阿霉素联合顺铂诱导宫颈癌细胞凋亡实验研究

目的:研究化疗药物阿霉素(ADM)联合顺铂(DDP)对宫颈癌CaSki细胞株的增殖及凋亡的影响,并探讨其可能的相互作用机制。方法:应用MTT比色法检测不同浓度阿霉素、顺铂单独和联合应用对宫颈癌CaSki细胞的增殖抑制作用;同时RT-PCR法在mRNA水平上检测Bcl-2和TNF-α基因表达量的变化。结果:两种药物单独应用均可抑制CaSki细胞的增殖,联合用药(<12μg/mL)时具有协同抑制作用并与各药物单一应用比较具有显著性差异(P<0.05);阿霉素、顺铂作用CaSki细胞后能上调TNF-α基因和下调Bcl-2基因的表达。结论:宫颈癌CaSki细胞在化疗药物阿霉素和顺铂两药联合作用下通过诱导TNF-α和Bcl-2 mRNA表达量的变化发挥协同抑制作用,同时TNF-α的高表达增强了化疗药物阿霉素和顺铂诱导肿瘤细胞凋亡的敏感性,其机制主要与凋亡诱导效应有关。     

c-myc表达对顺铂诱导人胶质瘤细胞系BT_(325)凋亡的影响

研究 c- m yc在化疗药物顺铂诱导肿瘤细胞凋亡中的作用。在原有已构建好 c- myc反义表达载体 pc DNA3- MYC并成功转染 BT32 5 细胞的基础上 ,用顺铂诱导细胞凋亡。采用 TUNEL技术并从光镜、电镜、荧光显微镜及激光共聚焦显微镜水平观察 BT32 5 细胞凋亡的形态学特征 ;流式细胞仪检测实验组 (即 pc DNA3- MYC转染的细胞 )及对照组 (包括未转染或仅转染空载体 pc DNA 3的细胞 )凋亡率的异同。结果显示 :与转染空载体 pc DNA3及未转染组的细胞相比 ,在转染 pc DNA 3-MYC的 BT32 5 细胞中 ,顺铂诱导的细胞凋亡作用明显受阻 ,结果表明 :肿瘤细胞内异常表达的 c- myc在顺铂诱导肿瘤细胞凋亡的过程中有重要作用。     

c—myc表达对顺铂诱导人胶质瘤细胞系BT325凋亡的影响

研究c-myc在化疗药物顺铂诱导肿瘤细胞凋亡中的作用。在原有已构建好c-myc反义表达载体pcDNA3-MYC并成功转染BT325细胞的基因上,用顺铂诱导细胞凋亡。采用TUNEL技术并从光镜、电镜、荧光微镜及激光内共聚焦显微镜水平观察BT325细胞凋亡的形态学特征;流式细胞仪检测实验组（即pcDNA3-MYC转染的细胞）及对照组（包括未转染或仅转染空载体pcDNA3的细胞）凋亡率的异同。结果显示：与转染pcDNA3及未转染组的细胞相比,在转染pcDNA3-MYC的BT325细胞中,顺铂诱导的细胞凋亡作用明显受阻,结果表明：肿瘤细胞内异常表达的c-myc在顺铂诱导肿瘤细胞凋亡的过程中有重要作用。     

LRP16影响宫颈癌Siha细胞对化疗药物的敏感性

目的:探讨抑制LRP16的表达对宫颈癌Siha细胞的化疗药物敏感性的影响。方法:将抑制LRP16表达的小干扰RNA:negativecontrol-si RNA(NC)、si RNA-374(si374)转染入Siha宫颈鳞癌细胞系中,通过顺铂(DDP)和紫杉醇(TAX)的处理后,采用CCK-8检测不同浓度紫杉醇、顺铂作用宫颈癌细胞系Siha48 h后,计算出细胞被抑制一半时顺铂、紫杉醇的药物浓度(IC50);使用Hoechst33342染色观察细胞凋亡,采用流式细胞仪检测顺铂IC50作用Siha细胞48小时后的细胞凋亡情况,紫杉醇IC50作用Siha细胞之后的细胞周期分布情况。结果:CCK-8检测转染的Siha细胞增殖活性受到抑制,Hoechst33342染色观察转染的Siha细胞凋亡明显增加,流式细胞仪检测凋亡显示,si374+顺铂的早期凋亡率22.15±2.24,NC+顺铂12.45±2.72,流式细胞仪检测周期显示G2/M(%),si374+紫杉醇29.94±1.87,NC+紫杉醇17.66±2.32。结论:LRP16基因表达下调之后,抑制Siha细胞的增殖、促进其凋亡,使细胞周期滞留于G2/M期,从而提高Siha细胞的化疗敏感性。     

靶向Survivin的反义寡核苷酸对肿瘤细胞增殖的抑制作用

 Survivin是新近克隆的一种凋亡抑制蛋白 (IAP)家族成员 ,在几乎所有肿瘤组织中特异性表达 ,而在正常成年终末分化组织中低表达甚至不表达 .采用四唑盐 (MTT)比色实验法比较 2 0条抗人survivin反义寡核苷酸对HeLa细胞增殖的抑制效果 ,并从中筛选效果显著的反义寡核苷酸 ,在体外水平进一步验证其抑制survivin表达的能力 .在用 4 0 0nmol L反义寡核苷酸转染HeLa细胞 4 8h后 ,有 4条反义寡核苷酸对细胞增殖的抑制率超过 4 0 %,其中 4 5号反义寡核苷酸的抑制率可达5 9%,而阳性对照序列ISIS2 372 2的抑制率仅达 30 %.Northern和Western印迹分析证明 :4 5号反义寡核苷酸可明显降低细胞中survivin基因的mRNA含量和蛋白水平 .4 5号反义寡核苷酸还可在较低浓度 (2 0 0nmol L)显著增强HeLa细胞对化疗药三尖杉酯碱的敏感性 .因此 ,4 5号反义寡核苷酸有望应用于survivin高表达肿瘤的辅助治疗之中     

AS-mCLB1重组质粒体外抗肿瘤及化疗增敏作用

研究反义全长小鼠细胞周期蛋白B1重组质粒(pAS-mCLB1)体外抗肿瘤及化疗增敏作用。扩增后少量抽提并纯化pAS-mCLB1,通过脂质体将其转染入小鼠露易丝肺癌(LL/2)细胞,RT-PCR和Western印迹测定细胞内细胞周期蛋白B1的表达,观察转染后细胞形态变化,MTT法检测细胞增殖活性,流式细胞仪检测细胞周期及凋亡。细胞转染48h后,用化疗药物健择(gemcitabine;0.2μmol/L)处理24h,MTT法测定健择对细胞的杀伤作用。研究提示pAS-mCLB1转染后LL/2细胞形态明显异常,细胞内细胞周期蛋白B1表达显著下调,细胞周期阻滞于G1期,增殖受抑,凋亡增加;健择对pAS-mCLB1转染后LL/2细胞的杀伤作用显著增强。重组质粒pAS-mCLB1体外具有明显的抗肿瘤作用,并能增强肿瘤细胞对化疗药物的敏感性,估计上述作用与其下调肿瘤细胞内细胞周期蛋白B1表达,从而诱导细胞周期阻滞及凋亡等作用相关。     

比较NRP-1反义寡核苷酸与VEGFR-2反义寡核苷酸对人胃癌SGC7901细胞增殖和凋亡的影响

目的:研究比较神经纤毛蛋白1(NRP-1)反义寡核苷酸(ASODN)与血管内皮生长因子受体2(VEGFR-2)反义寡核苷酸(ASODN)对人胃癌SGC7901细胞增殖活性及凋亡水平的影响。 方法:分别及同时将不同浓度经硫代磷酸化修饰的NRP-1 ASODN 和 VEGFR-2 ASODN 转染入人胃癌SGC7901细胞,逆转录-聚合酶链反应(RT-PCR)检测NRP-1基因和VEGFR-2 基因mRNA的转录水平;MTT比色法测量细胞的增殖活性;流式细胞仪测量细胞的凋亡水平。 结果:转染NRP-1 ASODN和VEGFR-2 ASODN后,人胃癌SGC7901细胞NRP-1基因和VEGFR-2 基因mRNA的转录水平均出现降低;NRP-1 ASODN和VEGFR-2 ASODN对SGC7901细胞有明显抑制增殖和促进凋亡的作用,且随着ASODN浓度升高而增强;分别转染时其作用无显著差别,联合转染时其作用明显增强。结论:NRP-1 ASODN和VEGFR-2 ASODN可抑制人胃癌SGC7901细胞 NRP-1基因和VEGFR-2 基因mRNA的转录水平及细胞增殖活性,促进细胞凋亡;与分别转染相比,两者联合转染作用明显增强。     

miR-17-92对急性白血病L1210/DDP细胞多药耐药性影响研究

目的:研究miR-17-92在白血病L1210/DDP细胞多药耐药形成中的作用.方法:首先构建L1210/DDP耐药细胞系,运用real-time PCR方法检测miR-17-92在L1210/DDP细胞与L1210细胞中的表达差异.利用脂质体Lipofectamine 2000将miR-17-92抑制物(miR-17-92sponge)及阴性对照(sponge vector)转染L1210/DDP细胞,构建miR-17-92表达下调的L1210/DDP细胞系.用MTS法检测转染后耐药细胞对顺铂和阿霉素体外药物敏感性.结果:miRNA-17-92在L1210/DDP耐药细胞系中高表达,上调倍数为(1.61±0.01)倍.体外药物敏感性实验表明,转染miR-17-92抑制物的实验组对顺铂和阿霉素的IC50分别为(3.29±0.51)、(1.35±0.13)g/ml,而转染阴性对照组对上述药物的IC50分别为(6.73± 0.82)、(2.66±0.42)g/ml,在耐药株中抑制miR-17-92在L1210/DDP细胞中的表达,显著增加细胞对顺铂和阿霉素的敏感性.结论:miR-17-92在白血病耐顺铂L1210/DDP细胞中高表达.抑制miR-17-92的表达可增加白血病L1210/DDP细胞对顺铂和阿霉素化疗药物的敏感性,部分逆转耐药.     

重组骆驼蓬脂转移蛋白与顺铂联合抑制黑色素瘤B16细胞增殖效应的研究

旨在探讨重组骆驼蓬脂转移蛋白(Recombinant Peganum harmala lipid transfer protein,r Ph LTP)与顺铂(DDP)联用对鼠黑色素瘤B16细胞增殖抑制和凋亡诱导作用。MTT法检测r Ph LTP、顺铂单独及联用后对鼠黑色素瘤B16细胞增殖的影响;流式细胞术检测细胞凋亡,活性氧(Reactive oxygen species,ROS)和线粒体膜电位(Δψm)的变化情况。结果显示,r Ph LTP和DDP联用后细胞生长抑制率和凋亡率显著高于DDP单独处理组(P0.01);而且胞内ROS升高的水平均高于单独处理组(P0.01),胞内Δψm水平均低于单独处理组,但无显著性差异。r Ph LTP可增强顺铂对B16细胞的生长抑制并促进细胞凋亡,与顺铂具有协同作用。     

康莱特联合顺铂对宫颈癌SiHa细胞增殖和凋亡的影响

目的:通过康莱特联合顺铂对宫颈癌SiHa细胞增殖和凋亡的影响,探讨其作用机制。方法:体外培养宫颈癌Siha细胞,分别将康莱特(浓度为1,2,4,6,8 mg/mL),顺铂(浓度梯度为1.5,3,6,9,12μg/mL),单独作用于宫颈癌SiHa细胞,加药24h、48h用噻唑蓝(MTT法)检测细胞增殖情况。用流式细胞术检测康莱特组和顺铂组细胞24h凋亡率,选取合适的药物浓度(康莱特6 mg/mL,顺铂3μg/mL),进行联合用药,加药24h、48h用MTT法检测细胞增殖情况,用流式细胞术检测24h细胞凋亡率。结果:①MTT法显示加药后两组的24h、48h,宫颈癌SiHa细胞的抑制率均高于对照组(P0.05),并且在一定程度上呈浓度和时间依赖性。②联合用药时,细胞的抑制率和凋亡率要显著高于单独用药(P0.01)。结论:康莱特、顺铂单独或联合作用均能抑制SiHa细胞的增殖,促进其凋亡,且康莱特联合顺铂的作用要显著高于单独用药,康莱特与化疗药物联合使用可提高肿瘤细胞对化疗药物的敏感性。     

MDM2反义寡核苷酸联合紫杉醇对乳腺癌MCF-7细胞株的作用

目的:探讨靶向MDM2反义寡核苷酸(ASON)联合紫杉醇对乳腺癌MCF-7细胞株的影响。方法:合成一段与MDM2 mRNA特异性结合的反义寡核苷酸和与反义寡核苷酸有4个碱基不同的的错义寡核苷酸(MON),脂质体2000介导不同浓度的MDM2ASON转染MCF-7乳腺癌细胞系,转染的乳腺癌细胞通过1μmol/L紫杉醇药物处理后,采用RT-PCR和Western Blot方法检测MDM2 ASON联合紫杉醇的协同作用及对乳腺癌MCF-7细胞株的抑制效率,MTT观察给药后MCF-7细胞的增殖能力和药物敏感性。结果:MDM2反义寡核苷酸联合紫杉醇明显下调MDM2 mRNA及MDM2蛋白表达水平,抑制MCF-7细胞的生长,随着MDM2 ASON浓度的增加,MDM2表达越来越低,协同作用越来越强,呈剂量依赖关系,A500联合紫杉醇的协同作用最明显,MTT显示紫杉醇处理的转染MCF-7细胞增殖抑制率明显增高,A500抑制增殖作用最明显,抑制率达(13.0±0.84)%。结论:不同浓度MDM2 ASON转染后的乳腺癌MCF-7细胞,等浓度紫杉醇处理后,乳腺癌MCF-7细胞MDM2表达明显降低,细胞凋亡增加,,MDM2 ASON联合紫杉醇对MCF-7细胞有协同作用,提高了乳腺癌MCF-7细胞对紫杉醇的药物敏感性。     

In Vitro and In Vivo Anti-influenza A Virus Activity of Antisense Oligonucleotides

Abstract

We have demonstrated that antisense phosphorothioate oligonucleotides (S-ODNs) inhibit influenza virus A replication in MDCK cells. The liposomally encapsulated and the free antisense phosphorothioate oligonucleotides with four target sites (PB1, PB2, PA, and NP) were tested for their abilities to inhibit virus-induced cytopathogenic effects by a MTT assay using MDCK cells. The liposomally encapsulated S-ODN complementary to the sites of the PB2-AUG initiation codon showed highly inhibitory effects. Therefore, the antiviral effects of S-ODN-PB2-AUG and PA-AUG were examined in a mouse model of influenza virus A infection. PB2-AUG oligomer treated i.v. significantly prolonged the mean survival time in day (MDS) and increased the survival rates with does dependent manner.     

Identification of basic fibroblast growth factor sensitivity and receptor and ligand expression in human colon tumor cell lines.

Basic fibroblast growth factor (bFGF) has been shown to be mitogenic to many different eukaryotic cell lines of mesodermal and neuroectodermal origin. Addition of exogenous bFGF to the chemically defined media of five characterized human colon tumor cell lines, cultured in the absence of epidermal growth factor (EGF), resulted in stimulation of growth from 24% to 146% in four of five cell lines, as measured by a colorimetric MTT assay. A positive dose-response relationship was observed when colon cells were treated with bFGF concentrations from 1 pM to 1 nM. bFGF showed a cumulative effect with EGF in stimulating the proliferation of colon tumor cells. The growth-inhibitory effect of exogenous transforming growth factor-beta (TGF-beta) on these cells was abolished by bFGF. When colon tumor cells were examined on immunoblots with a fibroblast growth factor (FGF) receptor-specific antibody, bands were detected at apparent molecular weights of 131 and 145 kDa. Conditioned media and cell lysates from the same human colon tumor cell lines were immunoprecipitated with a bFGF-specific antibody. An immunoreactive band was detected that comigrated with authentic human recombinant bFGF (16 kDa). Furthermore, preabsorption of anti-bFGF antibody with authentic ligand blocked immunodetection of the 16 kDa band on immunoblots. Documentation of a bFGF response, receptor, and ligand expression in human colon tumor cell lines is novel, and may represent a more widespread role for FGF that extends to epithelial cells and tumors of endodermal germ layer origin. The expression of both ligand and receptors by these cells indicates that bFGF could be involved in their growth regulation at the autocrine level.     

Glial cell line-derived neurotrophic factor (GDNF) is a proliferation factor for rat C6 glioma cells: evidence from antisense experiments

Growth factors play an important role in proliferation and differentiation of malignant brain gliomas in humans. Glial cell line-derived neurotrophic factor (GDNF) has been shown recently to be highly expressed in human glioblastomas and in rat glial cell lines B49 and C6. The aim of the present study was to knockdown GDNF, its receptor GFR-alpha1, and the related family member persephin by using antisense oligonucleotides and to observe the effects on cell proliferation. To enhance cellular uptake into C6 glioma cells, 15-mer phosphorothioate oligonucleotides were complexed with the cationic lipid Lipofectamine. The complex was applied for 3 x 12 hours to C6 glioma cells, and cells were allowed to recover for 24 hours after each transfection and then analyzed. This protocol markedly reduced GDNF and GFR-alpha1 protein levels in C6 glioma cells compared with control oligonucleotides. Knockdown of C6 cells with GDNF and GFR-alpha1 but not with persephin antisense oligonucleotides significantly decreased the number of C6 glioma cells and also inhibited the incorporation of bromodeoxyuridine as a sign of reduced DNA synthesis. In conclusion, it is shown that GDNF but not persephin is a potent proliferation factor for rat glioma cells. Knockdown of GDNF using antisense oligonucleotides complexed with lipids as carriers may be useful in gene therapeutic approaches in vitro and possibly also in vivo.     

Use of Cationic Lipids to Enhance the Biological Activity of Antisense Oligonucleotides

Abstract

Antisense oligonucleotides bind to specific mRNA or pre-mRNA sequences through Watson-Crick base pairing, resulting in decreased expression of the targeted protein. The use of cationic lipids to enhance cellular uptake of antisense oligonucleotides is reviewed herein. Cationic lipids such as N[1-(2,3-dioleyloxy)propyl]-N, N, N-trimethylammonium chloride (DOTMA) were found to enhance the biological activity of phosphorothioate oligonucleotides by at least 1000-fold in cell culture. Cationic lipid preparations enhanced both the rate and amount of oligonucleotide which associated with cells. In addition, DOTMA markedly changed the subcellular distribution of the oligonucleotide. In the absence of lipid, fluorescein labelled phosphorothioate oligonucleotides accumulated in discrete cytoplasmic structures. In the presence of cationic lipids, the oligonucleotides concentrated within the nucleus, were excluded from nucleoli, and localized in punctate cytoplasmic structures. The accumulation of the oligonucleotide in the nucleus was inhibited by incubation of the cells at 4°C and by monensin, but not by chloroquine, ammonium chloride, or nocodazole. Cell lines, both primary and transformed, differ markedly in their sensitivity to inhibition of gene expression with antisense oligonucleotides in the presence of cationic lipids. The differential sensitivity of the cells correlates with the amount of ³⁵S-labelled oligonucleotide associated with the cells and the number of cells in the population which take up the oligonucleotide. Our studies have demonstrated that several types of cationic lipids markedly enhance the activity of phosphorothioate oligonucleotides in cell culture models. We are currently investigating the ability of cationic lipids to enhance activity of antisense oligonucleotides in more complex systems such as organ cultures and in animals.     

Specific inhibition of transforming growth factor-beta2 expression in human osteoblast cells by antisense phosphorothioate oligonucleotides.

To elucidate the role of endogenous transforming growth factor (TGF)-beta2 on human osteoblast cell, antisense phosphorothioate oligonucleotides (S-ODNs) complementary to regions in mRNA of TGF-beta2 were synthesized and examined their effects on TGF-beta2 production and cell proliferation in a human osteoblast cell line ROS 17/2. Antisense S-ODNs were designated for three different target regions in the mRNA of TGF-beta2. Among several antisense S-ODN analyzed, an oligonucleotide (AS-11) complementary to the translation initiation site of mRNA of TGF-beta2 demonstrated a selective and strong inhibitory effect on TGF-beta2 production in osteoblast cells. Other antisense S-ODNs which were designated for other regions in mRNA of TGF-beta2 and one- or three-base mismatched analogs of AS-11 showed little or much less antisense activities than AS-11. Therefore, the most effective target site in mRNA of TGF-beta2 is at the initiation codon region. The antisense effects of AS-11 were observed without reduction of levels of mRNA of TGF-beta2. Furthermore, the inhibition of TGF-beta2 expression by antisense S-ODN appeared to enhance cell proliferation, demonstrating the growth inhibitory effect of autocrine TGF-beta2 in osteoblast cells.     

Antisense oligonucleotides with different backbones. Modification of splicing pathways and efficacy of uptake.

A novel, positive read-out assay that quantifies only sequence-specific nuclear activity of antisense oligonucleotides was used to evaluate morpholino and 2'-O-methyl sugar-phosphate oligonucleotides. The assay is based on modification of the splicing pathway of human beta-globin pre-mRNA. In addition, scrape-loading of cells with oligonucleotides allows the separate assessment of intracellular antisense activity of the oligonucleotides and their ability to penetrate the cell membrane barrier. The results show that, with scrape-loading, the morpholino oligonucleotides were approximately 3-fold more effective in their intrinsic antisense activity than alternating phosphodiester/phosphorothioate 2'-O-methyl-oligoribonucleotides and 6-9- and almost 200-fold more effective than the exclusively phosphorothioate and phosphodiester derivatives, respectively. The morpholino oligonucleotides were over 20-fold more effective than the phosphorothioate 2'-O-methyl-oligoribonucleotides in free uptake from the culture media. The antisense activity of the morpholino oligonucleotides was detectable not only in monolayer HeLa cells but also in suspension K562 cells. Time course experiments suggest that both the free uptake and efflux of morpholino oligonucleotides are slow.     

Suppression of basic fibroblast growth factor expression by antisense oligodeoxynucleotides inhibits the growth of transformed human astrocytes.

Basic fibroblast growth factor (bFGF) is a heparin-binding protein expressing potent mitogenic and angiogenic properties. Elevated levels of bFGF have recently been described in human glioma cell lines. The high degree of vascularity and invasiveness which characterize human gliomas suggest that activated expression of bFGF or similar proteins may be related to the aberrant growth patterns of these tumors. The influence of endogenous bFGF on glioma cell growth in vitro was evaluated in the present study by down-regulating bFGF expression using antisense oligonucleotide primers. The addition of 50 microM bFGF-specific antisense primer to the human glioma cell line SNB-19 resulted in an 80% inhibition in glioma growth. This effect was saturable and specific. Antisense primers directed to two different sites of bFGF mRNA were effective in suppressing SNB-19 growth, whereas sense strand primer was ineffective. Furthermore, only the antisense primer significantly reduced the specific activity of bFGF protein in SNB-19 cell extracts. Neither antisense or sense primers inhibited the growth of non-transformed human glia. bFGF mRNA was detected in both transformed and non-transformed human glia by polymerase chain reaction analysis suggesting that alterations in bFGF isoform content or activity may be specifically related to abnormal growth control in human gliomas.