肿瘤新生血管生成抑制剂-肿瘤抑素的研究进展

肿瘤的生长和转移都离不开新生血管的生成，肿瘤抑素是一种来源于基膜胶原Ⅳ（ColⅣ）的蛋白片断，具有抗肿瘤新生血管生成和抑制肿瘤细胞增殖的双重活性，能特异性地抑制肿瘤血管内皮细胞蛋白的合成。对其进一步的研究证实肿瘤抑素是一种内源性的病理血管生成的抑制剂，具有潜在的药用价值。本文主要概述肿瘤抑素的来源、结构和分布，活性研究，作用机制及其与基质金属蛋白酶（MMP）的关系等最新研究进展和应用前景。     

内皮抑素联合TACE治疗原发性肝癌的应用前景

原发性肝癌TACE后残存癌细胞血管内皮生长因子的表达能刺激新生血管的生长,有助于残存癌细胞的存活,并可能在TACE术后残癌组织血供重建中起重要的作用;内皮抑素可以特异性作用于新生血管的内皮细胞,抑制内皮细胞的迁移、诱导其凋亡而抑制肿瘤新生血管形成和肿瘤生长;内皮抑素联合TACE治疗原发性肝癌将有一个良好的临床应用前景。     

内皮抑素抗肿瘤作用研究进展

内皮抑素 (endostatin)是一种重要的内源性血管新生抑制因子 ,自 1997年被发现以来 ,因其高效、低毒和不产生耐药性而被视为最有希望的新型抗肿瘤药物。现从功能、结构和作用机制等各个角度对内皮抑素的抗肿瘤作用作一较为全面的介绍。血管新生 (angiogenesis)对于肿瘤的生长、浸润和转移都有重要的意义。研究表明 ,体积超过1mm3 的肿瘤不仅需要新生的血管维持营养供给和排泄代谢产物 ,还需要其提供通道进行转移。因此 ,抑制新生血管的形成就可使肿瘤细胞进入休眠状态并诱导其凋亡。诸多研究表明 ,肿瘤细胞的血管新生是一个多因子参与的多步…     

血管抑素抑制角膜新生血管

血管抑素（angiostatin，AS）是迄今发现的强的内源性血管生成抑制因子之一，它能特异性抑制内皮细胞的增殖和迁移，诱导细胞凋亡，从而抑制新生血管的形成。其在肿瘤血管治疗方面已取得较好实验结论。由于角膜新生血管形成和肿瘤诱导的新生血管形成由相同的生长因子所驱动，正因为此可将其用于角膜新生血管的治疗。本文就血管抑素的分子结构、功能及其抑制角膜新生血管的机制进行综述。     

血管抑素与肿瘤的抗血管治疗

肿瘤的存活、生长和转移与血管生成密切相关。抑制肿瘤的血管生成 ,遏制肿瘤细胞生长 ,以达到治疗肿瘤的目的 ,是近年来肿瘤研究的方向之一。血管抑素 (ａｎｇｉｏｓｔａｔｉｎ)是最近发现的血管生成抑制因子 ,通过强烈抑制肿瘤血管生成 ,对多种肿瘤具有抑制作用。本文对肿瘤血管生成的调节、血管抑素生物学特性及抗血管生成作用进行综述。1　肿瘤的血管生成及其调节新生血管生成是在已存在的血管中长出新生毛细血管。血管生成在正常情况下仅见于胚胎发育、生殖和伤口愈合过程中。然而 ,持续的血管生成见于某些病理情况如炎症和肿瘤。肿瘤的…     

肿瘤治疗的抗肿瘤血管生成策略

恶性肿瘤的生长与转移必须依靠新生血管提供足够的营养才能实现。因此 ,近年来以抑制或破坏肿瘤血管生成 (angiogenesis)为目的的肿瘤治疗新方法 ,已成为各国肿瘤医学领域里新的研究热点。其方法的主要优点是 :不易产生耐药性 ;药物最易达到作用部位 ,即血管的表面 ;对具有血管新生的肿瘤均有效 ;新生血管内皮细胞的有限损害可抑制或消减大量的肿瘤细胞等。近年来 ,一批靶向肿瘤新生血管的抑制因子如血管抑素 (angiostain)、内皮抑素 (endostatin)以及促进新生血管生成的血管内皮生长因子 (vascularendothelialgrowthfactor,VEGF)等 3 0多…     

内皮抑素基因治疗肾细胞癌的进展

肾细胞癌(RCC)是泌尿系统中常见的恶性肿瘤,其生物学行为极为复杂,对放疗和化疗均不敏感,肿瘤血管形成丰富为其主要特点.肿瘤新生血管形成在肿瘤生长、侵袭和转移过程中起着决定性的作用.内皮抑素(Endostatin,ES)是一种能够强烈抑制血管形成的因子,能够有效抑制RCC肿瘤细胞的生长和转移.本文就内皮抑素的发现、性质、结构、生物学功能,内皮抑素基因治疗RCC的抑制作用机理及其目前在临床上的应用进行了简要的总结和概括.     

内皮抑素治疗脑胶质瘤的研究进展

肿瘤的生长和转移与肿瘤区血管生长有着密切关系,如何抑制破坏肿瘤新生血管已成为实体瘤治疗的一种新策略。内皮抑素是目前发现最强的血管生长抑制剂,胶质瘤是富含血管的肿瘤,内皮抑素能否用于治疗胶质瘤,具体用什么形式治疗得到了广泛的研究。     

内皮抑素研究进展

血管生成是恶性肿瘤生长和转移的病理学基础,内皮抑素是胶原蛋白ⅩⅧ的羟基末端水解片断,研究表明它可以通过抑制新生血管生长进而抑制肿瘤生长和转移.本文介绍了内皮抑素的生物学特性及其抗肿瘤研究进展.     

内皮抑素与鼻咽癌关系的研究进展

肿瘤的生长依赖于血液供应及血管生成.所以,对于肿瘤治疗而言,血管新生预示着一个极有希望的治疗作用靶点.内皮抑素(Endostatin,ES)是胶原蛋白ⅩⅦ的分解产物,是至今公认的抑制效应最强的抗血管内皮生长因子,可以抑制肿瘤的血管生成和生长.现就内皮抑素与鼻咽癌之间的关系进行综述.     

Tumstatin肽对视网膜微血管内皮细胞增殖及ERK蛋白表达的影响

目的 观察Tumstatin肽对体外培养的视网膜微血管内皮细胞增殖及细胞外信号调节激酶(ERK) 蛋白表达的影响。方法　采用MTT比色法测定Tumstatin肽（T8肽）对血管内皮生长因子（VEGF）诱导下的恒河猴视网膜血管内皮细胞（RF/6A细胞）增殖的干预作用；采用Western-blot检测T8肽对VEGF刺激后15min、30min、45 min和60min的RF/6A细胞ERK1/2蛋白表达水平的变化。 结果　T8肽浓度在5μg/ml 以上时对RF/6A细胞有抑制作用，且抑制作用随着浓度的增大而增高，各组OD值和抑制率比较差异均有显著性意义（P均<0.05）；T8肽可抑制VEGF对RF/6A细胞的促增殖作用；在1～40μg /ml相同浓度条件下，T8肽对VEGF条件下的RF/6A细胞增殖抑制率显著大于无VEGF条件下的抑制率（P均<0.01）。VEGF可刺激RF/6A细胞ERK1/2蛋白表达增加，T8肽可抑制VEGF诱导的RF/6A细胞ERK1/2蛋白的表达。 结论　Tumstatin抑制视网膜微血管内皮细胞的增殖，机制可能与VEGF信号转导通路上的ERK1/2细胞信号转导通路有关。     

血管形成抑制剂Tumstatin在鼻咽癌中表达鉴定及基因克隆

目的:检测Tumstatin基因在鼻咽癌发病过程中的表达变化及克隆其编码序列。方法:利用半定量RT-PCR方法检测Tumstatin在鼻咽癌组织、鼻咽癌细胞和正常鼻咽组织中的表达,比较它们之间的表达差异。设计含酶切位点的PCR引物,利用RT-PCR方法从相对正常鼻咽组织中获取Tumstatin蛋白编码序列,T/A克隆入pMD18载体中。利用PCR和酶切鉴定获得阳性重组子。重组子最后经测序证实。结果:与相对正常鼻咽组织相比,Tumstatin在鼻咽癌组织和细胞中表达下调或缺失。RT-PCR法成功获得Tumstatin基因编码区全长cDNA序列。重组克隆质粒插入片段经DNA测序后与GenBank中Tumstatin基因相应序列比较,100%同源。结论:Tumstatin在鼻咽癌组织和细胞中表达下调或缺失。采用T/A技术成功克隆鼻咽组织中Tumstatin基因,为下一步将Tumstatin亚克隆入真核表达载体pEGFP-N1,探索其在鼻咽癌生长和转移中的作用奠定了基础。     

Effects of eukaryotic expression plasmid encoding human tumstatin gene on endothelial cells in vitro

Background Tumstatin is a novel endogenous angiogenesis inhibitor which is widely studied using purified protein.The current study evaluates the antiangiogenic effects of tumstatin-overexpression plasmid in vitro, reveals the mechanism underlying the vascular endothelial cell growth inhibition and searches for a novel method administering tumstatin persistently.Methods The eukaryotic expression plasmid pcDNA-tumstatin encoding tumstatin gene was constructed and transfected to human umbilical vein endothelial cell ECV304 and human renal carcinoma cell ACHN.Expression of tumstatin in the two cell lines was determined by RT-PCR and Western blotting.Vascular endothelial cell proliferation was assessed by CCK-8 assay and cell cycle was analyzed by flow cytometry.To investigate the mechanism by which pcDNA-tumstatin inhibited vascular endothelial cell proliferation in vitro, cyclin D1 protein was detected by Western blotting.Results DNA sequence confirmed that pcDNA-tumstatin was successfully constructed.RT-PCR and Western blotting indicated that tumstatin could express in the two cell lines effectively.After tumstatin gene transfer, ECV304 cell growth was significantly inhibited and the cell cycle was arrested in G1 phase.And Western blotting showed that pcDNA-tumstatin decreased the level of cyclin D1 protein.Conclusions Overexpression of tumstatin mediated by pcDNA 3.1 （＋） specially inhibited vascular endothelial cells by arresting vascular endothelial cell in G1 phase resulting from downregulation of cyclin D1 and administration of tumstatin using a gene therapy might be a novel strategy for cancer therapy.     

Effects of eukaryotic expression plasmid encoding human tumstatin gene on endothelial cells in vitro

摘要： 背景 肿瘤抑素（tumstatin）是一种新型的内源性血管生成抑制剂,目前的研究大都是用纯化的肿瘤抑素蛋白。本研究的目的是研究肿瘤抑素过表达质粒的体外抗血管生成作用,揭露其抑制血管内皮细胞生长的潜在机制,并寻找一种能够持续释放肿瘤抑素的方法。 方法：构建了肿瘤抑素真核表达质粒pcDNA-tumstatin并用它转染人脐静脉内皮细胞系ECV304细胞和人肾癌细胞系ACHN细胞。采用RT-PCR和Western blot技术分别检测tumstatin在两种细胞中的表达情况。通过细胞增殖实验（CCK-8）和细胞周期分析评价tumstatin 过表达对ECV304细胞增殖的影响。为研究pcDNA-tumstatin 体外抑制血管内皮细胞的机制,采用western blot技术检测细胞周期蛋白D1的蛋白水平。 结果 DNA测序确认了pcDNA-tumstatin质粒构建成功。RT-PCR和western blot 结果表明tumstatin能在这两种细胞中有效表达。Tumstatin基因转入后,ECV304细胞生长受到明显抑制,细胞周期停滞于G1期。我们的研究也表明,pcDNA-tumstatin可能通过下调cyclin D1蛋白的水平来抑制血管内皮细胞增殖。 结论 pcDNA3.1+介导的tumstatin过表达能够特异性的抑制血管内皮细胞的增殖,这可能是由于cyclin D1下调导致的细胞周期阻滞于G1期导致的。此外,基因治疗或许是持续释放tumstatin的一种新的策略。     

Effects of cloned tumstatin-related and angiogenesis-inhibitory peptides on proliferation and apoptosis of endothelial ceils

Background Tumstatin is a recently developed endogenous vascular endothelial growth inhibitor that can be applied as an anti-angiogenesis and antineoplastic agent.The study aimed to design and synthesize the small molecular angiogenesis inhibition-related peptide (peptide 21),to replicate the structural and functional features of the active zone of angiogenesis inhibition using tumstatin and to prove that synthesized peptide 21 has a similar activity:specifically inhibiting tumor angiogenesis like tumstatin.Methods Peptide 21 was designed and synthesized using biological engineering technology.To determine its biological action,the human umbilical vein endothelial cell line ECV304,the human ovarian cancer cell line SKOV-3 and the mouse embryo-derived NIH3T3 fibroblasts were used in in vitro experiments to determine the effect of peptide 21 on proliferation of the three cell lines using the MTT test and growth curves.Transmission electron microscopy (TEM) and flow cytometry (FCM) were applied to analyze the peptide 21-induced apoptosis of the three cell lines qualitatively and quantitatively.In animal experiments,tumor models in nude mice subcutaneously grafted with SKOV-3 were used to observe the effects of peptide 21 on tumor weight,size and microvessel density (MVD).To initially investigate the role of peptide 21,the effect of peptide 21 on the expression of vascular endothelial growth factors (VEGFs) by tumor tissue was semi-quantitatively analyzed.Results The in vitro MTT test and growth curves all indicated that cloned peptide 21 could specifically inhibit ECV304 proliferation in a dose-dependent manner (P ＜0.01);TEM and FCM showed that peptide 21 could specifically induce ECV304 apoptosis (P ＜0.01).Results of in vivo experiments showed that tumors in the peptide 21 group grew more slowly.The weight and size of the tumors after 21 days of treatment were smaller than those in the control group (P ＜0.05),with a mean tumor inhibition rate of 67.86%;MVD of the tumor tissue in the peptide 21 group was significantly lower than in the control group (P＜0.05);the number of cells positive for VEGF in the peptide 21 group was significantly fewer than in the control group (P ＜0.01).Conclusions Similar to the activity of tumstatin in specifically inhibiting tumor angiogenesis,peptide 21 may specifically inhibit tumor endothelial cell proliferation and induce their apoptosis,thereby suppressing tumor angiogenesis and indirectly inhibit the growth,infiltration and metastasis of tumors.Peptide 21 may exert its effect through down-regulating the VEGF expression of tumor cells and vascular endothelial cells.     

Tumstatin transfected into human glioma cell line U251 represses tumor growth by inhibiting angiogenesis

Background Angiogenesis is a prerequisite for tumor growth and plays an important role in rapidly growing tumors,such as malignant gliomas.A variety of factors controlling the angiogenic balance have b...     

Effects of cloned tumstatin-related and angiogenesis-inhibitory peptides on proliferation and apoptosis of endothelial cells

Background Tumstatin is a recently developed endogenous vascular endothelial growth inhibitor that can be applied as an anti-angiogenesis and antineoplastic agent. The study aimed to design and synthesize the small molecular angiogenesis inhibition-related peptide （peptide 21）, to replicate the structural and functional features of the active zone of angiogenesis inhibition using tumstatin and to prove that synthesized peptide 21 has a similar activity： specifically inhibiting tumor angiogenesis like tumstatin. Methods Peptide 21 was designed and synthesized using biological engineering technology. To determine its biological action, the human umbilical vein endothelial cell line ECV304, the human ovarian cancer cell line SKOV-3 and the mouse embryo-derived NIH3T3 fibroblasts were used in in vitro experiments to determine the effect of peptide 21 on proliferation of the three cell lines using the MTT test and growth curves. Transmission electron microscopy （TEM） and flow cytometry （FCM） were applied to analyze the peptide 21-induced apoptosis of the three cell lines qualitatively and quantitatively. In animal experiments, tumor models in nude mice subcutaneously grafted with SKOV-3 were used to observe the effects of peptide 21 on tumor weight, size and microvessel density （MVD）. To initially investigate the role of peptide 21, the effect of peptide 21 on the expression of vascular endothelial growth factors （VEGFs） by tumor tissue was semi-quantitatively analyzed. Results The in vitro Ml-r test and growth curves all indicated that cloned peptide 21 could specifically inhibit ECV304 proliferation in a dose-dependent manner （P 〈0.01）; TEM and FCM showed that peptide 21 could specifically induce ECV304 apoptosis （P 〈0.01）. Results of in vivo experiments showed that tumors in the peptide 21 group grew more slowly. The weight and size of the tumors after 21 days of treatment were smaller than those in the control group （P 〈0.05）, with a mean tumor inhibition rate of 67.86%; MVD     

Tumstatin-EGFP在CHO细胞中的表达和活性分析

目的分析融合蛋白tumstatin-EGFP在中国仓鼠卵巢细胞(CHO)中的表达及生物学活性。方法经过酶切和测序正确的真核表达载体PIRESneo3/STL-EGFP、PIRES-neo3/sig-EGFP和空载体稳定转染CHO细胞后,用Western blot从细胞上清液中检测融合蛋白的表达、倒置荧光显微镜观察绿色荧光蛋白的表达。通过生长曲线分析外源基因的导入对CHO细胞生长的影响,MTT法和管状形成抑制试验评估tumstatin-EGFP融合蛋白的tumstatin样活性,体外靶向试验验证tumstatin能否携带融合蛋白特异性结合内皮细胞。结果获得了稳定转染的CHO细胞系。生长曲线表明转染后的较未转染的CHO细胞生长缓慢,MTT试验和管状形成抑制试验分别证实了融合蛋白tumstatin-EGFP具有抑制内皮细胞增殖,明显抑制血管管状结构的形成的作用;体外靶向试验也验证了tumstatin-EGFP能够特异性结合内皮细胞。结论重组真核表达载体在CHO细胞获得稳定表达,融合蛋白tumstatin-EGFP的表达不影响tumstatin和EGFP各自的生物学活性,并且在体外tumstatin能特异性携带EGFP靶向内皮细胞,为进一步tumstatin及其融合蛋白的研究奠定基础。     

肺癌组织α3(Ⅳ)基因表达及其临床研究

目的利用Taqman技术,建立荧光定量逆转录聚合酶链反应(FQ-RT-PCR)方法,对肺癌组织中Ⅳ型胶原α3链[α3(Ⅳ]]mRNA表达进行定量及蛋白表达水平检测,评价其临床应用价值。方法在α3(Ⅳ)基因NC1结构域编码序列设计一对引物和一条Taqman探针;优化反应体系的条件,以不同质粒DNA含量为标准品和其循环阈值(Ct值)制作标准曲线,检测肺癌组织中α3(Ⅳ)mRNA含量;并对本方法进行方法学评价。Western blot法检测tumstatin蛋白表达情况。结果成功建立了α3(Ⅳ)基因mRNA表达实时荧光定量PCR检测方法,检测灵敏度为800拷贝/μl;在103～108拷贝/μl之间与Ct值具有很好的线性关系;PCR扩增效率为99.4%;肺癌组织中α3(Ⅳ)mRNA表达定量检测发现,48例肺癌患者肺癌组织α3(Ⅳ)mRNA组含量显著低于癌旁组织,差异有统计学意义(P<0.01),肺癌组织中tum-statin蛋白表达下调/缺失率显著高于癌旁组织(P<0.01)。临床监测发现肺癌患者癌组织α3(Ⅳ)mRNA含量检测和TNM分期程度具有相关性(P<0.05)。结论α3(Ⅳ)与肺癌的发生发展关系密切,FQ-RT-PCR检测α3(Ⅳ)mRNA的表达为肺癌转移、预后判断、临床治疗等奠定良好基础。     

人Tumstatin功能区基因原核表达载体的构建

【目的】构建人肿瘤抑素（Tumstatin）功能区Tum-5原核融合表达载体pET42a-Tum5。【方法】根据人Tumstatin基因序列以及大肠杆菌密码子的偏爱性设计合成多个寡核苷酸,经PCR拼接获得Tum-5基因;酶切目的基因与pET42a载体,回收纯化后做定向克隆连接,转化E.coliDH5α;重组质粒通过PCR、BglⅡ与XhoⅠ的双酶切、DNA测序加以鉴定。【结果】重组质粒PCR扩增出的产物与目的基因大小相同;重组质粒双酶切电泳分析,插入片段大小约320 bp,与预期结果一致;对连接点两端进行测序,结果显示接头两端序列连接正确,插入序列与载体读码框架相匹配。【结论】成功构建人Tum-5基因表达载体,该研究为抗血管药物治疗实体瘤奠定了基础。