肿瘤抑素活性肽段及其衍生物研究新进展

肿瘤抑素(tumstatin)是继内皮抑素(endostatin)和血管生成抑素(angiostatin)发现以来,来源于人血管基膜Ⅳ型胶原蛋白的极具潜力的肿瘤抑制因子。它强大的抗肿瘤新生血管生成、诱导肿瘤细胞和内皮细胞凋亡等生物活性,使其成为近年来的研究热点。本文详细介绍了肿瘤抑素活性肽段及其衍生物的研究进展。对Tum-5、T7肽和19肽以及这些活性肽段连接NGR导向肽或人源性IgG3铰链区等肽段后形成的衍生物作了较详细的介绍。肿瘤抑素的活性肽段及其衍生物,以高选择性、高活性、低毒性等优点正成为抗肿瘤药物研究的新希望。     

肿瘤抑素诱导喉癌Hep-2细胞凋亡的实验研究

目的探讨肿瘤抑素对体外培养的人喉癌Hep-2细胞系凋亡的影响。方法用MTT法和台盼蓝染色法观察肿瘤抑素对喉癌Hep-2细胞生长的抑制作用。实验分为对照组和治疗组．光镜、电镜观察细胞形态及超微结构变化，采用DNA琼脂糖凝胶电泳检测细胞凋亡的梯形条带，TUNEL标记法检测细胞凋亡情况。结果随着肿瘤抑素浓度的增加喉癌Hep-2细胞的存活率下降。在相同浓度下。随着时间延长存活细胞逐渐减少。电镜观察治疗组细胞出现明显凋亡，琼脂糖电泳结果可见治疗组产生了特征性的DNA梯形条带，TUNEL标记法也证实治疗组可见明显的棕黄色凋亡细胞。结论肿瘤抑素通过诱导凋亡对体外培养的人喉癌Hep-2细胞系产生杀伤作用。     

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.     

The anti-tumor properties of two tumstatin peptide fragments in human gastric carcinoma

Aim： The aim was to study the anti-tumor activities and mechanisms of two synthetic peptide fragments of tumstatin （alpha3 （iV） NCl domain） in human gastric carcinoma cells in vitro and in vivo. Methods： MTr assay and cell cycle assay were used to study the anti-tumor and anti-angiogenic activities of two peptide fragments in vitro. Apoptosis induced by the two peptide fragments was demonstrated by TUNEL assay and morphological observation. The orthotopic tumor model was established to investigate the activities of two peptide fragments in vivo. Intratumor vascularization and the expressions of VEGF, bFGF, Fas, FasL, Bax, Bcl-2, and caspase 3 were determined using immunohistochemistry and Western blot analysis. Results： Peptide 19 inhibited SGC-7901 proliferation and induced apoptosis both in vitro and in vivo. Notably, peptide 21 suppressed the proliferation of HUVEC-12 cells in vitro. Each peptide arrested both cell lines at the G0/G1 phase of the cell cycle, and they also synergistically suppressed in vitro and in vivo tumor growth. Immunohistochemistry and Western blot analysis revealed the strong expression of Fas, FasL and caspase 3 in orthotopic tumor tissues treated with peptide 19 alone or in combination with peptide 21. Decreased expressions of VEGF and bFGF and decreased microvessel density （MVD） in orthotopic tumor tissues were seen in mice treated with peptide 21 alone or in combination with peptide 19. Conclusion： Two tumstatin peptide fragments facilitate two unique antitumor activities. Thus, they are drug candidates in the treatment of gastric carcinoma.     

肿瘤抑素抑制血管生成作用的研究进展

肿瘤抑素是一种能够有效抑制肿瘤新生血管和肿瘤细胞增殖的生物活性物质,通过双重途径抑制肿瘤生长,具有较强的肿瘤生长抑制作用。肿瘤抑素的生物活性和作用机制逐渐得到揭示,文章对其抗新生血管的活性和作用机制进行综述,并展望其在眼科的应用前景。     

肿瘤抑素T7肽及其衍生物T7-NGR载体构建及表达

目的为了提高作用靶向性，将导向肽NGR与肿瘤抑素L肽的C端连接，获得L肽及其衍生物T广NGR的载体。方法通过PCR和合成基因序列方法分别构建了肿瘤抑素L肽及T7-NGR的克隆载体pMD-T7和pMD-T7N，经酶切和测序鉴定后，将2种载体分别双酶切，经低熔点琼脂糖凝胶电泳分离后，切下目的片段与酶切回收的质粒载体pET28a在低熔点琼脂糖中直接进行连接反应。阳性克隆经酶切和测序鉴定，转化感受态EcoliBL21（DE3），IPTG诱导表达。结果酶切和测序鉴定结果正确，分别成功构建表达载体pET-T7和pET-T，N，经IPTG诱导，完成了表达条件的优化。Tricine-SDS-PAGE凝胶电泳结果显示．当IPTG浓度为1mmol/L时．25℃诱导8h．分别获得了T7肽和T7-NGR的表达产物。结论已经成功构建T，肽和T7-NGR的表达载体．获得了表达产物,为下一步的小肽活性实验奠定了基础。     

Cyt-C 依赖的线粒体途径在T42诱导肝癌HepG2细胞凋亡中的作用

目的 检测重组肿瘤抑素42肽(T42)诱导肝癌HepG2细胞凋亡及与线粒体凋亡途径的关系,探讨T42诱导肿瘤细胞凋亡的可能机制.方法 吖啶橙/溴化乙锭(AO/EB)荧光染色观察细胞凋亡的形态学变化;流式细胞仪检测凋亡率;JC-1荧光染色检测线粒体膜电位的变化;Western印迹检测细胞色素C(Cyt-C)的分布.结果 18 μmol/LT42作用下,HepG2细胞出现明显凋亡形态学变化,凋亡率为22.4%,与对照组比较差异有统计学意义(t=7.75,P＜0.05);T42降低了HepG2细胞线粒体膜电势,明显减少了线粒体Cyt-C.结论 T42通过降低线粒体膜电势,促进Cyt-C由线粒体膜释放到胞浆中,激活 caspase-3途径诱导人肝癌细胞系HepG2细胞凋亡.

Abstract：

Objective The aim of the present article is to detect the apoptosis of hepatocarcinoma cells HepG2 induced by recombinant tumor endostatin 42 peptide (T42) ,with an emphasis on the signaling pathways involved. Methods Observed the morphological changes associated with the apoptosis of HepG2 cells by using AO/EB. Apoptosis rate were dentified by using flow cytometry. Mitochondrial membrane potential was evaluated by using JC-1 fluorescent staining. The distribution of cytochrome C(Cytc ) was estimated by using western blot. Results Compared with the control group, there was significant difference in apoptosis rate of cells HepG2 under 18μmol /L of T42. (22.4% vs 3.70% ,t =7.75, P＜0.05). Mitochondrial membrane potential was decreased by T42, and cytochrome c was reduced significantly compared with the control group. Conclusions The result demonstrated that the T42 enhanced the apoptosis of HepG2 cells and its potential mechanism was related to the decreased of mitochondrial membrane potential, an increase in Cytochrome C released into the cytosol, and reduced activation of Caspase-3 channels.     

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.     

重组人tumstatin的原核生物表达和多克隆抗体制备

目的原核生物表达可溶性的tumstatin抗原并制备相应的多克隆抗体。方法利用原核表达载体pMAL-tumstatin在大肠埃希菌BL21中表达tumstatin,经Amylose Resin亲和层析柱和Q Sepharose Fast Flow柱纯化tumstatin,以纯化蛋白免疫新西兰兔,获得抗tumstatin多克隆抗体。利用western blot和ELISA法对多克隆抗体进行特异性和效价检测。结果tumstatin蛋白表达成功。SDS-PAGE分析表明其为可溶性表达。成功获得了tumstatin纯品及兔抗tumstatin多克隆抗体。ELISA法表明多克隆抗体效价>5 000。western blot检测证明多克隆抗体的特异性良好。结论成功表达、纯化tumstatin蛋白,并获得高特异性、高效价兔抗tumstatin多克隆抗体,为其在临床免疫学检测应用奠定了基础。     

肿瘤抑素30肽的重组表达及抗肿瘤活性研究

目的：重组表达肿瘤抑素30肽并研究其抗肿瘤活性。方法设计并合成30肽基因序列,将此序列与融合蛋白表达载体 PTYB21重组,转化到大肠埃希菌 BL-21(DE3)。 IPTG 诱导表达,几丁质柱纯化得到30肽,经 SDS-PAGE 及 Tricine-SDS-PAGE 对纯化产物进行鉴定。利用 MTT 法、吖啶橙/溴化乙锭(AO/ EB)荧光染色法、小鼠 H22腹水转移型肝癌实体瘤抑瘤实验,研究30肽的抗肿瘤活性。结果成功重组并表达肿瘤抑素30肽。体外实验显示,30肽具有抑制 HGC-27胃癌细胞、 HUVEC 人脐静脉细胞增殖和促进这两种细胞凋亡的作用。体内实验显示,30肽对小鼠 H22腹水型肝癌抑瘤率达43.18 ％ 。结论重组的肿瘤抑素30肽具有较强的抗肿瘤活性。