反义寡核苷酸抑制肿瘤细胞表达VEGF的研究

研究用反义寡核苷酸（ＯＤＮ）抑制肿瘤细胞ＶＥＧＦ的表达，探讨发展新的抗肿瘤药物的可能性。方法Ｓ１８０细胞培养上清液有促进牛脐血管内皮细胞生长的作用。用ＶＥＧＦ的反义ＯＤＮｓ分别加入Ｓ１８０细胞培养液，检查各上清液刺激内皮细胞生长的受抑情况，即可了解ＯＤＮｓ抑制肿瘤细胞表达ＶＥＧＦ的情况。结果证明ＶＥＧＦ反义ＯＤＮｓ确有抑制ＶＥＧＦ表达的作用。结论反义ＶＥＧＦ的ＯＤＮｓ有望成为新一代的抗肿瘤药物。     

VEGF反义寡核苷酸对C_6胶质瘤细胞VEGF表达和内皮细胞生长的抑制作用

目的　探讨VEGF反义寡核苷酸作为抗肿瘤血管生成治疗新药的可能性。方法　观察VEGF反义、正义、错义寡核苷酸制备的条件培养基对牛血管内皮细胞生长的抑制作用。应用免疫荧光流式细胞技术观察三者分别对C6胶质瘤细胞VEGF表达的影响。结果　反义寡核苷酸对C6胶质瘤细胞VEGF的表达和内皮细胞生长有明显抑制作用 ,而且抑制作用存在浓度依赖性。结论　反义VEGF寡核苷酸通过抑制C6胶质瘤细胞VEGF的表达 ,进而抑制内皮细胞的生长。     

儿童恶性胶质瘤VEGF表达与血管生成及细胞增殖的关系

研究血管内皮细胞生长因子在儿童恶性胶质瘤的表达及其与血管生成,细胞增殖的关系。方法免疫组化方法检测３３例儿童恶性胶质瘤组织中ＶＥＧＦ表达与微血管数、ＰＣＮＡ标记指数。结果３３例儿童恶性胶质瘤ＶＥＧＦ表达阳性２３例;     

VEGF反寡核苷酸对C6胶质瘤细胞VEGF表达和内皮细胞生长的抑制作用

目的 探讨VEGF反认寡核苷酸作为抗肿瘤血管生成治疗新药的可能性。方法 观察VEGF反义、正义、错义寡核苷酸制备的条件培养基对牛血管内皮细胞生长的抑制作用。应用免疫荧光流式细胞技术观察三者分别C6胶质瘤细胞VEGF表达的影响。结果 反义寡核苷酸对C6胶质瘤细胞VEGF的表达和内皮细胞生长有明显抑制作用,而且抑制作用存在浓度依赖性。结论 反义VEGF寡核苷酸通过抑制C6胶质瘤细胞VEGF的表达,进而抑制内皮细胞的生长。     

VEGF反义寡核苷酸对Lewis肺癌细胞的抑制作用

目的:〖HT5"SS〗 探讨血管内皮生长因子（vascular endothelial growth factor, VEGF）反义寡核苷酸对C57BL/6小鼠肺癌细胞的抑制作用。〖HT5W〗方法:〖HT5"SS〗 制作C57BL/6小鼠皮下肺癌模型40只,分为VEGF反义寡核苷酸（ASODN）治疗组、VEGF正义寡核苷酸(SODN)治疗组、VEGF错义寡核苷酸（MODN）治疗组及对照组。接种Lewis肺癌细胞后24 h内,用ASODN、SODN及MODN皮下注射进行治疗,对照组只注射生理盐水,观察小鼠肿瘤的生长情况以及组织形态学改变,标本常规石蜡切片,HE染色,用免疫组化方法检测VEGF蛋白表达。〖HT5W〗结果:〖HT5"SS〗 对照组、ASODN组、SODN组、MODN组平均瘤重分别为（7.33±0.71）g、（4.56±0.38）g、(7.59±0.32)g和（7.62±0.39）g,ASODN组、SODN组、MODN组抑瘤率分别为43.8%、5.5%、3.1%。光镜下观察, VEGFASODN 能明显抑制肿瘤细胞生长,降低增殖活性。 免疫组化结果表明,ASODN组VEGF的表达水平明显低于SODN组、MODN组及对照组（P<0.05）。CD34免疫组化结果表明,ASODN组MVD为8.25±2.12,与对照组(14.78±3.51)、SODN组(13.71±3.62)及MODN组(12.81±2.56)比较,ASODN组MVD明显减少（P<0.01）。〖HT5W〗结论: 〖HT5"SS〗原位注射VEGF反义寡核苷酸能抑制小鼠肺癌生长。     

VEGF反义RNA 抑制裸鼠体内食管癌细胞生长的实验研究

 目的 探讨VEGF反义RNA对食管癌细胞在裸鼠体内生长的影响。方法 观察转染VEGF反义RNA的食管癌细胞TE-1在裸鼠体内成瘤性；应用免疫组化法、RT—PCR法检测移植瘤组织中VEGF蛋白及mRNA表达和MVD；用流式细胞仪和透射电镜检测细胞凋亡情况。结果 转染VEGF反义RNA的TE-1细胞在裸鼠体内肿瘤形成的潜伏期明显延长，所形成肿瘤的重量和体积小于对照组及空载体组。肿瘤组织中VEGF蛋白和mRNA的表达降低，MVD降低；肿瘤细胞发生明显的凋亡形态学改变；G0／G1期细胞明显增多，S期细胞明显减少，细胞增殖能力降低。结论 VEGF反义RNA能抑制裸鼠体内TE-1细胞的生长，减少肿瘤内血管的生成，增加细胞凋亡；为食管癌的基因治疗提供了一定的实验依据。     

阳离子脂质体VEGF反义寡核苷酸对白血病细胞凋亡的影响

目的观察阳离子脂质体VEGF反义寡核苷酸对白血病细胞凋亡的作用。方法应用阳离子脂质体VEGF ASODN转染H160细胞48h后，观察细胞形态，电泳法检测DNA梯状带和流式细胞仪Annexin V—FTC／PI检测细胞凋亡。结果VEGF ASODN组VEGF mRNA的表达完全抑制，VEGF MSODN组和空白对照组的VEGF、mRNA的表达没有明显变化，细胞生长受抑制可见细胞固缩，DNA电泳出现典型的梯状条形带，流式细胞仪分析细胞凋亡率可达23．28％，明显高于VEGF MSODN组的8．21％和空白对照组。结论VEGF ASODN可抑制白血病细胞VEGF mRNA的表达，导致白血病细胞的凋亡，VEGF基因与白血病细胞的凋亡有关。     

反义寡核苷酸对 Lewis肺癌细胞合成VEGF的抑制作用

背景与目的 血管内皮生长因子(VEGF)是一种特异地作用于血管内皮细胞的生长因子,它能促进血管内皮细胞增殖及新生血管的生成.本研究的目的是探讨脂质体介导的VEGF反义硫代寡核苷酸(ASODN)对培养的Lewis肺癌细胞合成VEGF的影响.方法 将经脂质体包裹并经部分硫代修饰后的VEGF ASODN、正义寡核苷酸(SODN)加入培养的Lewis肺癌细胞中,采用RT-PCR技术和免疫组织化学SP法检测肺癌细胞中VEGF mRNA和VEGF蛋白表达,同时测定各上清液刺激下血管内皮细胞生长的受抑情况.结果 VEGF ASODN能明显抑制Lewis肺癌细胞VEGF mRNA和VEGF蛋白表达,而且可以显著抑制内皮细胞的生长.结论 VEGF ASODN能抑制肺癌细胞表达VEGF,可以成为肺癌基因治疗的一种新的途径.     

125IUdR偶联c-myb反义寡核苷酸对大鼠C6脑胶质瘤细胞增殖的影响及其相关机制

目的:探讨125IUdR偶联c-myb反义寡核苷酸对C6脑胶质瘤细胞增殖和凋亡的影响及可能的机制。方法:60只大鼠随机余数法分成对照组、c-myb-ASODN处理组和125IUdR-ASODN处理组。MTT和TUNEL法检测各组大鼠肿瘤细胞增殖抑制率和凋亡率;免疫组化法检测Bax和Bcl-xl蛋白表达;RT-PCR法检测BaxmRNA和Bcl-xlmRNA含量;Western-blot法检测c-myb蛋白表达。结果:125IUdR-ASODN处理组C6胶质瘤细胞的增殖抑制率和细胞凋亡率(69·81%和11·03%)增高均较c-myb-ASODN处理组(40·37%和6·48%)增高,P均<0·01;与对照组凋亡率(0·95%)比较,差异有统计学意义,P均<0·01。c-myb-ASODN处理组和125IUdR-ASODN处理组C6胶质瘤细胞的Bax蛋白194·6±6·25增高及Bcl-xl蛋白59·0±3·56下降较ASODN组Bax蛋白147·4±5·23增高及Bcl-xl蛋白83·9±3·93下降,P均<0·01;125IUdR-ASODN和ASODN处理组BaxmRNA表达随着时间延长而显著升高,Bcl-xlmRNA及c-myb蛋白表达随着时间延长而显著下降,P均<0·01;且125IUdR-ASODN的作用明显强于c-myb-ASODN,P<0·01。结论:c-myb-ASODN和125IUdR-ASODN均具有促进C6胶质瘤细胞凋亡和抑制其增殖的作用,且125IUdR-ASODN的作用更为显著;c-myb-ASODN和125IUdR-ASODN可能通过Bax/Bcl-xl途径诱导C6胶质瘤细胞凋亡,其对Bax和Bcl-xl的调节发生在转录水平前的某一环节。     

Inhibition effect of vascular endothelial growth factor antisense oligodeoxynucleotides on C6 glioma

Objective: To explore the probability of vascular endothelial growth factor (VEGF) antisense as a developing new therapeutic strategy for glioma. Methods: VEGF protein expression was detected by S-P immunohistochemical technique.Tumor cell apoptosis was observed by TUNEL method.Results: Compared with control, VEGF protein expression was inhibited by antisense oligodeoxynucleotides in vitro.And the inhibitory effects increased with the increasing concentration. VEGF positive rate was 82.10% in control group, while in 2.5, 5, 10 μmol/L AODN groups, they were 70.00%, 57.85%, 53.20% respectively. No inhibition effect was found in the cell lines treated with missense and senseolig odeoxynucleotides. In vivo, antisense oligodeoxy-nucleotides therapy also inhibited VEGF protein expression and induced the increase of apoptotic tumor cells. However,it has no effect on tumor cell proliferation. Conclusion: It is hopeful that VEGF antisense oligodeoxynucleotides may be a new gene therapy method to glioma through its antiangiogenesis effect by inhibition of VEGF.     

Experimental study on the gene therapy of malignant glioma with antisense VEGF RNA

Objective: To study the effect of antisense VEGF RNA on rat C6 gliomas in vivo and find out the feasibility of antiangiogenesis therapy with antisense VEGF RNA formalignant gliomas. Methods: Parental rat C6 glioma cells and C6 cells transfected with antisense VEGF cDNA were implanted intracerebrally and subcutaneously into SD rats as control and transfected group. Rats bearing cerebral and subcutaneous C6 gliomas were treated with antisense VEGF cDNA as treated group and sense VEGF cDNA and empty vector as control of treated group. The general manifestation, survival time, MRI and histopathological changes of all rats were observed. The volume of subcutaneously implanted tumors was determined regularly. In situ hybridization and immunohistochemical staining were used for detection of VEGF gene expression of gliomas while PCNA immunostaining and TUNEL method for examination of proliferation activity and apoptosis of gliomas, respectively. Results: The survival of the rats in transfected and treated group was prolonged.There were two rats surviving over 90 d in the treated group and their tumors disappeared. The VEGF gene expression, the number of microvessels and the proliferation activity were decreased and a large amount of apoptotic cells could be found in cerebral and subcutaneous gliomas in treated and transfected groups. Conclusion:VEGF is one of the candidate genes for gene therapy of malignant gliomas. Antisense VEGF RNA combined with other therapies should be studied further for enhancing the therapeutic effect of malignant gliomas.     

可溶性VEGF受体基因sflt-1与反义VEGF核苷酸对新生血管形成的影响

背景与目的:肿瘤组织中新生血管提供的大量营养物质和生长因子是肿瘤快速生长的关键,因此如何抑制肿瘤组织中新生血管的形成、促使肿瘤组织坏死也是肿瘤治疗中的一条值得探讨的途径。本文拟研究和观察可溶性血管内皮细胞生长因子(vascularendothelialgrowthfactor,VEGF)受体基因sflt-1与反义VEGF对新生血管形成的影响。方法:用Ad-反义VEGF感染肝癌细胞株MM45T.Li后,观察反义VEGF对MM45T.Li分泌VEGF的影响;将人工重组的3'ΔFlt-1蛋白(C末端缺失的Flt-1蛋白)加入条件培养液中,观察3'ΔFlt-1对VEGF刺激的脐静脉血管内皮细胞(humanumbilicalvascularendotheliumcell,HUVEC)增殖的影响;并将Ad-反义VEGF、Ad-sflt-1注射于鸡胚尿囊绒毛膜中,观察sflt-1、反义VEGF对鸡胚新生血管形成的影响。结果:反义VEGF重组腺病毒感染MM45T.Li细胞后,细胞培养上清中VEGF浓度仅为对照组中VEGF浓度的15%(P<0.01);在含有3'ΔFlt-1蛋白的调价培养液中,HUVEC的增殖明显减慢,在一定的范围内与剂量呈负相关;两者都能有效地抑制鸡胚新生血管的形成。结论:sflt-1与反义VEGF均能有效抑制新生血管的形成,两者联合能增强抑制效果,但其作用机制不同。     

Celastrol inhibits the growth of human glioma xenografts in nude mice through suppressing VEGFR expression

Celastrol, a compound purified from Tripterygium wilfordii whose preparations have been used for clinical treatment for rheumatoid arthritis, has been demonstrated to have antiangiogenic activity, and be inhibitory against mice tumor growth by a few recent studies. However, whether its antiangiogenic activity plays a role in the celastrol-mediated suppression of tumor growth and the molecular basis of anti-tumor activity are poorly understood. In this study, we found that celastrol inhibited the growth of human glioma xenografts in mice, which concurred with the suppression of angiogenesis. Interestingly, while celastrol had no effect on either the expression of VEGF or its mRNA levels, celastrol treatment lowered the expression levels of its receptors (VEGFR-1 and VEGFR-2) and their mRNA levels. These findings suggest that celastrol have potential to be used as an antiangiogenesis drug through its role in suppressing VEGF receptors expression that might consequently reduce the signal transduction between VEGF and VEGFR.     

The effect of antisense epidermal growth factor receptor (EGFR) RNA on the proliferation of human glioma cells and induction of cell apoptosis

Malignantgliomaisoneofthemostdevastatingdiseases.Theprognosisofpatientshasnotbeenimprovedevenwhenthecurrentlyavailablec0mbinedtherapiesareused.Astheknowledge0ftumorbi0l0gyandmoleculargeneticshasgreatlyincreasedoverthepastdecades,ithasbeenshownthattumorigenesisbasicallyresultsfromtheactivation0fprotooncogenesandinactivati0n0ftumorsuppressorgenes.Sincegenesencodinggrowthfactorsandtheirreceptorsarecloselyrelatedtooncogenes,muchattentionhasbeenfocusedontheroleofgrowthfactorsandtheirreceptorsinthep…     

Treatment of the T98G glioblastoma cell line with antisense oligonucleotides directed toward mRNA encoding transforming growth factor-α and the epidermal growth factor receptor

Antisense oligonucleotides (oligos) complementary to mRNA encoding transforming growth factor-α (TGF-α) and its target, the epidermal growth factor receptor (EGFR), are efficacious against human prostate and breast cancers carried in athymic nude mice. Glioblastomas, also regulated by EGFR expression, would appear to be similarly susceptible, and we now employ them against the T98G tumor model. T98G cells were distributed into wells and allowed to adhere prior to addition of oligos (12.5 μM) directed against TGF-α and/or EGFR for 6 d of treatment before thymidine radiolabeling. Supplemental media and oligos (25 μM final concentration) were added after d 3. Statistically significant inhibition by oligos directed against TGF-α, EGFR, and their combination was 13.8%, 26.3%, and 18.1%, respectively. In a subsequent experiment cells were incubated with increasing amounts of each oligo and their combination for 3 d prior to radiolabeling. Statistically significant inhibition of growth for either oligo at every concentration was found. Cells incubated with 6.25, 12.5, 25, and 50 μM antisense directed against TGF-α had a mean inhibition of 29.3%, 33.3%, 21.7%, and 46.6%, respectively. Cells similarly treated with oligos against EGFR had a mean inhibition of 77.9%, 80.3%, 82.0%, and 83.7%, respectively, and cells incubated with 6.25, 12.5, 25 and 50 μM of each oligo had a mean inhibition of 74.7%, 70.6%, 70.8%, and 76.3%, respectively. Lastly, in a paired experiment, cells treated with 0, 0.39, 0.78, 1.56, 3.125, and 6.25 μM of oligos, either specifically directed against EGFR or a random control, for 3 d were evaluated for both thymidine incorporation and EGFR expression. Statistically significant inhibition of ³H-thymidine incorporation was seen in cells with the oligo specifically directed against EGFR at 3.125 μM and 6.25 μM when compared to non-oligo containing controls. This was accompanied by a comparable significantly decreased expression of a low-MW reactive derivative of EGFR at 3.125 μM and 6.25 μM in Western blots, and of a high-MW reactive EGFR at 6.25 μM. The significant effect against high-MW EGFR was observed vs both the non-oligo containing control and the random sequence. Oligo concentrations between 0.78 and 1.5 μM also resulted in decreased expression of the low-MW form, but not significant differences in thymidine radiolabeling. In recovery experiments, cells treated initially with greater oligo concentrations required significantly increased time to recover, particularly in cells treated with EGFR directed oligos. Intracellular uptake and nuclear localization was demonstrated with FITC tagged oligos. In summary, even at relatively low oligo concentrations and short exposure, oligos against TGF-α, and particularly EGFR, significantly inhibit in vitro growth of the T98G glioblastoma, possibly mediated by decreased EGFR expression.     

联合VEGF-AON和PDGF-TFO对大鼠脑胶质瘤增殖和细胞凋亡的作用效果

目的观察PDGFB链基因的TFO和VEGF的反义寡核苷酸AON对荷瘤大鼠脑胶质瘤增殖和细胞凋亡的影响。方法所有大鼠均在立体定向导引下行右尾状核区微量灌注20μL生理盐水中含1×106C6胶质瘤细胞。在细胞接种后第8天,实验Ⅰ组原位注射含TFO1·0mg的20μL生理盐水,实验Ⅱ和Ⅲ组则分别原位注射含TFO1·0mg AON0·125mg和TFO1·0mg AON0·250mg的20μL生理盐水。以后每隔72h原位注射相同剂量的药物,共注射3次。对照组仅在相同时间原位注射20μL生理盐水。实验3周时处死所有的大鼠,观察肿瘤的生长情况,流式细胞仪观察PDGFB链基因(PDGF-B)、VEGF、PCNA及细胞凋亡的变化。结果实验Ⅰ组的成瘤抑制率为53·1%,实验Ⅱ组为81·4%,实验Ⅲ组为93·1%,三组肿瘤体积比较差异有统计学意义,P<0·01。TFO对胶质瘤细胞PDGF-B、VEGF和PCNA表达有明显的抑制作用;TFO能使胶质瘤细胞凋亡增加。联合应用TFO和AON能更好地抑制PDGF-B、VEGF和PC-NA的表达,使胶质瘤细胞增殖能力进一步降低,细胞凋亡进一步增加。结论联合应用TFO和AON能更有效地抑制肿瘤生长和细胞增殖,促进胶质瘤细胞凋亡。     

VEGF及其受体Flt和KDR水平变化与胶质瘤关系及临床意义

 目的 探讨胶质瘤患者血清血管内皮生长因子（VEGF）及其受体Flt-1和KDR水平变化，为评价胶质瘤治疗效果，判断预后寻找一种科学的生物学标志物。方法 收集治疗前后胶质瘤患者、脑转移癌患者和健康对照者血清，进行VEGF，Flt-1和KDR水平的检测，SPSS11.5统计软件包对数据进行t检验和相关分析。结果 （1）治疗前脑胶质瘤和脑转移瘤患者血清VEGF水平明显高于健康对照组； 脑胶质瘤患者组和脑转移瘤患者组血清Flt-1水平明显高于健康对照组；脑胶质瘤患者组血清KDR水平明显高于健康对照组； 治疗后缓解的胶质瘤患者组VEGF和Flt-1水平明显低于治疗前。差异均有统计学意义。（2） VEGF与Flt-1和KDR有显著的相关性。结论 胶质瘤患者血清中VEGF及其受体的检测，可作为胶质瘤辅助诊断、治疗效果监测和预后判断的重要生物学指标。     

COX-2反义寡核苷酸对胰腺癌PC-3细胞株COX-2 mRNA和蛋白表达的影响

目的探讨环氧合酶(COX)-2反义寡核苷酸(ODNs)对胰腺癌PC-3细胞株COX-2 mRNA和蛋白表达的影响,并进一步阐明COX-2反义ODNs基因治疗胰腺癌的可行性.方法设计和合成COX-2反义ODNs,体外转染胰腺癌PC-3细胞,然后通过荧光显微镜、RT-PCR及Western blotting证实转染效果. 结果转染COX-2反义ODNs后,PC-3细胞COX-2 mRNA和蛋白表达均下降,且体现出一定的剂量和时间依赖性关系. 结论 COX-2反义ODNs转染胰腺癌PC-3细胞株后,可下调细胞中COX-2 mRNA和蛋白表达,COX-2反义ODNs有可能达到胰腺癌基因治疗的效果.