Survivin基因RNA干涉对HeLa宫颈癌细胞凋亡及顺铂敏感性的影响

背景与目的:Survivin基因是凋亡抑制因子家族的新成员,它在人类多种恶性肿瘤,包括宫颈癌中均高表达,其表达下调可提高恶性肿瘤的化疗敏感性。本研究观察了survivin基因RNA干涉对宫颈癌细胞HeLa凋亡及顺铂敏感性的影响。方法:通过脂质体介导将含人survivin基因siRNA的重组真核表达质粒pS ilencer2.1-s2转染宫颈癌细胞系HeLa,G418筛选阳性克隆,半定量PCR、W estern b lot分别检测survivin mRNA和蛋白表达,激酶活性检测法测定半胱氨蛋白水解酶-3(caspase-3)活性变化,流式细胞仪、Hoechst染色观察细胞凋亡情况,四甲基偶氮唑蓝(MTT)比色法检测细胞存活率并计算顺铂的50%抑制浓度(IC50)。结果:G418筛选24天后出现阳性克隆,与转染阴性对照质粒(HeLa-NC)、空载质粒(HeLa-U6 neo)及未转染(HeLa)细胞比较,转染pS ilencer2.1-s2质粒者survivin mRNA和蛋白表达明显下降,表达抑制率分别为:62.8%和60.1%;caspase-3激酶活性增强,A405达1.26±0.04;细胞凋亡率明显升高,为(29.2±1.4)%(P<0.05);在同一药物浓度下,细胞存活率明显下降,顺铂的IC50值降低为0.87±0.02ug/m l(P<0.05)。结论:Survivin基因RNA干涉可通过阻抑HeLa细胞中survivin表达,增强caspase-3激酶活性,诱导细胞凋亡并显著提高宫颈癌细胞对顺铂的敏感性。     

survivin shRNA重组腺病毒的构建及其对人肺癌细胞A549的作用

 目的 构建携带人生存素survivin短发夹RNA（short hairpin RNA,shRNA）表达载体的重组腺病毒,以此介导小干扰RNA（small interfering RNA,siRNA）药物对人A549细胞肺癌的治疗,为下一步动物实验研究提供基础。 方法 将前期实验中构建并筛选的重组质粒pShutte-survivin与携带LacZ标签的腺病毒骨架质粒共转染HEK-293 T细胞,经同源重组产生重组腺病毒Ad-survivin;经PCR鉴定并测定病毒滴度;β-gal染色检测病毒对人肺癌细胞A549的感染效率;MTT实验和流式细胞术检测其对A549细胞的作用;半定量RT-PCR检测RNAi效果;Hoechst染色观察凋亡情况。 结果 PCR证实重组腺病毒Ad-survivin构建成功;β-gal染色表明感染效率明显;A549细胞增殖受到抑制并阻滞于G₂/M期;survivin mRNA的表达受到抑制;荧光染色发现有凋亡细胞。 结论 成功构建人survivin基因shRNA 表达载体的重组腺病毒,下调survivin表达后能诱导A549 细胞凋亡,为研究肺癌RNAi 途径的基因治疗奠定基础。     

慢病毒介导的USP9X基因RNA干扰对人胰腺癌细胞株裸鼠移植瘤生长的影响

目的研究慢病毒介导的连锁的泛素特异肽酶9(USP9X)基因RNA干扰对人胰腺癌细胞系裸鼠皮下移植瘤的影响。方法将靶向USP9X基因的干扰小RNA(siRNA)慢病毒(LV-siRNA-USP9X)和阴性对照慢病毒(LV-siRNA-NC)转染至SW1990细胞,并分别作为siUSP9X组和siNC组,未转染细胞作为对照组,分别将3组细胞系接种至裸鼠皮下,比较3组裸鼠接种第30天的移植瘤生长情况和瘤体重量。蛋白质印记(Western blot)法和免疫组织化染色检测3组裸鼠皮下移植瘤中survivin蛋白的表达情况,TUNEL法检测细胞凋亡指数(AI)。结果siUSP9X组USP9X蛋白的相对表达量均明显低于siNC组和对照组,差异均有统计学意义(P<0.01),干扰效率约为90%。siUSP9X组裸鼠的肿瘤体积明显小于siNC组和对照组裸鼠,且随着接种时间的延长,差异越来越明显。在接种第30天时,siUSP9X组裸鼠瘤体重量均小于siNC组和对照组裸鼠,抑瘤率为52%。siUSP9X组survivin蛋白的相对表达量和阳性表达率均低于siNC组和对照组,AI均高于siNC组和对照组,差异均有统计学意义(P<0.05)。结论慢病毒介导的USP9X基因沉默可明显抑制人胰腺癌细胞系裸鼠皮下移植瘤的生长,该现象可能与其下调survivin蛋白的表达并促进细胞凋亡有关,USP9X有可能成为胰腺癌基因治疗的一个新靶点。     

Telomerase inhibition by stable RNA interference impairs tumor growth and angiogenesis in glioblastoma xenografts

Telomerase is highly expressed in advanced stages of most cancers where it allows the clonal expansion of transformed cells by counteracting telomere erosion. Telomerase may also contribute to tumor progression through still undefined cell growth-promoting functions. Here, we inhibited telomerase activity in 2 human glioblastoma (GBM) cell lines, TB10 and U87MG, by targeting the catalytic subunit, hTERT, via stable RNA interference (RNAi). Although the reduction in telomerase activity had no effect on GBM cell growth in vitro, the development of tumors in subcutaneously and intracranially grafted nude mice was significantly inhibited by antitelomerase RNAi. The in vivo effect was observed within a relatively small number of population doublings, suggesting that telomerase inhibition may hinder cancer cell growth in vivo prior to a substantial shortening of telomere length. Tumor xenografts that arose from telomerase-inhibited GBM cells also showed a less-malignant phenotype due both to the absence of massive necrosis and to reduced angiogenesis.     

Survivin、MVD在人脑胶质瘤中的表达及意义

目的　探讨Survivin基因在人脑胶质瘤中的表达 ,及其与MVD的相关性。方法　采用免疫组织化学SABC法检测Survivin、CD34在 4 1例胶质瘤及 10例正常脑组织中的表达。结果　Survivin蛋白在正常脑组织中无表达 ,4 1例胶质瘤中 ,2 5例呈阳性表达 ,占 6 1 0 % ,Ⅰ～Ⅱ级和Ⅲ～Ⅳ级间的表达有显著性差异P<0 .0 5 ) ;胶质瘤组织中Survivin蛋白的表达与MVD的表达密切相关P <0 .0 1,rs=0 .70 7)。结论　Survivin蛋白异常表达在胶质瘤的恶化中起重要作用 ,它可能促进血管内皮细胞的增生 ,从而促进肿瘤细胞的增殖     

survivin基因RNA干涉对宫颈癌细胞凋亡和放射敏感性的影响

目的：观察survivin基因siRNA干涉对宫颈癌细胞HeLa凋亡和放射敏感性的影响。方法：通过脂质体介导将含人survivin基因siRNA的重组真核表达质粒pSilencer2.1-s2转染宫颈癌细胞系HeLa,G418筛选阳性克隆,半定量PCR、Western blot分别检测survivin mRNA和蛋白表达,激酶活性检测法测定半胱氨蛋白水解酶-3(caspase-3)活性变化,流式细胞仪检测细胞凋亡情况,平板克隆形成实验、多靶单击模型拟合细胞存活曲线观察细胞放射敏感性变化。结果：G418筛选形成稳定转染阳性克隆,建立了3组稳定转染细胞系：HeLa-s2(转染重组质粒pSileneer2.1-s2)、HeLa-NC(转染阴性对照质粒pSi- lencer2.1-NC)和HeLa-U6 neo(转染空载质粒pSileneer2.1-U6 neo)。经与HeLa-NC、HeLa-U6 neo和未转染HeLa细胞比较,HeLa-s2 survivin mRNA和蛋白表达明显下降,与HeLa细胞相比,表达抑制率分别为：62.8%和60.1%；caspase-3激酶活性增强,D_(405)达1.26±0.04(P＜0.05)；细胞凋亡率为：(29.2±1.4)%,明显升高(P＜0.05)；同一剂量X线照射下,平板克隆形成率显著降低(P＜0.05)；细胞存活曲线显示：D_0、D_q值显著下降,分别为：3.15、1.21(P＜0.05),放射增敏比分别为：2.01 (D_0值比)、1.77(D_q值比)。结论：survivin基因siRNA干涉可通过阻抑HeLa细胞中survivin表达,增强caspase-3激酶活性,诱导细胞凋亡,并显著提高细胞的放射敏感性。     

survivin基因的RNA干涉抑制人乳腺癌SKBr-3细胞体外增殖的作用

目的:研究RNA干涉(RNAinterference,RNAi)抑制survivin基因表达后对人乳腺癌SKBr3细胞体外增殖能力的影响。方法:通过RTPCR和间接免疫荧光检测转染survivin靶向的小干涉RNA表达载体pSUPERS1后SKBr3细胞中survivin基因的表达;集落形成实验、MTT比色法检测细胞体外增殖能力;流式细胞仪检测细胞周期。结果:转染pSUPERS1后,SKBr3细胞中survivin的mRNA和蛋白表达水平明显降低;克隆形成率(38±16.70)%比对照组(90.3±4.04)%显著降低;细胞增殖减慢,主要阻滞于G1期(74.03±8.91)%。结论:RNA干涉沉寂survivin基因表达后明显抑制了人乳腺癌SKBr3细胞的体外增殖能力。     

TRAIL conjugated to nanoparticles exhibits increased anti-tumor activities in glioma cells and glioma stem cells in vitro and in vivo

Glioblastomas (GBM) are characterized by resistance to chemotherapy and radiotherapy, and therefore, alternative therapeutic approaches are needed. TRAIL induces apoptosis in cancer but not in normal cells and is considered to be a promising anti-tumor agent. However, its short in vivo half-life and lack of efficient administration modes are serious impediments to its therapeutic efficacy. Nanoparticles (NP) have been used as effective delivery tools for various anticancer drugs. TRAIL was conjugated to magnetic ferric oxide NP by binding the TRAIL primary amino groups to activated double bonds on the surface of the NP. The effect of NP-TRAIL was examined on the apoptosis of glioma cells and self-renewal of glioma stem cells (GSCs). In addition, the ability of the NP-TRAIL to track U251 cell–derived glioma xenografts and to affect cell apoptosis, tumor volume, and survival among xenografted rats was also examined. Conjugation of TRAIL to NP increased its apoptotic activity against different human glioma cells and GSCs, as compared with free recombinant TRAIL. Combined treatment with NP-TRAIL and γ-radiation or bortezomib sensitized TRAIL-resistant GSCs to NP-TRAIL. Using rhodamine-labeled NP and U251 glioma cell–derived xenografts, we demonstrated that the NP-TRAIL were found in the tumor site and induced a significant increase in glioma cell apoptosis, a decrease in tumor volume, and increased animal survival. In summary, conjugation of TRAIL to NP increased its apoptotic activity both in vitro and in vivo. Therefore, NP-TRAIL represents a targeted anticancer agent with more efficient action for the treatment of GBM and the eradication of GSCs.     

RNAi沉默survivin表达对A549细胞凋亡及紫杉醇敏感性的影响

目的利用RNAi （RNA interference,RNAi）沉默抗凋亡基因survivin,观察其对人肺腺癌细胞A549凋亡以及紫杉醇药物敏感性的影响。方法构建重组质粒,将其导入A549细胞,检测转染前后survivin的表达情况,TUNEL法检测细胞凋亡情况,MTT法检测转染后A549细胞对紫杉醇的敏感性变化。结果成功构建重组质粒。转染重组质粒后,survivin表达明显降低;细胞凋亡率增加。转染前紫杉醇对A549细胞的IC50为转染后的11.9倍,P<0.05。结论构建的重组质粒能有效抑制survivin基因表达,诱导细胞凋亡,增强A549细胞对紫杉醇的敏感性。     

Short hairpin RNA targeting survivin inhibits growth and angiogenesis of glioma U251 cells

Survivin is a novel tumor-associated gene, its overexpression mostly associates with carcinogenesis and development. Nevertheless, the precise role of survivin in initiation and progression of gliomas is still not completely clear. We constructed here three short hairpin RNA (shRNA) targeting survivin plasmid vectors and introduced them into glioma U251 cells. The three shRNAs were efficiently and specifically able to knockdown the survivin expression in transiently transfected U251 cells. The stable transfectants expressing the shRNA having the strongest inhibitory effect against survivin exhibited decreased cell growth, increased spontaneous apoptosis, mitotic catastrophe and cell cycle arrest. Furthermore, in nude mice xenografts, the stable transfectants presented decreased de novo glioma formation and reduced development of angiogenesis. Results from this study indicate that survivin plays an important role in malignant proliferation, antiapoptosis and angiogenesis of gliomas, which may become an attractive target for gene therapy of gliomas, while RNA interference (RNAi) mediated by shRNA may become a new promising strategy for cancer gene therapy.     

生存素基因RNAi对宫颈癌裸鼠移植瘤生长、凋亡和放射敏感性的影响

背景与目的：生存素基因是近年来肿瘤研究的焦点之一,本研究旨在观察生存素基因RNAi对宫颈癌裸鼠移植瘤生长、凋亡和放射敏感性的影响。方法：随机选择雌性BALB／c—nu／nu裸小鼠24只,细胞接种法建立4组人宫颈癌裸鼠皮下移植瘤模型,每天观察裸鼠一般状况及肿瘤生长情况,通过绘制肿瘤生长曲线并计算肿瘤生长抑制率,观察生存素基因RNAi对人宫颈癌裸鼠皮下移植瘤生长的影响;通过免疫组化SP法检测各组移植瘤组织中生存素蛋白表达情况,Ⅷ因子相关抗原（factor Ⅷ related antigen,FⅧRAg）表达情况并计算微血管密度（MVD）,HE染色及TUNEL染色观察生存素基因RNAi对人宫颈癌裸鼠皮下移植瘤凋亡的影响;当肿瘤体积达0．2cm^3时予X线放射治疗观察生存素基因RNAi对人宫颈癌裸鼠皮下移植瘤放疗敏感性的影响。结果：成功建立4组人宫颈癌裸鼠皮下移植瘤模型,接种HeLa-s2组裸鼠肿瘤体积在每个检测点均明显小于接种HeLa组。观察结束时,接种HeLa—s2组裸鼠瘤重明显小于接种HeLa组,分别为（0．369&#177;0．043）g和（1．150&#177;0．136）g（P〈0．05）;接种HeLa—s2组裸鼠肿瘤生长抑制率为67．9％。免疫组化结果显示：接种HeLa-s2组裸鼠生存素蛋白表达显著下降;接种HeLa—s2组裸鼠FⅧRag表达亦显著下降,MVD值降至23．4&#177;3．1。HE染色、TUNEL染色结果显示：接种HeLa—s2组裸鼠细胞凋亡明显增多,AI值达（22．7&#177;1．4）％。X线放疗后不同检测点接种HeLa—s2组裸鼠肿瘤体积明显小于接种HeLa组,肿瘤生长明显受抑。观察结束后,接种HeLa～s2组裸鼠瘤重明显小于接种HeLa组,分别为（0．41&#177;0．06）g和（1．38&#177;0．29）g（P〈0．05）。接种HeLa—s2组裸鼠肿瘤细胞凋亡明显增多,与接种HeLa组AI比较,接种HeLa—s2组AI明显升高,分别为（30．06&#177;0．98）％和（4．17&#177;0．64）％（P〈0．05）。结论：生存素基因RNAi可通过下调移植瘤组织生存素蛋白表达降低其MVD,从而抑制移植瘤生长并促进其凋亡,并通过增加X线放射治疗诱导的细胞凋亡,增强放射治疗对移植瘤的生长抑制,进而提高移植瘤的放射敏感性。     

Quercetin promotes degradation of survivin and thereby enhances death-receptor-mediated apoptosis in glioma cells

The flavonoid quercetin has been reported to inhibit the proliferation of cancer cells, whereas it has no effect on nonneoplastic cells. U87-MG, U251, A172, LN229, and U373 malignant glioma cells were treated with quercetin (50-200 microM). Quercetin did not cause cytotoxicity 24 h after treatment. Combining quercetin with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) strongly augmented TRAIL-mediated apoptosis in U87-MG, U251, A172, and LN229 glioma cells; U373 cells could not be sensitized by quercetin to TRAIL-mediated apoptosis. TRAIL-induced apoptosis was enhanced by quercetin-induced reduction of survivin protein levels. Upon treatment with quercetin, the protein level of survivin was strongly suppressed in U87-MG, U251, and A172 but not in U373 glioma cells. Quercetin exposure resulted in proteasomal degradation of survivin. TRAIL-quercetin-induced apoptosis was markedly reduced by overexpression of survivin. In addition, upon treatment with quercetin, downregulation of survivin was also regulated by the Akt pathway. Taken together, the results of the present study suggest that quercetin sensitizes glioma cells to death-receptor-mediated apoptosis by suppression of inhibitor of the apoptosis protein survivin.     

Survivin knockdown and concurrent 4-HPR treatment controlled human glioblastoma in vitro and in vivo

Survivin is highly expressed in most cancers, including glioblastoma, and it plays a significant role in inhibiting apoptosis and promoting tumor growth. Treatment of cancer cells with N-(4-hydroxyphenyl) retinamide (4-HPR) induces apoptosis through destabilization of mitochondrial membrane and activation of caspase-mediated apoptotic pathways. We studied the efficacy of a combination of survivin knockdown and 4-HPR treatment to induce apoptosis and inhibit invasion, angiogenesis, and growth of human glioblastomas in vitro and in vivo. Using a plasmid encoding survivin shRNA, we downregulated survivin in glioblastoma U251MG and U118MG cells and simultaneously treated with 1 µM 4-HPR for 48 hours. Cells following treatments were subjected to the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and invasion assays. In vivo angiogenesis and tumor regression studies were performed in nude mice. TUNEL assay demonstrated apoptosis in more than 80% of cells after survivin knockdown and 4-HPR treatment. Matrigel invasion assays demonstrated marked decreases in tumor cell invasion. In vivo angiogenesis studies depicted a remarkable inhibition of neovascularization due to the knockdown of survivin and 4-HPR treatment. Imaging of intracerebral tumorigenesis and longitudinal studies on subcutaneous solid tumor formation showed dramatic decreases in tumorigenesis and solid tumor progression, respectively, after treatment with the combination. Studies to elucidate the molecular mechanisms of the inhibition of angiogenesis and tumor regression demonstrated marked decreases in proliferating cell nuclear antigen, metalloproteinase-9, vascular endothelial growth factor, basic fibroblast growth factor, and CD31 in solid tumors. Our data demonstrated that survivin knockdown and concurrent 4-HPR treatment could be a novel therapeutic strategy for controlling growth of human glioblastomas.     

siRNA介导的survivin基因沉默诱导人膀胱癌BIU-87细胞凋亡的研究

[目的]研究siRNA（small interference RNA）对人膀胱癌BIU-87细胞凋亡和survivin基因表达的影响。[方法]利用Ambion公司设计软件，设计并体外转录合成针对survivin基因的3个特异性siRNA，通过脂质体将siRNA转入BIU-87细胞，分别采用MTT和DNA原位末端标记（TUNEL）法检测siRNA对BIU-87细胞生长抑制率（IR）和凋亡指数（AI），半定量逆转录聚合酶链反应（RT-PCR）和Western Blotting检测siRNA对survivin mRNA及其蛋白表达的影响。[结果]转染后的siRNA1～3组的BIU-87细胞的IR（41.84%、56.21%、54.13%）和AI（21.98%、36.54%、31.34%）均分别显著高于正常对照组（1.98%和3.17%）（P＜0.05），survivin mRNA及其蛋白表达水平均显著低于对照组；其中siRNA2～3对BIU-87细胞的IR、AI和survivin表达的抑制作用均显著高于siRNA1。[结论]体外转录合成的siRNA可抑制BIU-87细胞survivin的表达，诱导BIU-87细胞凋亡，从而抑制BIU-87细胞生长，为siRNA介导的膀胱肿瘤基因沉默提供实验依据。     

survivin基因RNAi逆转录病毒载体设计与构建方法实验研究

目的　设计和构建survivin基因的表达si RNA逆转录病毒重组载体,探讨胶质瘤分子病因及用于基因治疗的可行性。方法　利用在线软件si Direct设计干扰survivin基因靶序列,合成回文DNA序列退火后克隆至线性化p SUPER质粒载体,重组质粒载体双酶切电泳鉴定和测序分析,再转染phoenix细胞产生病毒转染低分化SHG4 4 - 9胶质瘤细胞株。利用NIH3T3细胞测定病毒滴度。Western blot测定转染后SHG4 4 - 9细胞survivin表达量。结果　p SU PER表达si RNA重组质粒载体经双酶切电泳鉴定和DNA测序分析,证实插入6 0 bp序列与原序列一致,位置正确。测定p SUPER.retro- S1、p SU PER.retro- S2病毒滴度值分别为5 .5×10 5CFU/ m l和5 .75×10 5CFU/ ml,干扰效率分别为70 .5 %和接近10 0 .0 %。结论　survivin基因的表达si RNA逆转录病毒重组载体的构建成功,不但为研究胶质瘤分子病因和基因治疗提供了有用工具,而且也为研究高表达survivin的其它肿瘤构建了新的平台。     

Efficient inhibition of human colorectal carcinoma growth by RNA interference targeting polo-like kinase 1 in vitro and in vivo

Polo-like kinase 1 (PLK1) showing a high expression in various kinds of tumors is considered a candidate target for cancer therapy. The aim of our study was to explore the effects of silencing PLK1 gene on human colorectal carcinoma cell line HCT-116 in vitro and in vivo. In vitro, the plasmids generating short hairpin RNA (shRNA)-targeting PLK1 were transfected into HCT-116 by using FugeneHD reagent, and the silencing potency was measured by RT-PCR, western blot, flow cytometry, and Caspase-Glo 3/7 assay, respectively. In vivo, the growth inhibition capacity of PLK1-shRNA on HCT-116 xenograft was measured in nude mice. Then, the silencing effect of PLK1 was analyzed by RT-PCR, western blot, and immunohistochemistry, respectively. Apoptosis, angiogenesis, and proliferation in tumor tissues were measured by TUNEL, CD31, and PCNA stain, respectively. The RNA interference targeting PLK1 significantly decreased the expression of PLK1 in vitro. More importantly, anti-PLK1 treatment in HCT-116 xenograft decreased tumor weight by 81.58% compared with the control group (p<0.001), accompanied with decreased PLK1 mRNA and protein expression, increased cell apoptosis, and reduced angiogenesis and proliferation (p<0.001). Our study showed that knockdown of PLK1 by shRNA might be the potential therapeutic approach against human colorectal carcinoma.     

RNA干扰技术抗非小细胞肺癌作用的体内实验研究

Objective  

Lung cancer has emerged as a leading cause of cancer death in the world. Current therapies are ineffective, thus new approaches are needed to improve the therapeutic ratio. RNA interference (RNAi) has shown promise in gene silencing in vitro, the potential of which in developing new methods for the therapy of non-small-cell lung cancer (NSCLC) needs to be further tested in vivo. In this study, chemically synthesized double-stranded RNA (dsRNA) targeting epidermal growth factor receptor (EGFR) was transfected into NSCLC cell line SPC-A1 cells and established the tumor burdened athymic nude mice model to investigate whether dsRNA could induce gene silencing in NSCLC cells in vivo.     

生存素siRNA表达质粒对结肠癌细胞侵袭和增殖能力的抑制作用

 目的 构建针对生存素的小分子RNA干扰表达质粒，研究siRNA表达质粒沉默生存素基因后对大肠癌细胞株SW480侵袭性和增殖性的影响。方法 用pRNAT U6.1/Neo载体构建生存素 siRNA表达质粒（pRNAT/sur siRNA），该质粒转染SW480细胞株48小时后，用Westeron blot和半定量 RT PCR分析生存素蛋白和mRNA表达的变化。G418 400μg/L筛选阳性克隆细胞株（sur siRNA/SW480），MTT法检测SW480细胞体外增殖的变化；细胞侵袭性实验研究SW480细胞的侵袭性变化。结果 成功地构建了pRNAT/sur siRNA表达质粒，并且该质粒明显下调SW480细胞生存素蛋白和mRNA的表达水平，分别下调85%，80%。G418 400μg/L筛选出转染pRNAT/sur siRNA的SW480细胞株（sur siRNA/SW480）；sur siRNA/SW480细胞增殖受到显著抑制，抑制率为37.4% (P<0.01)；细胞侵袭实验示sur siRNA/SW480细胞的穿透数为(153±66)个，而对照组pRNAT/SW480、SW480细胞的穿透数为(505±65)，(578±98)个细胞，sur siRNA/SW480细胞的穿透数与对照组相比显著减少(P<0.01)。结论 针对生存素siRNA可以显著降低生存素的表达，抑制肿瘤细胞的增殖和侵袭，该生存素siRNA序列可能成为治疗结肠癌的一种有效手段。     

Survivin反义RNA真核表达载体的构建及鉴定

目的 为了特异封闭白血病细胞survivin的表达，抑制其功能，本实验构建了survivin反义核酸载体并导入白血病细胞系中。方法 应用RT—PCR获得survivin的cDNA片段，反向插入pcDNA3质粒载体中；经限制性酶切和测序鉴定所构建的反义核酸是否正确；采用电转染方法将重组体导入HL—60细胞中；RT—PCR技术检测转染细胞survivin表达的变化。结果 经限制性酶切和测序鉴定证明survivin反义核酸已成功构建；RT—PCR产物电泳结果显示，与转染前细胞、空质粒转染细胞相比，转染survivin反义核酸的细胞survivin mRNA水平明显降低。结论 本实验已成功建立了survivin反义核酸真核表达载体，而且在白血病细胞系中发挥了特异封闭作用，为进一步研究survivin反义核酸在白血病治疗中的作用提供了实验基础。