反义血管内皮生长因子对食管癌细胞体外放射增敏作用

[目的]研究TE-1细胞转染VEGF反义寡核苷酸，观察转染后肿瘤细胞VEGF表达情况及其对放射敏感性的影响。[方法]将反义VEGF寡核苷酸经Lipofectamine^TM2000介导转染TE-1细胞；并经γ线照射。采用MTT法检测细胞生长状况，RT-PCR、Western blotting检测细胞照射前后VEGF基因的表达，流式细胞术检测细胞周期分布情况和凋亡率；克隆形成实验反映转染后细胞放射敏感性的变化。[结果]将反义VEGF寡核苷酸成功地转染人食管癌TE-1细胞中，VEGF表达水平明显降低，体外增殖反应无明显差异，细胞周期分布情况相似．未见明显凋亡发生。不同剂量照射后增殖情况无统计学差异；反义组细胞凋亡率略有上升．转染反义组细胞较未转染组细胞放射敏感性增加。[结论]转染反义VEGF寡核苷酸可抑制食管癌TE-1细胞VEGF表达，联合放射治疗，对TE-1细胞增殖无明显作用，而其放射敏感性有增强作用。     

血管内皮生长因子反义RNA对裸鼠食管癌细胞增殖和凋亡的影响意义

目的：研究血管内皮生长因子（VEGF）反义RNA对裸鼠食管癌移植瘤细胞增殖和凋亡的影响。方法：将转染VEGF反义eDNA和空载体peDNA3．1的食管癌细胞株TE-1分别接种在裸鼠体内；应用免疫组化法、RT-PCR法检测移植瘤组织中VEGF蛋白和mRNA表达情况；流式细胞仪和透射电镜检测肿瘤细胞增殖及凋亡情况。结果：反义组肿瘤组织中VEGF蛋白和mRNA的表达降低，肿瘤细胞的细胞器发生扩张和肿胀、核染色质边集、凝集成块等凋亡形态学改变；Go／G，期细胞明显增多，S期细胞明显减少，细胞增殖指数降低；对照组和空载体组细胞凋亡较少。结论：VEGF反义RNA能抑制裸鼠食管癌细胞的增殖，诱导细胞凋亡，为食管癌基因治疗基础研究提供依据。     

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.     

反义寡核苷酸抑制survivin基因表达及肝癌细胞生长的研究

目的:采用反义寡核苷酸抑制肝癌细胞中survivin基因的表达,研究其对肝癌细胞生长的作用.方法:采用脂质体介导survivin反义寡核苷酸转染人肝癌细胞株SMMC-7721细胞,Western blot及原位杂交方法检测survivin蛋白及mRNA表达,流式细胞仪检测凋亡细胞比率,检测转染前后细胞贴壁率变化,并绘制细胞生长曲线.结果:survivin反义寡核苷酸转染后,肝癌细胞survivin蛋白及mRNA表达均显著降低,细胞贴壁率显著降低,细胞增殖活性明显受抑,细胞凋亡率显著增加.结论:survivin反义寡核苷酸转染可以有效降低细胞内survivin基因的表达,诱导细胞发生凋亡,抑制肝癌细胞生长.     

人核糖体蛋白S13(RPS13)编码基因的克隆及其正反义核酸转染胃癌细胞的初步研究

目的：扩增人核糖体蛋白S13（RPS13）编码基因序列，构建其正，反义真核表达载体，分别转染胃癌细胞SGC7901及胃癌耐药细胞SGC7901/VCR，以进一步研究RPS13基因与胃癌多药耐药的关系。方法：采用RT-PCR法扩增RPS13cDNA片段编码区全长序列，利用DNA重组技术的建正，反义真核表达载体，经脂质体介导转染胃癌细胞SGC7901及胃癌耐药细胞SGC7901/VCR，筛选正，反义基因稳定转染细胞，RNA斑点杂交法检测正，反义稳定转染细胞mRNA水平的变化。结果：从高表达RPS13的SGC7901/VCR耐药细胞中提取细胞总RNA，RT-PCR法成功扩增了RPS13cDNA片段编码区全长序列，并经测序证实；目的基因因按正，反两个方向亚克隆至真核表达载体pcDNA3.1( ),并经酶切鉴定证实；经脂质体介导将正义真核表达载体转染SGC7901细胞，反义真核表达载体转染SGC7901/VCR细胞，G418筛选获得稳定转染细胞；RNA斑点杂交试验证实；正义稳定转染细胞RPS13mRNA水平上调，反义稳定转染细胞RPS13mRNA水平下调。结论：成功克隆了人RPS13编码基因序列，并构建了其正，反义真核表达载体，在胃癌细胞系SGC7901及长春新碱耐药细胞SGC7901/VCR中得到稳定转染细胞，正义转染细胞中RPS13mRNA表达明显增加，反义转染细胞中表达明显受抑。     

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。     

雷公藤甲素对肺癌细胞Spc-A1 VEGF表达的影响

目的研究雷公藤甲素(TP)对人肺癌细胞Spc-A1增殖及血管内皮生长因子(VEGF)表达的影响。方法细胞生长增殖抑制试验(MTT法)检测TP对Spc-A1细胞的增殖抑制作用；反转录聚合酶链式反应法(RT-PCR)检测TP对VEGF信使核糖核酸(VEGF mRNA)表达的影响；Western blot检测TP对VEGF蛋白表达的影响。结果TP对人肺癌细胞Spc-A1的生长和增殖都具有一定程度的抑制作用,药物作用24 h抑制率为2．16±2．10～13．33±1．43,药物作用48 h抑制率为7．97±2．13～29．27±7．28,与对照组相比差异有显著意义(P＜0．05)；TP作用于人肺癌细胞Spc-A1 24 h,下调VEGF mRNA及蛋白的表达。结论不同浓度的TP干预Spc-A1细胞24 h、48 h后,Spc-A1细胞的增殖受到一定程度的抑制,抑制率随浓度(剂量)和时间的增加而增加；不同浓度的TP作用24 h后的Spc-A1细胞VEGF mRNA及蛋白表达均明显减少。     

脂质体介导反义C-FLIP对BGC823细胞凋亡的影响

目的：探讨脂质体介导下c-FLIP（Cellular—FLICE Inhibitory Protein）反义寡核苷酸对胃癌细胞系BGC823的作用。方法：RT—PCR和Western Blot方法检测c-FLIPL、c—FLIPs的mRNA和蛋白的表达；MTT法检测细胞抑制率：原位末端标记法、流式细胞术检测细胞凋亡：Western Blot检测蛋白表达变化。结果：c-FLIP基因编码的2种mRNA和蛋白在Hela、BGC823细胞中呈阳性表达。MTT显示脂质体介导反义寡核苷酸可显著抑制BGC823细胞增殖：反义寡核苷酸作用细胞36h后．TUNEL检测可见明显阳性染色，流式细胞分析发现在G1峰前出现凋亡峰；Western Blot可见c-FLIPL和c-FLIPs蛋白表达显著下降。结论：c-FLIPL、c—FLIPs mRNA和蛋白在Hela、BGC823细胞中表达。脂质体介导c-FLIP反义寡核苷酸转染BGC823细胞后抑制目的蛋白表达，抑制细胞生长、促进凋亡．其作用呈剂量依赖性。研究靶向c-FLIP的反义基因治疗药物可能为胃癌的临床治疗提供新的方法和思路。     

核苷酸切除修复基因XPB反义RNA表达质粒的构建及其功能的初步研究

背景与目的：核苷酸切除修复机制是细胞修复损伤DNA的重要途径,肿瘤细胞的耐药常伴随DNA损伤修复基因表达增强,采取反义策略降低细胞的DNA损伤修复能力可以增加肿瘤细胞的药物敏感性。本研究拟构建能在哺乳动物细胞中表达XPB反义RNA的表达质粒pcDNA-XPB/AS(XPB：着色性干皮病B基因),并初步探讨其对肺癌细胞DNA损伤修复能力及抗肿瘤药物耐药性的影响。方法：采用RT-PCR技术扩增的XPB cDNA5′端一段69--520bp的序列被反向插入表达质粒pcDNA3．1／His。将该重组质粒瞬时转染肺癌A549细胞。单细胞凝胶电泳(SCGE)比较阿霉素诱导的转染前后细胞DNA损伤修复情况。MTT法检测转染前后细胞对阿霉素的敏感性。结果：酶切图谱分析和基因测序证实反义表达质粒构建成功。RT-PCR显示转染细胞XPB mRNA表达水平下降。以4．Oμg／ml阿霉素诱导细胞DNA损伤,SCGE显示转染细胞修复DNA损伤能力受到抑制。MTT显示未转染细胞与转染细胞对阿霉素的敏感性存在差别,但无统计学意义。结论：构建的反义表达质粒能下调转染细胞XPB mRNA表达,抑制细胞DNA损伤修复能力,为进一步研究XPB基因功能奠定了基础。     

p27kip1的过表达对肾癌细胞增殖及凋亡的影响

目的：研究外源性p27kip1基因对肾癌细胞周期、细胞凋亡及增殖的影响。方法：将含p27cDNA的质粒在脂质体介导下在体外转染肾癌GRC-1细胞．Western Blot、FCM及MTT检测分析外源性p27kip1蛋白在细胞内的表达．观察其对细胞周期、凋亡及细胞增殖的影响结果：体外转染GRC-1细胞48h后．p27蛋白在p27kip1转染后的GRC-1细胞中高表达。FCM检测表明．细胞停滞于G1期，实验组G1期细胞占66．9%．并出现亚G1凋亡峰：而空白对照组和转染空载体组G1期细胞分别为32.2%和33.8%。MTT法检测表明．转染p27cDNA后，实验组细胞增殖明显受抑。结论：p27kip1基因在体外转染GRC-1细胞并过表达p27kip1蛋白，抑制细胞由G1期向S期过渡．从而抑制细胞增殖．诱导细胞凋亡。     

Estradiol and nicotine exposure enhances A549 bronchioloalveolar carcinoma xenograft growth in mice through the stimulation of angiogenesis

Angiogenesis is required for lung cancer growth, which is mediated by various growth factors such as vascular endothelial growth factor (VEGF). Increases in VEGF and angiogenesis have been correlated with poor prognosis and survival in patients with lung cancer. In addition, recent reports show that estradiol and nicotine play important roles in lung tumor initiation and progression. In this report, we demonstrate that estradiol and nicotine exposure enhances the growth of A549 bronchioloalveolar carcinoma xenografts in mice through the stimulation of cell proliferation, VEGF secretion and angiogenesis. We detect a four-fold increase in microvascular density in tumors from mice exposed to estradiol and nicotine compared to control tumors resulting in an increase in tumor growth. Intriguingly, the effects on angiogenesis and tumor growth by the combination of agents were additive when compared to either agent alone. Furthermore, estradiol promotes VEGF secretion from various non-small cell lung carcinoma (NSCLC) cells and this effect is augmented by nicotine in a tumor xenograft model. These results indicate that aside from their roles in promoting cell proliferation, estradiol and nicotine appear to have additive effects on the induction of angiogenesis through the stimulation of VEGF secretion during NSCLC progression.     

Membrane ganglioside enrichment lowers the threshold for vascular endothelial cell angiogenic signaling

Malignant tumor progression depends on angiogenesis, requiring vascular endothelial cell migration, and proliferation, triggered by tumor-derived vascular endothelial cell growth factor (VEGF). We show that gangliosides, which are actively shed by tumor cells and bind to normal cells in the tumor microenvironment, have the potential to sensitize vascular endothelial cells to respond to subthreshold levels of VEGF: Ganglioside enrichment of human umbilical vein vascular endothelial cells (HUVEC) caused very low, normally barely stimulatory, VEGF concentrations to trigger robust VEGF receptor dimerization and autophosphorylation, as well as activation of downstream signaling pathways, and cell proliferation and migration. Thus, by dramatically lowering the threshold for growth factor activation of contiguous normal stromal cells, shed tumor gangliosides may promote tumor progression by causing these normal cells to become increasingly autonomous from growth factor requirements by a process that we term tumor-induced progression of the microenvironment.     

Inhibition of K562 leukemia angiogenesis and growth by expression of antisense vascular endothelial growth factor (VEGF) sequence

Vascular endothelial growth factor (VEGF), a major angiogenic factor, plays a key role in the growth of solid tumor. Recently, expression of VEGF and its receptors has been found on leukemic cells as well as on endothelial cells. VEGF may fulfill a fundamental role in promoting tumor angiogenesis and proliferation by stimulating both endothelial cells and leukemic cells. To investigate the role of VEGF in the angiogenesis and growth of leukemic cell, we used an antisense strategy to downregulate VEGF expression in K562 cells, a human erythroleukemia cell line. Expression of antisense-VEGF in K562 cells reduced the secretion of VEGF protein and inhibited cell survival. The proliferation and migration of human umbilical vein endothelial cells were decreased in response to the conditioned medium (CM) from K562 cells expressed antisense-VEGF, compared to CM from K562 cells transfected with vector control. Moreover, subcutaneous injection of nude mice with antisense-VEGF K562 cells inhibited tumor growth with a reduction of the density of microvessels and an increased apoptosis in those tumors, compared to vector control K562 cells. These results suggest that the efficient downregulation of the VEGF production in leukemic cells using antisense-VEGF may constitute a novel strategy of treatment in leukemia.     

Administration of VEGF receptor tyrosine kinase inhibitor increases VEGF production causing angiogenesis in human small-cell lung cancer xenografts

Angiogenesis is mediated mainly by vascular endothelial growth factor (VEGF), and VEGF causes rapid growth in cancers, including human small-cell lung cancer (SCLC). The anti-angiogenic strategy of treating cancer using VEGF receptor (VEGFR) inhibition is currently of great interest. We tested the effects of the VEGFR2 tyrosine kinase inhibitor (TKI) vandetanib on the proliferation of two kinds of SCLC cell lines: SBC-1 cells, with detectable VEGFR2 expression and MS-1-L cells, without detectable VEGFR2 expression. To evaluate the anti-tumor and anti-angiogenic effects of vandetanib in vivo, we grafted SBC-1 and MS-1-L cells into mice. After a 3-week treatment, we measured the tumor size and histologically evaluated necrosis and apoptosis using H&E and TUNEL staining, respectively. The microvessels in the xenografts were also quantified by immunostaining of CD31. Vandetanib did not affect the proliferation of SBC-1 cells, but stimulated the growth of MS-1-L cells. In the SCLC xenograft model, vandetanib inhibited growth and tumor angiogenesis in a dose-dependent manner in SBC-1 xenografts. Vandetanib inhibited the growth of MS-1-L xenografts at a low dose (<12.5 mg/kg/day), but it did not affect tumor size or change microvessel counts at a higher dose. Interestingly, secretion of VEGF increased significantly in the MS-1-L cell line in the presence of a high dose of vandetanib in vitro. The effects of vandetanib on tumor angiogenesis were different in SBC-1 and MS-1-L cell lines. Production of angiogenic factors such as VEGF by the tumor potentially stimulates tumor angiogenesis and results in the acquisition of resistance to VEGFR TKI.     

人乳腺癌细胞系MCF—7原位移植血管生成超微结构观察及其相关因素表达

目的：探讨人乳腺癌细胞增殖与血管生成方式和时间的超微结构特点,明确人乳腺癌生长中VEGF、F8因子表达水平及其意义,方法：应用人乳腺癌细胞系MCF－7原位移植SCID鼠,光镜、电镜动态观察肿瘤生长与血管生成的细胞形态,结合免疫组化检测VEGF、F8因子的表达水平。结果：人乳腺癌细胞系MCF－7移植后1、2天生存靠固有组织的血管滋养生长,肿瘤组织中未见新生血管生成。肿瘤生长第3天时,肉眼可见周围血管血移植的肿瘤表面延伸,电镜下观察在 存活的癌细胞中可见血管母 细胞出现。第6天时,在癌细胞增生活跃区血管母细胞演化为血管内皮细胞,此时癌细胞群与新生的肿瘤微血管紧密相连。微血管邻旁周边可见极少量的蛋白质网架。第8－10天,内皮细胞有较丰富的线性粒体,粗面内质网增多,池内含有中等量蛋白质,血管内皮的腔面小球增多,光镜下可见肿瘤周围的血管与肿瘤新生血管相通。VEGF、F8因子的表达与肿瘤增殖,侵袭、新生血管生成有关。结论：移植初期人乳腺癌细胞何分泌血管生成因子,肿瘤周围的血管母细胞被激活,快速增殖期癌细胞可诱导血管内上细胞增生形成肿瘤新生血管,逐步演化的肿瘤新生血管与肿瘤边缘固有的血管相连通。     

VEGF受体KDR胞外Ⅴ—Ⅶ区的克隆,单抗制备及KDR在不同来源肿瘤组织 …

目的 利用制备的血管内皮细胞增殖因子（ＶＥＧＦ）受体ＫＤＲ抗体,通过免疫组湍不同来 的表达,为探讨ＶＥＧＦ及其受体对肿瘤生长及转移的作用提供可能方法 采用ＲＴ－ＰＣＲ方法,从人胎儿脐静脉内皮细胞克隆ＫＤＲ胞外Ⅴ－Ⅶ区基因片段,在大肠杆菌中表达,并用传统的杂交瘤融合技术制备小鼠ＫＤＲ单克隆抗体。通过组化及Ｗｅｓｔｅｒｎ－ｂｌｏｔ鉴定ＫＤＲ单抗的特生,最后采用Ｓ－Ｐ免疫组化法,分别对不同来源的１１５例     

Blockade of insulin-like growth factor I receptor function inhibits growth and angiogenesis of colon cancer.

Purpose and Experimental Design: Insulin-like growth factors (IGFs) I and II and their principle receptor, IGF-I receptor (IGF-IR), are frequently expressed in human colon cancers and play a role in preventing apoptosis, enhancing cell proliferation, and inducing expression of vascular endothelial growth factor (VEGF). To elucidate the in vitro and in vivo effects of IGF-IR in human colon cancer growth and angiogenesis, HT29 cells were transfected with a truncated dominant-negative (DN) form of IGF-IR or vector alone. RESULTS: IGF-I increased VEGF expression in parental and vector-transfected cells, whereas IGF-I induction of VEGF mRNA and protein was abrogated in IGF-IR DN cells. The IGF-IR DN cells demonstrated inhibited growth in both monolayer culture and soft agar (P < 0.05). s.c. injections of IGF-IR DN cells in nude mice led to significantly decreased tumor growth (P < 0.05). Immunohistochemical analyses revealed that IGF-I DN tumors demonstrated decreased tumor cell proliferation, VEGF expression, and vessel count and increased tumor cell apoptosis (P < 0.05 for all parameters compared with controls). Furthermore, IGF-IR DN-transfected cells yielded significantly decreased tumorigenicity and growth in the liver. CONCLUSIONS: These studies demonstrate that the IGF ligand-receptor system plays an important role in multiple mechanisms that mediate human colon cancer growth including regulation of VEGF and angiogenesis.     

ZD6474, an inhibitor of vascular endothelial growth factor receptor tyrosine kinase, inhibits growth of experimental lung metastasis and production of malignant pleural effusions in a non-small cell lung cancer model

ZD6474 is a novel, orally active inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase, with some additional activity against epidermal growth factor receptor (EGFR) tyrosine kinase. The purpose of this study was to determine the potential of ZD6474 in the control of established experimental lung metastasis and pleural effusions produced by human non-small cell lung cancer (NSCLC) cells. PC14PE6 (adenocarcinoma) and H226 (squamous cell carcinoma) cells express high levels of EGFR and only PC14PE6 cells overexpress VEGF. Neither ZD6474 nor the EGFR tyrosine kinase inhibitor gefitinib inhibit proliferation of PC14PE6 or H226 cells in vitro. Both PC14PE6 and H226 cells inoculated intravenously into nude mice induced multiple lung nodules after 5-7 weeks. In addition, PC14PE6 cells produced bloody pleural effusions. Daily oral treatment with ZD6474 did not reduce the number of lung nodules produced by PC14PE6 or H226 cells, but did reduce the lung weight and the size of lung nodules. ZD6474 also inhibited the production of pleural effusions by PC14PE6 cells. Histological analyses of lung lesions revealed that ZD6474 treatment inhibited activation of VEGFR-2 and reduced tumor vascularization and tumor cell proliferation. Therapeutic effects of ZD6474 were considered likely to be due to inhibition of VEGFR-2 tyrosine kinase because gefitinib was inactive in this model. These results indicate that ZD6474, an inhibitor of VEGFR-2, may be useful in controlling the growth of established lung metastasis and pleural effusions by NSCLC.