CH50多肽真核表达载体pCH510的构建、表达及体内趋化和抑瘤作用

目的：探讨血管内皮生长因子的表达与肿瘤血管生成的关系。方法：将VEGF165正、反义RNA表达载体导入人胃癌细胞,观察接种VEGF高表达和低表达胃癌细胞裸鼠移植瘤的生长情况,并对移植瘤进行组织学检查,检测其血管密度、组织增生及环死程度等变化。结果：VEGF正义转染细胞所致移植瘤的生长速度明显快于反义转染细胞所致的移植瘤;组织学检查发现,正义转染细胞移植瘤的血管密度显著高于的转染细胞所致的肿瘤。结论：血管皮生长因子通过启动血管生成而促进肿瘤的生长,阻断血管内皮生长因子的产生可以抑制肿瘤的生长。     

血管内皮生长因子的表达对人胃癌细胞生物学表型的影响

 目的　探讨血管内皮生长因子对人胃癌细胞生物学表型的影响。方法　将 VEGF16 5正、反义 RNA表达载体导入人胃癌细胞 ,并观察其细胞周期、增殖、裸鼠致瘤生长等生物学指标。结果　与 VEGF正义转染细胞相比 ,在反义转染细胞的细胞周期中 G1期细胞数增加了 1 7.3% ,而S期细胞减少了 43.6 % ;正义转染细胞的克隆形成率明显高于反义转染细胞 ;正义转染细胞组的肿瘤生长速度及瘤体积明显高于其他组。结论　 VEGF可以加强肿瘤组织血管生成 ;VEGF反义RNA可以防治肿瘤生长 ;VEGF可能与肿瘤细胞增殖能力有关.     

血管内皮生长因子反义cDNA对人肺癌的抑瘤作用

血管内皮生长因子(vascular endothelial growth factor,VEGF)与血管生成有主要关联,体内肿瘤生长依赖于包括VEGF在内的血管生成因子诱导的血管生成[1].有研究表明VEGF高表达与肺癌的分期、转移、患者的预后等有较强的相关性[2,3],阻断VEGF合成可使肿瘤生长受抑制.本研究以卡铂化疗为对照,探讨VEGF反义cDNA对人肺癌裸鼠皮下移植瘤的抑瘤作用.     

血管内皮生长因子反义寡核苷酸对体外及裸鼠体内前列腺癌细胞PC3生物学特性影响的研究

 目的　探讨血管内皮生长因子（VEGF）反义寡核苷酸对前列腺癌细胞PC3在体外和裸鼠体内生长特性的影响,以及局部注射反义寡核苷酸治疗裸鼠皮下移植肿瘤的疗效。方法　采用Oligofectamine携带VEGF反义寡核苷酸转染前列腺癌细胞PC3,实验分为反义寡核苷酸组、正义寡核苷酸组和对照组。软琼脂糖凝胶实验和细胞侵袭实验检测转染反义寡核苷酸的肿瘤细胞成瘤性和侵袭性。裸鼠成瘤实验观察VEGF反义寡核苷酸对体内PC3细胞增殖的影响。建立裸鼠前列腺癌种植瘤模型,局部注射VEGF反义核酸治疗,测定体内抑瘤率。结果　对照组、正义寡核苷酸组和反义寡核苷酸组的PC3细胞每组阳性克隆数分别为53.67±5.86、52.33±6.43和26±4.58（F＝13.73,P＜0.01）,反义组体外形成克隆数显著减少;三组细胞侵袭至下室的细胞数分别为45.60±5.53、42.35±6.21和18.37±3.52（F＝14.18,P＜0.01）;转染VEGF反义核酸的细胞移植瘤生长较慢,接种28 d后三组的肿瘤体积分别为（1330.32±81.38）、（1267.64±120.26）和（641.83±58.34）mm3（F＝17.26,P＜0.01）;局部注射治疗4周后,三组肿瘤质量分别为（1.25±0.08）g、（1.17±0.06）g和（0.41±0.05）g,与对照组比较,正义组和反义组肿瘤抑制率分别为6.4 %和67.2 %,差异有统计学意义（χ2＝17.72,P＜0.005）。结论　VEGF反义寡核苷酸可以抑制前列腺癌细胞PC3 VEGF的表达,进而促进细胞凋亡,降低其成瘤性,抑制侵袭能力。转染VEGF反义寡核苷酸的前列腺癌细胞PC3移植瘤在裸鼠体内生长受抑制,局部注射反义核酸可显著抑制移植瘤的生长。     

VEGF反义RNA对人食管癌细胞生长转移的抑制作用

目的:探讨研究血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)反义RNA在抑制恶性肿瘤生长和转移及抗肿瘤血管生成治疗中的意义。方法:采用脂质体法将反义VEGFcDNA质粒转染入食管癌细胞TE1;MTT法检测细胞增殖情况;原位杂交和RTPCR技术检测VEGF的表达水平,FCM分析细胞周期,并对转染前后细胞进行裸鼠体内生长转移等生物学行为实验。结果:转染反义VEGFcDNA质粒的TE1细胞中VEGF表达水平降低,与对照组比较,裸鼠体内成瘤时间延长,肿瘤生长速度减慢,质量和体积差异有显著性意义(P<0.05)。结论:VEGF反义RNA能抑制食管癌TE1细胞VEGF表达,对裸鼠体内TE1细胞的生长有抑制作用,有望成为食管癌基因治疗的优选基因之一。     

紫杉醇节律化疗联合EGCG抑制胃癌生长和肿瘤血管生成的实验研究

目的:探讨低剂量紫杉醇联合表没食子儿茶素没食子酸酯(EGCG)对胃癌肿瘤血管生成的影响,并观察其抑瘤效果。方法:建立裸鼠人胃癌移植瘤模型,分别给予0.9%NaCl溶液、最大耐受剂量(MTD)紫杉醇、低剂量紫杉醇、EGCG及低剂量紫杉醇联合EGCG腹腔注射,观察肿瘤生长情况。免疫组化染色,测定CD31和血管内皮生长因子(VEGF)表达情况。结果:对照组肿瘤生长迅速,其他治疗组肿瘤生长都受到了抑制,联合治疗组抑瘤效果最明显,各组比较差异有统计学意义,P<0.05。与对照组及MTD组相比,低剂量紫杉醇组肿瘤组织内的微血管密度(MVD)及VEGF表达均下降,合用EGCG后,MVD和VEGF表达下降更明显,P<0.05。结论:低剂量紫杉醇化疗能够有效抑制裸鼠胃癌移植瘤的生长及微血管的形成,与EGCG联合应用时效果增强。     

血管内皮生长因子反义cDNA对人肺癌抑瘤作用的体外实验研究

目的：研究血管内皮生长因子(VEGF)反义cDNA对肿瘤细胞VEGF mRNA和蛋白表达的影响，并探讨其对肿瘤细胞生长、增殖过程的作用。方法：以脂质体介导VEGF反义cDNA转染肺癌细胞，通过原位杂交法检测细胞VEGF mRNA．免疫组化法检测细胞VEGF蛋白和PCNA增殖活性，ELISA法检测分泌型VEGF蛋白，MTT法测定细胞生长率．研究VEGF反义cDNA对肿瘤细胞的作用。结果：转染后肺癌细胞内源性VEGF mRNA和蛋白的表达受抑．细胞生长及增殖受抑。结论：VEGF反义cDNA转染可降调节肿瘤细胞的VEGF生成并抑制其生长与增殖．     

血管内皮生长因子介导的卵巢恶性肿瘤浸润生长的实验研究

 目的探讨血管内皮生长因子(VEGF)在卵巢癌细胞肿瘤血管形成及浸润中的作用。方法脂质体介导VEGFcDNA转染卵巢癌细胞OVCAR3,并经Northernblot和Westernblot法检测转染VEGFcDNA前后卵巢癌细胞中的VEGFmRNA和蛋白表达水平。将VEGF表达阳性的细胞接种于裸鼠皮下观察负荷瘤生长速度、重量并采用免疫组化的方法检测其微血管密度(MVD)。结果(1)VEGFcDNA转染后的卵巢癌细胞系VEGFmRNA和蛋白表达水平明显高于转染前(P<0.05)。(2)体外实验显示转染与非转染细胞系生长曲线基本相同。(3)VEGFcDNA转染后的卵巢癌细胞系接种裸鼠后其负荷瘤生长速度和重量均明显高于未转染的裸鼠负荷瘤(P<0.05)。(4)VEGF表达阳性的裸鼠负荷瘤其MVD也明显高于VEGF表达阴性的裸鼠负荷瘤(P<0.05)。结论VEGF通过促进肿瘤新生血管的增多进而在卵巢癌浸润生长中发挥作用。     

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。     

双调蛋白反义核酸表达对裸鼠乳腺癌血管生成的抑制作用

目的 研究双调蛋白（AR）反义RNA表达对乳腺癌血管生成的抑制作用。方法 乳腺癌NS2T2A1细胞经AR反义cDNA质粒转染后,经筛选获得表达AR反义RNA的AR-AS1及AR-AS3克隆,转染空载体获得NS2T2A1 V对照细胞,接种裸鼠皮下形成肿瘤。研究条件培养液对人微血管内皮细胞（HMEC）增殖的影响,以EL1SA法测定细胞血管内皮生长因子（VEGF）分泌量。以定量RT-PCR分析肿瘤组织的VEGF表达水平。以免疫组化法标记CD31研究肿瘤内血管数量。结果 HMEC在AR-AS1和AR-AS3细胞条件培养液中的增殖比例明显降低。AR-AS1和AR-AS3细胞VEGF分泌量亦降低。AR-AS1和AR-AS3肿瘤的血管数量仅有对照组的50％左右,且VEGF表达显著降低。结论 AR反义RNA表达可有效抑制乳腺癌的血管生成,应作为新治疗靶点进一步研究．     

反义VEGF硫代寡脱氧核苷酸抑制裸鼠人肝癌移植瘤生长的研究

目的 :检测硫代修饰的反义脱氧寡核苷酸 (ASPODN)抑制人肝癌血管形成和生长的作用 ,以探讨ASPODN的临床应用前景。方法 :传代培养的肝癌细胞系HepG2 ,接种于裸鼠颈背部皮下 ,自 4 8h始局部注射ASPODN和等量的PBS ,彩色多普勒能量图 (CDE)检测移植瘤内新生血管的数目和分布 ;LSAB法免疫组化染色检测移植瘤中微血管密度 (MVD) ;FCM分析裸鼠移植瘤细胞周期动力学和凋亡率。结果 :局部注射ASPODN组和PBS对照组 ,裸鼠人肝癌移植瘤瘤重及MVD平均值分别为 ( 1.2 8±0 .2 6)g、2 6.90± 7.4 2和 ( 3.4 6± 0 .86)g、50 .36± 10 .2 7(P <0 .0 1) ;局部注射ASPODN组 ,用CDE动态检测到移植瘤内的血管数目及血流丰富程度明显低于PBS组P <0 .0 1;经ASPODN治疗后的裸鼠人肝癌移植瘤细胞凋亡率 ( 7.4 8± 1.64) %高于PBS对照组 ( 2 .75± 0 .2 2 ) % (P <0 .0 1)。结论 :VEGFAS PODN明量抑制肝癌血管形成和生长 ,有望成为理想的抗肝癌血管生成基因治疗的新药     

A small interfering RNA targeting vascular endothelial growth factor inhibits Ewing's sarcoma growth in a xenograft mouse model.

Angiogenesis plays an essential role in tumor growth and metastasis and is a promising therapeutic target for cancer. Vascular endothelial growth factor (VEGF) is a key regulator in vasculogenesis as well as in angiogenesis. TC71 human Ewing's sarcoma cells overexpress VEGF, with a shift in isoform production from membrane-bound VEGF189 to the more soluble VEGF165. Transfection of TC71 cells with a vector-based VEGF targeted small interfering RNA expression system (VEGFsi) inhibited VEGF165 expression by 80% and VEGF165 protein production by 98%, with no alteration in VEGF189 expression. Human microvascular endothelial cell proliferation and migration induced by conditioned medium from VEGFsi-transfected TC71 cells was significantly less than that induced by conditioned medium from TC71 cells and control vector-transfected TC71 cells. Furthermore, after s.c. injection into athymic nu/nu mice, the tumor growth of VEGFsi-expressing TC71 cells was significantly less than that of parental or control vector-transfected cells. Vessel density as assessed by CD31 immunohistochemical analysis and VEGF165 expression as assessed by Northern blotting were also decreased. Intratumor gene therapy with polyethylenimine/VEGFsi also resulted in tumor growth suppression. When inoculated into the tibias of nude mice, VEGFsi-expressing TC71 cells induced osteolytic bone lesions that were less severe than those induced by control groups. These data suggest that targeting VEGF165 may provide a therapeutic option for Ewing's sarcoma.     

nm23H1对肝癌细胞增殖及体内肿瘤形成能力的影响

目的 研究ｎｍ２３Ｈ１对肝癌细胞增殖、体内肿瘤形成和转移的影响。方法 构建正反义ｎｍ２３Ｈ１ ｃＤＮＡ表达载体并转染肝癌细胞ＳＭＭＣ－７７２１,得到ｎｍ２３Ｈ１稳定最高和最低表达的两种细胞克隆,并进行细胞生长曲线测定、裸鼠皮下及脾包膜下移植试验。结果 转染反义表达载体后,肝癌细胞ｍＲＮＡ和蛋白表达下降,在体内、体外增殖加速;转染正义表达载体后,出现与之相反的结果。正义表达细胞接种组裸鼠肿瘤结节形成     

Antisense oligodeoxynucleotide targeted to Midkine, a heparin-binding growth factor, suppresses tumorigenicity of mouse rectal carcinoma cells.

Midkine (MK), a heparin-binding growth factor, is overexpressed in a wide range of human carcinomas and is believed to contribute to tumorigenesis and tumor progression. To develop an antitumor reagent, we designed a phosphorothioate antisense oligodeoxynucleotide molecule based on the secondary structure of MK mRNA. The antisense MK at the dosage of 5 microM suppressed MK production by CMT-93 mouse rectal carcinoma cells after cationic liposome-mediated transfection, to 13% of that in control cultures. The growth of CMT-93 cells and their colony formation in soft agar were inhibited by the addition of the antisense MK, whereas the control reagent, the sense MK, showed no effects. On s.c. injection into nude mice, CMT-93 cells transfected with the antisense MK formed tumors much smaller than those by control cells. Finally, untreated CMT-93 cells were inoculated to nude mice, and 7 days later the antisense MK (50 microM) with atelocollagen was directly injected into the preformed tumor region to evaluate the curative effect; the injection was repeated at the interval of 2 weeks. During the period of 10-41 days after initiation of therapy, the rate of increase of tumor volume treated with the antisense MK was found to be about 4.2-fold lower than that seen after treatment with the sense MK. On this occasion, proliferation of tumor cells as estimated by 5-bromodeoxyuridine incorporation was strongly inhibited, whereas angiogenesis was less affected. These findings strongly suggested the usefulness of MK antisense oligodeoxynucleotide as a new reagent for cancer therapy.     

罗非昔布对胰腺癌 BXPC-3细胞裸鼠移植瘤血管形成的影响

背景与目的:肿瘤血管形成在肿瘤细胞浸润性生长中起了重要作用,增加表达的环氧合酶-2与快速生长肿瘤中的血管形成有关.本研究目的是观察选择性环氧合酶-2抑制剂-罗非昔布抑制胰腺癌生长的体内效应,以及对胰腺癌移植瘤相关血管形成的影响.方法:将表达有环氧合酶-2的人胰腺癌细胞 BXPC-3种植入裸鼠皮下,形成移植瘤.经口灌入罗非昔布 30 mg· (kg· d)- 1,共 8周,记录肿瘤大小,采用Ⅷ因子免疫组化显示血管密度, BXPC-3细胞上的血管内皮生长因子 (VEGF)检测亦用免疫细胞化学染色.采用 RT-PCR和明胶酶法检测胰腺癌基质金属蛋白酶-2(MMP-2)mRNA的表达及酶活性的变化.结果:罗非昔布对裸鼠胰腺癌移植瘤的肿瘤重量抑瘤率为 73.64%,肿瘤体积抑瘤率为 87.74%.实验组胰腺癌组织微血管密度 [(1.5± 0.2)个 /200倍放大视野 ]明显低于对照组 [(4.7± 1.5)个 /200倍放大视野 ].与对照组比较,罗非昔布显著降低了胰腺癌 BXPC-3细胞中 VEGF、 MMP-2 mRNA的表达以及酶活性.结论:减少胰腺癌相关的血管形成是罗非昔布阻止胰腺癌生长的机制之一.     

YC-1对人肝细胞癌裸鼠移植瘤的影响及其机制

目的探讨缺氧诱导因子抑制剂（YC-1）对人肝癌细胞裸鼠皮下移植瘤的影响及其相关机制。方法用人肝癌细胞株SMMC-7721建立人肝癌裸鼠皮下移植瘤模型,待肿瘤生长至约（100~150）mm3时,将荷瘤裸鼠随机分为两组：实验组和对照组,分别给予YC-1和二甲基亚砜,观察两组裸鼠肿瘤生长情况;应用RT-PCR、Western blot及免疫组织化学方法检测移植瘤组织HIF-1α和VEGF表达。结果YC-1治疗组各时点肿瘤体积显著低于对照组(P<0. 05 );与对照组比较,实验组移植瘤组织HIF-1α mRNA表达差异无统计学意义(P>0.05),而HIF-1α蛋白的表达显著降低 (P<0. 05 );移植瘤组织VEGFmRNA及蛋白表达均显著低于对照组 (P<0. 05 )。结论YC-1可能通过下调HIF-1α和VEGF的表达来抑制肝癌的生长。     

Stimulation of tumor growth by human soluble intercellular adhesion molecule-1

Because serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) are elevated in cancer and sICAM-1 is angiogenic, we tested the ability of sICAM-1 to promote tumor growth. Our preliminary experiments showed that exogenous sICAM-1 significantly stimulated the growth of human tumors in vivo. Human fibrosarcoma transfectants, which express ICAM-1, produce ICAM-1 on the cell surface and release sICAM-1 into the medium without any apparent effect on cell growth in vitro. We found that conditioned medium from sense ICAM-1 transfectants compared with mock or antisense ICAM-1 transfectants stimulates endothelial cell migration in vitro and neovascularization in the chick chorioallantoic membrane assay. Tumor cells transfected with sense constructs form faster growing tumors than mock- and antisense-transfected cells in both chick embryos and nude mice models. Serum levels of human sICAM-1 from nude mice bearing sense ICAM-1 transfectants correlate positively with tumor weight. Sense ICAM-1 transfectants are more proliferative and induce more blood vessel formation than mock and antisense transfectants in nude mice. Because expression of ICAM-1 does not affect tumor cell growth in vitro, the angiogenic activity of sICAM-1 produced by sense ICAM-1 transfectants may be involved in the stimulation of tumor growth. Therefore, sICAM-1 may perform dual functions that are essential for tumor growth: angiogenesis and escape from immune surveillance.     

VEGF receptor antisense therapy inhibits angiogenesis and peritoneal dissemination of human gastric cancer in nude mice

The efficacy of a phosphorothioate antisense oligonucleotide (ASO) for KDR/Flk-1 (KDR/Flk-1-ASO), an endothelial cell-specific vascular endothelial growth factor (VEGF) receptor, was investigated on the peritoneal dissemination and angiogenesis of a human gastric cancer cell line in nude mice. Green fluorescent protein (GFP)-transduced NUGC-4 (NUGC-4-GFP) human gastric cancer cells were implanted into the peritoneal cavity of nude mice. KDR/Flk-1-ASO, -SO, or phosphate-buffered saline was administrated from days 7 to 14, 200 microg/mouse, once a day. The mice were sacrificed on day 28. Disseminated peritoneal tumor nodules expressing GFP were visualized by fluorescence microscopy. KDR/Flk-1-ASO significantly decreased the extent of peritoneal dissemination of the tumors. The number of cells undergoing apoptosis was significantly increased in the KDR/Flk-1-ASO-treated tumors. Microvessel density was significantly reduced in the KDR/Flk-1-ASO-treated tumor nodules. The KDR/Flk-1 antisense strategy, therefore, decreases tumor dissemination apparently by inhibiting angiogenesis.