端粒酶反义hTR和反义hTERT合用对宫颈癌HeLa细胞凋亡的影响

目的探讨端粒酶反义hTR和反义hTERT联合作用于宫颈癌HeLa细胞,对HeLa细胞凋亡的影响。方法实验分空白对照组、脂质体对照组、正义hTR组、正义hTERT组、反义hTR组、反义hTERT组及反义hTR+反义hTERT组。分别采用四甲基偶氮唑蓝法(MTT)、逆转录聚合酶链反应技术-端粒重复序列扩增(TRAP)酶联免疫法(ELISA)、吖啶橙染色法、流式细胞术检测宫颈癌HeLa细胞转染反义hTR和反义hTERT后细胞的端粒酶活性、形态学、凋亡和周期的改变。结果宫颈癌HeLa细胞转染端粒酶反义核酸后,出现明显的细胞凋亡形态学改变,端粒酶活性抑制率、凋亡率增高及细胞周期阻滞,与空白对照组、脂质体对照组及正义核酸组比较,统计学上差异有显著性(P<0.01)。HeLa细胞转染0.2μmol·L-1反义hTR+反义hTERT72h后,端粒酶活性抑制率、细胞凋亡率分别为80.5%、28.6%,与反义hTR组和反义hTERT组比较(端粒酶活性抑制率、细胞凋亡率分别为hTR:62.7%、13.2%;hTERT:66.5%、13.7%),差异有显著性(P<0.01,q值分别为0.919、1.075)。反义hTR+反义hTERT组比反义hTR组和反义hTERT组细胞凋亡的形态学改变更明显,反义hTR+反义hTERT组G0/G1期细胞比例增加(G1期细胞比例为57.8%,空白对照组G1期细胞比例为50.7%)。结论端粒酶反义核酸能通过抑制端粒酶活性诱导细胞凋亡,端粒酶反义hTERT和反义hTR有协同效应。     

靶向Bcl-2、Bcl-xl、Mcl-1、Bcl-w、A1反义核酸抑制消化系肿瘤细胞增殖效果的差异

目的比较靶向Bcl-2、Bcl-xl、Mcl-1、Bcl-w、A1反义核酸(antisense oligodeoxynucleotide,ASO)对消化系统肿瘤细胞(肝癌HepG2细胞、胃癌MGC-803细胞、结肠癌Lovo细胞)的增殖抑制作用和致凋亡作用的差异。方法分别合成靶向Bcl-2、Bcl-xl、Mcl-1、Bcl-w、A1的反义核酸和随机序列反义核酸(randomolig odeoxynucleotide,RODN),采用脂质体Li-pofectamineTM 2000转染细胞,WST法检测相同浓度的5种ASOs对肝癌HepG2细胞、胃癌MGC-803细胞、结肠癌Lovo细胞的增殖抑制作用和致凋亡作用。结果 Bcl-2、Bcl-xl、Mcl-1、Bcl-w、A1等5种ASOs中,不论是对细胞增殖抑制还是致凋亡作用,均以Bcl-xlASO、Mcl-1ASO的作用效果较好。结论在Bcl-2、Bcl-xl、Mcl-1、Bcl-w、A1等5种ASOs中,Bcl-xlASO、Mcl-1ASO可明显抑制消化系肿瘤细胞增殖,诱导细胞凋亡。     

半乳糖糖基转移酶反义核酸诱导卵巢癌细胞凋亡的实验研究

目的研究α-1,4半乳糖糖基转移酶(α-1,4galactosyl transferase,α-1,4Gal-T)基因反义核酸诱导卵巢癌细胞COC1凋亡的作用。方法人工合成α1,4Gal-T基因的反义寡脱氧核苷酸,采用脂质体介导的基因转染方法将反义寡脱氧核苷酸转入卵巢癌细胞COC1中。用流式细胞仪检测其红细胞三糖基神经酰胺(Globotriaosyl ceramide,Gb3)的表达,以及卵巢癌细胞COC1的凋亡率。结果脂质体介导的反义寡脱氧核苷酸转入卵巢癌细胞COC1后,其红细胞三糖基神经酰胺含量显著下降(P<0.05),卵巢癌细胞COC1的凋亡率显著升高(P<0.05)。结论1,4Gal-T基因反义核酸能够诱导卵巢癌细胞COC1的凋亡。     

柳珊瑚来源的愈创木薁倍半萜生物碱化合物Muriceidine A体外抗肿瘤活性的研究

目的 对从高领类尖柳珊瑚Muriceides collaris中分离得到的1种愈创木薁倍半萜生物碱Muriceidine A进行体外抗肿瘤活性研究。方法 MTT法测定Muriceidine A对多种肿瘤细胞株的增殖抑制作用。流式细胞仪检测Muriceidine A对HeLa细胞周期及凋亡的影响。Western Blot检测Muriceidine A对HeLa细胞中周期相关蛋白、凋亡相关蛋白、E6/E7蛋白及其下游信号分子的影响。结果 Muriceidine A体外对多种肿瘤细胞均有增殖抑制作用,其中对HeLa 细胞的抑制作用最强,IC50为17.40 μmol/L。流式细胞仪分析及Western Blot检测显示,Muriceidine A 能够以剂量依赖性的方式使HeLa细胞阻滞在G2/M期,可通过激活Caspase依赖的线粒体凋亡途径诱导HeLa细胞凋亡。同时,Western Blot检测显示Muriceidine A能够通过降低HeLa细胞中E6/E7的水平,抑制ERK和STAT3的磷酸化。结论 Muriceidine A体外对人宫颈腺癌细胞HeLa具有较强的细胞毒作用。本研究为该小分子化合物今后的合成与改造提供思路与方向。     

Livin mRNA的反义核酸诱导MCF-7乳癌细胞凋亡作用

目的：以livin mRNA为靶点设计反义硫代脱氧寡核苷酸(PS—ODNs)，探讨其体外抗肿瘤效应及其机制。方法：设计以livin mRNA为靶点的反义核酸并作用于MCF-7乳癌细胞，用MTT、RT—PCR和流式细胞仪检测等方法对其进行评价研究。结果：在设计的一些反义核酸中，YMZ05能够有效地抑制livin基因的表达，增加caspase-3的活性，诱导MCF-7细胞凋亡，明显抑制其生长，结论：通过抑制livin基因的表达能够抑制MCF-7乳癌细胞生长、诱导其凋亡，livin可能会成为抗肿瘤的新靶点。     

MiR-24反义核酸通过BIM-Smac/Diablo途径促进多柔比星对结肠癌细胞的凋亡诱导效应

目的 探讨miR-24在结肠癌中发挥的作用并研究其是否和多柔比星的体外治疗有关。方法 用RT-qPCR法检测miR-24在结肠癌患者血浆中及结肠癌细胞系中的表达水平。MTT法检测miR-24反义核酸对多柔比星杀伤结肠癌细胞能力的影响。利用生物信息学、定量PCR及western blot法验证miR-24是否调节结肠癌细胞BIM的表达。运用JC-1染色、Annexin V染色及western blot法研究miR-24反义核酸影响多柔比星疗效的信号通路。结果 结肠癌患者血浆及结肠癌细胞系中miR-24表达水平显著升高。miR-24反义核酸可显著增强多柔比星对SW480细胞的杀伤活性。定量PCR及western blot实验表明miR-24的靶基因可能为BIM。miR-24反义核酸联合多柔比星可引起SW480细胞线粒体膜电位的丧失并诱导线粒体内Smac/DIABLO的释放,进而引起caspase-3的活化和凋亡的发生。转染BIM siRNA后miR-24反义核酸联合多柔比星对SW480细胞的凋亡诱导效应显著降低。结论 MiR-24反义核酸通过BIM-Smac/Diablo途径促进多柔比星对结肠癌细胞的凋亡诱导效应。     

反义基因治疗在消化系统肿瘤中的研究进展

反义基因治疗(antisensegenetherapy)即利用反义核酸与其靶基因或基因产物互补形成一种特殊的“基因封条结构,在转录或翻译水平阻断靶基因的异常表达,促进细胞正常分化或诱导细胞凋亡,以达到治疗肿瘤的目的。反义核酸所针对的靶点应该是在肿瘤发生中起关键作用的一种或几种癌基因、抗癌基因、自分泌生长因子及其受体基因。1载体选择有效的基因转移是基因治疗成功的关键,目前应用的载体主要有病毒载体和非病毒载体两大类。(1)病毒载体:①逆转录病毒:感染增殖分裂细胞,能整合到细胞染色体基因组中持续稳定表达,靶向性相对强,操作较方便,适合于…     

米非司酮诱导宫颈癌HeLa细胞凋亡及对OMI/HTRA2蛋白的影响

目的观察米非司酮、Omi/HtrA2对人宫颈癌Hela细胞凋亡的作用,探讨在宫颈癌HeLa细胞中米非司酮对Omi/HtrA2的影响。方法①对Omi/HtrA2重组质粒DNA的提取;②通过细胞转染,利用共聚焦显微镜观察Omi/HtrA2在细胞内的表达和定位;③应用流式细胞仪方法研究米非司酮、Omi/HtrA2对宫颈癌Hela细胞凋亡的作用。结果①融合蛋白EGFP-Omi/HtrA2、DsRed2-Omi/HtrA2在HeLa细胞中的定位为弥漫分布在细胞核和细胞浆;②米非司酮（40umol.L-1）促进Hela细胞凋亡,凋亡率为（46.16%）,转染pDsRed2-Omi/HtrA2凋亡率为32.69%,米非司酮＋转染质粒pDsRed2-Omi/HtrA2的凋亡率为78.85%。结论米非司酮能够促进HeLa细胞凋亡,DsRed2-Omi/HtrA2融合蛋白能够促进HeLa细胞凋亡,米非司酮能增加Omi/HtrA2促凋亡作用,Omi/HtrA2与米非司酮在HeLa细胞凋亡中存在协同作用。Omi/HtrA2可能是治疗宫颈癌的一个新靶点,米非司酮可能成为治疗宫颈癌的新方法。     

白藜芦醇诱导HeLa细胞凋亡的实验研究

目的:观察白藜芦醇对宫颈癌HeLa细胞凋亡作用.方法:应用四甲基偶氮唑蓝(MTT)比色法检测白藜芦醇对HeLa细胞的抑制效应,并采用末端标记法(TUNEL)对凋亡细胞进行定量检测.结果:白藜芦醇对HeLa细胞48h的抑制效应为20.15%,72h的抑制效应为38.60%.TUNEL检测结果为白藜芦醇促进HeLa细胞凋亡.结论:白藜芦醇抑制HeLa细胞增殖,对指导临床药物治疗宫颈癌具有重要意义.     

X染色体连锁的凋亡抑制蛋白对米非司酮诱导宫颈癌HeLa细胞凋亡的影响

目的观察X染色体连锁的凋亡抑制蛋白(XIAP蛋白)对米非司酮诱导宫颈癌HeLa细胞凋亡的影响。方法 XIAP重组质粒转染HeLa细胞,共聚焦显微镜观察其在细胞内的分布;流式细胞仪检测米非司酮处理和未处理的各组细胞凋亡率。结果 XIAP在HeLa细胞中弥漫分布于细胞核和细胞浆,米非司酮可诱导HeLa细胞凋亡,XIAP能抑制由米非司酮诱导的HeLa细胞凋亡。结论米非司酮能促进HeLa细胞凋亡,XIAP能抑制由米非司酮诱导的HeLa细胞凋亡。     

Silencing livin gene by siRNA leads to apoptosis induction, cell cycle arrest, and proliferation inhibition in malignant melanoma LiBr cells

Aim： The aim of the present study was to investigate the effects of silencing the livin gene by small interfering RNA （siRNA） on the expression of livin and the effects on apoptosis, cell cycle, and proliferation in human malignant melanoma LiBr cells. Methods： Three chemically-synthetic siRNA duplexes targeting livin were transiently transfected into the LiBr cells, and the effects on livin expression were detected both at the mRNA level by real-time RT-PCR and at the protein level by Western blotting. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay, flow cytometric analysis, and the expression of procaspase-3 and activated caspase-3 analysis by Western blotting. Cell cycle was analyzed by flow cytometry. Cell proliferation was determined by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide. Results： One of the 3 designed siRNA could effectively knock down the livin expression both at the mRNA and protein levels in dose- and time-dependent manners; 100 nmol/L with maximum downregulation on mRNA at 48 h, and on the protein at 72 h after transfection. Silencing livin could significantly induce apoptosis, arrest cell cycle at the Go/G1 phase, and inhibit proliferation in LiBr cells. Meanwhile, caspase-3 was activated. Conclusion： The livin gene could serve as a potential molecular target for gene therapy by siRNA for malignant melanoma.     

Antisense oligonucleotides targeting midkine induced apoptosis and increased chemosensitivity in hepatocellular carcinoma cells

AIM: Overexpression of midkine (MK) has been observed in many malignancies. This aim of this study is to screen for suitable antisense oligonucleotides (ASODN) targeting MK in hepatocellular carcinoma (HCC) cells and evaluate its antitumor activity. METHODS: Ten ASODN targeting MK were designed and synthesized. After transfection with ASODN, cell proliferation was analyzed with MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay. In addition, MK mRNA, protein levels, as well as apoptosis and caspase-3 activity were also examined in HepG2 cells. Cell proliferation was then analyzed after treatment with both ASODN and chemotherapeutic drugs. RESULTS: In this experiment, the ASODN5 among the 10 ASODN showed higher inhibitory activity against proliferation of hepatocellular carcinoma cells in a dose-dependent manner. In HepG2 cells, ASODN5 could significantly reduce the MK mRNA level and protein content. After transfection with ASODN5 for 48 h, accompanied with a decline of survivin and Bcl-2 protein content, a remarkable increase of apoptosis and caspase-3 activity was observed in HepG2 cells. Furthermore, ASODN5 transfer can significantly increase chemosensitivity in HepG2 cells. CONCLUSION: Antisense oligonucleotides targeting MK shows therapeutic effects on HCC; ASODN5 has the possibility to be developed as an effective antitumor agent.     

小剂量顺铂对Hela细胞凋亡的实验研究

目的：实验研究小剂量顺铂对放疗诱导Hela细胞凋亡的作用。方法：将细胞分为空白对照组、单纯化疗组、单纯放疗组、放疗加化疗组4组,采用TUNEL法分另1检测各组细胞的凋亡率。结果：单纯放疗、化疗都可诱导Hela细胞凋亡,顺铂(化疗)可以明显增强放疗诱导的细胞凋亡。结论：小剂量顺铂可明显增加放疗诱导的Hela细胞凋亡,为临床应用小剂量顺铂使放疗增效提供了理论依据。     

Targeting tumor cell resistance to apoptosis induction with antisense oligonucleotides: progress and therapeutic potential.

Despite the use of combination chemotherapy and immunotherapy, the survival rate of adult cancer patients has only moderately increased. Diminished apoptosis, due to overexpression of anti-apoptotic proteins, is involved in tumorigenesis and treatment resistance. Antisense oligonucleotides can be used to specifically inhibit unwanted gene expression and hence target the molecular basis of genetic diseases. Recently developed antisense oligonucleotides with the ability to inhibit the expression of anti-apoptotic proteins, including Bcl-2, Bcl-xL, FLIP and surviving, have been shown to facilitate tumor cell apoptosis and sensitize tumor cells to cytotoxic treatments. This suggests their use in combination with conventional treatments as an approach to more effective cancer therapy.     

硫芥诱导Hela细胞发生凋亡及坏死

目的 研究硫芥诱导的Ｈｅｌａ细胞凋亡的作用。方法 生长在ＤＭＥＭ培养基中的Ｈｅｌａ细胞与不同浓度的硫芥作用３小时,凋亡用电镜,电泳及流式术检测。结果 低浓度硫芥（１μｍｏｌ．Ｌ＾－１）抑制细胞生长;较高浓度（１－１００μｍｏｌ．Ｌ＾－１）俣细胞主要在Ｇ１期阻滞,发生典型的凋亡形态改变,提了细胞ＤＮＡ进行琼脂糖凝胶电泳,出现“ＤＮＡＬａｄｄｅｒ”,流式术观察表明硫芥处理３小时后,细胞撤药培养１２小时     

双黄连抑制甲1型流感病毒诱导细胞凋亡的机制研究

目的:研究双黄连抑制甲1型流感病毒诱导细胞凋亡的机制。方法:将宫颈癌(Hela)细胞分成4组:病毒对照组、实验组、细胞对照组、药物对照组,采用流式细胞术计算细胞凋亡的百分率。结果:实验组细胞凋亡的百分率与病毒对照组比较有非常显著性差异(P<0.01)。结论:双黄连可抑制甲1型流感病毒诱导Hela细胞凋亡。     

Highly loaded nanoparticulate carrier using an hydrophobic antisense oligonucleotide complex.

Antisense oligonucleotides, and particularly those with phosphorothioate backbones, have emerged as potential gene specific therapeutic agents and are currently undergoing evaluation in clinical trials for a variety of diseases. In the area of HIV-1 therapeutics, targeting of oligonucleotides to infected cells, such as macrophages, would be highly desirable. The present study was designed to prepare and characterize oligonucleotide-loaded nanoparticles for this purpose. Due to their hydrophilic characteristics, oligonucleotides are difficult to entrap in polymeric particles. Here, the oligonucleotides were first complexed with cetyltrimethylammonium bromide. The oligonucleotide-loaded nanoparticles were prepared by the emulsification-diffusion method and subsequently purified. In comparison with previous studies, a high oligonucleotide-loading was achieved; 2.5, 5 and 10% oligonucleotide loading were assessed. If the initial oligonucleotide content was 4%, this method produced a final oligonucleotide loading of 1.9% with an entrapment efficiency of 47%. The integrity of the oligonucleotide and of the polymer, in the final freeze-dried product, was retained.     

女贞子血清药理对Hela细胞增殖抑制的实验研究

目的：观察女贞子血清药理对宫颈癌Hela细胞增殖抑制作用。方法：应用血清药理学方法研究中药的体外效应,用四甲基偶氮唑蓝(MTT)比色法检测含女贞子兔血清对Hela细胞的抑制效应,并采用末端标记法(TUNEL)对凋亡细胞进行定量检测。结果：中药兔血清对Hela检测结果为女贞子血清促进Hela细胞凋亡。结论：女贞子血清药理抑制Hela细胞增殖,对指导临床药物治疗宫颈癌具有重要意义。     

D-24851衍生物YB-N12诱导Hela细胞凋亡及其机制

目的:研究D-24851衍生物YB-N12能否诱导宫颈癌细胞株(Hela细胞)发生凋亡及其作用机制。方法:对数生长细胞培养于24孔培养板内24h,给药组加入不同浓度的YB-N12,对照组加入与给药组等体积的DMSO的培养液,培养24h后检测细胞凋亡数。采用MTT、荧光显微镜检测凋亡细胞、DNA ladder方法进行凋亡检测,用RT-PCR方法检测凋亡过程中相关基因表达的变化。结果:D-24851衍生物YB-N12在体外试验中对Hela细胞的杀伤力强,荧光显微镜观察到了凋亡小体,DNA ladder法检测到凋亡时DNA降解形成的梯带。在凋亡过程中,凋亡相关基因p53表达明显增加(P<0.05),而bcl-2表达下降(P<0.05)。结论:D-24851衍生物YB-N12能诱导Hela细胞发生凋亡,其机制可能与p53基因表达上调及bcl-2基因表达下调有关。