新型重组人血管内皮抑素恩度治疗恶性浆膜腔积液的研究进展

Since recombinant human endostatin (rh-endostatin; Endostar) has been listed 5 years, clinicians have combined it with chemotherapy for the treatment of lung cancers and other malignant tumors, and proved its effect and safety. A number of scholars have explored the application of Endostar alone or in combination with chemotherapy for treatment of malignant serous effusion, finding its high efficiency and low toxicity; and that hydrops controlling is stronger, and that it can significantly improve patients' quality of life. It is worthy of conducting prospective, randomized and multi-center clinical studies and basic researches to clarify the mechanism.     

pEgr-ssEndostatin的构建及其在体外的辐射诱导表达

为探讨pEgr-ssEndostatin重组质粒转染B16细胞后接受不同辐射剂量以及接受同一剂量不同时程endostatin表达的规律。采用RT-PCR方法从鼠肝脏中扩增出Endostatin cDNA,连入信号肽,构建了pEgr-ssEndostatin重组质粒,用脂质体介导的转染法转染小鼠B16细胞,用ELISA方法检测B16细胞上清中endostatin的表达水平。结果表明的分泌型Endostatin序列与报道完全一致,Egr-1启动子和Endostatin cDNA正确插入表达载体pcDNA3．1;pEfr-ssEndostatin具有辐射诱导表达特性。提示小鼠Endostatin基因成功地被克隆和表达,Egr-1启动子具有辐射激活和诱导下游基因表达增强的功能。     

Co-delivery of docetaxel and endostatin by a biodegradable nanoparticle for the synergistic treatment of cervical cancer

Cervical cancer remains a major problem in women''s health worldwide. In this research, a novel biodegradable d-α-tocopheryl polyethylene glycol 1000 succinate-b-poly(ε-caprolactone-ran-glycolide) (TPGS-b-(PCL-ran-PGA)) nanoparticle (NP) was developed as a co-delivery system of docetaxel and endostatin for the synergistic treatment of cervical cancer. Docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPs were prepared and further modified by polyethyleneimine for coating plasmid pShuttle2-endostatin. All NPs were characterized in size, surface charge, morphology, and in vitro release of docetaxel and pDNA. The uptake of coumarin 6-loaded TPGS-b-(PCL-ran-PGA)/PEI-pDsRED by HeLa cells was observed via fluorescent microscopy and confocal laser scanning microscopy. Endostatin expression in HeLa cells transfected by TPGS-b-(PCL-ran-PGA)/PEI-pShuttle2-endostatin NPs was detected using Western blot analysis, and the cell viability of different NP-treated HeLa cells was determined by MTT assay. The HeLa cells from the tumor model, nude mice, were treated with various NPs including docetaxel-loaded-TPGS-b-(PCL-ran-PGA)/PEI-endostatin NPs, and their survival time, tumor volume and body weight were monitored during regimen process. The tumor tissue histopathology was analyzed using hematoxylin and eosin staining, and microvessel density in tumor tissue was evaluated immunohistochemically. The results showed that the TPGS-b-(PCL-ran-PGA)/PEI NPs can efficiently and simultaneously deliver both coumarin-6 and plasmids into HeLa cells, and the expression of endostatin was verified via Western blot analysis. Compared with control groups, the TPGS-b-(PCL-ran-PGA)/PEI-pShuttle2-endostatin NPs significantly decreased the cell viability of HeLa cells (p < 0.01), inhibited the growth of tumors, and even eradicated the tumors. The underlying mechanism is attributed to synergistic anti-tumor effects by the combined use of docetaxel, endostatin, and TPGS released from NPs. The TPGS-b-(PCL-ran-PGA) NPs could function as multifunctional carrier for chemotherapeutic drugs and genetic material delivery, and offer considerable potential as an ideal candidate for in vivo cancer therapy.     

恩度联合TACE治疗原发性肝癌的疗效

目的 探讨重组人血管内皮抑制素（恩度）联合经肝动脉化疗栓塞（TACE）治疗原发性肝癌的临床疗效及安全性.方法 对64例原发性肝癌患者采用恩度联合TACE治疗.TACE治疗方案：吡柔比星60 mg＋奥沙利铂100 mg＋超液化碘油10-20 mL经三通阀门充分乳化混合,部分患者加用明胶海绵栓塞;TACE术后第2天给予恩度15 mg·d-1静脉滴注,d2-15.每例患者至少完成1次治疗,每周复查血常规、生化,4～6周后复查AFP及腹部CT或MRI,根据复查结果评估疗效并确定是否再次治疗,2次治疗TACE间隔4～6周.结果 64例患者共接受176次治疗,总有效率为73.4%,其中 CR（完全缓解） 5例（7.8%）、PR（部分缓解） 42例（65.6%）、SD（稳定）10例（15.6%）、PD（疾病进展） 7例（10.9%）.TTP（疾病进展时间）为9.0个月,OS（总生存期）为11.6个月.不良反应较轻微,主要为1-2级,其中发热54例（84.4%）、恶心21例（32.8%）、呕吐23例（35.9%）、腹痛41例（64.1%）、便秘21例（32.8%）、白细胞减少20例（31.3%）、转氨酶升高44例（68.8%）,无严重不良反应.结论 恩度联合TACE是原发性肝癌安全有效的治疗方式.     

姜黄素对Lewis肺癌小鼠血管内皮成长因子和endostatin表达的影响

目的:探讨姜黄素对Lewis肺癌组织中血管内皮成长因子（VEGF）和血管生成抑制因子抑制素（endostatin）表达的影响。方法: 本实验分为3组：空白对照组、模型组、姜黄素组（100 mg/kg）。空白对照组设10只C57BL/6纯系小鼠,另20只小鼠右腋皮下接种Lewis瘤细胞,随机分为2组：模型组、姜黄素组（100 mg/kg）,每组10只。接种成功后,空白对照组与模型组灌胃0.5%羟甲基纤维素钠溶液,姜黄素组灌胃100 mg/kg,灌胃容积为0.2 mL/10 g,连续灌胃14 d,每天1次。末次给药后处死小鼠,称其体重,剥离肿瘤组织称重后采用Western Blot法和Q-PCR法检测VEGF、endostatin蛋白及基因的表达。结果:给予Lewis肺癌小鼠姜黄素治疗后肿瘤体积与质量明显减少,VEGF的表达显著减少和endostatin的表达明显增加。 结论:姜黄素抑制肺癌肿瘤的生长,其作用可能是通过下调VEGF与上调endostatin的表达,从而抑制肿瘤血管的生长。     

Disruption of the KEX1 gene in Pichia pastoris allows expression of full‐length murine and human endostatin

Endostatin is a potent angiogenesis inhibitor. In order to isolate sufficient quantities of soluble protein for in vivo studies in mice, we expressed murine endostatin in Pichia pastoris. Analysis of the expressed protein by mass spectrometry indicated that the protein was truncated. N‐terminal sequence analysis determined that the N‐terminus was intact, suggesting that the C‐terminal lysine was missing. In Saccharomyces cerevisiae, Kex1p can cleave lysine and arginine residues from the C‐terminus of peptides and proteins. We hypothesized that the KEX1 homologue in P. pastoris is responsible for the loss of the C‐terminal lysine of endostatin. To test this hypothesis, we cloned and disrupted the P. pastoris KEX1 gene. Although the overall amino acid identity between the P. pastoris and the S. cerevisae Kex1p is only 36%, the amino acid residues involved in the catalytic activity or close to the active residues are highly conserved. Disruption of the KEX1 reading frame allowed expression of murine and human endostatin with the C‐terminal lysine. The KEX1 disruption strain may be a useful tool for the expression of other proteins with a C‐terminal basic amino acid. Addition of a lysine to the C‐terminus of recombinant proteins may protect the C‐terminus from degradation by other carboxypeptidases. 3·5 kb of the P. pastoris KEX1 gene locus have been deposited in the GeneBank database and are available under Accession No. AF095574. Copyright © 1999 John Wiley & Sons, Ltd.     

持续泵注恩度联合化疗新辅助治疗局部晚期直肠癌的对比研究

目的: 探讨持续泵注重组人血管内皮抑制素（恩度）联合mFOLOFX6方案新辅助治疗局部晚期直肠癌的临床有效率、病理缓解率及安全性。方法: 62例符合入组标准患者被随机分配到mFOLFOX6组（对照组）和恩度联合mFOLFOX6组（试验组）。对照组具体用法：奥沙利铂,85 mg/m2,第1天,静脉滴注2 h;亚叶酸钙,200 mg/m2,第1天,静脉滴注2 h;氟尿嘧啶,0.4 g/m2,静脉推注;氟尿嘧啶,2.6 g/m2,亚叶酸钙滴注结束后,立即持续泵入46 h,每2周重复;试验组具体用法：在mFOLFOX6基础上,恩度7.5 mg·m-2·d-1,每天持续24 h静脉泵入,连续7 d,每2周重复;治疗4到6周期后接受根治性手术。结果: 62例患者一共接受 310个周期的治疗,均可评价疗效;试验组的总有效率为62.5%;对照组为36.6%,试验组的总有效率高于对照组,两组差异有统计学意义（P<0.05）;试验组病理完全缓解率为28.1%,对照组为6.7%,试验组的病理完全缓解率高于对照组,两组差异有统计学意义（P<0.05）;试验组保肛率为81.3%,对照组为56.7%,试验组的保肛率高于对照组,两组差异有统计学意义（P<0.05）;两组的毒性反应较轻,均以Ⅰ、Ⅱ级为主,两组差异无统计学意义（P>0.05）;两组患者的手术并发症发生率均较低,两组差异无统计学意义（P>0.05）。结论: 重组人血管内皮抑制素（恩度）联合mFOLOFX6方案新辅助治疗局部晚期直肠癌可以提高总有效率、病理完全缓解率和保肛率,不增加不良反应和并发症,值得临床推广应用。     

人内皮抑素基因的定点突变及其在大肠杆菌中的表达

目的对人内皮抑素(hES)基因进行定点突变,提高其在大肠杆菌中的可溶性表达量。方法根据Internet网站提供的关于hES的结构信息及其结构与功能方面的研究文献,设计突变位点;利用生物信息学的相关网站,对hES突变体进行结构预测,验证突变位点设计的合理性;采用重叠延伸PCR方法进行hES基因的定点突变,并将突变基因和未突变基因分别亚克隆至表达载体pGEX-4T-3,在大肠杆菌中进行融合表达,比较突变前后目的蛋白的可溶性表达量。结果三级结构预测结果表明突变位点设计合理,序列分析结果表明实现了hES基因的定点突变。突变基因的表达产物中,可溶性部分约占总表达量的40%,而未突变基因的表达产物几乎全部为包涵体。结论已成功对hES基因进行了定点突变,突变后的hES可溶性表达量得到了提高。     

人与鼠源内皮抑制素不同标签重组蛋白的表达纯化及活性分析

根据文献报道和NCBI数据库确定人源内皮抑制素(HE)和鼠源内皮抑制素(ME)DNA序列,以DNA合成法分别获得长度为554 bp的人源内和长度为565 bp的鼠源内皮抑素基因;分别构建携带谷胱甘肽巯基转移酶(GST)表达标签的原核表达质粒pGEX-4T1-HE和携带硫氧还蛋白A(TrxA)表达标签的原核表达质粒pET-32a-ME,在大肠杆菌BL21(DE3)中诱导表达,获得不同表达标签的HE、ME重组蛋白. 最后通过鸡胚绒毛尿囊膜(CAM)试验验证重组蛋白的抗血管生成活性. 结果表明,构建了人与鼠源内皮抑制素基因不同标签表达载体,在大肠杆菌中成功诱导表达. 获得具有活性的人与鼠源ES重组蛋白. 纯化复性后的重组蛋白能明显抑制新生血管的生长. GST和TrxA标签对增进重组ES可溶表达没有显著差异,两组标签的重组蛋白都主要存在于包涵体内.