反义寡核苷酸抑制肝癌血管内皮生长因子表达

目的 探讨不同序列的反义寡核苷酸对肝癌细胞血管内皮生长因子(VEGF)表达的抑制效果。 方法 SMMC7721细胞于正常氧和缺氧条件下培养24 h,观察缺氧对VEGF mRNA表达的影响;SMMC7721细胞接种于6孔板后,各加入不同的反义寡核苷酸(反帽子结构A06513、反翻译起始部位A06514、反第三外显子A06515、反翻译终止部位A06516和空白组),缺氧培养24 h,然后收集细胞提取总RNA并作逆转录聚合酶链反应分析,同时以管家基因GAPDH(3-磷酸甘油醛脱氢酶)表达为内对照。 结果 缺氧条件下,SMMC7721细胞VEGF mRNA表达明显增强,而有氧条件下未见表达;反义寡核苷酸具有明显抑制VEGF mRNA表达,和空白对照组相比,A06513、A06514、A06515及A06516的VEGF/GAPDH值分别为0.49±0.08、0.71±0.12、0.72±0.11及0.86±0.12(F=12.21,P<0.05)。帽子结构的反义基因表现最强的抑制作用(P<0.0 1)。 结论 与VEGF帽子结构作用的反义寡核苷酸,有望成为肝癌反义基因治疗的新选择。     

VEGF反义寡核苷酸对体外生长的大肠癌HT-29细胞的抑制作用

目的:探讨血管内皮生长因子(VEGF)反义寡核苷酸(ASODN)对体外生长的人大肠癌HT-29细胞的抑制作用.方法:实验设空白对照组、脂质体转染组、错义链转染组(SODN组)和不同浓度反义链转染组(ASODN组).用LipofectamineTM2000介导的VEGF ASODN和错义寡核苷酸(SODN)转染人大肠癌细胞株HT-29,半定量RT-PCR检测各组细胞VEGF mRNA的表达:Western blot测定转染48、72 h后VEGF蛋白表达:MTT法和流式细胞术检测细胞增殖和凋亡.结果:转染48 h后,ASOND组的VEGF mRNA表达水平明显低于脂质体对照组和SODN组(0.455±0.032 vs 0.934±0.031,0.915±0.004,p<0.01);脂质体对照组与SODN组之间无显著差异.细胞转染48、72 h后,ASODN组蛋白表达明显弱于脂质体对照组和SODN组,且72h弱于48 h.与对照组比较.VEGF ASODN对HT-29细胞有明显的生长抑制作用,并且抑制呈剂量和时间依赖性(P<0.05).结论:VEGF ASODN通过抑制VEGF的基因表达,对体外生长的人大肠癌HT-29细胞的增殖进行抑制.     

反义VEGF腺病毒的制备及其对类风湿关节炎滑膜细胞VEGF分泌的影响

目的 制备反义血管内皮生长因子（VEGF）腺病毒表达载体，为反义VEGF转基因治疗类风湿关节炎（RA）奠定基础。方法 采用基因克隆的方法制备高滴度的反义VEGF腺病毒，并将其感染培养的RA滑膜细胞，观察期对RA滑膜细胞分泌VEGF的影响。结果 成功制备了反义VEGF腺病毒，并观察到，在一定浓度范围内，反义VEGF腺病毒可以有效抑制RA滑膜细胞分泌VEGF，浓度越高，抑制效果越明显。结论 制备的反义VEGF腺病毒可以高效抑制RA滑膜细胞分泌VEGF，为探索RA的反义VEGF基因疗法奠定了基础。     

VEGF反义寡核苷酸对胆囊癌细胞VEGF,Flt-1及KDRmRNA表达和VEGF蛋白分泌的影响

目的:体外研究Oligofectamine介导的VEGF反义寡核苷酸(antisense oligodeoxynucleotide,ASODN)转染对人胆囊癌GBC-SD细胞VEGF,Flt-1及KDR mRNA表达和VEGF蛋白分泌的影响.方法:运用Oligofectamine介导的VEGF反义寡核苷酸(ASODN)和错义寡核苷酸(Scrambled Oligodeoxynucleotide,SODN)转染人胆囊癌细胞GBC-SD,半定量RT-PCR检测转染后各组细胞不同时间的VEGF,Flt-1及KDR mRNA表达变化,ELISA测定转染后各组细胞培养上清液VEGF蛋白浓度.结果:半定量RT-PCR发现ASODN组及ASODN Oligofectamine组24,48,72,96 h VEGF(ASODN组VEGF165:0.686±0.033,0.569±0.049,0.489±0.036,0.716±0.017;ASODN组VEGF121:0.462±0.046,0.338±0.034,0.219±0.022,0.471±0.038;ASODN Oligofectamine组VEGF165:0.601±0.021,0.465±0.042,0.416±0.023,0.662±0.035:ASODN Oligofectamine组VEGF121:0.408±0.014,0.286±0.019,0.157±0.021,0.418±0.037)、Flt-1(ASODN组:0.694±0.019,0.562±0.045,0.435±0.042,0.724±0.026;ASODN Oligofectamine组:0.609±0.018,0.442±0.049,0.314±0.015,0.614±0.029)及KDR(ASODN组:0.667±0.063,0.490±0.033,0.301±0.029,0.665±0.068;ASODN Oligofectamine组:0.523±0.048,0.432±0.027,0.218±0.036,0.524±0.037) mRNA的表达显著低于Control组(P<0.05),且ASODN Oligofectamine的抑制作用比ASODN强(P>0.05).ELISA测定结果显示ASODN组(281.26±18.62,526.44±34.95,791.13±20.99)及ASODN Oligofectamine组(250.7±14.57,506.09±19.14,711.79±19.91)24,48,72 h VEGF蛋白的分泌浓度均显著低于Control组(394.23±16.26,711.6±26.21,933.85±28.65)(P<0.05),且ASODN Oligofectamine的抑制作用比ASODN强(P>0.05).结论:Oligofectamine介导的VEGF ASODN能抑制GBC-SD细胞VEGF,Flt-1及KDR mRNA表达和VEGF蛋白分泌.     

VEGF165正反义表达载体的构建及其对人胃癌细胞VEGF表达的调节

目的构建VEGF165(vascular endothelial growth factor,血管内皮生长因子)正、反义表达载体,将其转染人胃癌细胞,观察其对胃癌细胞VEGF在mRNA和蛋白质水平表达的调节作用.方法用限制性内切酶将VEGF165 cDNA从pGEM-hVEGF165克隆载体上切下来,以正、反方向插入质粒pCDNA3中,构建VEGF165正、反义真核表达载体;用限制性内切酶和Sanger双脱氧终止DNA测序法证明VEGF165正、反义表达载体目的基因的序列和方向的正确性;用脂质体将VEGF165正、反义表达载体转染人胃癌细胞,用RNA斑点杂交及免疫荧光流式细胞仪检测VEGF mRNA和蛋白质的表达水平.结果酶谱分析及DNA测序表明VFGF165正、反义真核表达载体目的基因的序列及方向正确;转染正义表达载体后VEGF mRNA和蛋白质表达水平增加(蛋白免疫强度为75.4%;P＜0.05vs对照组,31.6%),转染反义表达载体后VEGF mRNA和蛋白表达水平降低(蛋白免疫强度为8.9%;P＜0.05vs对照组).结论成功地构建了VEGF165正、反义真核表达载体;构建的载体能在胃癌细胞内有效表达,调节血管内皮生长因子的水平.     

IL-21对人上皮性卵巢癌SKOV3细胞增殖及VEGF表达的影响

通过噻唑兰(MTT)比色法检测观察白细胞介素21(IL-21)对人上皮性卵巢癌细胞SKOV3细胞生长的影响;流式细胞仪观察IL-21对SKOV3细胞周期的作用;免疫细胞化学检测IL-21对血管内皮生长因子(VEGF)表达水平的影响.发现IL-21对SKOV3细胞生长有明显抑制作用,呈剂量和时间依赖关系;随着IL-21浓度升高,VEGF的表达水平逐步下降.认为IL-21可以抑制人上皮性卵巢癌细胞的生长和增殖,其机制可能与降低VEGF的表达有关.     

反义血管内皮生长因子腺病毒基因感染对类风湿关节炎滑膜细胞VEGF基因转录的影响

目的 观察反义血管内皮生长因子(VEGF)165腺病毒对滑膜细胞VEGF基因转录的影响。方法 将反义VEGF165腺病毒转染滑膜细胞，通过Northern-blot的方法检测其对VEGF的基因转录的影响。结果 Northem杂交结果显示：反义基因转染滑膜细胞3d后，VEGF165基因的转录即受到明显抑制，4d抑制更加明显，5d后几乎检测不出阳性杂交信号。结论 反义VEGF165腺病毒感染滑膜细胞后可以阻断VEGF基因的转录过程，从而抑制VEGF蛋白质的翻译和表达。     

粘着斑激酶反义寡核苷酸与胃癌细胞生长及凋亡的关系

根据FAKcDNA序列设计合成与FAKmRNA碱基序列互补的FAK反义寡核苷酸,导入BGC－823胃癌细胞,通过MTT比色法、细胞免疫化学染色法、逆转录－聚合酶链反应（RT－PCR）、细胞周期及电镜等方法,检测FAK反义寡核苷酸对人胃癌细胞的影响。结果示：①胃癌细胞生长明显受到抑制,抑制效应呈浓度和时间依赖性。②细胞内P125蛋白和FAKmRNA表达均明显下降。③经FAK反义寡核苷酸处理后BGC－823胃癌细胞发生凋亡,FCM普上可见明显的凋亡峰,电镜观察呈现凋亡早期形态改变,认为FAK反义寡核甘酸导入BGC－823胃癌细胞后,使BGC－823胃癌细胞FAKmPNA及P125蛋白表达水平下降,抑制PGC－823胃癌细胞增列,同时诱导BGC－823胃癌细胞发生凋亡。     

bcl-2反义寡核苷酸与VP16协同抑制小细胞肺癌细胞增殖的研究

目的 研究bcl-2反义寡核苷酸与足叶乙甙（VPl6）联用对小细胞肺癌细胞NCI—H69增殖的影响。方法 通过脂质体将不同寡核苷酸导入NCI—H69细胞中，分为反义寡核苷酸组、正义寡核苷酸组、无义寡核苷酸组和空白对照组4组。Western Blot法检测细胞bcl-2蛋白的表达。不同浓度的VP16作用于4组转染后的细胞，MTT法测定细胞存活分数。结果 4组细胞中。与空白对照组相比较，反义寡核苷酸组的bcl-2蛋白表达明显受到抑制。bcl-2反义寡核苷酸转染细胞与VP16作用后，反义寡核苷酸组细胞在各个浓度的存活分数均低于对照组，且有统计学意义（P〈0．01）。结论 bcl-2反义寡核苷酸与VP16能协同抑制小细胞肺癌细胞的增殖。     

齐墩果酸对K562细胞系VEGF表达影响的实验研究

目的观察齐墩果酸(Oleanolic Acid,OA)对慢性粒细胞白血病系K562细胞中血管内皮生长因子(VEGF)表达的影响。方法采用MTT实验观察OA对体外培养K562细胞增殖抑制作用,用RT-PCR以及Western印迹法研究OA处理后K562细胞中VEGF基因表达的变化。并用ELISA方法检测培养液中VEGF的表达。结果OA能抑制K562细胞生长,呈一定的量效特征;并能抑制K562细胞中VEGF的表达。结论OA通过下调VEGF表达,抑制白血病细胞的增殖。     

膀胱移行细胞癌VEGF和VEGFR的表达及相关性的探讨

目的　探讨膀胱移行细胞癌 (BTCC)中血管内皮生长因子 (VEGF)及其受体 (VEGFR)的表达及与两者之间的关系。方法　采用免疫组织化学链霉菌抗生物素 过氧化物酶连接法 (S P法 )对 30例BTCC及 1 0例正常膀胱黏膜组织中VEGF及VEGFR的表达进行检测。结果　VEGF和VEGFR在绝大多数BTCC中呈阳性表达 ,平均表达率分别为 87%和 73 %。随肿瘤分期和分级的升高其表达水平升高 ,但在正常膀胱组织中未见表达。结论　BTCC中VEGF和VEGFR表达阳性 ,提示其在BTCC的血管生成和侵袭进展过程中起着重要作用 ,并将有可能为BTCC抗血管形成治疗及预防提供新的思路     

VEGF165 antisense RNA suppresses oncogenic properties of human esophageal squamous cell carcinoma

AIM: To investigate the effect of antisense RNA to vascularendothelial growth factor165 (VEGF165) on human esophagealsquamous cell carcinoma call line EC109 and the feasibilityof gene therapy for esophageal carcinoma.METHODS: Using subclone technique, the full length ofVEGF165 amino acid cDNA, which was cut from pGEM-3Zf( + ), was cloned inversely into the eukaryotic expressionvector pCEP4 . The recombinant plasmid pCEP-AVEGF165was transfected into EC109 cell with Iipofectamine. After astable transfection, dot Blot, enzyme-linked immunosorbentassay (ELISA), laser confocal imaging system analysis,transmission electron microscopy and flow cytometry wereperformed to determine the biological characteristics ofEC109 cell line before and after transfection in vitro andwhether there was a reversion in the tumorigenic propertiesof the EC109 cell in vivo.RESULTS: The eukaryotic expression vector pCEP-AVEGF165was successfully constructed and transfected into EC109cells. The expression of VEGF165 was significantly decreasedin the transfected cells while the biological characteristics ofthe cells were not influenced by the expression of antisensegene. The tumorigenic and angiogenic capabilities weregreatly reduced in nude mice, as demonstrated by reducedtumorend volume (820 ± 112.5)mm3 vs (7930 ± 1035) mn3and (7850 ± 950) mm3, P ＜ 0.01) and microvessel density(8.5 ± 1.2)mm-2 vs (44.3 ± 9.4)mm-2 and (46.4 ± 12.6)mm-2, P ＜ 0.01) in comparison between experimentalgroup, empty vector transfected group and control group.CONCLUSION: The angiogenesis and tumorigenicity ofhuman esophageal squamous cell carcinoma were effectivelyinhibited by VEGF165 antisense RNA. Antisense RNA toVEGF1, can potentially be used as an adjuvant therapy forsolid tumors.     

奥曲肽对人子宫内膜癌HEC-1-B细胞增殖及其血管内皮生长因子表达的影响

目的探讨奥曲肽对人子宫内膜癌细胞增殖及其血管内皮生长因子（VEGF）表达的影响。方法加入不同浓度的奥曲肽对HEC-1-B细胞进行培养。细胞增殖用MTT法检测，细胞中VEGF的表达采用RT-PCR技术及酶联免疫吸附试验（ELISA）检测。结果加入奥曲肽培养后，HEC-1-B细胞的增殖受抑制．而且其VEGFmRNA指数降低，VEGF蛋白表达减少。结论奥曲肽能抑制人子宫内膜癌细胞增殖及VEGF的分泌。     

Vascular endothelial growth factor/vascular permeability factor is an autocrine growth factor for AIDS–Kaposi sarcoma

Kaposi sarcoma (KS) is the most common tumor associated with HIV-1 infection and develops in nearly 30% of cases. The principal features of this tumor are abnormal vascularization and the proliferation of endothelial cells and spindle (tumor) cells. KS-derived spindle cells induce vascular lesions and display enhanced vascular permeability when inoculated subcutaneously in the nude mouse. This finding suggests that angiogenesis and capillary permeability play a central role in the development and progression of KS. In this study, we show that AIDS–KS cell lines express higher levels of vascular endothelial growth factor/vascular permeability factor (VEGF/VGF) than either human umbilical vein endothelial cells or human aortic smooth muscle cells. AIDS–KS cells and primary tumor tissues also expressed high levels of Flt-1 and KDR, the receptors for VEGF, while the normal skin of the same patients did not show any expression. We further demonstrate that VEGF antisense oligonucleotides AS-1 and AS-3 specifically block VEGF mRNA and protein production and inhibit KS cell growth in a dose-dependent manner. Furthermore, growth of KS cells in nude mice was specifically inhibited by VEGF antisense oligonucleotides. These results show that VEGF is an autocrine growth factor for AIDS–KS cells. To our knowledge, this is the first report that shows that VEGF acts as a growth stimulator in a human tumor. Inhibitors of VEGF or its cognate receptors may thus be candidates for therapeutic intervention.     

Vascular endothelial growth factor (VEGF) is an autocrine growth factor for VEGF receptor-positive human tumors

Angiogenesis is required for the progression of tumors from a benign to a malignant phenotype and for metastasis. Malignant tumor cells secrete factors such as vascular endothelial growth factor (VEGF), which bind to their cognate receptors on endothelial cells to induce angiogenesis. Here it is shown that several tumor types express VEGF receptors (VEGFRs) and that inhibition of VEGF (VEGF antisense oligonucleotide AS-3) or VEGFRs (neutralizing antibodies) inhibited the proliferation of these cell lines in vitro. Furthermore, this effect was abrogated by exogenous VEGF. Thus, VEGF is an autocrine growth factor for tumor cell lines that express VEGFRs. A modified form of VEGF AS-3 (AS-3m), in which flanking 4 nucleotides were substituted with 2-O-methylnucleosides (mixed backbone oligonucleotides), retained specificity and was active when given orally or systemically in vitro and in murine tumor models. In VEGFR-2-expressing tumors, VEGF inhibition may have dual functions: direct inhibition of tumor cell growth and inhibition of angiogenesis.     

血管内皮生长因子反义寡核苷酸抑制甲状腺癌血管生成的效应

目的探讨反义寡核苷酸(ASODN)抑制甲状腺癌细胞血管内皮生长因子(VEGF)表达及内皮细胞生长的效应。方法设计合成靶向VEGF的ASODN转染人髓状甲状腺癌细胞系(TT)细胞,并制备相应条件培养基作用内皮细胞ECV304,设正义寡核苷酸(SODN)和空白对照组进行比较。观察细胞生长状态,RT PCR、免疫细胞化学法检测TT细胞VEGFmRNA和蛋白表达,四氮唑蓝法检测TT和ECV304细胞生长抑制率(IR),流式细胞仪、吖啶橙/溴化乙锭染色法检测ECV304细胞凋亡状态。结果ASODN组TT细胞VEGFmRNA和蛋白表达显著低于SODN和对照组(P<0.01),但IR差异无统计学意义(P>0.05);各转染组ECV304细胞IR差异亦无统计学意义(P>0.05);而经各ASODN组TT细胞条件培养基作用的ECV304细胞生长明显受抑,IR(分别为0.21±0.03、0.31±0.01、0.42±0.22)显著高于SODN组(0.05±0.03,P<0.01),并伴明显细胞凋亡,上述效应呈浓度依赖性。结论ASODN可通过特异性封闭甲状腺癌细胞VEGF表达,抑制内皮细胞生长,干扰肿瘤血管生成。     

Blocking effects of siRNA on VEGF expression in human colorectal cancer cells

AIM:To investigate the expression of vascular endothelial cell growth factor (VEGF) and its receptors Fmslike tyrosine kinase 1 (FLT-1) and fetal liver kinase 1 (FLK-1) in colorectal carcinoma (CRC),and the blocking effects of small interfering RNAs (siRNAs) on VEGF expression in human colorectal cancer HCT116 cells.METHODS:Immunohistochemical staining for VEGF,FLT-1 and FLK-1 proteins was performed in 82 cases of CRC and 14 normal colorectal mucosae.A siRNA targeting VEGF was synthesized and transfected into HCT116 cells using lipofectamine 2000.Immunocytochemical staining and Western blotting analyses were performed to detect the expression of VEGF protein.The suppressive effect of the siRNA on cell proliferation was detected using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltertrazolium bromide (MTT) assay.Cellular apoptosis was detected using flow cytometry (FCM).RESULTS:The expression of VEGF,FLT-1 and FLK-1 in tumor tissues was significantly higher than that in normal tissues (P=0.008,P=0.000,P=0.000).The expression of VEGF was positively correlated with both lymph node metastasis and clinical stage (P=0.009 and P=0.025,respectively).Immunocytochemistry showed that the expression of VEGF was weakly positive and Western blotting indicated a significant reduction in VEGF-siRNA cell protein levels.VEGF-siRNA cell growth inhibition was assessed by the MTT assay,and the tumor cell proliferation rate was significantly different at 24,48,and 72 h after transfection.FCM results showed that the VEGF-siRNA group had an apparent aneuploid peak.CONCLUSION:VEGF,FLT-1 and FLK-1 are associated with colorectal carcinogenesis.siRNA silencing of the VEGF gene suppresses proliferation,and induces apoptosis in HCT116 cells.The results suggest that VEGF may be a new gene therapy target for colorectal cancer.