重组血管抑制剂CHM-Ⅰ对骨肉瘤的治疗作用

目的观察重组人软骨调节素(chondromodulin,CHM)蛋白对骨肿瘤的治疗作用.方法利用RT-PCR技术从人软骨组织中扩增出CHM-Ⅰ基因片段,并将其克隆到原核表达载体pET28a,在大肠杆菌中诱导表达和纯化重组人软骨调节素蛋白,将重组蛋白与人脐静脉上皮细胞或MG-63骨肉瘤细胞共培养,观察其对内皮细胞形成管样结构和MG-63骨肉瘤细胞生长的影响;将重组蛋白局部注射裸鼠皮下肿瘤,观察其对肿瘤形成的影响.结果获得纯化的重组人软骨调节素蛋白,该蛋白在体外可以抑制血管内皮细胞管样结构的形成而对肿瘤细胞的生长无影响;重组蛋白瘤内注射可以抑制肿瘤在裸鼠体内的生长.结论重组人软骨调节素蛋白通过抑制血管形成而抑制肿瘤生长,具有良好的临床应用价值.     

重组人血管内皮抑制素对裸鼠骨肉瘤的抑瘤作用

目的 研究Rh-Endostatin对人骨肉瘤细胞裸鼠移植瘤的抑瘤作用.方法 建立人骨肉瘤细胞OS-732皮下移植瘤裸鼠动物模型,分为4组,分别给予(a)生理盐水;(b、c、d) Rh-Endostatin低、中和高剂量,分别为2.5、5.0和10mg/kg;为腹腔注射给药,1次/天,4周后裸鼠全部处死,称量肿瘤重量计算抑瘤率,用药疗效.对肿瘤标本进行微血管密度计数和细胞凋亡指数检测.结果 Rh-Endostatin单药2.5、5和10mg/kg治疗抑瘤率分别为25.3%、34.1%和35.7%;微血管密度:所有治疗组与对照组比较,差异均具有统计学意义(P＜0.01).凋亡指数:治疗组与对照组比较,差异均具有统计学意义(P＜0.01).结论 Rh-Endostatin单药对骨肉瘤具有明显的抑瘤作用,抗血管生成治疗骨肉瘤具有潜在的临床应用价值,值得进一步临床试验评估其疗效.     

阿霉素联合重组人血管内皮抑制素对人骨肉瘤细胞OS-732作用的体外实验研究

目的确定阿霉素和重组人血管内皮抑制素两种药物联合对人骨肉瘤细胞的体外作用。方法阿霉素、重组人血管内皮抑制素及两药联用处理人骨肉瘤细胞OS-732,分别在24h、72h及144h应用MTT法测定其细胞毒性,同时用倒置相差显微镜观察细胞形态学改变。结果在阿霉素10μg/ml和100μg/ml作用72h和144h后,细胞增殖活性受抑制率达到50%以上,重组人血管内皮抑制素在第72h时对骨肉瘤细胞抑制率为7.3%～9.2%,联合用药组细胞增殖活性与阿霉素单药组无显著性差异。阿霉素和联合用药组细胞均发生细胞变小,核碎裂,失去细胞正常形态,而重组人血管内皮抑制素单药组细胞形态与对照组相似。结论重组人血管内皮抑制素仅在给药后短期内对人骨肉瘤细胞有轻度的抑制作用,重组人血管内皮抑制素不影响阿霉素对骨肉瘤细胞的细胞毒作用。     

人类骨肉瘤细胞的血管形成因子研究

Objective To partially purify the angiogenesis factor of human osteosarcoma(HuOs) and study its biological features. Methods The active peptide with a molecular weight of 8000-10000 Da in the conditioned medium obtained from the cultivation of Hu-Os cells(osteoblastic osteosarcoma) was partially purified by ultrafiltration, chromatography and dialysis.The angiogenic effects of the fractions were assessed by proliferation assay of human umbilical vein and pig thoracic aorta endothelial cells. Results The chromatography fractions 4-6 could significantly promote the proliferation of the endothelial cells.Conclusion The HuOs cells could synthesize and secrete angiogenesis factor with a molecular weight of 8000-10000 Da.     

蛋白酶体抑制剂Z-LLL-CHO对人骨肉瘤细胞株MG-63作用的体外研究

目的　探讨蛋白酶体抑制剂Z LLL CHO对人骨肉瘤细胞株MG 63的作用 ,并探讨其作用机制。方法　将不同浓度的Z LLL CHO作用于骨肉瘤细胞株MG 63 ,采用荧光显微镜观察细胞形态改变、电镜观察细胞超微结构变化、MTT法检测细胞增殖活力、琼脂糖凝胶电泳检测细胞凋亡、流式细胞仪分析细胞凋亡率及细胞周期改变。结果　Z LLL CHO可有效抑制MG 63细胞株的生长 ,并诱导细胞发生凋亡。凋亡细胞表现为浓染致密的颗粒块状荧光 ,胞核固缩、染色质凝聚并边缘化 ,DNA呈“阶梯状”排列的条带。流式细胞仪分析显示 ,Z LLL CHO在 1.0 μmol/L作用 2 4h、3 6h、48h后 ,细胞凋亡率分别为 5 .4%、2 0 .5 %、5 2 .7%。随药物浓度增高及作用时间延长 ,细胞周期被阻滞于G2 /M期。结论　蛋白酶体抑制剂Z LLL CHO可有效抑制人骨肉瘤MG 63细胞株的生长增殖 ,阻滞细胞周期及诱导细胞凋亡可能起重要作用。     

肿瘤血管抑制剂的研究进展

肿瘤生长和转移具有血管依赖性，应用肿瘤血管抑制剂改变肿瘤血管生长促进因子和抑制因子的平衡可有效地抑制肿瘤生长、转移和复发，是近年来肿瘤研究的新热点之一，也是肿瘤防治的又一条新途径。本文对肿瘤血管抑制剂的种类、各自的作用机理以及临床应用的研究进展作一综述。     

血管生长抑制因子

恶性实体肿瘤的生长必须依赖新生血管持续不断地为其提供营养和排除代谢产物。因此，如能抑制或破坏这些新生血管就可防止恶性肿瘤的生长与转移，这是近年来癌症研究的一个新领域。国外为寻找效果好、毒性低的血管生长抑制因子进行了大量研究。血管生长抑制因子对癌症治疗具有良好前景。     

血管抑素、bFGF与骨肉瘤血管形成

血管生成是微血管内皮细胞的一系列连续过程。生理性血管的生成对于胚胎发育、创伤愈合和月经周期是必需的，在时间性和空间性上都受到严密调控。而肿瘤血管生成则是持续性、不受控制和无序的。肿瘤的发生发展由血管生成因子和抗血管生成因子共同控制，因此探讨血管形成在肿瘤发生发展中的作用，不仅有助于防治骨肉瘤的研究，也有助于寻找骨肉瘤转移和侵袭的机制，并有利于开发特异性靶向于骨肉瘤血管组织的血管生成抑制剂。     

重组腺病毒介导小鼠内皮抑素对荷骨肉瘤MG-63细胞裸鼠肺转移的抑制

目的: 构建携小鼠内皮抑素(endostatin， ES)基因的重组腺病毒载体，观察其对荷骨肉瘤裸鼠肺转移的抑制，探讨内皮抑素表达水平与骨肉瘤肺转移的关系。方法:构建pDC315mEndo表达质粒，同源重组产生重组腺病毒AdmEndo。裸鼠右前肢皮下注射骨肉瘤MG63细胞建立移植瘤裸鼠模型；随机分为4组：小鼠内皮抑素腺病毒（AdmEndo）组，携带EGFP基因腺病毒（AdEGFP）组， PBS组，未接种肿瘤细胞裸鼠空白对照组。各组裸鼠每周分别注射相应药物200 μl，连续5次，观察各组动物移植瘤体积、瘤组织病理，ELISA法检测各组裸鼠血ES水平；7周后处死动物，观察有无肺转移及肺转移灶病理。结果：AdEGFP组肿瘤体积为（1.53±0.05） cm3,PBS组为（1.56±0.07） cm3, AdmEndo组为（0.91±0 .03） cm3，AdmEndo治疗的抑瘤率达40.7％。AdmEndo组裸鼠血内皮抑素表达水平明显高于AdEGFP组和PBS组（P<0.05）。AdmEndo组裸鼠肺部未发现肿瘤转移灶，其他两组肺部见大量散在转移灶，肺转移率分别为80%和90%。未发生肺转移裸鼠的ES水平显著高于发生肺转移的裸鼠（P<0.05）。结论：腺病毒介导的小鼠内皮抑素显著抑制了荷骨肉瘤裸鼠肺转移，内皮抑素表达水平与肺转移有着直接的关系。     

PDGF-BB通过外泌体促进人骨肉瘤的血管新生

目的:探索PDGF-BB是否可以通过外泌体传递,并且验证其在人骨肉瘤中的促血管新生的功能.方法:分离多种人骨肉瘤细胞中的外泌体,WB法检测PDGF-BB在人骨肉瘤细胞及其外泌体中的表达,将骨肉瘤SJSA-1细胞来源的外泌体与HUVEC共孵育,免疫荧光和激光共聚焦扫描显微镜观察外泌体PDGF-BB进入细胞的方式;慢病毒感...     

Effect of the histone deacetylase inhibitor SNDX-275 on Fas signaling in osteosarcoma cells and the feasibility of its topical application for the treatment of osteosarcoma lung metastases

BACKGROUND:

Patients with lung metastases from osteosarcoma (OS) have poor response to salvage therapy. Understanding the mechanisms involved in the metastatic process of OS may lead to new effective therapeutic approaches. The authors reported previously that up‐regulation of the Fas receptor by transfecting OS cells with Fas plasmid inhibited the in vivo growth of metastases in the lungs.

METHODS:

In the current study, the authors treated OS cells with the histone deacetylase inhibitor SNDX‐275 and studied its cytotoxicity and its effect on Fas signaling in vitro and in vivo.

RESULTS:

Subtoxic doses of SNDX‐275 were able to activate the Fas pathway in OS cells by increasing the expression of Fas messenger RNA; however, the increased expression was not always followed by increased levels of Fas receptor expression on the cell surface. The treatment of cells with a combination of SNDX‐275 and Fas ligand (FasL) had a stronger cytotoxic effect on tested OS cells than either agent alone. Inhibition of the Fas pathway in cells by inhibition of the Fas‐associated death domain (FADD) molecule eliminated this combination effect, indicating that activity of FADD is important for the efficacy of this agent in the FasL‐expressing environment of the lungs. Intranasal administration of SNDX‐275 in mice with OS lung metastases revealed that SNDX‐275 may inhibit metastatic growth at a dose of 0.13 mg/kg, which is approximately 200‐fold lower than the therapeutically effective oral dose reported previously.

CONCLUSIONS:

The current findings indicated that SNDX‐275 can activate Fas signaling in OS cells in vitro and in vivo and that the administration of SDNX‐275 by inhalation is feasible as a treatment for OS metastases and warrants its further investigation. Cancer 2011. © 2011 American Cancer Society.     

血清血管生成因子在骨肉瘤组织中的表达及其临床意义

目的:探讨血清血管形成相关因子在骨肉瘤组织中的表达,及其与临床病理和微血管密度(MVD)的关系。方法:免疫组化SP法检测80例骨肉瘤组织中VEGF、CD34和FⅧRag等蛋白表达,并根据CD34和FⅧRag免疫组化染色结果计数MVD。同时采用酶联免疫分析定量检测36例骨肉瘤患者血清中的血管生成因子:血管内皮生成因子(VEGF)、碱性成纤维因子(bFGF)、转化生长因子(TGFβ1)以及内皮抑制素(edostatin,ES)的表达。结果:1)术前血清VEGF(1706ng/dL)和ES(302.4ng/dL)水平明显高于正常对照组,是VEGF的升高与肿瘤的微血高管密度、患者早期复发及其组织高表达密切相关;2)血清VEGF水平(1706ng/dL)和ES(302.4ng/dL)水平呈正相关;3)术后VEGF(490.0ng/dL)和ES(32.7ng/dL)水平明显下降,VEGF复发组较未复发组高,但差异无统计学意义,而复发组的ES水平明显低于未复发组。结论:术前血清VEGF和术后ES水平能反映骨肉瘤血管生成特性,对骨肉瘤早期复发具有重要预测价值,可作为抗血管生成的重要靶标。     

癌基因MSP58在骨肉瘤组织中的表达及其对骨肉瘤细胞增殖的影响

目的:研究MSP58基因在骨肉瘤组织中的表达,探讨干涉MSP58基因对体外培养的U2OS骨肉瘤细胞增殖的影响。方法:利用Real-time PCR（n=15）和Western blot（n=7）方法分别检测新鲜骨肉瘤及癌旁组织中MSP58 mRNA和蛋白的表达情况,并以癌旁组织作为对照。用癌基因MSP58特异的siRNA转染U2OS骨肉瘤细胞,而后采用MTT法检测细胞增殖情况,流式细胞术检测细胞周期。结果:与癌旁组织相比,MSP58基因mRNA和蛋白水平在骨肉瘤组织中的表达明显升高（P<0.05）。干涉MSP58基因表达明显抑制U2OS骨肉瘤细胞增殖,同时G1期细胞明显增多,G2/M期和S期细胞明显减少（P<0.05）。结论:MSP58基因在骨肉瘤肿瘤组织中过度表达,下调MSP58表达可抑制骨肉瘤细胞增殖。     

Increased pre-therapeutic serum vascular endothelial growth factor in patients with early clinical relapse of osteosarcoma

To investigate the clinical significance of circulating angiogenic factors, especially in association with early relapse of osteosarcoma, we quantified pre-therapeutic levels of vascular endothelial growth factor, basic fibroblast growth factor and placenta growth factor in the sera of 16 patients with osteosarcoma using an enzyme-linked immunosorbent assay. After a 1-year follow-up, the serum level of angiogenic factors was analysed with respect to microvessel density of the biopsy specimen and clinical disease relapse. The serum vascular endothelial growth factor levels were positively correlated with the microvessel density with statistical significance (P=0.004; Spearman rank correlation) and also significantly higher in seven patients who developed pulmonary metastasis than the remaining nine patients without detectable disease relapse (P=0.0009; The Mann-Whitney U-test). In contrast, the serum levels of basic fibroblast growth factor or placenta growth factor failed to show significant correlation with the microvessel density or relapse of the disease. Although there was no significant correlation between serum vascular endothelial growth factor levels and the tumour volume, the serum vascular endothelial growth factor levels were significantly higher in patients with a vascular endothelial growth factor-positive tumour than those with a vascular endothelial growth factor-negative tumour. These findings suggest that the pre-therapeutic serum vascular endothelial growth factor level reflects the angiogenic property of primary tumour and may have a predictive value on early disease relapse of osteosarcoma.     

人骨肉瘤鸡胚移植瘤模型的生物学特性研究

目的:建立人骨肉瘤鸡胚移植瘤模型,研究其形态学及生物学特性。方法:将人骨肉瘤细胞接种于鸡胚绒毛尿囊膜(chick embryo chorioallantoic membrane,CAM),观察影响骨肉瘤鸡胚移植瘤成活的因素、移植瘤生长特性、形态学特征和生物学性状。结果:建立了人骨肉瘤鸡胚移植瘤模型。移植瘤易于生长,具有较强的血管诱导作用。光镜下移植瘤具有与人骨肉瘤类似的组织结构。结论:该模型易于复制,能动态观察骨肉瘤诱导的血管生成过程,可用于骨肉瘤的生物学行为研究、药物筛选等领域。     

骨肉瘤CT灌注成像与血管生成的关系

Objective  

The aim of the study was to explore the application of 64-slice spiral computed tomography perfusion imaging (CTPI) in evaluating angiogenesis in human osteosarcoma.