共查询到19条相似文献,搜索用时 78 毫秒
1.
KDR是血管内皮生长因子(VEGF)的主要受体之一,它在介导VEGF刺激内皮细胞增殖及血管通透性等生物学活性中起重要作用.为了获得人重组的有VEGF结合活性的KDR,通过RT-PCR从人胎儿脐静脉内皮细胞(EC)扩增出编码KDR胞外Ⅰ~Ⅳ区片段,将其克隆在谷胱甘肽转移酶(GST)融合蛋白表达载体pGEX2T中,并在大肠杆菌中获得稳定表达.表达的融合蛋白GST-KDR以包涵体形式存在,其分子质量约66 ku左右.经碱变性法大量提取,及制备胶电泳获得纯化的KDR,为进一步的研究奠定了基础. 相似文献
2.
利用T4噬菌体展示猪瘟病毒E2抗原 总被引:3,自引:0,他引:3
利用重组PCR技术将猪瘟病毒 (CSFV )E2蛋白主要抗原编码区基因 (mE2 )与T4噬菌体SOC基因融合 ,构建了大小为 643bp的SOC/mE2融合基因 ,再将其插入携带T4溶菌酶基因 (e)和(denV)基因的T4重组载体 (PRH) ,构建了重组载体pRsmE2。通过重组载体与缺失突变型T4发生同源重组 ,可将SOC/mE2融合基因整合入T4的基因组中 ,并成功地将大小约 2 1 5aaSOC/mE2融合蛋白展示于T4噬菌体衣壳表面。经Westernblot、胶体金免疫电镜等免疫学检测证实 ,展示于T4表面的mE2融合蛋白具有CSFV免疫学活性。 相似文献
3.
VEGF(血管内皮细胞生长因子,Vascular Endothelial Growth Factor)是刺激内皮细胞增殖和新生血管形成的最重要因子,与多种实体瘤的生长和转移密切相关。应用其可溶性受体阻断它的病理作用是一个非常有前景的课题。将VEGF受体KDR胞外区前三个Loop 969碱基对的cDNA片段克隆到杆状病毒表达载体pFastBacI,与杆状病毒表达载体Bacmid同源重组后,转染昆虫细胞SF9,获得重组杆状病毒并证明了目的基因的高效表达。经Western blot证实表达产物的特异性。经ELISA和体外生物学活性检测表明表达产物可阻断VEGF的生物学活性,抑制鸡胚CAM血管的生长。 相似文献
4.
新生血管大量形成在实体瘤的生长和转移中起着关键的作用。血管内皮生长因子(VEGF)是介导肿瘤血管生成的最主要因素。从原代培养的人脐静脉血管内皮细胞(HUVEC)提取细胞总RNA,采用逆转录PCR(RT-PCR)方法得到VEGF受体KDR胞外区cDNA片段。将获得的受体基因克隆到AAV基因治疗载体pSNAV中,得到重组质粒pSNAV/KDR。重组质粒转染BHK细胞,加入辅助病毒后,获得了表达目的蛋白的重组AAV。重组病毒表达的KDR在体外实验中具有与VEGF结合的活性。在体内实验中,重组AAV感染的黑色素瘤细胞在小鼠中形成肿瘤的血管化程度明显低于对照组。 相似文献
5.
用腺相关病毒载体介导人血管内皮生长因子受体KDR胞外区基因的有达 总被引:1,自引:0,他引:1
新生血管大量形成在实体瘤的生长和转移中起着关键的作用。血管内皮生长因子 (VEGF)是介导肿瘤血管生成的最主要因素。从原代培养的人脐静脉血管内皮细胞 (HUVEC)提取细胞总RNA ,采用逆转录PCR(RT PCR)方法得到VEGF受体KDR胞外区cDNA片段。将获得的受体基因克隆到AAV基因治疗载体pSNAV中 ,得到重组质粒pSNAV KDR。重组质粒转染BHK细胞 ,加入辅助病毒后 ,获得了表达目的蛋白的重组AAV。重组病毒表达的KDR在体外实验中具有与VEGF结合的活性。在体内实验中 ,重组AAV感染的黑色素瘤细胞在小鼠中形成肿瘤的血管化程度明显低于对照组。 相似文献
6.
目的探讨血管内皮生长因子(VEGF)及其受体(Flt-1)、转化生长因子β1(TGF-β1)在非小细胞肺癌(non-smallcelllungcancer,NSCLC)组织血管形成中的意义及与肿瘤临床、生物学行为的关系。方法免疫组化染色观察VEGF、血管内皮生长因子受体-1(Flt-1)、TGF-β1在NSCLC组织中的表达,以CD34免疫组化染色来显示NSCLC组织中微血管生成情况和进行微血管密度判断。结果NSCLC组织中癌细胞不同程度地表达VEGF、血管内皮生长因子受体因子、TGF-β1,而对照组肺组织基本不表达(P<0·05);VEGF、Flt-1、TGF-β1阳性表达的NSCLC组织内微血管计数明显高于VEGF、Flt-1、TGF-β1表达阴性的NSCLC组织(P<0·05);NSCLC组织中VEGF、Flt-1、TGF-β1的阳性表达率和表达强度均与肿瘤淋巴结转移密切相关;随年龄增长,VEGF及其受体Flt-1的阳性表达率有所降低(P<0·05)。结论VEGF、Flt-1、TGF-β1的表达与NSCLC组织内肿瘤血管生成和淋巴结转移有密切关系,VEGF、Flt-1的表达与患者的年龄有一定关系。 相似文献
7.
EGFR基因重组T7噬菌体疫苗抗Lewis肺癌的实验研究 总被引:1,自引:0,他引:1
本研究中制备了表达表皮生长因子受体(EGFR)部分肽段的基因重组T7噬菌体疫苗,并开展了诱导小鼠产生内源性抗EGFR抗体的实验性抗肿瘤作用研究。由T7噬菌体展示系统将7个经筛选的异种属(人源、鸡源)EGFR膜外区片段展示在其壳体次要头蛋白(P10B)上,用所制备的基因重组噬茵体疫苗免疫小鼠,免疫4W后皮下接种Lewis肺癌细胞,10d后分离瘤体并称重,观察各实验组的抗肿瘤效果。Western Blot检测重组的融合壳蛋白均有EGFR抗原性:高表达EGFR的A431 细胞与免疫3W的小鼠抗血清结合并被荧光二抗标记,流式细胞仪检测法确认有抗EGFR抗体产生;各实验组肿瘤均重统计结果显示,P-CL1-670组、P-cp1-130组、P-cp2-136组、P-cp3-145组、 P-cp4-142组与空白噬菌体组差异性显著。说明表达EGFR的基因重组噬菌体疫苗诱导产生的内源性抗体.在一定程度上抑制了EGFR阳性肿瘤的生长.为诱导型内源性抗EGFR抗体的肿瘤靶向治疗研究开辟了新的途径。 相似文献
8.
目的:检测趋化因子受体4(CXCR4)及血管内皮生长因子(VEGF)在肾母细胞瘤中的表达,探讨其在肾母细胞瘤的发生、发展、浸润和转移中的作用。方法:选择临床及病理资料齐全的存档肾母细胞瘤标本30例,另取正常肾脏标本10例作对照。采用免疫组化(SABC)方法检测CXCR4及VEGF的表达。结果:CXCR4在肾母细胞瘤组织中高表达(66.67%),在正常肾脏组织中低表达(30.0%),两者相比有统计学意义(P<0.05),VEGF在肾母细胞瘤组织中高表达(73.33%),在正常肾脏组织中低表达(20.0%),两者相比同样有统计学意义(P<0.05),肾母细胞瘤组织中CXCR4与VEGF的表达两者之间呈正相关性(r=0.392,P<0.05)。结论:肾母细胞瘤组织中CXCR4和VEGF均高表达。CXCR4及VEGF可能成为预测肾母细胞瘤浸润转移的重要指标。 相似文献
9.
目的:探讨2-甲氧基雌二醇(2-Methoxyestradiol,2ME2)对乳腺癌MCF-7细胞生长及缺氧诱导因子-1α(Hypoxia-inducible factor1 α,HIF-1α)、趋化因子受体-4(CXC chemokine receptor-4,CXCR4)、血管内皮生长因子(Vascular endothelial growth factor,VEGF)表达的影响.方法:采用MTT法检测不同浓度2ME2对MCF-7细胞的增殖抑制作用;Hoechest 33258染色观察细胞凋亡形态学改变;RT-PCR、Western blot分别检测不同浓度2ME2对乳腺癌MCF-7细胞中HIF-1α、CXCR4、VEGF mRNA及蛋白表达水平的影响.结果:2ME2可较强的抑制MCF-7细胞增殖,并呈时间和剂量依赖性;经2ME2作用48h后MCF-7细胞表现出典型的凋亡形态特征;不同浓度2ME2作用于MCF-7细胞48小时后,随着药物浓度的增加细胞中HIF-1α、CXCR4、VEGF在mRNA及蛋白表达水平逐渐降低,与对照组相比有统计学意义(P<0.05).结论:2ME2可通过降低HIF-1α、CXCR4、VEGFmRNA及蛋白表达,抑制乳腺癌MCF-7细胞的增殖及肿瘤血管生成和侵袭转移相关因子的表达. 相似文献
10.
目的:研究RGD肽对肺癌A549细胞增殖凋亡及侵袭迁移的影响,并探讨其作用机制。方法:不同浓度RGD肽处理肺癌A549细胞后,MTT检测肺癌细胞的增殖能力,流式细胞仪检测肺癌细胞凋亡及周期分布,Transwell检测其迁移及侵袭能力的变化,Western blot检测RGD肽对肺癌A549细胞MMP2、MMP9的表达水平影响。结果:当RGD肽浓度增加至50 mg/L时,肺癌A549细胞增殖明显受到抑制,且这种抑制作用呈剂量依赖关系;RGD肽组A549细胞G0/G1期细胞比例增高,细胞凋亡率由(6.1±0.1)%增至(15.2±0.5)%;在迁移和侵袭试验中,RGD肽组A549细胞的穿膜细胞数分别由123±10和43±10降至45±5和18±5;RGD肽组A549细胞MMP2、MMP9表达水平显著降低。结论:RGD肽对肺癌A549细胞的增殖有明显抑制作用,并促进其凋亡,可能与RGD肽改变其周期分布有关,RGD肽可明显抑制A549细胞的迁移及侵袭,可能与其下调MMP2、MMP9的表达相关。 相似文献
11.
Kawakami JK Martinez Y Sasaki B Harris M Kurata WE Lau AF 《Bioorganic & medicinal chemistry letters》2011,21(5):1371-1375
12.
VEGF-E activates endothelial nitric oxide synthase to induce angiogenesis via cGMP and PKG-independent pathways 总被引:1,自引:0,他引:1
Cudmore M Ahmad S Al-Ani B Hewett P Ahmed S Ahmed A 《Biochemical and biophysical research communications》2006,345(4):1275-1282
Vascular endothelial growth factor-A (VEGF), which binds to both VEGF receptor-1 (Flt1) and VEGFR-2 (KDR/Flk-1), requires nitric oxide (NO) to induce angiogenesis in a cGMP-dependent manner. Here we show that VEGF-E, a VEGFR-2-selective ligand stimulates NO release and tube formation in human umbilical vein endothelial cells (HUVEC). Inhibition of phospholipase Cgamma (PLCgamma) with U73122 abrogated VEGF-E induced endothelial cell migration, tube formation and NO release. Inhibition of endothelial nitric oxide synthase (eNOS) using l-NNA blocked VEGF-E-induced NO release and angiogenesis. Pre-incubation of HUVEC with the soluble guanylate cyclase inhibitor, ODQ, or the protein kinase G (PKG) inhibitor, KT-5823, had no effect on angiogenesis suggesting that the action of VEGF-E is cGMP-independent. Our data provide the first demonstration that VEGFR-2-mediated NO signaling and subsequent angiogenesis is through a mechanism that is dependent on PLCgamma but independent of cGMP and PKG. 相似文献
13.
Bauer D Whittington DA Coxon A Bready J Harriman SP Patel VF Polverino A Harmange JC 《Bioorganic & medicinal chemistry letters》2008,18(17):4844-4848
A novel class of potent and selective inhibitors of KDR incorporating an indazole moiety 1 is reported. The discovery, synthesis, and structure–activity relationships of this series of inhibitors have been investigated. The most promising compounds were also profiled to determine their pharmacokinetic properties and evaluated in a VEGF-induced vascular permeability assay. 相似文献
14.
目的:研究转录因子WSTF对肺癌细胞增殖和侵袭作用的影响。方法:采用慢病毒介导的基因转染方法建立A549细胞WSTF高表达细胞系A549-WSTF和空质粒对照细胞系A549-control。细胞增殖实验和克隆形成实验观察ING5过表达对肺癌A549细胞增殖能力的影响;Trans-well迁移实验和侵袭实验观察WSTF对肺癌细胞迁移和侵袭能力的影响。结果:Western blot验证A549-WSTF细胞WSTF蛋白水平显著高于对照细胞A549-control,P=0.0004。WSTF高表达明显促进了肺癌细胞的增殖能力(1-4天P值分别为0.002、0.0004、0.0002和3.21×10-5)和克隆形成能力(P=0.004);WSTF过表达还显著促进了肺癌细胞从trans-well小室迁移到下室的作用,其OD570值分别为0.626±0.013(A549-WSTF)和0.322±0.010(A549-control),P=2.37×10-5;WSTF还促进肺癌细胞穿透基质胶迁移到下室,其OD570值分别为0.600±0.027(A549-WSTF)和0.333±0.017(A549-control),P=0.0004。结论:WSTF可以促进肺癌细胞的增殖和侵袭能力而发挥促癌作用。 相似文献
15.
Dabrowska K Zembala M Boratynski J Switala-Jelen K Wietrzyk J Opolski A Szczaurska K Kujawa M Godlewska J Gorski A 《Archives of microbiology》2007,187(6):489-498
We previously investigated the biological, non-antibacterial effects of bacteriophage T4 in mammals (binding to cancer cells
in vitro and attenuating tumour growth and metastases in vivo); we selected the phage mutant HAP1 that was significantly more
effective than T4. In this study we describe a non-sense mutation in the hoc gene that differentiates bacteriophage HAP1 and its parental strain T4. We found no substantial effects of the mutation on
the mutant morphology, and its effects on electrophoretic mobility and hydrodynamic size were moderate. Only the high ionic
strength of the environment resulted in a size difference of about 10 nm between T4 and HAP1. We compared the antimetastatic
activity of the T2 phage, which does not express protein Hoc, with those of T4 and HAP1 (B16 melanoma lung colonies). We found
that HAP1 and T2 decreased metastases with equal effect, more strongly than did T4. We also investigated concentrations of
T4 and HAP1 in the murine blood, tumour (B16), spleen, liver, or muscle. We found that HAP1 was rapidly cleared from the organism,
most probably by the liver. Although HAP1 was previously defined to bind cancer cells more effectively (than T4), its rapid
elimination precluded its higher concentration in tumours.
Maria Zembala and Janusz Boratynski contributed equally to this work. 相似文献
16.
Summary The bacteriophage T4 uvsX gene encodes a 43 kDa, single-stranded DNA-dependent ATPase, double-stranded DNA-binding protein involved in DNA recombination, repair and mutagenesis. Mutants of uvsX have a DNA-arrest phenotype and reduced burst size. Western blot immunoassay of UvsX peptides made by a number of amber mutants revealed amber peptides ranging from 25–32 kDa. Wild-type UvsX protein was also detected in lysates of cells infected with uvsX amber mutants, suggesting that their mutations are suppressed by translational ambiguity. We investigated the effects of mutations near the 5 end of uvsX. A frameshift mutation was engineered at codon 33. Western immunoblots for UvsX protein demonstrated that the frameshift mutant expresses no detectable wild-type UvsX; instead, a 37 kDa reactive peptide was detected. In order to determine if this peptide represents truncated UvsX protein, the mutation was regenerated in the cloned uvsX gene and expressed in transformed Escherichia coli. Endopeptidase digestion of the 37 kDa protein from the cloned gene generated peptide fragments indistinguishable from those obtained from wild-type UvsX. A double-amber mutant of uvsX was also generated by oligonucleotide site-directed mutagenesis. No UvsX protein was detected in lysates of cells infected with the uvsX-am64am67 double mutant. Plaque size and sensitivity to UV inactivation for both the double-amber and the frame-shift mutants were indistinguishable from those of other uvsX mutants. Mutations in uvsY had no demonstrable effect on efficiency of plating or UV sensitivity of uvsX mutants. Thus, null mutants of uvsX are viable. 相似文献
17.
Kingkan Sanphanya Suvara K. Wattanapitayakul Suwadee Phowichit Valery V. Fokin Opa Vajragupta 《Bioorganic & medicinal chemistry letters》2013,23(10):2962-2967
We report a novel VEGFR-2 inhibitor, developed by the back-to-front approach. Docking experiments indicated that the 3-chloromethylphenylurea motif of the lead compound occupied the back pocket of VEGFR-2 kinase. An attempt was made to enhance the binding affinity of 1 by expanding the structure to access the front pocket using a triazole linker. A library of 1,4-(disubstituted)-1H-1,2,3-triazoles were screened in silico, and one compound (VH02) was identified with an IC50 against VEGFR-2 of 0.56 μM. VH02 showed antiangiogenic effects, inhibiting tube formation in HUVEC cells (EA.hy926) at 0.3 μM, 13 times lower than its cytotoxic dose. These enzymatic and cellular activities suggest that VH02 has potential as a lead for further optimization. 相似文献
18.
Jingpeng Jin Shanshan Zhou Changfeng Li Ruisi Xu Lingling Zu Jiacong You Bin Zhang 《Life sciences》2014
Aims
Aberrant expression of microRNAs (miRNAs) results in alterations of various biological processes (e.g., cell cycle, cell differentiation, and apoptosis) and cell transformation. Altered miRNAs expression was associated with lung carcinogenesis and tumor progression. This study aimed to investigate the function and underlying molecular events of miR-517a-3p on regulation of lung cancer cell proliferation and invasion.Main methods
Transfected miR-517a-3p mimics or inhibitors into 95D and 95C cells respectively, the effects of miR-517a-3p on lung cancer cell proliferation, migration, and invasion were detected. Bioinformatics software forecasted potential target genes of miR-517a-3p and dual luciferase reporter gene system and western blot verified whether miR-517a-3p regulates FOXJ3 expression directly.Key findings
MiR-517a-3p was differentially expressed in lung cancer 95D and 95C cell lines that have different metastatic potential. Manipulation of miR-517a-3p expression changed lung cancer cell proliferation, migration and invasion capacity. MiR-517a-3p directly regulated FOXJ3 expression by binding to FOXJ3 promoter.Significance
This study demonstrated that miR-517a-3p promoted lung cancer cell proliferation and invasion by targeting of FOXJ3 expression. 相似文献19.
Se Hui Sohn Hyun Kyu Song Kyeongsik Min Seung-Je Cho Jinho Moon Jae Young Lee Hyung Jun Ahn Changsoo Chang Hie-Joon Kim Se Won Suh 《Acta Crystallographica. Section D, Structural Biology》1999,55(5):1061-1063
Deoxycytidylate hydroxymethylase from bacteriophage T4 is a homodimeric enzyme in which each polypeptide chain consists of 246 amino-acid residues. It has been crystallized in the presence of its substrate, deoxycytidine monophosphate, at room temperature using sodium citrate as precipitant. The crystals are monoclinic, belonging to space group C2, with unit-cell parameters a = 174.22, b = 53.12, c = 75.17 Å, β = 115.29°. The asymmetric unit contains one homodimer, with a corresponding Vm of 2.65 Å3 Da−1 and solvent content of 54%. Native diffraction data to 1.6 Å resolution have been collected from two crystals using synchrotron radiation. 相似文献