基质金属蛋白酶-9与骨肉瘤鸡胚移植瘤血管生成的关系

目的 建立人骨肉瘤鸡胚移植瘤模型，探讨基质金属蛋白酶-9（Matrix Metalloproteinase-9，MMP-9）在移植瘤模型中的表达及其与肿瘤血管生成的关系。方法 人骨肉瘤细胞接种于鸡胚绒毛尿囊膜（chick embryo chorioallantoic membrane，CAM），建立人骨肉瘤鸡胚移植瘤模型，观察移植瘤生长特性和生物学性状；采用免疫组化法检测移植瘤组织中MMP-9的表达及微血管密度（microvessel density，MVD）。结果 建立了人骨肉瘤鸡胚移植瘤模型，移植瘤具有较强的诱导血管生成作用，骨肉瘤鸡胚移植瘤中，MMP-9的表达水平与MVD呈显著性正相关（P〈0．001，r=0．855）。结论 人骨肉瘤鸡胚移植瘤模型易于复制、操作简单、适用范围广，是研究骨肉瘤诱导血管生成的较好动物模型；人骨肉瘤鸡胚移植瘤中MMP-9表达水平与骨肉瘤诱导的血管生成密切相关。     

仿血管通道结构在大鼠和裸鼠骨肉瘤组织微血管形成中的作用

目的:研究肿瘤仿血管通道在骨肉瘤裸鼠移植瘤组织中的结构特点及其在肿瘤微血管形成中的作用。方法:建立荷人骨肉瘤MG-63细胞裸鼠移植瘤和荷骨肉瘤UMR106细胞大鼠移植瘤模型,以CD34、CD147免疫荧光和GFP荧光法观察移植瘤组织中肿瘤仿血管通道的结构特点及其与肿瘤微血管的关系。结果:骨肉瘤MG-63细胞移植瘤和UMR106细胞移植瘤组织CD147染色均呈阳性,血管内皮细胞CD34染色均阳性。肿瘤仿血管通道主要位于移植瘤组织中心区域,主要由骨肉瘤细胞构成,是具有功能的类血管通道;肿瘤微血管主要位于移植瘤周边区域,主要由宿主组织的血管内皮细胞组成,是与肿瘤仿血管通道存在直接相通的管道样结构。结论:大鼠和裸鼠骨肉瘤细胞移植瘤组织中存在的肿瘤仿血管通道具有类血管功能,并与来源于宿主组织的肿瘤微血管相互联通,诱导肿瘤微血管侵入肿瘤内部生长。     

Roquinimex治疗人大肠癌的抗血管生成作用研究

目的：观察Roquinimex对人大肠癌细胞系SW1116的体内抑瘤效应,并探讨其可能的作用机制。方法：建立人大肠癌裸鼠移植瘤模型,比较各剂量Roquinimex的抑瘤效应的强弱。同时运用免疫组化方法,检测人大肠癌裸鼠移植瘤块的瘤灶内微血管密度（MVD）,并观察Roquinimex对鸡胚尿膜囊血管生长的影响。结果：体内实验显示Roquinimex能明显抑制SW1116裸鼠移植瘤的生长,各剂量Roquinimex治疗组的瘤块平均体积均小于空白对照组（P＜0．01）。各剂量Roquinimex治疗组瘤块微血管密度明显低于未经治疗组（P＜0．05）。在鸡胚尿膜囊血管生长模型中,Roquinimex明显抑制了鸡胚尿膜囊血管的生长。结论：Roquinimex能明显降低瘤块内微血管密度,抑制大鼠癌裸鼠移植瘤的体内生长,是一种有效的血管生长抑制剂。     

去甲斑蝥素对人乳腺癌血管生成的抑制作用

背景与目的:去甲斑蝥素(norcantharidin,NCTD)具有抑瘤作用,本文研究它对血管生成的作用,以及对人乳腺癌MCF-7细胞鸡胚绒毛尿囊膜(chorioallantoci membrane,CAM)移植肿瘤血管生成的影响。方法:①建立鸡胚绒毛尿囊膜模型,将明胶海绵载体置于两条前卵黄静脉间血管的CAM上,将不同剂量的NCTD加在载体上,以生理盐水对照,观察记录载体周围血管分支点数,计算血管生成抑制率。②建立人乳腺癌MCF-7细胞鸡胚移植模型。给予不同剂量的NCTD20μl[72、36、18μg/(胚.d)],以生理盐水对照,计算血管生成抑制率。结果:NCTD对以明胶海绵为载体的鸡胚绒毛尿囊膜血管生成具抑制作用,72、36、18、9μg NCTD组血管生成抑制率分别为77.7%、62.9%、50.6%及33.0%,并且呈剂量依赖性。移植肿瘤可诱发血管生成,NCTD能明显抑制MCF-7鸡胚移植肿瘤血管生成,72、36、18μg NCTD对移植瘤诱导的血管抑制率分别为66.2%、39.3%和22.8%。结论:NCTD能明显抑制鸡胚绒毛尿囊膜新生血管的生成,同时能抑制人乳腺癌MCF-7细胞鸡胚移植肿瘤的血管生成。其抗肿瘤作用与抑制肿瘤血管生成有关。     

人骨肉瘤细胞系OS—732 CAM移植瘤中VEGF、bFGF的表达及意义

目的：研究人骨肉瘤细胞系OS－732血管生成相关作用。方法：应用鸡胚绒毛尿囊膜模型,通过解剖显微镜及透射电镜观察该细胞系促血管生成特点,并以免疫组化方法检测人骨肉瘤细胞系OS－732鸡胚绒毛尿囊膜移植瘤中血管内皮生长因子（vascular endothelial growth factor,VEGF),碱性成纤维细胞生长因子（Basic fibroblast growth factor,bFGF)的表达。结果：该细胞系具较强的诱导血管生成能力,解剖显微镜下可见血管辐辏现象,透射电镜下可见新生血管壁由单层内皮细胞构成,内皮细胞裂隙增宽,基底膜不完整,缺乏平滑肌成分,移植瘤组织发VEGF和bFGF均呈阳性表达,其中VEGF呈高表达。结论：肿瘤诱导的新生血管在病理,生理及形态功能方面都具有特征性,其诱导血管生成过程中可能有多种血管生长因子的共同参与,针对血管生长因子为靶点进行抗血管生成治疗对改善骨肉瘤预后可能具有重要意义。     

人骨肉瘤原位移植模型的建立及生物学特征

目的 用人骨肉瘤细胞系HOS-98建立人骨肉瘤裸鼠胫骨原位移植模型,以探讨宿主器官微环境对人骨肉瘤细胞侵袭及转移等生物学行为的影响。方法 将人骨肉瘤细胞系HOS-98接种于裸鼠皮下,形成移植瘤,用传代移植瘤组织作为移植材料,进行胫骨原位移植及皮下移植。分别于移植后4周和8周处死小鼠,进行病理形态学检查,并对两种方法在成瘤率、生长方式及侵袭、转移等生物学行为比较。结果 两种移植方式在成瘤率及形态学上无明显不同,胫骨原位移植的潜伏期较短,并且生长快于皮下移植方式。皮下移植瘤呈局限性膨胀生长,有不完整的纤维包膜,瘤内类骨基质较少见,未见肺转移,观察8周时无明显消瘦;而胫骨原位移植瘤侵袭周围组织,可见发生肺转移,8周明显消瘦。原位移植的裸鼠血清ALP水平高于皮下移植者。原位移植的X线检查有明显的类似于人的骨性反应。结论 用人骨肿瘤细胞系HOS-98皮下接种的移植瘤作为移植材料是建立肿瘤异位移植的可行途径,裸鼠胫骨微环境较皮下组织更适合于人骨肉瘤的侵袭及转移表达,裸鼠胫骨原位移植模型的恶性生物学行为更接近临床骨肉瘤患者的体内侵袭及转移实际,该原位移植模型为今后的实验研究提供了更加接近患者实际的实验模型。     

桑黄灵芝UE-1对肿瘤生长及血管新生的抑制作用

目的观察桑黄灵芝UE-1对Lewis肺癌生长及血管新生的抑制作用。方法建立Lewis肺癌实体瘤模型,观察其抗肿瘤作用;通过免疫组织化学染色,检测肿瘤微血管密度;采用新生血管计数实验检测UE-1对肿瘤组织血管生成的影响;通过鸡胚尿囊膜血管新生实验观察UE-1多糖的抗血管新生作用。结果实体瘤模型中,UE-1组移植瘤重量和体积明显低于对照组（P<0.05）,且微血管密度值也低于对照组（P<0.05）;肿瘤诱导新生血管中UE-1组肿瘤周围的血管数明显少于对照组（P<0.05）;鸡胚尿囊膜血管新生实验显示UE-1多糖具有抑制血管新生作用。结论桑黄灵芝UE-1可抑制Lewis肺癌的生长,可能是通过抑制肿瘤血管新生来发挥其抑瘤作用。     

人肺癌高转移动物模型的筛选及其细胞系的建立

目的:建立人肺癌小鼠高转移模型及高转移细胞系,同时观察相关生物学特性,为肺癌转移机制和防治等研究提供有用的实验工具。方法:切除首代小鼠移植瘤,以延长动物生存时间而获得转移灶,从第二代起采用肺转移灶→皮下移植→肺转移灶→皮下移植的体内循环筛选方法建立NOD/SCID小鼠人肺癌细胞SPC-A-1皮下移植瘤高转移模型,并进行肿瘤的生长和转移情况、组织病理学观察,同时建立相应的高转移细胞系,进行各种相关生物学特性观察。结果:第一代移植瘤切除后转移率达66.7%,通过4代体内反复筛选建立了100%肺转移NOD/SCID小鼠模型及相应高转移细胞系,细胞生长行为和染色体分析等生物学特性观察表明该细胞系保持了原有的人肺腺癌的生物学特性。结论:应用体内筛选的方法成功建立了人肺癌皮下移植瘤高转移模型及高转移细胞系,为肺癌防治研究及抗转移实验治疗提供了理想的动物模型。     

人子宫内膜癌细胞系HECCL-1的建立及其生物学特性的初步观察

目的 :建立人子宫内膜癌细胞系 ,并研究其生物学特性。方法 :将传代稳定后的人子宫内膜癌裸小鼠移植瘤标本用贴壁培养法建立细胞系 ,用光学显微镜、电子显微镜观察细胞系的形态 ,研究细胞系的生长及生物学特性。结果 :成功建立 1株人子宫内膜癌细胞系 ,已传代 6 0代 ,命名为HECCL 1。其生物学特征为 :形态学具有腺上皮癌细胞的特点 ,细胞生长旺盛 ,DNA为非整倍体 ,连续传代后仍保留人类肿瘤染色体的特点 ,异种动物移植阳性 ,雌、孕激素受体表达均阴性。结论 :HECCL 1细胞系的建立 ,为开展人类子宫内膜癌的基础及临床研究提供了理想的实验工具。     

人子宫内膜癌瘤组织块活体移植瘤动物模型的建立

目的:研究建立人子宫内膜癌的组织块活体移植动物模型方法。方法:将生长状态良好的Ishikawa细胞浓度调整至1.5×107/ml,接种于BALB/c nude裸鼠腋下皮下(Ⅰ组),Ⅱ组生理盐水对照,观察其生长情况,待成瘤5-10mm时建立成瘤模型,收集Ⅰ组瘤组织块行活体移植于Ⅲ组小鼠,留取组织标本行病理检查。结果:成功建立了人子宫内膜癌Ishikawa细胞的BABL/c裸鼠皮下移植瘤模型和活体移植瘤模型,形成的肿瘤具备人肿瘤生物学特点。结论:建立的瘤组织块活体二次传代移植人子宫内膜癌的BABL/c nude小鼠皮下移植瘤模型具有人子宫内膜癌特征且成瘤率更高,为大批建立子宫内膜癌单位模型研究子宫内膜癌的发病机制和药物治疗提供了实验动物模型。     

X-rays modulate extracellular matrix in vivo.

X-rays have an antiangiogenic effect in the chicken embryo chorioallantoic membrane (CAM) model of in vivo angiogenesis. Our study demonstrates that X-rays induce an early apoptosis of CAM cells, modulate the synthesis and deposition of extracellular matrix (ECM) proteins involved in regulating angiogenesis and affect angiogenesis induced by tumour cells implanted onto the CAM. Apoptosis was evident within 1-2 hr, but not later than 6 hr after irradiation. Fibronectin, laminin, collagen type I, integrin alpha(v)beta3 and MMP-2 protein amounts were all decreased 6 hr after irradiation. In contrast, collagen type IV, which is restricted to basement membrane, was not affected by irradiation of the CAM. There was a similar decrease of gene expression for fibronectin, laminin, collagen type I and MMP-2, 6 hr after irradiation. The levels of mRNA for integrin alpha(v)beta3 and collagen type IV were unaffected up to 24 hr after irradiation. The decrease in both protein and mRNA levels was reversed at later time points and 48 hr after irradiation, there was a significant increase in the expression of all the genes studied. When C6 glioma tumour cells were implanted on irradiated CAMs, there was a significant increase in the angiogenesis induced by tumour cells, compared to that in non-irradiated CAMs. Therefore, although X-rays have an initial inhibitory effect on angiogenesis, their action on the ECM enhances new vessel formation induced by glioma cells implanted on the tissue.     

Cell adhesion molecules of the immunoglobulin supergene family and their role in malignant transformation and progression to metastatic disease

Summary Cell adhesion molecules (CAMs) of the immunoglobulin supergene family may play important roles in tumorigenesis and the development of metastatic disease. In a variety of human malignancies, tumor progression has been observed to be associated with changes in CAM expression. An early event in colorectal tumorigenesis appears to be the down regulation of a normally expressed CAM, DCC. Over-expression of a second CAM, carcinoembryonic antigen, is associated with colorectal tumors which have a high risk for metastasis development. Several tumors, including Wilms tumors and neuroblastoma, have been found to express a developmentally regulated form of NCAM which inhibits a variety of cell-cell interactions. Malignant cells not only show aberrations in the expression of their CAMS and thus their normal cell-cell interactions, but establish new adhesive interactions. The development of metastatic potential in cutaneous melanoma is associated with the de novo expression of two CAMs, one of which is ICAM-1, a molecule mediating adhesion between the tumor cells and leukocytes.     

Weibel-Palade bodies as a marker for neovascularization induced by tumor and rheumatoid angiogenesis factors

An analysis was made of ultrastructural changes in capillary endothelial cells in experimentally induced angiogenesis and in a human pathological situation known to involve increased angiogenesis. Chick chorioalloantoic membrane (CAM) showing a positive angiogenic response to low-molecular weight angiogenesis factor isolated from rat Walker sarcoma or from human rheumatoid joint was compared with untreated CAM. Serotonin-treated CAM provided an additional control in that serotonin has the capacity to stimulate endothelial cell growth in vitro but did not induce angiogenesis on the CAM. Human rheumatoid joints were studied using normal healthy human joints as controls. The number of Weibel-Palade (W-P) bodies per unit of cytoplasmic area were higher in tumor angiogenesis factor-treated CAMs (not significant) and rheumatoid angiogenesis factor-treated CAMs (P less than 0.008) than in untreated controls. These differences were more pronounced if W-P body volumetric density was analyzed (P in both cases less than 0.008). Serotonin-treated control CAMs did not show higher numbers of W-P body or greater WPV than untreated controls. Numbers of W-P body and W-P body volumetric density were higher (P less than 0.008) in rheumatoid joints than normal joints. Median values for W-P body number were 16-fold higher and, for W-P body volumetric density, they were up to 30-fold higher in rheumatoid joints.     

The effect of AD-VEGF-siRNA on the expression of vascular endothelial growth factor in osteosarcoma-bearing nude mice

Objective: To explore the influence of AD-VEGF-siRNA on the expression of vascular endothelial growth factor (VEGF) in neoplasm and blood serum.Methods: Transplantable model of human osteosarcoma was successfully established by the way of subcutaneous injection of VEGF highly expressed human MG63 osteosarcoma cells.These mice were divided randomly into three groups: AD-VEGF-siRNA group, 15 mice; AD-EGFP group, 15 mice; PBS group, 15 mice.Three mice were additionally raised without any treatment.The drug was injected intratumorally 200 IJL at each time, once a day.The total dose of virus was 2×109 pfu.Three osteosarcoma-bearing mice of each group were sacrificed at 11th, 14th ,17th day after the implantation of MG63 cells.The expression of VEGF in implanted tumors and blood serum was detected by ELISA methods.Then the left mice were all sacrificed at the end of experiment (19th day).The expression of VEGF in implanted tumors was detected by RT-PCR and immune histochemistry methods, and that in implanted tumors and blood serum was detected by ELISA methods.Results: (1) Tumors in mice could be seen at 5th day from the implantation of MG63 ceils.(2)The expression of VEGF could be detected in all groups by RT-PCR and immune histochemistry, Which was much lower in the group receiving AD-VEGF-siRNA therapy than two control groups (P＜0.05).(3) The expression of VEGF in blood serum of osteosarcoma-bearing mice was much higher than that of three healthy mice by ELISA (P＜0.05).(4) The expression of VEGF in blood serum and neoplasm in AD-VEGF-siRNA group was much lower than that in two control groups (P＜0.05).Conclusion: AD-VEGF-siRNA could effectively inhibited VEGF expression in vivo.This technology would bring some good references for our therapy of antiangiogenesis in osteosarcoma.     

腺病毒介导干扰RNA对荷人骨肉瘤裸鼠VEGF表达的影响

目的: 探讨AD-VEGF-siRNA对荷人骨肉瘤裸鼠瘤体组织以及血液中VEGF表达的影响。 方法: 应用VEGF高表达人骨肉瘤细胞系MG-63制备荷骨肉瘤裸鼠模型;成瘤组动物随机分为3组,AD-VEGF-siRNA组15只;AD-EGFP组15只;PBS组15只。另留取3只裸小鼠,未接种肿瘤细胞,未注射药物。成瘤裸鼠瘤体内分别注射上述各组药物,隔天1次,200μl/次,共5次,病毒总量2×10⁹PFU。接种后第11天、14天、17天实验各组分别处死3只裸小鼠,用ELISA法检测各组荷瘤小鼠血液和瘤体组织中VEGF蛋白表达情况。试验结束(第19天)处死剩余裸小鼠,用RT-PCR及免疫组化技术检测裸小鼠瘤体组织VEGF的表达,用ELISA法检测各组荷瘤小鼠血液和瘤体组织中VEGF蛋白的表达。 结果: 1)各组裸小鼠约在接种5天成瘤;2)用AD-VEGF-siRNA处理后,RT-PCR检测发现裸小鼠瘤体组织的VEGFmRNA表达水平明显低于两对照组(P<0.05);3)免疫组化检测发现AD-VEGF-siRNA组VEGF蛋白表达明显减少;4)ELISA检测发现成瘤组小鼠血清VEGF蛋白水平明显高于未作任何处理的健康裸小鼠组(P<0.05),在各时间段AD-VEGF-siRNA组血液及瘤体组织中VEGF蛋白的表达水平明显低于两对照组(P<0.05)。 结论: AD-VEGF-siRNA在体内可以抑制荷骨肉瘤裸小鼠瘤体VEGF基因的表达及血液中VEGF的水平;应用siRNA腺病毒表达载体技术为骨肉瘤抗血管生成的治疗提供了借鉴。     

A fully human antimelanoma cellular adhesion molecule/MUC18 antibody inhibits spontaneous pulmonary metastasis of osteosarcoma cells in vivo.

PURPOSE: The melanoma cellular adhesion molecule, also known as MUC18, is highly expressed on several tumors, including bone sarcomas. The level of MUC18 expression has been found to correlate directly with tumor progression and metastatic potential. These observations have established MUC18 as a candidate mediator of tumor growth and metastasis, and suggest that blockade of MUC18 might be a potential target for immunotherapy against several MUC18-expressing tumors, including human bone sarcomas. EXPERIMENTAL DESIGN: To investigate whether blockade of MUC18 might be a potential target for immunotherapy against osteosarcoma, we have recently developed a fully human anti-MUC18 antibody, ABX-MA1. We studied the effect of ABX-MA1 on growth, adhesion, invasion, and metastasis of human osteosaroma cells both in vitro and in vivo. RESULTS: MUC18 was widely expressed on both osteosarcoma and Ewing's sarcoma cells. ABX-MA1 had no effect on the proliferation of osteosarcoma cells in vitro, nor did it significantly inhibit the growth of KRIB human osteosarcoma cells when they were orthotopically implanted into the tibias of nude mice. However, after 6 weeks, significantly fewer ABX-MA1-treated mice developed spontaneous pulmonary metastases than did IgG-treated control mice. Additionally, ABX-MA1 decreased the invasion of osteosarcoma cells through Matrigel-coated filters and disrupted homotypic adhesion between osteosarcoma cells and their heterotypic interaction with human vascular endothelial cells. CONCLUSIONS: Our findings demonstrate that MUC18 plays a central role in the metastasis of osteosarcoma and suggest that targeted inhibition of this antigen by ABX-MA1 may be a novel immunotherapeutic approach in the management of this tumor.     

Complementary medicine use before and 1 year following genetic testing for BRCA1/2 mutations.

PURPOSE: We explored change in complementary and alternative medicine (CAM) use by unaffected women and cancer survivors from enrollment into a randomized BRCA1/2 testing program to CAM use 1 year following results disclosure.METHODS: A cohort of 243 high-risk women completed questionnaires at enrollment into a BRCA1/2 randomized trial and 1 year post results disclosure. Uses of several CAMs for cancer prevention were explored, including ingestible, behavioral, and physical modalities. Assessment of the change in CAM use from baseline to 1 year follow-up was conducted using a repeated self-administered questionnaire. Correlates of the number of CAMs used at 1 year were explored using multivariable linear regression models.RESULTS: Among the subset of women who changed their CAM behavior from enrollment to 1 year following BRCA1/2 results disclosure, there was a significantly higher proportion who changed from no CAM use to CAM use among the overall cohort (P=0.01), among women without cancer at enrollment (P=0.003), among women found to be BRCA1/2 carriers (P=0.03), and among women randomized to the genetic counseling intervention arm of the study (P=0.009). Number of CAMs used at 1 year was positively associated with number of CAMs used at baseline, sunscreen use, and BRCA1/2 mutation status.CONCLUSION: High-risk women who have received BRCA1/2 counseling and testing frequently adopt new CAM use in the first year after learning their genetic status. Mutation carriers frequently initiate CAM use after learning their genetic status as part of their cancer preventive regimen. Further studies are warranted to determine the efficacy of CAM-related strategies for cancer prevention.     

Systemically administered PEDF against primary and secondary tumours in a clinically relevant osteosarcoma model

Background:

Pigment epithelium-derived factor (PEDF) is an endogenous glycoprotein with a potential role as a therapeutic for osteosarcoma. Animal studies have demonstrated the biological effects of PEDF on osteosarcoma; however, these results are difficult to extrapolate for human use due to the chosen study design and drug delivery methods.

Methods:

In this study we have attempted to replicate the human presentation and treatment of osteosarcoma using a murine orthotopic model of osteosarcoma. The effects of PEDF on osteosarcoma cell lines were evaluated in vitro prior to animal experimentation. Orthotopic tumours were induced by intra-tibial injection of SaOS-2 osteosarcoma cells. Treatment with PEDF was delayed until after the macroscopic appearance of primary tumours. Pigment epithelium-derived factor was administered systemically via an implanted intraperitoneal micro-osmotic pump.

Results:

In vitro, PEDF inhibited proliferation, induced apoptosis and inhibited cell cycling of osteosarcoma cells. Pigment epithelium-derived factor promoted adhesion to Collagen I and inhibited invasion through Collagen I. In vivo, treatment with PEDF caused a reduction in both primary tumour volume and burden of pulmonary metastases. Systemic administration of PEDF did not cause toxic effects on normal tissues.

Conclusion:

Systemically delivered PEDF is effective in suppressing the size of primary and secondary tumours in an orthotopic murine model of osteosarcoma.