血管生成素相互作用蛋白的发现及其在哺乳动物细胞中的验证

通过RT-PCR从人外周血白细胞中钓取血管生成素(angiogenin,Ang)cDNA,构建诱饵蛋白载体pAS-2-1-Ang,对其自身转录激活活性进行鉴定后,通过酵母双杂交系统筛选人肝细胞cDNA文库,获得两个双阳性克隆.序列分析和同源检索表明所获候选蛋白分别为人上皮细胞素和λ晶体蛋白.构建Ang及候选蛋白标签融合表达载体并共转染COS-7细胞,利用免疫共沉淀和蛋白质印迹方法在哺乳动物细胞中验证了Ang与候选蛋白间的相互作用.为阐明Ａng促血管新生的分子机制创造了条件.     

重组人色素上皮细胞衍生因子融合蛋白的制备及其抑制新生血管

目的：人色素上皮细胞衍生因子 (pigment epithelium-derived factor, PEDF)是一种有效的新生血管形成抑制因子和神经营养因子。本文通过原核细胞表达人PEDF蛋白,鉴定其抑制新生血管的生物学活性。方法：采用PCR法扩增人PEDFcDNA,将其克隆到pET32a载体中,在大肠杆菌BL21中表达人PEDF蛋白。经SDS-PAGE和Western-blot鉴定后,镍柱亲和层析法变性条件下纯化重组融合蛋白。Bradford法测蛋白浓度,采用鸡胚脲囊膜法测其对新生血管形成的影响。结果：成功构建了pET32a-PEDF表达载体。重组人PEDF蛋白在BL21宿主菌中获得了稳定高效表达,鸡胚脲囊膜实验结果显示在重组蛋白浓度为0.4、0.04 ng/ml时均有对新生血管的显著抑制作用（P<0.01）,而在4 ng/ml时无抑制作用。结论： 成功高效表达及纯化了重组人PEDF蛋白,鉴定其抑制新生血管的生物学活性,并且证实该活性在一定范围内有效,为进一步研究其功能及推广应用奠定了基础。     

人血管生成素cDNA的克隆与表达

血管生成素 ( angiogenin,ANG)广泛存在于多种肿瘤组织中 ,在肿瘤发生的不同阶段刺激新生血管的形成 .利用 RT- PCR方法从培养的人肺癌细胞系 A549扩增得到了 ANG c DNA片段 ,测序正确后克隆入融合表达载体 p RSETB中 ,构建了原核表达菌株 .经 IPTG诱导 ,表达了 N端融合His6的 ANG融合蛋白 ,表达量占菌体总蛋白的 1 0 % ,纯化后的血管生成素体外能够有效地刺激鸡胚绒毛尿囊膜的血管形成 .     

NOS和PTEN在血管紧张素Ⅱ诱导心肌细胞肥大中的作用

目的: 研究NOS和PTEN在AngⅡ诱导的心肌细胞肥大中的作用及其机制.方法: 采用AngⅡ诱导新生大鼠心肌细胞肥大模型,应用RT-PCR、Western blot及免疫组化等方法,分别检测各组心肌细胞eNOS、iNOS、PTEN mRNA表达和PTEN蛋白表达的变化,以及PTEN蛋白定位.结果: ①应用AngⅡ培养1 d的心肌细胞,心肌细胞蛋白质含量未见明显变化,但eNOS mRNA表达显著减少,iNOS mRNA表达显著增加.②应用AngⅡ培养5 d的心肌细胞,心肌细胞蛋白质含量显著增加;eNOS mRNA和iNOS mRNA表达未见明显变化;心肌细胞PTEN蛋白表达显著减少.③免疫组化结果显示,心肌细胞核内有棕黄色细颗粒状的免疫产物生成.结论: NOS和PTEN参与了血管紧张素Ⅱ诱导心肌细胞肥大的发生发展过程.     

血管新生及丝蛋白材料血管化过程

基于医用生物材料开发及组织工程中血管化问题的重要性,本文就与生物材料血管化紧密相关的血管发生和血管新生有关研究做一综述,分析了芽式和套迭式血管新生的模式及机制,特别是对丝蛋白材料的血管化过程进行了分析与探讨.通过深入探讨血管新生的模式和机制,进而阐明丝蛋白材料中毛细血管生长与生物材料微结构之间的关系,有助于设计出适合于细胞黏附、组织生长、血管化顺利进行的生物材料,促进生物材料的临床应用及组织工程血管化研究的深化.     

靶向毒素DT-VEGF的构建、表达与活性分析

肿瘤的快速生长依赖于新生血管的形成。血管内皮生长因子（VEGF）是血管发生和形成过程中的主要介质,其特异性受体在正常组织和肿瘤组织的表达率存在数个数量级的差异,因此可以将毒素分子转运至增生的肿瘤上皮组织中抑制肿瘤血管增生,从而抑制肿瘤的生长。将白喉毒素的前389个氨基酸基因片段与VEGF165通过一短肽相连构建为融合蛋白基因,在大肠杆菌中表达,获得纯化蛋白。实验证实该融合蛋白对血管内皮细胞有特异性杀伤作用,并研究了其对鸡胚尿囊膜新生血管的抑制作用。     

L-型钙电流在血管紧张素Ⅱ诱导新生大鼠心肌细胞肥大中的作用

目的:探讨血管紧张素II(Ang II)诱导的新生大鼠肥大心肌细胞中L-型钙电流的功能在分子水平改变。方法:在血管紧张素II诱导的新生大鼠肥大心肌细胞中,应用全细胞膜片钳技术检测L-型钙电流的密度及门控动力学变化;应用半定量RT-PCR技术检测L-型Ca2+通道α1C亚单位mRNA的表达量。结果:Ang II在引起新生大鼠心肌肥大的同时,也增加了心肌细胞ICa,L电流密度,但并不影响ICa,L电流的激活、失活和复活特征。另外,Ang II还增加了L-型Ca2+通道α1C亚单位mRNA表达量。Ang II的这些作用都可被其1型受体阻断剂losartan所抑制。结论:在Ang II诱导的新生大鼠肥大心肌中,L-型Ca2+通道的功能在分子水平发生了显著变化,这些变化是通过激活心肌细胞上Ang II 1型受体所介导的。     

人内皮抑素基因的克隆以及在大肠杆菌中的可溶性表达研究

用PCR的方法从人胎肝cDNA文库中得到人内皮抑素基因 ,克隆测序正确后连接到硫氧还蛋白融合表达载体上 ,转化大肠杆菌BL2 1 (DE3)得到表达人内皮抑素的工程菌。用IPTG诱导表达 ,表达量达到全菌蛋白的 64%。经分析硫氧还蛋白可以辅助内皮抑素可溶性表达 ,表达的融合蛋白保持了天然蛋白的免疫学特性。而且表面带有多聚组氨酸的突变的硫氧还蛋白还简化了蛋白纯化的步骤 ,使融合蛋白可以通过固相金属螯和层析 (IMAC)的方法纯化。纯化后的融合蛋白经IgA蛋白酶的切割可得到大小正确的重组人内皮抑素 ,用此方法获得的重组人内皮抑素可以在CAM试验中抑制新生血管的形成。高效可溶型表达内皮抑素的工程菌的构建成功 ,为内皮抑素的生产应用打下了良好的基础。     

血管舒-缩肽在血管平滑肌细胞中的表达与调控

为探讨血管舒 缩肽表达的调控机制及血管平滑肌细胞 (VSMC)在该网络平衡中的地位 ,以血管紧张素Ⅱ (AngⅡ )为诱发因素刺激培养的大鼠VSMC ,用RT PCR和放射免疫分析观察内皮素 1(ET 1)、AngⅡ、心钠素 (ANF)和肾上腺髓质素 (ADM)在VSMC中的表达与释放及相互关系 ,用电泳迁移率改变分析 (EMSA)和染色质免疫沉淀 (ChIP)分析揭示其分子机制 .在被AngⅡ处理的VSMC中 ,4种血管活性肽的表达活性均升高 ,其中缩血管肽基因表达被迅速诱导 ,而舒血管肽则是先降后升 .但刺激前后舒 缩血管肽之间的平衡关系无明显改变 .放免分析证实 ,AngⅡ可程度不同地促进 4种血管活性肽合成 ,使胞内 4种活性肽水平升高 ;对培养液中 4种活性肽进行检测的结果显示 ,AngⅡ可促进ET 1、AngⅡ释放 ,抑制舒血管肽释放 ,尤以ANF的胞内水平明显高于胞外 .EMSA分析显示 ,在AngⅡ诱导 4种肽表达的同时 ,与细胞增殖有关的转录调控因子转录激活蛋白(AP 1)与 4种活性肽基因启动子的结合活性明显增强 .ChIP结果表明 ,AP 1在染色质靶位点的募集与血管活性肽基因的表达上调有直接关系 .结果提示 ,AP 1与特异DNA顺式作用元件的相互作用参与了血管活性肽的转录激活 .VSMC不仅作为它们的效应器 ,而且还通过调节AP 1与靶基因中的共有顺式元件——     

人血纤维蛋白溶酶原K5环的基因合成、表达、纯化与活性鉴定

 根据大肠杆菌遗传密码的偏爱性 ,人工合成人血纤维蛋白溶酶原 K5全基因 ,并在原核系统中以硫氧还蛋白融合蛋白的形式实现了高效表达 .重组蛋白通过 Ni2 +金属螯合层析得到初步纯化 ,通过肠激酶切割去除了融合标签 .应用鸡胚尿囊膜实验检测切割后的 rh K5的生物学活性 ,发现与对照组相比 ,rh K5能明显地降低血管管径、血管总面积以及血管总面积与视野面积的比值 ,表明切割后的产物具有显著抑制新生血管生成的生物学活性 .为进一步研究和开发抗血管生成药物奠定了基础 .     

Expression, purification and characterization of recombinant human angiogenin in Pichia pastoris

The potential of angiogenin (Ang) for clinical use has been highlighted in view of its important roles in inducing angiogenesis, facilitating cell proliferation, and inhibiting cell apoptosis. To produce soluble, correctly folded recombinant protein with a high yield, a DNA fragment encoding human Ang was inserted into eukaryotic expression vector pPIC9 and transformed into Pichia pastoris. The expression of recombinant human Ang (rhAng) accounted for about 70% of total secreted proteins. Purifying the Ang from the culture supernatant yielded 30 mg/L at 90% purity by chromatography with a SP Sepharose FF column. Biological assays indicated that rhAng can induce new blood-vessel formation, promote HeLa cell proliferation, increase Erk1/2 phosphorylation, and upregulate c-myc expression. Preparation of bioactive rhAng might lay the basis for further functional study, and might provide an effective strategy for large-scale production of soluble human Ang.     

Expression,Purification and Characterization of Recombinant Human Angiogenin in Pichia pastoris

The potential of angiogenin (Ang) for clinical use has been highlighted in view of its important roles in inducing angiogenesis, facilitating cell proliferation, and inhibiting cell apoptosis. To produce soluble, correctly folded recombinant protein with a high yield, a DNA fragment encoding human Ang was inserted into eukaryotic expression vector pPIC9 and transformed into Pichia pastoris. The expression of recombinant human Ang (rhAng) accounted for about 70% of total secreted proteins. Purifying the Ang from the culture supernatant yielded 30 mg/L at 90% purity by chromatography with a SP Sepharose FF column. Biological assays indicated that rhAng can induce new blood-vessel formation, promote HeLa cell proliferation, increase Erk1/2 phosphorylation, and upregulate c-myc expression. Preparation of bioactive rhAng might lay the basis for further functional study, and might provide an effective strategy for large-scale production of soluble human Ang.     

C-terminal angiogenin peptides inhibit the biological and enzymatic activities of angiogenin

Synthetic peptides corresponding to the C-terminal region of angiogenin (Ang) inhibit the enzymatic and biological activities of the molecule while peptides from the N-terminal region do not affect either activity. The peptide Ang(108-121) transiently abolishes the inhibition of cell-free protein synthesis caused by angiogenin coincidentally with its cleavage of reticulocyte RNA. Several C-terminal peptides also inhibit nuclease activity of angiogenin when tRNA is the substrate. Furthermore, peptide Ang(108-123) significantly decreases neovascularization elicited by angiogenin in the chick chorioallantoic membrane assay.     

Prokaryotic expression,purification and functional characterization of human FHL3

Four and a half LIM domain protein 3 (FHL3) is a member of the family of LIM proteins and is involved in myogenesis, cytoskeleton reconstruction, cell growth and differentiation. The full-length FHL3 cDNA was cloned from human spleen cDNA library and inserted in a prokaryotic expression vector pBV220 and then the recombinant plasmid was transformed into E. coli JM109. The expression of the recombinant protein was induced at 42°C. SDS-PAGE analysis showed that recombinant human FHL3 (rhFHL3) was mainly expressed as an inclusion body. After purification by HisTrap FF crude, the rhFHL3 was renatured by dialysis against renaturing buffer and identified by Western blot analysis using human FHL3 polyclonal antibody. The MTT assay showed that the purified rhFHL3 could inhibit HepG2 cell growth but promote the proliferation of ECV304 cells. In addition, the expression of angiogenin (Ang) gene was increased when ECV304 cells were pretreated with rhFHL3.     

Enzymatically active angiogenin/ribonuclease A hybrids formed by peptide interchange

The primary structures of the blood vessel inducing protein human angiogenin and human pancreatic ribonuclease (RNase) are 35% identical. Angiogenin catalyzes the limited cleavage of ribosomal RNA (18 and 28 S), yielding a characteristic pattern of polynucleotide products, but shows no significant activity toward conventional pancreatic RNase substrates [Shapiro, R., Riordan, J. F., & Vallee, B. L. (1986) Biochemistry 25, 3527-3532]. Angiogenin/RNase hybrid enzymes--wherein particular regions of primary structure in RNase are replaced by the corresponding segments of angiogenin--serve to explore the structural features underlying angiogenin's characteristic activities. Herein we show that synthetic angiogenin peptides, Ang(1-21) and Ang(108-123), form noncovalent complexes with inactive fragments of bovine RNase A--RNase(21-124) (i.e., S-protein) and RNase(1-118), respectively--with regeneration of activity toward conventional RNase substrates. Maximal activities for the Ang(1-21)/S-protein complex (Kd = 1.0 microM) are 52%, 45%, and 15% toward cytidine cyclic 2',3'-phosphate, cytidylyl(3'----5')adenosine, and yeast RNA, respectively. In contrast, activities of the RNase(1-118)/Ang(108-123) hybrid (Kd = 25 microM) are 1-2 orders of magnitude lower toward cyclic nucleotides and dinucleoside phosphates. However, substitution of phenylalanine for Leu-115 in Ang(108-123) increases activity up to 100-fold. Both His-13 and His-114 in the angiogenin peptides are required for activity since their substitution by alanine yields inactive complexes. Importantly, the pattern of polynucleotide products formed during cleavage of ribosomal RNA by the Ang(1-21)/S-protein hybrid shows a striking resemblance to that formed by angiogenin, demonstrating that the hybrid retains features of both angiogenin and RNase A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence of the cDNA and gene for angiogenin, a human angiogenesis factor

Human cDNAs coding for angiogenin, a human tumor derived angiogenesis factor, were isolated from a cDNA library prepared from human liver poly(A) mRNA employing a synthetic oligonucleotide as a hybridization probe. The largest cDNA insert (697 base pairs) contained a short 5'-noncoding sequence followed by a sequence coding for a signal peptide of 24 (or 22) amino acids, 369 nucleotides coding for the mature protein of 123 amino acids, a stop codon, a 3'-noncoding sequence of 175 nucleotides, and a poly(A) tail. The gene coding for human angiogenin was then isolated from a genomic lambda Charon 4A bacteriophage library employing the cDNA as a probe. The nucleotide sequence of the gene and the adjacent 5'- and 3'-flanking regions (4688 base pairs) was then determined. The coding and 3'-noncoding regions of the gene for human angiogenin were found to be free of introns, and the DNA sequence for the gene agreed well with that of the cDNA. The gene contained a potential TATA box in the 5' end in addition to two Alu repetitive sequences immediately flanking the 5' and 3' ends of the gene. The third Alu sequence was also found about 500 nucleotides downstream from the Alu sequence at the 3' end of the gene. The amino acid sequence of human angiogenin as predicted from the gene sequence was in complete agreement with that determined by amino acid sequence analysis. It is about 35% homologous with human pancreatic ribonuclease, and the amino acid residues that are essential for the activity of ribonuclease are also conserved in angiogenin. This provocative finding is thought to have important physiological implications.     

Angiogenin and Its Functions in Angiogenesis

The review is devoted to angiogenin, one of the factors that induce formation of blood vessels, which is unique in that it is a ribonuclease. Consideration is given to the tertiary structure of human angiogenin; the catalytic and cell receptor binding sites, their significance for angiogenic activity; the human angiogenin gene structure, chromosomal localization, and expression; the specificity of angiogenin as a ribonuclease and abolishment of protein synthesis; the nuclear localization of angiogenin in proliferating endothelial cells and its significance for angiogenic activity; angiogenin binding to cell surface actin as a plausible mechanism of inducing neovascularization (enhancement of plasminogen activation by actin, stimulation of the cell-associated proteolytic activity; promotion of the cultured cell invasiveness); modulation of mitogenic stimuli in endothelial, smooth muscle, and fibroblast cells by angiogenin. The importance of angiogenin as an adhesive molecule for endothelial and tumor cells is discussed too, as well as the modulation of tubular morphogenesis by bovine angiogenin, prevention of tumor growth in vivoby angiogenin antagonists, prospects of the use of angiogenin and angiogenin-encoding recombinant plasmids and vaccinia virus in therapeutic practice.     

Engineered human angiogenin mutations in the placental ribonuclease inhibitor complex for anticancer therapy: Insights from enhanced sampling simulations

Targeted human cytolytic fusion proteins (hCFPs) represent a new generation of immunotoxins (ITs) for the specific targeting and elimination of malignant cell populations. Unlike conventional ITs, hCFPs comprise a human/humanized target cell‐specific binding moiety (e.g., an antibody or a fragment thereof) fused to a human proapoptotic protein as the cytotoxic domain (effector domain). Therefore, hCFPs are humanized ITs expected to have low immunogenicity. This reduces side effects and allows long‐term application. The human ribonuclease angiogenin (Ang) has been shown to be a promising effector domain candidate. However, the application of Ang‐based hCFPs is largely hampered by the intracellular placental ribonuclease inhibitor (RNH1). It rapidly binds and inactivates Ang. Mutations altering Ang's affinity for RNH1 modulate the cytotoxicity of Ang‐based hCFPs. Here we perform in total 2.7 µs replica‐exchange molecular dynamics simulations to investigate some of these mutations—G85R/G86R (GGRR_mut), Q117G (QG_mut), and G85R/G86R/Q117G (GGRR/QG_mut). GGRR_mut turns out to perturb greatly the overall Ang‐RNH1 interactions, whereas QG_mut optimizes them. Combining QG_mut with GGRR_mut compensates the effects of the latter. Our results explain the in vitro finding that, while Ang GGRR_mut‐based hCFPs resist RNH1 inhibition remarkably, Ang WT‐ and Ang QG_mut‐based ones are similarly sensitive to RNH1 inhibition, whereas Ang GGRR/QG_mut‐based ones are only slightly resistant. This work may help design novel Ang mutants with reduced affinity for RNH1 and improved cytotoxicity.     

The complete amino acid sequence of bovine milk angiogenin

The amino acid sequence of angiogenin isolated from bovine milk was deduced by gas-phase sequencing of the protein and its fragments. The protein contains 125 residues and has a calculated molecular mass of 14,577 Da. The sequence is highly homologous (65% identity) to the sequence of human angiogenin, most of the differences being the result of conservative replacements. Like human angiogenin, the bovine protein is also homologous to bovine pancreatic RNase A (34% identity) and the three major active site residues known to be involved in the catalytic process, His-14, Lys-41 and His-115, are conserved. When tested against conventional substrates for RNase A activity, bovine angiogenin displays the same selective ribonucleolytic activity as human angiogenin. The sequence of bovine angiogenin contains the cell recognition tripeptide Arg-Gly-Asp which is not present in the human protein.