时间呈正相关。     

罗格列酮对人肝癌细胞HepG2裸鼠移植瘤的影响及机制

目的探讨罗格列酮对人肝癌Hep G2细胞裸鼠皮下移植瘤的影响和可能机制。方法建立人肝癌裸鼠皮下移植瘤模型,随机分为罗格列酮组及对照组。观察移植瘤生长情况,HE染色观察移植瘤细胞形态,流式细胞术(FCM)分析细胞周期及凋亡情况,Western印迹法检测移植瘤细胞中第10号染色体缺失的磷酸酶张力蛋白同源物基因(PTEN)、磷酸化蛋白激酶B(p Akt)、S期激酶相关蛋白2(Skp2)及细胞周期蛋白激酶抑制剂P27kip1蛋白的表达情况。结果研究结束时,罗格列酮组移植瘤体积与重量均明显小于对照组,体积抑制率为52.13%,重量抑制率为65.63%。罗格列酮组移植瘤细胞G0/G1期比例及凋亡率均显著高于对照组(P<0.05)。罗格列酮组移植瘤细胞PTEN及P27kip1蛋白的表达量明显高于对照组,p Akt及Skp2蛋白表达量明显低于对照组(P<0.05)。结论罗格列酮对人肝癌Hep G2细胞裸鼠移植瘤的生长有抑制作用,可引发移植瘤细胞G0/G1期阻滞并诱导其凋亡,其机制可能与罗格列酮上调PTEN蛋白的表达,抑制PI3K/Akt信号通路,下调Skp2蛋白,导致P27kip1蛋白水平升高有关。     

p27kip1基因转染联合化疗对胃癌细胞生长的影响

背景：p27kipl是一种抑癌基因，早期研究显示p27kipl基因转染能明显抑制胃癌、食管癌细胞生长．表明该基因疗法可能是治疗胃癌、食管癌的新途径，深入阐明p27kipl的抑癌机制，可为p27kipl基因治疗胃癌、食管癌提供可靠的理论基础。目的：研究p27kipl基因转染联合化疗对胃癌细胞生长的影响。方法：将p27kipl重组腺病毒（Ad-p27kipl）转染胃癌细胞SGC-7901，免疫细胞化学法检测p27kipl的表达。加入5-氟尿嘧啶（5-FU）和顺铂（DDP）后．甲基噻唑基四唑（MrIT）法测定细胞生长抑制率,^3H-胸腺嘧啶核苷（^3H-TdR）掺入试验测定DNA的合成。结果：Ad-p27kipl转染胃癌细胞后，p27kipl表达明显增强。加入5-FU和DDP后，细胞生长抑制率显著增高．^3H-TdR掺人量显著降低（P〈0．01）。结论：p27kipl基因转染联合化疗能明显抑制胃癌细胞的生长。     

重组腺病毒介导的人p27kipl基因高表达对体外培养的大鼠主动脉血管平滑肌细胞迁移的影响

目的探讨重组腺病毒介导的p27kip1基因及其蛋白产物高表达对血管平滑肌细胞(VSMCs)迁移的抑制作用.方法将含人p27kip1cDNA的重组腺病毒(Adhp27kip1)及含β-半乳糖苷酶基因的重组腺病毒(AdLacZ)在体外转染原代大鼠主动脉VSMCs,用Western blot及Boyden趋化小室检测外源性p27kip1蛋白在细胞内的表达及对VSMCS迁移的影响.结果转染后24h,Adhp27kip1转染的VSMCs内p27kip1蛋白高表达,而AdLacZ转染的VSMCs内仅显示极低水平的内源性p27kip1蛋白表达;Boyden趋化小室检测显示未转染的VSMCs、AdLacZ及Adhp27kip1转染的VSMCs血清诱导后的迁移细胞数分别为139±26、106±16及68±14.结论外源性p27kip1基因及蛋白产物在VSMCs内高表达可显著抑制VSMCs的迁移.     

p16INK4α重表达对人食管癌细胞系EC109生长的抑制作用

目的探讨用腺病毒介导野生型的p16基因在食管癌细胞系表达,研究重表达野生型p16基因对食管癌细胞系EC109生长的抑制作用,为寻找食管癌致癌机制的研究及其基因治疗提供理论基础.方法用基因重组技术,将pcDNA3-p16中的野生型p16基因用Kpn I/BamH I进行双酶切,克隆入腺病毒表达载体pAdCMV中,将重组质粒pad-CMV-p16与腺病毒质粒JM17用Lipofectamime 2000共转染293细胞,产生重组腺病毒.用重组腺病毒感染人食管癌细胞系EC109,在感染后的不同时段,用免疫荧光、打点杂交方法检测p16基因在细胞中的表达用MTT法及流式细胞仪观察p16基因的表达对食管癌细胞株生长的影响.结果经酶切鉴定野生型p16基因克隆入腺病毒表达载体中,与JM17共转染293细胞后,可产生具有感染活性的重组腺病毒,用重组腺病毒感染食管癌细胞株EC109细胞后,可抑制食管癌细胞的生长,同对照组相比其最大抑制率可达52.7%.Multipcycle分析软件分析对细胞周期影响表明,处于G0～G1期的细胞为41%～63%感染后的细胞经Dot blot和Westernblot杂交证实,有外源的p16mRNA及蛋白的表达.结论重组腺病毒可将野生型p16基因导入人食管癌细胞系EC109中,野生型p16基因在p16基因功能缺失的细胞中重表达能抑制癌细胞恶性生长.p16基因功能缺失是食管癌致癌因素之一     

真核翻译延长因子1A2对人胰腺癌裸鼠移植瘤生长和血管形成作用的研究

人类真核翻译延伸因子1A2（EEF1A2）是一种管家基因，可能作为一种新的潜在癌基因。参与肿瘤的发生、发展。目的：探讨EEFIA2对人胰腺癌裸鼠移植瘤生长和血管形成的作用。方法：以聚合酶链反应（PCR）扩增目的基因片段，应用基因重组技术构建Ad5／F35。EEF1A2重组腺病毒载体。15只裸鼠建立人胰腺癌移植瘤模型．随机分为对照组、绿色荧光蛋白（GFP）组和EEF1A2组，分别注射PBS0．1ml、Ad5／F35-GFP0．1ml（1×10^8PFU）和Ad5／F35-EEF1A20．1ml（1×10^8 PFU）。测定各组肿瘤体积和质量，应用免疫组化方法检测各组增殖细胞核抗原（PCNA）和CD31的表达。结果：成功构建了表达EEF1A2的重组腺病毒载体。与对照组和GFP组相比，EEF1A2组裸鼠肿瘤体积和质量显著增加（P〈O．05），PCNA和CD31表达显著增高（P〈O．05）。结论：EEF1A2可显著促进人胰腺癌裸鼠移植瘤生长，细胞增殖和肿瘤血管形成可能是其重要作用机制。     

p16-INK4alpha重表达对人食管癌细胞系EC109生长的抑制作用

目的探讨用腺病毒介导野生型的p16基因在食管癌细胞系表达,研究重表达野生型p16基因对食管癌细胞系EC109生长的抑制作用,为寻找食管癌致癌机制的研究及其基因治疗提供理论基础.方法用基因重组技术,将pcDNA3-p16中的野生型p16基因用Kpn I/BamH I进行双酶切,克隆入腺病毒表达载体pAdCMV中,将重组质粒pad-CMV-p16与腺病毒质粒JM17用Lipofectamime 2000共转染293细胞,产生重组腺病毒.用重组腺病毒感染人食管癌细胞系EC109,在感染后的不同时段,用免疫荧光、打点杂交方法检测p16基因在细胞中的表达用MTT法及流式细胞仪观察p16基因的表达对食管癌细胞株生长的影响.结果经酶切鉴定野生型p16基因克隆入腺病毒表达载体中,与JM17共转染293细胞后,可产生具有感染活性的重组腺病毒,用重组腺病毒感染食管癌细胞株EC109细胞后,可抑制食管癌细胞的生长,同对照组相比其最大抑制率可达52.7%.Multipcycle分析软件分析对细胞周期影响表明,处于G0～G1期的细胞为41%～63%感染后的细胞经Dot blot和Westernblot杂交证实,有外源的p16mRNA及蛋白的表达.结论重组腺病毒可将野生型p16基因导入人食管癌细胞系EC109中,野生型p16基因在p16基因功能缺失的细胞中重表达能抑制癌细胞恶性生长.p16基因功能缺失是食管癌致癌因素之一     

Adenovirus expressing p27kip1 suppresses growth of established esophageal carcinoma xenograffs

AIM: To investigate the growth suppression of adenovirus expressing p27kip1 on established esophageal tumors in nude mice.METHODS: Esophageal carcinoma xenografts in nude mice were established by tumor tissue mass transplantation. The successfully constructed recombinant adenoviral vectors carrying p27kip1 gene (Adp27kip1) were directly injected into the esophageal tumors in nude mice. Compared to control group, the growth curve of tumor was drawn and the growth inhibition rate of tumor was calculated. The histology of tumors was examined by hematoxylin and eosin (H&E) staining. The expression of p27kip1 and survivin was detected in tumors by immunohistochemical technique.RESULTS: The growth of tumors in gene therapy group with Ad-p27kip1 was obviously suppressed compared to control group (0.42±0.08 g vs 1.17±0.30 g, t=6.39,P＜0.01), the inhibition rate of tumor growth reached 64.1%. Pathological detection showed that the tumors in nude mice were poorly differentiated esophageal squamous carcinoma. In addition, the expression of p27kip1 was increased, while the expression of survivin was decreased in tumors after being transfected with Ad-p27kip1.CONCLUSION: p27kip1 gene therapy mediated by adenovirus vector has a significant inhibitory effect on esophageal carcinoma in vivo. Up-regulated p27kip1expression and down-regulated survivin expression may be its important mechanisms.     

Adenovirus expressing p27kip1 suppresses growth of established esophageal carcinoma xenografts

AIM: To investigate the growth suppression of ade novirus expressing p27kip1 on established esophageal tumors in nude mice. METHODS: Esophageal carcinoma xenografts in nude mice were established by tumor tissue mass transplantation. The successfully constructed reco mbinant adenoviral vectors carrying p27kip1 gene (Ad p27kip1) were directly injected into the esophageal tumors in nude mice. Compared to control group, the growth curve of tumor was drawn and the growth inhibition rate of tumor was calculated. The histology of tumors was examined by hematoxylin and eosin (H&E) staining. The expression of p27kip1 and survivin was detected in tumors by immunohistochemical technique. RESULTS: The growth of tumors in gene therapy group with Ad-p27kip1 was obviously suppressed compared to control group (0.42±0.08 g vs 1.17±0.30 g, t=6.39, P<0.01), the inhibition rate of tumor growth reached 64.1%. Pathological detection showed that the tumors in nude mice were poorly differentiated esophageal squamous carcinoma. In addition, the expression of P27kip1 was increased, while the expression of survivin was decreased in tumors after being transfected with Ad-p27kip1. CONCLUSION: p27kip1 gene therapy mediated by adenovirus vector has a significant inhibitory effect on esophageal carcinoma in vivo. Up-regulated p27kip1 expression and down-regulated survivin expression may be its important mechanisms.     

Inhibition of adenovirus-mediated p27kip1 gene on growth of esophageal carcinoma cell strain

AIM: To investigate the inhibition of p27kip1 gene on the growth of esophageal carcinoma cell strain (EC9706). METHODS: Recombinant adenovirus Ad-p27kip1 was constructed and transfected into esophageal carcinoma cell EC-9706, and its effect on p27kip1 expression, the growth of esophageal carcinoma cell, DNA replication, protein synthesis, cell multiplication and apoptosis were explored by means of cell growth count, 3H-TdR, 3H-Leucine incorporation, flow cytometry, DNA fragment analysis and TUNEL. RESULTS: Recombinant adenovirus Ad-p27kip1 was successfully constructed with a virus titer of 1.24 X 10(12) pfu/ml. p27kip protein expression increased markedly after EC-9706 transfection, while incorporation quantity of 3H-TdR and 3H-Leucine decreased significantly. The growth of esophageal carcinoma cell was inhibited obviously. Testing of flow cytometry displayed a typical apoptosis peak, and DNA gel electrophoresis showed a typical apoptosis ladder. TUNEL showed the apoptosis rate of Ad-p27kip1 group and control group to be 37.3% and 1.26% (P<0.001) respectively. CONCLUSION: Ad-p27kip1 can inhibit the growth and multiplication of esophageal carcinoma cells and induce apoptosis. Therefore, enhanced p27kip1 expression may be a new way to treat esophageal carcinoma.     

Molecular therapy with recombinant antisense c-myc adenovirus for human gastric carcinoma cells in vitro and in vivo

BACKGROUND AND AIMS: This study used a recombinant antisense c-myc adenovirus (Ad-ASc-myc) to evaluate how alterations of c-myc expression in the SGC7901 human gastric carcinoma cells could influence the proliferation, apoptosis and the growth of human gastric tumors in nude mice. METHODS: The human gastric carcinoma cell line, SGC7901, treated with Ad-ASc-myc or adenovirus recombinants carrying LacZ gene (Ad-LacZ) were analyzed by using X-gal stain, MTT, DNA ladder, TUNEL assay, flow cytometric analysis, polymerase chain reaction and western blot in vitro. The tumorigenicity and experimental therapy in nude mice models were assessed in vivo. RESULTS: The Ad-ASc-myc could strongly inhibit cell growth and induce apoptosis in SGC7901 cells. The proliferation of the Ad-ASc-myc-infected SGC7901 cells was reduced by 44.1%. The mechanism of killing gastric carcinoma cells by Ad-ASc-myc was found to be apoptosis, which was detected by the use of a DNA ladder, TUNEL and flow cytometric analysis. Infection of Ad-ASc-myc in nude mice showed that all three mice failed to form tumors from the 7 to 30 day period, compared with injection of Ad-LacZ and parent SGC7901 cells. Experimental therapy on the nude mice bearing subcutaneous tumors of SGC7901 cells showed that intratumor instillation of Ad-ASc-myc inhibited the growth of the tumors. Recombinant antisense c-myc adenovirus-treated tumors were inhibited by 68.9%, compared with tumors injected with Ad-LacZ and control (LacZ and phosphate-buffered saline). CONCLUSION: The expression of Ad-ASc-myc can inhibit growth and induce apoptosis of gastric cancer cells in vitro and in vivo and thus is a potential clinical utility in gene therapy for the treatment of gastric carcinoma.     

转染kiss-1基因对人食管癌EC9706细胞裸鼠移植瘤的抑制作用

目的:研究转染kiss-1基因对人食管癌EC9706细胞裸鼠皮下移植瘤的作用,探讨其在食管癌基因治疗中的可行性和特异性.方法:在食管癌细胞系EC9706中转染kiss-1基因,经G418筛选,建立稳定高表达Kiss-1蛋白的细胞系.稳定表达该基因的细胞为转染kiss-1基因组,转染空质粒细胞及未处理细胞为对照组,建立裸鼠荷瘤模型;监测肿瘤生长变化,HE染色观察肿瘤病理学变化,RT-PCR、Western blot方法检测kiaa-1 mRNA和蛋白变化.结果:转染kiss-1基因组肿瘤生长受到显著抑制:HE染色显示转染kiss-1基因组及转染空质粒纽肿瘤组织内坏死均较空白对照组多;RT-PCR、Western blot结果表明转染kiss-1基因组裸鼠肿瘤组织kiss-1 mRNA和蛋白表达均显著升高,三组间比较差异具有统计学意义(F=72.685,24.807,均P<0.05).结论:转染kiss-1基因能抑制人食管癌EC9706细胞裸鼠皮下移植瘤的形成,且能有效上调kiss-1 mRNA和蛋白的表达,可为食管癌的基因治疗提供新的靶点、开辟新的思路.     

Effect of mutant p27kip1 gene on human cholangiocarcinoma cell line, QBC939

AIM:To investigate the effects of exogenously mutated p27kip1 (p27) on proliferation and apoptosis of human cholangiocarcinoma cell line,QBC939 in vivo.METHODS:Adenviral vectors were used to transfect mutated p27 cDNA into human QBC939 cell line.Expression of p27 was detected by RT-PCR.Western blot.Cell growth,morphological change,cell cycle,apoptosis and cloning formation were determined by MTT assay and flow cytometry.RESULTS:The expression of p27 protein and mRNA was increased significantly in QBC939 cell line transfected with Ad-p27mt.The transfer of Adp27mt could significantly inhibit the growth of QBC939cells,decrease the cloning formation rate and induce apoptosis,p27 over expression caused cell cycle arrest at G0/G1 phase 72 h after infection with Adp27mt.CONCLUSION:p27 may cause cell cycle arrest at G0/G1 phase and subsequently lead to apoptosis.Recombinant adenovirus expressing mutant p27 may be potentially useful in gene therapy for cholangiocarcinoma.     

重组腺病毒介导的人p27kip1基因高表达对体外培养的大鼠主动脉血管平滑肌细胞迁移的影响

目的　探讨重组腺病毒介导的p2 7kip1基因及其蛋白产物高表达对血管平滑肌细胞 (VSMCs)迁移的抑制作用。方法　将含人p2 7kip1cDNA的重组腺病毒 (Adhp2 7kip1)及含 β 半乳糖苷酶基因的重组腺病毒 (AdLacZ)在体外转染原代大鼠主动脉VSMCs,用Westernblot及Boyden趋化小室检测外源性p2 7kip1蛋白在细胞内的表达及对VSM Cs迁移的影响。结果　转染后 2 4h ,Adhp2 7kip1转染的VSMCs内p2 7kip1蛋白高表达 ,而AdLacZ转染的VSMCs内仅显示极低水平的内源性p2 7kip1蛋白表达 ;Boyden趋化小室检测显示未转染的VSMCs、AdLacZ及Adhp2 7kip1转染的VSMCs血清诱导后的迁移细胞数分别为 139± 2 6、10 6± 16及 6 8± 14。结论　外源性p2 7kip1基因及蛋白产物在VSMCs内高表达可显著抑制VSMCs的迁移     

腺病毒介导的p27kip1基因对胃癌细胞周期和DNA合成的影响

背景：近年来胃癌基因治疗的研究取得了一定的进展，但总体疗效尚不尽人意，目前正积极寻求组织特异性基因作为胃癌基因治疗的突破点。目的：以腺病毒为载体，研究p27kipl基因对胃癌细胞周期和DNA合成的影响。方法：将成功构建的携带人p27kipl基因的重组腺病毒载体Ad-p27kipl和LacZ重组腺病毒Ad-LacZ转染胃癌细胞系SGC-7901，并观察细胞形态的变化，流式细胞仪检测细胞周期和凋亡，^3H-胸腺嘧啶核苷(TdR)掺入实验测定细胞DNA合成。结果：Ad-p27kip1转染SGC-7901细胞后，细胞变圆、呈葡萄串样聚集以致脱落，G0／G1期细胞比例增加，8期、G2／M期细胞比例降低，并有凋亡发生，^3H-TdR掺入量亦显著降低。结论：腺病毒介导的p27kipl基因能使SGC-7901细胞产生G0／G1期阻滞，并能诱导细胞凋亡，抑制DNA合成，表明该基因疗法能有效抑制体外胃癌细胞的生长。