原核表达融合型血管生成抑制剂Kringle 5发酵条件的优化和初步纯化

在构建融合型Kringle 5原核表达重组菌BL21(DE3)pET32a/K5的基础上,通过改变培养基的成分,确定了适宜于重组菌生长和融合Kringle 5蛋白表达的培养基.采用正变实验方法,对融合型血管生成抑制剂Kringle 5原核表达重组菌的发酵条件进行了优化,通过发酵罐中重组菌的分批培养,确定了培养液中的最佳溶氧量.采用优化后的发酵工艺,在20 L发酵罐中融合Kringle 5的表达量可达0.59 g·L-1.最后在Cu2 螯合的Sepharose Fast Flow层析介质上对融合Kringle 5进行了初步纯化,其纯度约为80%.     

可溶型非融合血管生长抑制因子Kringle 5基因工程菌发酵培养和诱导表达条件的优化

在实验室试管培养条件下优化了基因工程菌E.coli BL21（DE3）pET15b/K5发酵产可溶型非融合血管生长抑制因子Kringle 5的培养基和诱导表达条件。经菌体干重测定和SDS-PAGE检测,确定最佳培养基（g.L-1）为：胰蛋白胨10.0,酵母提取物5.0,NaCl 10.0,葡萄糖6.0,NH4Cl 2.6,NaH2PO45.0,Na2HPO46.0;优化的诱导表达条件为：诱导剂浓度0.01mmol.L-1,诱导时间6h,诱导温度37℃,摇床转速220r.min-1。在优化的培养基和诱导表达条件下,菌体干重为1.8g.L-1,Kringle 5表达量为360mg.L-1,占总蛋白含量的20%,与基础LB培养基相比,Krin-gle 5表达量提高了1.18倍。     

人血管生成抑制素基因的克隆、表达及纯化

目的 构建携带人血管生成抑制素基因的原核表达载体,诱导表达具有活性的血管生成抑制素。方法 用PCR法从人纤溶酶原cDNA中扩增出Kringle 1-3基因,克隆入pET21a(+)载体中,在大肠杆菌BL 21中表达,表达产物经亲和层析纯化。结果 所构建的原核表达载体为高效表达系统,所表达的产物经层析纯化后可获得重组人血管生成抑制素。结论 可获得大量纯化人血管生成抑制素,为进一步临床应用奠定基础。     

环氧合酶抑制剂塞来昔布对神经胶质瘤细胞促血管新生作用的影响

目的探讨环氧合酶抑制剂塞来昔布对神经胶质瘤细胞促血管新生作用的影响及作用机制。方法体外分离培养人脐带静脉内皮细胞和人神经胶质瘤细胞,应用Matrigel建立血管形成的体外培养体系。结果塞来昔布具有抑制肿瘤细胞诱导血管新生作用,并且在一定范围内呈时间、浓度依赖效应。结论COX-2选择性抑制剂塞来昔布可以抑制神经胶质瘤细胞诱导的肿瘤血管新生。     

反义核酸药物与抗肿瘤研究

现在各国正在利用反义技术发展一类由核酸组成的新型药物,即反义核酸药物。我们合成了5种血管内皮生长因子(VEGF)的反义寡聚脱氧核苷酸(ODN_S)。测定了它们在S180肿瘤细胞中对VEGF表达的抑制作用,还测定了它们在鼠角膜肿瘤模型上抑制由肿瘤引起的新生血管形成和肿瘤的生长。结果表明,部分硫代的或发夹结构修饰的ODNs均可有效抑制VEGF的表达、肿瘤的生长和新生血管的形成。线性的ODN效果不好。两种作用于不同位点的ODN联合使用可得到最好的效果。因此反义VEGF寡聚脱氧核苷酸有望成为抗肿瘤和抗新生血管形成的新型药物。     

抗肿瘤血管新生的靶向药物研究进展

抗肿瘤研究一直以来都是医学界的研究热点,近年来随着医学研究的不断发展,抗肿瘤血管新生成为一种新型的靶向肿瘤治疗方法,这种方法具有诸多优势,所以迅速引起了业内外的广泛关注。就肿瘤血管生成机制及抗肿瘤血管新生的靶向药物研究进展进行了综述。     

抗肿瘤新生血管生成抑制剂临床研究概况

肿瘤的生长和转移与血管有着密切的关系,抑制肿瘤血管生成可以调节肿瘤的生长。近年来,抑制肿瘤新生血管的药物已成为药学领域的研究热点,一些新生血管抑制剂已经进入临床试验阶段,本文对抗肿瘤血管生成的理论进行了概述,并总结了新生血管生成抑制剂的临床研究情况。     

黄腐酸盐在制备促血管新生药物中的应用

<正>申请(专利权)人:昆明理工大学申请日期:2014.03.17申请(专利)号:CN201410097114主分类号:A61P9/10;A61K35/02本发明公开了黄腐酸盐在制备促血管新生药物中的应用,经临床研究表明,血管新生为缺血性血管疾病的血运重建开辟了新的治疗途径。实验结果显示,本发明所述的黄腐酸盐具有脐静脉     

人纤溶酶原饼环区5蛋白的原核表达体系的建立

目的　构建人纤溶酶原饼环区 5 (hPK 5 )蛋白原核表达载体 ,并进行表达和鉴定 ,为获取大量高纯度、具有生物活性的hPK 5蛋白奠定基础。方法　以纤溶酶原cDNA为模板 ,PCR扩增hPK 5基因 ,经酶切后构建表达载体pBV2 2 0 /hPK 5 ,转入大肠杆菌JM10 9进行温控诱导表达 ,表达产物经纯化、复性后 ,检测其抑制鸡胚绒毛尿囊膜 (CAM)血管新生的生物学活性。结果　带有pBV2 2 0 /hPK 5的大肠杆菌表达的目的蛋白占菌体总蛋白的34 8% ,其表达蛋白具有His tag抗原活性和野生活性。结论　已成功构建了pBV2 2 0 /hPK 5重组质粒 ,并在大肠杆菌中获得高效表达。     

废橡胶再生工艺及废橡胶新生剂

由李大年申请的专利 (专利号　 0 0 1 3 5 4 80 ,公布日期　 2 0 0 1 0 8 1 5 )“废橡胶再生工艺及废橡胶新生剂”涉及废硫化胶的再生工艺及所用新生剂。在炼胶机中 ,在低于 75℃的辊温下 ,废硫化胶与新生剂混合、薄通后 ,废硫化胶网状弹性结构中的硫交联键断裂 (主链断裂较少 ) ,废硫化胶变成可模压硫化的长链状塑性 (化合物 )物料。本发明新生剂具有开链效率和再生工效高、再生成本低的特点 ,再生后的硫化胶能保持原胶 6 5 %～ 75 %的性能。本再生工艺操作简便、能耗低、无污染、经济效益显著废橡胶再生工艺及废橡胶新生剂$杭州市科技情…     

An Update on Glioblastoma Biology,Genetics, and Current Therapies: Novel Inhibitors of the G Protein-Coupled Receptor CCR5

The mechanisms governing therapeutic resistance of the most aggressive and lethal primary brain tumor in adults, glioblastoma, have increasingly focused on tumor stem cells. These cells, protected by the periarteriolar hypoxic GSC niche, contribute to the poor efficacy of standard of care treatment of glioblastoma. Integrated proteogenomic and metabolomic analyses of glioblastoma tissues and single cells have revealed insights into the complex heterogeneity of glioblastoma and stromal cells, comprising its tumor microenvironment (TME). An additional factor, which isdriving poor therapy response is the distinct genetic drivers in each patient’s tumor, providing the rationale for a more individualized or personalized approach to treatment. We recently reported that the G protein-coupled receptor CCR5, which contributes to stem cell expansion in other cancers, is overexpressed in glioblastoma cells. Overexpression of the CCR5 ligand CCL5 (RANTES) in glioblastoma completes a potential autocrine activation loop to promote tumor proliferation and invasion. CCL5 was not expressed in glioblastoma stem cells, suggesting a need for paracrine activation of CCR5 signaling by the stromal cells. TME-associated immune cells, such as resident microglia, infiltrating macrophages, T cells, and mesenchymal stem cells, possibly release CCR5 ligands, providing heterologous signaling between stromal and glioblastoma stem cells. Herein, we review current therapies for glioblastoma, the role of CCR5 in other cancers, and the potential role for CCR5 inhibitors in the treatment of glioblastoma.     

载5-氟尿嘧啶纳米球的表征

5-氟尿嘧啶广泛用于治疗肝癌、乳腺癌等各种肿瘤,但却严重损伤骨髓、心脏等重要器官。为降低其毒副作用,以可生物降解的α-氰基丙烯酸正丁酯为载体,采用乳液聚合技术,制备了5-氟尿嘧啶聚α-氰基丙烯酸正丁酯纳米球。扫描电镜显示纳米球呈规则球形,平均粒径(181±17)nm,有利于通过网状内皮系统的摄取和肿瘤的高渗透和截留效应,实现肝及肝部肿瘤组织的被动靶向。紫外分光光度计测得纳米球载药量为41%。体外实验显示,纳米球体外释放呈两相特征,缓释相约9 h,表明α-氰基丙烯酸正丁酯纳米球能作为5-氟尿嘧啶的缓释载体,这将推动5-氟尿嘧啶新剂型的合成与发展。     

CYLD Inhibits the Development of Skin Squamous Cell Tumors in Immunocompetent Mice

Cylindromatosis (CYLD) is a deubiquitinase (DUB) enzyme that was initially characterized as a tumor suppressor of adnexal skin tumors in patients with CYLD syndrome. Later, it was also shown that the expression of functionally inactive mutated forms of CYLD promoted tumor development and progression of non-melanoma skin cancer (NMSC). However, the ability of wild-type CYLD to inhibit skin tumorigenesis in vivo in immunocompetent mice has not been proved. Herein, we generated transgenic mice that express the wild type form of CYLD under the control of the keratin 5 (K5) promoter (K5-CYLDwt mice) and analyzed the skin properties of these transgenic mice by WB and immunohistochemistry, studied the survival and proliferating characteristics of primary keratinocytes, and performed chemical skin carcinogenesis experiments. As a result, we found a reduced activation of the nuclear factor kappa B (NF-κB) pathway in the skin of K5-CYLDwt mice in response to tumor necrosis factor-α (TNF-α); accordingly, when subjected to insults, K5-CYLDwt keratinocytes are prone to apoptosis and are protected from excessive hyperproliferation. Skin carcinogenesis assays showed inhibition of tumor development in K5-CYLDwt mice. As a mechanism of this tumor suppressor activity, we found that a moderate increase in CYLD expression levels reduced NF-κB activation, which favored the differentiation of tumor epidermal cells and inhibited its proliferation; moreover, it decreased tumor angiogenesis and inflammation. Altogether, our results suggest that increased levels of CYLD may be useful for anti-skin cancer therapy.     

TRAIL-Inspired Multivalent Dextran Conjugates Efficiently Induce Apoptosis upon DR5 Receptor Clustering

Triggering apoptosis of tumor cells has been in focus of cancer-inspired research since decades. As clustering of death receptor 5 (DR5), which is overexpressed on various cancer cells, leads to formation of the death-inducing signaling cascade (DISC), DR5 has recently become a promising target for tumor treatment. Herein, we demonstrate that covalent multimerization of a death receptor targeting peptide (DR5TP) on a dextran scaffold generates potent apoptosis-inducing conjugates (EC₅₀=2–20 nm ). A higher conformational flexibility compared to reported DR5TP multimerization approaches, introduced by the polysaccharide framework compensates the reported need for the defined ligand orientation that was considered as essential prerequisite for effective receptor clustering and apoptosis induction. Enzyme-catalyzed ligation of a hydrophilic dextran conjugate bearing multiple DR5-targeting sites to a human fragment crystallizable (Fc) receptor did not affect the potency (EC₅₀=2–7 nm ), providing an option for improved in vivo half-life and prospective conjugation to an antibody of interest in view of bispecific tumor targeting.     

The Anti-Cancer Effects of a Zotarolimus and 5-Fluorouracil Combination Treatment on A549 Cell-Derived Tumors in BALB/c Nude Mice

Zotarolimus is a semi-synthetic derivative of rapamycin and a novel immunosuppressive agent used to prevent graft rejection. The pharmacological pathway of zotarolimus restricts the kinase activity of the mammalian target of rapamycin (mTOR), which potentially leads to reductions in cell division, cell growth, cell proliferation, and inflammation. These pathways have a critical influence on tumorigenesis. This study aims to examine the anti-tumor effect of zotarolimus or zotarolimus combined with 5-fluorouracil (5-FU) on A549 human lung adenocarcinoma cell line implanted in BALB/c nude mice by estimating tumor growth, apoptosis expression, inflammation, and metastasis. We established A549 xenografts in nude mice, following which we randomly divided the mice into four groups: control, 5-FU (100 mg/kg/week), zotarolimus (2 mg/kg/day), and zotarolimus combined with 5-FU. Compared the results with those for control mice, we found that mice treated with zotarolimus or zotarolimus combined with 5-FU retarded tumor growth; increased tumor apoptosis through the enhanced expression of cleaved caspase 3 and extracellular signal-regulated kinase (ERK) phosphorylation; decreased inflammation cytokines levels (e.g., IL-1β, TNF-α, and IL-6); reduced inflammation-related factors such as cyclooxygenase-2 (COX-2) protein and nuclear factor-κB (NF-κB) mRNA; enhanced anti-inflammation-related factors including IL-10 and inhibitor of NF-κB kinase α (IκBα) mRNA; and inhibited metastasis-related factors such as transforming growth factor β (TGF-β), CD44, epidermal growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF). Notably, mice treated with zotarolimus combined with 5-FU had significantly retarded tumor growth, reduced tumor size, and increased tumor inhibition compared with the groups of mice treated with 5-FU or zotarolimus alone. The in vivo study confirmed that zotarolimus or zotarolimus combined with 5-FU could retard lung adenocarcinoma growth and inhibit tumorigenesis. Zotarolimus and 5-FU were found to have an obvious synergistic tumor-inhibiting effect on lung adenocarcinoma. Therefore, both zotarolimus alone and zotarolimus combined with 5-FU may be potential anti-tumor agents for treatment of human lung adenocarcinoma.     

Stromal CCL5 Promotes Breast Cancer Progression by Interacting with CCR3 in Tumor Cells

Chemokines secreted from stromal cells have important roles for interactions with carcinoma cells and regulating tumor progression. C-C motif chemokine ligand (CCL) 5 is expressed in various types of stromal cells and associated with tumor progression, interacting with C-C chemokine receptor (CCR) 1, 3 and 5 expressed in tumor cells. However, the expression on CCL5 and its receptors have so far not been well-examined in human breast carcinoma tissues. We therefore immunolocalized CCL5, as well as CCR1, 3 and 5, in 111 human breast carcinoma tissues and correlated them with clinicopathological characteristics. Stromal CCL5 immunoreactivity was significantly correlated with the aggressive phenotype of breast carcinomas. Importantly, this tendency was observed especially in the CCR3-positive group. Furthermore, the risk of recurrence was significantly higher in the patients with breast carcinomas positive for CCL5 and CCR3 but negative for CCR1 and CCR5, as compared with other patients. In summary, the CCL5-CCR3 axis might contribute to a worse prognosis in breast cancer patients, and these findings will contribute to a better understanding of the significance of the CCL5/CCRs axis in breast carcinoma microenvironment.     

Efficacy of 5-FU Combined to Na[trans-RuCl(4)(DMSO)Im], A Novel Selective Antimetastatic Agent, on the Survival Time of Mice With P388 Leukemia, P388/DDP subline and MCa Mammary Carcinoma

The combinational treatment between the selective antimetastatic agent, sodium-trans-rutheniumtetrachloridedimethylsulfoxideimidazole, Na[trans-RuCl(4)(DMSO)Im], and the cytotoxic drug 5-fluorouracil (5-FU) on primary tumor growth and on the survival time of experimental tumors results in an effect significantly greater than that of each single agent used alone either with the solid metastasizing MCa mammary carcinoma of the CBA mouse or with the lymphocytic leukemia P388 and its platinum resistant P388/DDP subline. Thus the inorganic compound Na[trans-RuCl(4)(DMSO)Im], known for its potent and selective antimetastatic effects, positively interacts with the antitumor action of an organic anticancer agent such as 5-FU on both a solid metastasizing tumor and a tumor of lymphoproliferative type. In particular, the effects of the combinational treatment on the survival time of tumor bearing mice seem to be related to the selective antimetastatic activity of the ruthenium complex that joins the potent cytotoxicity of 5-FU for the tumor. Moreover, these data show that Na[trans-RuCl(4)(DMSO)Im] is almost as effective on the subline of P388 made resistant to cisplatin as it was on the parental line.     

Dietary menhaden oil enhances mitomycin C antitumor activity toward human mammary carcinoma MX-1

In the present study, we investigated the effects of high levels of dietary fish oil on the growth of MX-1 human mammary carcinoma and its response to mitomycin C (MC) treatment in athymic mice. We found that high levels of dietary fish oil (20% menhaden oil+5% corn oil, w/w) compared to a control diet (5% corn oil, w/w) not only lowered the tumor growth rate, but also increased the tumor response to MC treatment. We also found that high levels of dietary fish oil significantly increased the activities of tumor xanthine oxidase and DT-diaphorase, which are proposed to be involved in the bioreductive activation of MC. Since menhaden oil is highly unsaturated, its intake caused a significant increase in the degree of fatty acid unsaturation in tumor membrane phospholipids. This alteration in tumor membrane phospholipids made the tumor more susceptible to oxidative stress, as indicated by the increased levels of both endogenous lipid peroxidation and protein oxidation after feeding the host animals the menhaden oil diet. In addition, the tumor antioxidant enzyme activities, catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPOx), and glutathione S-transferase peroxidase (GSTPx), were all significantly enhanced by feeding a diet high in fish oil. MC treatment caused further increases in tumor lipid peroxidation and protein oxidation, as well as in the activities of CAT, SOD, GPOx, and GSTPx, suggesting that MC causes oxidative stress in this tumor model which is exacerbated by feeding a diet high in menhaden oil. Thus, feeding a diet rich in menhaden oil decreased the growth of human mammary carcinoma MX-1, increased its responsiveness to MC, and increased its susceptibility to endogenous and MC-induced oxidative stress, and increased the tumor activities of two enzymes proposed to be involved in the bioactivation of MC, that is, DT-diaphorase and xanthine oxidase. These findings support a role of these two enzymes in the bioactivating of MC and indicate that the type of dietary fat may be important in tumor response to therapy.