siRNA沉默survivin基因表达对恶性胶质瘤细胞放射敏感性的影响

目的探讨针对人survivin基因的siRNA（small interfering RNA）对恶性胶质瘤细胞放疗敏感性的影响。方法设计并合成针对survivin基因的siRNA片断,构建干涉质粒载体pSil4.1-shsurvivin1和pSil4.1-shsurvivin2（pSil4.1-shcontrol作为阴性对照）。利用脂质体辅助转染恶性胶质瘤细胞（U251）,以G418筛选稳定表达干涉载体的细胞系,RT-PCR和W estern blot试验检测survivin的m RNA和蛋白表达,并筛选survivin基因显著抑制的稳定转染细胞系（U251-s2）。通过克隆形成试验和皮下移植瘤试验分别在体外和体内检测恶性胶质瘤细胞放射敏感性的变化。结果相对于未转染和阴性对照组细胞,U251-s2细胞中survivin mRNA和蛋白表达分别显著下调了40.5%和51.8%;同时,survivin基因表达下调能够显著增强恶性胶质瘤细胞的体内外放射敏感性。结论Survivin基因过表达和恶性胶质瘤的放疗抗性密切相关,抑制其表达可以显著增强恶性胶质瘤细胞的放疗敏感性,具有潜在的临床应用前景。     

Survivin siRNA对卵巢癌细胞侵袭的影响

目的：探讨survivin靶向小分子干扰RNA（siRNA）对卵巢癌细胞侵袭的影响。方法：在survivin基因的编码区内根据干涉位点的设计原则,体外转录合成survivin siRNA,转染人卵巢上皮性癌细胞株SKOV3细胞,用Westernblot方法鉴定干涉位点对survivin基因蛋白的干扰效果;细胞侵袭实验（transwell）检测细胞体外侵袭能力的变化。结果：survivin siRNA能有效抑制survivin蛋白的表达,siRNA组survivin蛋白的相对表达水平分别与空白对照组、非特异性组相比,差异均有统计学意义（P〈0.05）。特异性干涉组细胞的侵袭能力明显低于非特异性干涉组及空白对照组,差异均具有统计学意义（P〈0.001）。结论：特异性的survivin siRNA能有效降低卵巢癌细胞survivin的表达,抑制细胞侵袭能力,这为卵巢癌的基因治疗提供了一种新策略。     

survivin特异性RNAi表达载体的构建及其干扰效果鉴定

目的构建survivin基因的RNAi真核表达载体并观察其对转染腺样囊性癌细胞株ACC-2后survivin表达变化以及对细胞凋亡的影响。方法设计、合成siRNA（small interfering RNA）,构建表达载体pGenesibshRNA-survivin,将重组质粒导入人腺样囊性癌ACC-2细胞株。采用逆转录-聚合酶链反应（RT-PCR）法检测转染后的ACC-2细胞中survivinmRNA表达的变化,用TUNEL检测细胞凋亡的变化。结果经测序模版序列和设计序列完全正确,在pGenesil—shRNA-survivin转染ACC-2细胞株中,survivinmRNA表达明显降低,转染的ACC-2细胞凋亡显著增加。结论成功构建了survivin基因的RNAi真核表达载体,且其对腺样囊性癌细胞有一定的干扰效果。     

表皮生长因子受体通路基因干扰与放线菌素D联合对胶质瘤生长抑制作用的研究

目的探讨靶向性表皮生长因子受体（EGFR）信号转导通路关键基因丝氨酸苏氨酸激酶（AKT）小分子干扰RNA构建体与化疗药放线菌素D（ACTD）联合对胶质瘤生长的抑制作用。方法选择AKT的siRNA靶序列构建靶向AKT的siRNA表达质粒,并以空载siRNA表达质粒为对照进行脂质体介导的小分子干扰siRNA表达质粒单独及分别与化疗药物ACTD对胶质瘤细胞C6系生长抑制作用的研究。TUNEL法分析细胞凋亡;流式细胞术分析细胞周期变化,^3H-TdR掺人法分析细胞增殖。结果与c6细胞和转染空载体的C6细胞比较,转染siRNA表达质粒组AKT表达下调72％。TUNEL法发现C6组和空载转染组几乎没有凋亡细胞,而siRNA表达质粒组的凋亡率明显增加（Χ^2=35．26,P〈0．01）;流式细胞分析发现siRNA表达载体转染后S期指数较对照细胞减少,而加入化疗药物ACTD后S期指数较对照细胞明显减少^3H-TdR掺人法发现与C6组和空载转染组比较,转染组细胞自第2天起存活率均明显下降（P〈0．01）,且加入ACTD后治疗各组之间存活率也有明显差异（均P〈0．01）;以siRNA表达载体转染组联合ACTD组减低最显著。结论靶向AKT的siRNA表达质粒联合化疗药ACTD显著抑制肿瘤细胞增殖并诱导细胞凋亡。因此,质粒介导的siRNA表达载体为胶质瘤靶向性基因治疗与化疗药物联合可以成为胶质瘤治疗的一种新策略。     

端粒酶催化亚基启动子调控caspase-3基因表达对瘤细胞抑制作用的研究

通过构建端粒酶催化亚基（hTERT）启动子调控caspase3基因的真核表达载体,采用RT-PCR、短时转染、MTT、流式细胞和动物实验等研究方法,探讨hTERT启动子调控caspase3基因在端粒酶阳性的瘤细胞中表达的特异性及在体内外的抑瘤作用。结果表明,hTERT启动子调控caspase3基因在端粒酶阳性的瘤细胞HepG2中明显表达,而在正常人胚肺成纤维细胞（human embryonic lung fibroblast,HEL）中未见表达;caspase3的表达能明显对端粒酶阳性的瘤细胞HepG2、HeLa、Glc和A549的增殖产生明显的抑制作用,抑制率为10.5%～37.9%;同时也明显诱导HepG2、HeLa、Glc和A549细胞发生凋亡,凋亡率为15.39%～35.19%;而对端粒酶阴性的正常细胞HEL没有明显的影响。动物实验结果显示,caspase3基因转染对HepG2细胞的体内生长产生了明显的抑制作用。     

siRNA沉默CD147基因对肝癌细胞的影响

目的 观察小分子干扰RNA(siRNA)沉默CD147基因在肝癌细胞HepG2中的表达,探讨CD147基因对肝癌细胞生长、凋亡的影响.方法 根据CD147 cDNA序列设计具有短发夹结构的两条DNA序列,与载体pSileneerTM4.1-CMV neo构建重组表达载体,鉴定后转染至HepG2细胞,Western blotting法检测抑制效果,MTT法检测细胞增殖情况,流式细胞术检测肿瘤细胞凋亡情况.结果 成功构建了针对CD147基因表达的干扰质粒,有效抑制了HepG2细胞增殖,促进了HepG2细胞凋亡.结论 CD147靶向RNA干扰重组表达栽体为肝癌的基因治疗提供了可能.     

Survivin基因沉默对裸鼠人卵巢癌细胞移植瘤凋亡的影响

目的探讨survivin基因沉默对人卵巢癌细胞株SKOV3裸鼠移植瘤凋亡的影响。方法将重组survivin shRNA plasmid转染人卵巢癌细胞株SKOV3细胞,通过裸鼠移植瘤实验比较survivin基因沉默对裸鼠移植瘤的生长抑制作用,荧光定量聚合酶链反应(PCR)和蛋白印迹法检测与凋亡密切相关的cytochrome-c、caspase-9和caspase-3基因的表达。结果裸鼠移植瘤实验显示,与对照组(空载体组和未转染组)相比,survivin shRNA plasmid转染组人卵巢癌细胞株SKOV3细胞生长速度减慢,肿瘤体积缩小(P<0.05);荧光定量PCR和蛋白印迹法结果都显示,与对照组相比,survivin shRNA plasmid转染组cytochrome-c、caspase-9和caspase-3的表达明显升高(P<0.05)。结论 survivin基因沉默可通过促进肿瘤细胞的凋亡而抑制裸鼠移植瘤生长。     

RNAi对大肠癌SW620细胞survivin基因的影响

目的：探讨RNAi（RNA interference）对survivin在大肠癌细胞SW620表达的影响及siRNA干扰后诱导肿瘤survivin表达降低的机制。方法：构建survivin特异性的RNA封闭性载体survivinl通过培养大肠癌细胞SW620，survivin封闭性载体转染SW620细胞；Hoechest染色大肠癌细胞，通过荧光显微镜观察细胞形态学情况；流式细胞仪分析肿瘤细胞的凋亡情况。结果：DNA测序证实表达质粒构建成功，转染后的大肠癌细胞核明显可见凋亡小体；流式细胞仪分析大肠癌细胞系SW620的凋亡率survivinl为10．03％。结论：RNAi能够下调survivin，并促进大肠癌细胞系SW620的细胞凋亡。     

靶向Survivin基因siRNA诱导肝癌细胞凋亡的实验

目的探讨靶向survivin的siRNA转染肝癌细胞阻抑survivin表达,及其对肝癌细胞凋亡、增殖与细胞化疗敏感性的影响。方法设计合成特异性靶向survivin的siRNA序列。肝癌细胞株HepG2接种于6孔培养板内,分为5组:空白对照组、阴性对照组和低、中、高浓度转染组(分别给予50,100和200nmol.L-1siRNA转染)。作用36h后收集各组细胞。Westernblot法检测各组细胞survivin蛋白表达情况,逆转录-聚合酶链反应(RT-PCR)检测survivinmRNA表达水平,流式细胞术检测各组细胞增殖和凋亡指数,四氮唑盐(MTT)法检测氟尿嘧啶(5-FU)和顺铂(DDP)对各组细胞的生长抑制率。结果各浓度siRNA转染组细胞survivin蛋白和mRNA表达有不同程度减弱。各浓度siRNA转染组细胞凋亡指数明显高于对照组(P<0.05),高浓度转染组最明显(P<0.05)。各浓度siRNA转染组细胞增殖指数明显低于各对照组(P<0.05),高浓度转染组最明显(P<0.05)。等浓度化疗药物5-FU和DDP对各浓度siRNA转染组细胞的抑制率明显高于对照组(P<0.05),高浓度转染组最明显(P<0.05)。结论不同浓度survivinsiRNA转染能下调survivin蛋白和mRNA表达,诱导肝癌细胞凋亡,抑制细胞增殖,增加肝癌细胞对化疗药物的敏感性。     

Bcl-XL siRNA对肺腺癌细胞A549/DDP药物敏感性及凋亡的影响

目的：探讨小干扰RNA沉默Bcl-XL对肺腺癌耐药细胞株A549／DDP药物敏感性的影响及其凋亡机制。方法：将Bcl-XL siRNA质粒载体转染至A549／DDP细胞，MTT、流式细胞仪检测细胞药物敏感性的改变；丫啶橙／溴化乙锭（AO／EB）染色法检测细胞凋亡；RT-PCR检测Bcl-XL mRNA水平；Western-Blot检测Bcl—XL、caspase-3蛋白表达的变化。结果：分别用0．2、2、20、200μg／mL顺铂处理细胞48h，MTT显示转染Bcl-XL siRNA细胞组A570吸光度值较阴性siRNA细胞组和未转染对照组明显降低，细胞生长抑制率明显增高；用20μg/mL顺铂处理细胞48h，流式细胞仪检测，转染Bcl-XL siRNA组凋亡率较转染阴性siRNA组和未转染组增加；AO／EB染色显示转染Bcl-XL siRNA的细胞较转染阴性siRNA及未转染者凋亡率增加；转染Bcl-XL siRNA降低了Bcl-XL mRNA和蛋白水平，升高了caspase-3活性形式蛋白表达。结论：Bcl-XL siRNA特异性下调A549／DDP细胞Bcl-XL的表达，活化caspase-3蛋白，促进A549／DDP细胞凋亡，增强该肺腺瘤细胞对顺铂的敏感性。     

Survivin-siRNA在体外诱导Panc-1细胞凋亡的实验研究

目的:探讨RNA干扰技术抑制Survivin对人胰癌细胞株Panc-1细胞凋亡的影响。方法:体外设计、合成针对sur-vivin基因的小分子干扰RNA(survivin-siRNA),应用脂质体介导survivin-siRNA转染Panc-1细胞后,MTT试验测定转染细胞的增殖抑制率,RT-PCR检测细胞survivin mRNA表达,琼脂糖凝胶电泳分析其对Panc-1细胞凋亡诱导作用。结果:转染survivin-siR-NA的Panc-1细胞增殖明显受抑制,与对照进行比较,具有显著性差异(P<0.01);经琼脂糖凝胶电泳,转染survivin-siRNA的Panc-1细胞可见到DNA梯形条带,而对照细胞未见到。结论:survivin-siRNA能够显著诱导Panc-1细胞凋亡。     

Inhibitory effect of siRNA targeting survivin in gastric cancer MGC-803 cells

Gastric cancer is the fourth most common cancer in the world and the leading cause of cancer death in China. It is necessary to find a safe and effective way to treat this disease. A sustained overexpression of survivin is a characteristic feature of gastric cancer as it gives cancer cells a survival and growth advantage. RNA interference (RNAi), which has been proven to be a powerful tool for suppressing gene expression, may provide a promising way forward in gastric cancer therapy. Few studies have been conducted on the inhibitory effects of small interfering RNA (siRNA) against survivin in gastric cancer. In this study, we constructed the recombinant Psilencer 2.1-survivin siRNA plasmids and transfected them into gastric cancer MGC-803 cells in vitro, and also injected MGC-803/Silence (+) cells into nude mice to observe the inhibitory effects in vivo. The transfection of Psilencer 2.1-survivin (+) plasmid led to remarkable inhibition of survivin mRNA expression, the intensity of survivin mRNA was lower (P<0.05), the expression of survivin protein was strongly suppressed, the amount of survivin protein was also lower (P<0.05) and the percentage of apoptotic cells was much higher (P<0.05). The tumor size and growth ratio were lower in nude mice injected with MGC-803/Silence (+) cells and the inhibition ratio of survivin expression was higher (P<0.05). In summary, siRNA targeting of survivin can effectively inhibit the growth of gastric cancer cells and may be used as a potent therapy.     

腺病毒介导的FasL基因诱导肺癌细胞凋亡

目的研究外源性FasL基因对人肺癌细胞凋亡的影响以及FasL基因治疗肺癌的可能性.方法构建腺病毒载体介导FasL基因转移到低水平表达FasL的人肺腺癌细胞系A549,逆转录聚合酶链反应(RT-PCR)、流式细胞术(FCM)检测FasL基因的表达,并观察A549肺癌细胞凋亡和生长,对肿瘤生长模型的治疗效果进行分析.结果外源性FasL基因在A549肺癌细胞获得高水平的表达,FasL能显著抑制A549的生长和集落形成,流式细胞仪检测显示细胞G1期阻滞并发生了凋亡.瘤内注射Ad-FasL对裸鼠体内移植瘤有较强的抑瘤作用.结论FasL基因参与了肺癌细胞凋亡的诱导,能抑制肺腺癌细胞的生长,因此该基因在肿瘤的基因治疗上有广泛的应用前景.     

Bcl-2 siRNA induced apoptosis and increased sensitivity to 5-fluorouracil and HCPT in HepG2 cells

To investigate the changes in drug sensitivity of Bcl-2 siRNA transfected HepG2 cells. Bcl-2 siRNA and negative siRNA expression vector were constructed and stably transfected into HepG2 cells. RT-PCR and Immunofluorescence were used to detect the target gene expression. Western Blotting was used to detect Bcl-2, Bax and caspase-3 protein expressiom. Drug sensitivity of the cells to 5-fluorouracil (5-FU) and 10-hydroxycamptothecin (HCPT) were analyzed with MTT and flow cytometry. Results were following: (1) the mRNA and protein expression level of Bcl-2 in Bcl-2 siRNA stable transfectants were reduced compared with negative siRNA transfected or untreated cells. Accordingly, Bax protein expression had no change and caspase-3 protein expression showed significantly be up regulated; (2) MTT results showed that Bcl-2 siRNA transfectants had higher cell inhibitory rates after treated with 5-FU or HCPT; (3) flow cytometry results demonstrated that sub G1 population increased in Bcl-2 siRNA transfected cells compared with negative siRNA or untreated cells. After addition 5-FU (1300 mg/l) and HCPT (0.72 mg/l), Bcl-2 siRNA cells showed higher sub G1 population than negative siRNA or untreated cells. siRNA targeting Bcl-2 gene can specifically down-regulate Bcl-2 expression, increased Bax/Bcl-2 ratio expression and caspase-3 activity in HepG2 cells, which lead to increase cells spontaneous apoptosis and sensitize cells to 5-FU or HCPT. Bcl-2 siRNA may be a potential therapy agent against human hepatoblastoma.     

Bcl-2 siRNA induced apoptosis and increased sensitivity to 5-fluorouracil and HCPT in HepG2 cells

To investigate the changes in drug sensitivity of Bcl-2 siRNA transfected HepG2 cells. Bcl-2 siRNA and negative siRNA expression vector were constructed and stably transfected into HepG2 cells. RT-PCR and Immunofluorescence were used to detect the target gene expression. Western Blotting was used to detect Bcl-2, Bax and caspase-3 protein expressiom. Drug sensitivity of the cells to 5-fluorouracil (5-FU) and 10-hydroxycamptothecin (HCPT) were analyzed with MTT and flow cytometry. Results were following: (1) the mRNA and protein expression level of Bcl-2 in Bcl-2 siRNA stable transfectants were reduced compared with negative siRNA transfected or untreated cells. Accordingly, Bax protein expression had no change and caspase-3 protein expression showed significantly be up regulated; (2) MTT results showed that Bcl-2 siRNA transfectants had higher cell inhibitory rates after treated with 5-FU or HCPT; (3) flow cytometry results demonstrated that sub G1 population increased in Bcl-2 siRNA transfected cells compared with negative siRNA or untreated cells. After addition 5-FU (1300 mg/l) and HCPT (0.72 mg/l), Bcl-2 siRNA cells showed higher sub G1 population than negative siRNA or untreated cells. siRNA targeting Bcl-2 gene can specifically down-regulate Bcl-2 expression, increased Bax/Bcl-2 ratio expression and caspase-3 activity in HepG2 cells, which lead to increase cells spontaneous apoptosis and sensitize cells to 5-FU or HCPT. Bcl-2 siRNA may be a potential therapy agent against human hepatoblastoma.     

The effect of survivin on multidrug resistance mediated by P-glycoprotein in MCF-7 and its adriamycin resistant cells

Although anticancer chemotherapeutic drugs have been designed to inhibit the growth of tumor cells, chemotherapy frequently fails due to the development of multidrug resistance (MDR). In this paper, the effect of survivin on multidrug resistance mediated by P-glycoprotein (Pgp) was investigated in breast cancer cells. Overexpression of survivin in MCF-7 cells transfected with survivin expression vector pEGFP/survivin results in decreasing sensitivity to anticancer drugs and activation of Pgp to export drug out of cells. Down regulation of survivin in MCF-7/adriamycin (ADR) transfected with RNAi directed against survivin vector psh1/survivin could increase the drug accumulation in cells by inhibiting Pgp. Downregulation of the expression of the Pgp with the specific inhibitor verapamil could markedly suppress the survivin mRNA expression, whereas the reverse impact was not observed. Survivin might modulate the turnover of Pgp or transport by Pgp in cells, which result in anti-apoptosis and drug resistance. Our results suggest that survivin might play a key role in MDR in the presence of Pgp, and this might represent a novel strategy for modulating MDR in cancer cells.     

hIAP-2-siRNA 对hepG2 肝癌细胞抑制作用

目的合成凋亡抑制蛋白（hIAP-2）的si-RNA,观察其对hepG2细胞增殖、凋亡的影响。方法化学合成三条hIAP-2-siRNA干扰片段并用脂质体法转染hepG2细胞,同时设立脂质体对照。RT-PCR和western blotting检测hIAP-2-siRNA作用后hIAP-2mRNA和蛋白的表达,MTT检测细胞增殖,AV-PI流式细胞术检测细胞的凋亡。结果 RT-PCR及Western blootting检测显示3条siRNA均能有效抑制hIAP-2mRNA及蛋白表达（P〈0.05）,以siRNA1作用效果最显著（P〈0.05）。MTT检测显示hIAP-2-siRNA80nmoL·L-1终浓度转染后的hepG2细胞的增殖受到显著抑制（P〈0.01）,其中以80nmoL·L-1转染72h时增殖性下降最明显（P〈0.01）。流式细胞术检测结果显示hIAP-2-siRNA转染hepG2细胞后凋亡率增加（P〈0.05）。结论 hIAP-2-siRNA可明显抑制该肿瘤细胞的增殖,促进hepG2肝癌细胞的凋亡。