Tumor blood vessels formation in osteosarcoma: vasculogenesis mimicry

Background Osteosarcoma is characterized by high neovascularization and a high propensity for metastasis through bloodstream. This study was to examine whether there is evidence for vasculogenic mimicry in osteosarooma and to illustrate mechanism of tumor blood vessels formation in osteosarcoma. Methods Osteosarcoma cell lines (U-2OS) were tested for their ability to form tubular networks in three-dimensional culture containing type I collagen. The structures of the tubular networks were observed with phase contrast microscope and transmission electron microscope (TEM). Morphometric studies using hematoxylin and eosin (HE) stain and CD31 immunohistochemical stain to show tumor-lined channels in human osteosarooma were also performed. Results Observation with light microscope and TEM showed that highly aggressive osteosarcoma cell lines (U-2OS) formed networks containing channels when grown in three-dimensional culture containing type I collagen, in the absence of endothelial cells or fibroblasts. Morphometric observation using HE stain and CD31 immunohistochemical stain showed that tumor cell-lined channels were also detected in vivo in osteosarcoma; by comparison, all vascular areas in the pedicel of osteochondroma or outside osteochondroma were endothelial-lined. Conclusion These observations strongly suggest that aggressive osteosarcoma cells may generate vascular channels that facilitate tumor perfusion independent of tumor angiogenesis and have the ability of vasculogenic mimicry.  

Effect of human osteopontin on proliferation, transmigration and expression of MMP-2 and MMP-9 in osteosarcoma cells

Background To explore the effect of human osteopontin (hOPN) on the proliferation, transmigration and expression of matrix metallproteinase-2 (MMP-2) and matrix metallproteinase-9 (MMP-9) in osteosarcoma (OS) cells in vitro.Methods The prokaryotic-expression vector of hOPN was produced, hOPN was then subcloned into E. coli BL21 (DE3) cells and purified with ProBond^TM Columns. The proliferation, cell cycle and the expression of cycUn A in OS cells were investigated by using MTT assay, flow cytometry and Western blot respectively. The transmigration of OS cells was checked by using transwell cell culture chamber.The micro-pore-filter-membrane system was used to study the chemiotaxis of hOPN to OS cells. The levels of total protein were examined according to Coomassie Brilliant Blue manuals. The expression of MMP-2 and MMP-9 were evaluated by detecting the volume of degradation of gelatin on SDS-PAGE gel.Results The prokaryotic-expression vector of hOPN and purified hOPN protein were achieved hOPN promoted OS cells proliferation in a dose-dependent manner, and stimulated cyclin A expression in OS cells to accelerate cell division cycle, hOPN facilitated the trans-membrane migration of OS cells.hOPN also enhanced the secretion of MMP-2 and MMP-9 in OS cells.Conclusion hOPN could stimulate cyclin A expression in OS cells, hOPN has chemiotaxis to OS cells and increases their transmigration, hOPN enhances the secretion of MMP-2 and MMP-9 in OS cells.  

利用基因芯片技术筛选不同转移能力骨肉瘤细胞差异表达基因

目的：运用基因芯片技术研究骨肉瘤转移相关基因表达．方法：提取具有不同转移特性的骨肉瘤细胞总RNA，反转录制备杂交探针，应用基因芯片筛选与骨肉瘤转移相关的基因．杂交信号用ScanArray3000扫描，结果用ImaCene3．0软件分析．结果：对原位及转移骨肉瘤细胞基因表达诺分析，发现两种肿瘤细胞中存在差异表达基因，其中有抑癌基因、转移相关蛋白基因、细胞周期蛋白基因等，与肿瘤发生发展密切相关．结论：基因芯片技术对肿瘤转移机制研究有重要意义。  

环氧合酶-2在骨肉瘤中的表达及其意义

目的　探讨环氧合酶 2 (COX 2 )在骨肉瘤中的表达及其意义。方法　应用免疫组织化学染色研究人骨肉瘤组织、骨类肿瘤样病变 (骨纤维结构不良 )及正常骨组织中COX 2的表达 ,分析其表达差异性。结果　COX 2在正常骨组织中不表达 ,在骨肉瘤中表达阳性率为 6 7.7% ,骨纤维结构不良表达阳性率为 2 3.5 % ,后两者间的差异具有极显著性意义 (P <0 .0 1) ;在发生转移的骨肉瘤中COX 2表达阳性率为 82 .4 % ,明显高于未发生转移组 (5 0 .0 % ) ,两者差异有显著性意义 (P <0 .0 5 )。结论　骨肉瘤组织中存在COX 2的高表达 ,COX 2与骨肉瘤的发生发展有密切关系。  

巨噬细胞融合瘤苗对大鼠骨肉瘤的主动性免疫治疗

目的：探讨融合瘤苗在骨肉瘤免疫治疗中的应用潜力。方法：用ＵＭＲ１０６细胞系建立大鼠骨肉瘤动物模型，然后将该细胞系与大鼠巨噬细胞融合作为瘤苗应用于荷瘤大鼠，观察其抑制肿瘤生长和肺转移的效果。结果：在治疗６ＯＫ时治疗组和对照组相比，瘤重明显减小（Ｐ〈０．０１）；肺转移发生率降低（Ｐ〈０．０１）。结论：骨肉瘤细胞与巨噬细胞的两便瘤苗可望的骨肉瘤的治疗中发挥作用。  

RNA干扰沉默缺氧诱导因子-1α治疗骨肉瘤的实验研究

目的　观察靶向缺氧诱导因子 (HIF)- 1α的短发夹状小干扰RNA基因转染对骨肉瘤生长的影响。方法　针对HIF- 1α构建短发夹状siRNA真核表达载体 (pSilencer) HIF并转染骨肉瘤细胞系(SaOS) 2。骨肉瘤细胞置于缺氧诱导培养基中培养。逆转录 聚合酶链反应 (RT -PCR)和免疫印迹沉淀观察HIF -1α的基因沉默效果。四甲基偶氮唑盐 (MTT)比色分析检测细胞体外增殖活力,透射电镜,原位末端标记TUNEL法,AnnexinV/PI双标记法检测细胞的凋亡。同时,通过裸鼠移植瘤模型观察HIF- 1α基因沉默后对骨肉瘤体内治疗效果。结果　测序证实成功构建HIF- 1α的短发夹状siRNA真核表达载体pSilencer HIF,转染骨肉瘤细胞后,HIF -1α基因表达显著抑制 ( 90% )。与对照组比较,pSilencer HIF转染组骨肉瘤细胞缺氧状态下的增殖活性显著降低。缺氧培养 72h后,透射电镜、TUNEL法显示细胞典型凋亡特征,AnnexinV/PI双标检测转染组细胞凋亡率为 18. 71% ±0. 98%,明显高于pSilencer neo空载体转染组和未转染组 (P< 0 .01 )。裸鼠移植瘤实验显示pSilencer HIF转染组肿瘤生长减慢,成瘤率下降,肿瘤组织中可见大量坏死组织。结论　靶向HIF- 1α基因的短发夹状siRNA可以有效阻断骨肉瘤缺氧耐受转导通路,抑制骨肉瘤细胞的生长。  

蜂毒素诱导骨肉瘤细胞U2OS凋亡与坏死的实验研究

目的:研究蜂毒素是否能够诱导骨肉瘤细胞U2OS凋亡与坏死.方法:U2OS经不同浓度蜂毒素处理后,采用台盼蓝排染法测定细胞增殖抑制率,利用单克隆抗体,通过流式细胞仪检测AnnexinV、Apo2.7及Fas蛋白的表达.结果:64 mg/L蜂毒素作用U2OS 12 h、24 h,其坏死率在80%以上.16 mg/L、32 mg/L蜂毒素对细胞增殖有明显抑制作用,并且可诱导细胞凋亡和坏死的产生,增加细胞Aap2.7及 Fas 蛋白的表达.结论:蜂毒素高剂量有明显杀伤U2OS的作用,低剂量具有促细胞凋亡作用,并能上调Fas蛋白的表达.  

骨肉瘤血管内皮生长因子表达及微血管密度与肿瘤预后

目的：研究骨肉瘤中血管内皮生长因子（ＶＥＧＦ）的表达与微血管密度（ＭＶＤ）及其与临床预后的关系。方法：对６９例骨肉瘤患者的临床病理资料进行回顾性研究，应用ＣＤ３４，ＶＥＧＦ（ＪＨ１２１?mＡb对其组织切片分别进行免疫组化染色，并将上述资料与患者预后进行统计学分析。结果：骨肉瘤ＶＥＧＦ表达与ＭＶＤ相关（Ｐ〈０．０５）。骨肉瘤ＶＥＧＦ表达、ＭＶＤ同临床肿瘤大小、边界微小病灶、Ｅｎｎｅｋｉｎｇ外科分期一样  

人骨肉瘤细胞系OS-9901的建立及其生物学特性

目的：建立人骨肉瘤细胞系，为骨肉瘤研究提供新的实验模型。方法：取经病理证实的新鲜骨肉瘤手术标本，进行体外原代组织块培养。对存活细胞进行形态学观察、组织化学梁 色、细胞周期检查、核型分析、电镝观察和异种移植等。结果：建成细胞系ＯＳ－９９１０，其形态学表现、组化染色、电镜观察和异种移植等均符合骨肉瘤细胞特征。经半年多体外培养，已连续传代７０余次，细胞倍增时间为３３h，细胞周期测定：Ｇ１期６０．２％、Ｔ  

蜂毒素对骨肉瘤细胞系细胞增殖的影响

为了解蜂毒素对骨肉瘤细胞生长、增殖的作用。以骨肉瘤U2OS细胞体外培养，采用台盘蓝排染法观察蜂毒素对细胞生长的影响，MTT法测定细胞增殖抑制率，流式细胞仪检测细胞周期和增殖细胞核抗原（PCNA）的表达。结果：蜂毒素可明显抑制U2OS细胞生长、增殖，下调PCNA的表达，对细胞周期的影响表现为S期细胞的阻滞。提示蜂毒素具有抑制骨肉瘤U2OS细胞株生长、增殖的作用。