骨肉瘤血管内皮细胞特异性结合肽的功能验证
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摘要
研究背景
骨肉瘤是起源于间叶组织的恶性骨肿瘤,多发于儿童和青少年。现阶段大剂量的化疗是主要的治疗方法,该方法虽有效但无选择性,当化疗药物进入机体后,对正常的细胞亦有杀伤作用,引起了很严重的副作用。因此,增强化疗药物针对骨肉瘤组织的靶向性,成为目前急待解决的一个课题。肿瘤的血管生成是肿瘤发展的重要环节,肿瘤必须通过形成新的血管系统来提供足够的营养,以支持其继续生长,因此将肿瘤新生血管内皮上的某些特异性分子作为药物作用的新靶点,日益受到研究者的密切关注。为了获取能与上述靶点特异性结合的配体,必须具备有效的筛选手段。噬菌体展示技术的应用为实现此目的提供了一个全新的工具。
噬菌体展示技术是20世纪90年代发展起来并得到广泛应用的新技术。采用该技术,可以在欲筛选的靶标分子结构不明确的情况下,无需事先知道它们之间相互作用的区域及相互作用的性质来进行筛选,从而筛选出一些能与靶分子结合的特异性短肽。近几年兴起的噬菌体体内展示技术更是创造性地将常规的噬菌体展示技术与动物模型相结合,是寻找组织、器官特异性结合多肽的有效手段。此方法可在受体分子尚不清楚的情况下,以受体天然存在的环境——组织器官为配基,利用噬菌体短肽的抗原特异性,寻找未知的靶分子,确定其结构域。
本课题组管明强于2009年利用鼠骨肉瘤细胞UMR-106制作骨肉瘤裸鼠模型,然后利用噬菌体7肽肽库进行体内筛选,得到一条能特异性结合UMR-106骨肉瘤血管内皮细胞的七肽(序列为：TKPDKGY),将表达该序列的噬菌体克隆进行体内回输验证发现,该克隆在鼠骨肉瘤组织的聚集显著高于其它正常组织,免疫组化亦显示该克隆在骨肉瘤血管内皮表面大量聚积,在心脏、肺脏、肾脏及脑组织内仅有少量的聚积,具有明显的鼠骨肉瘤血管内皮细胞特异亲合性,TKPDKGY有可能成为骨肉瘤血管靶向治疗的特异性短肽,为本研究的顺利进行奠定了良好的基础。
目的
验证骨肉瘤血管内皮细胞特异性结合肽(TKPDKGY)对体外培养的骨肉瘤血管内皮细胞和荷瘤裸鼠模型体内的血管内皮细胞的亲和性。
方法
1.血管内皮细胞的原代培养与鉴定采取血管内壁贴壁消化法,分别进行裸鼠主动脉血管内皮细胞和骨肉痈血管内皮细胞的原代培养,同时进行Ⅷ因子免疫组化鉴定。
2.短肽靶向性的体外验证
合成骨肉瘤血管内皮细胞特异性结合肽TKPDKGY,并在其N端加载荧光基团FITC。将短肽荧光复合物与OAVECs结合后于荧光显微镜和流式细胞仪下观察,同时以AECs和UMR-106细胞为对照组,以评估短肽针对OAVECs的靶向性。
3.短肽靶向性的体内验证
将前述合成的短肽荧光复合物注入动物血管内,通过小动物整体荧光成像来观察短肽在活体动物内的分布,同时定时处死动物,切取重要器官和肿瘤部位使用LSCM来评价其动物体内与OAVECs的亲和度。
结果
1.血管内皮细胞的原代培养与鉴定
正常裸鼠于原代培养后的6d后可见明显内皮细胞集落,肿瘤血管内皮细胞则4d后即可见细胞集落。正常血管内皮细胞初期呈团块状排列,细胞较小但形态规则,呈短梭形、多角形或不规则形,核清晰,呈圆或椭圆形,细胞生长迅速。约7-9d后细胞汇合,胞体呈梭形或多角形,互相嵌合,细胞间界限不清。此时细胞生长缓慢,传3-4代时,细胞开始变性、变形、脱落,不再继续生长。OAVECs和AECs形态上无明显差别。Ⅷ因子免疫组化鉴定显示：两种细胞胞质均呈黄褐色着色(阳性细胞率：AECs98%; OAVECs 99%),而不加单抗的对照组无特殊显色。
2.短肽靶向性的体外验证
荧光显微镜示OAVECs与FITC-TKPDKGY组荧光显微镜下细胞表面显示较强且清晰的荧光,表现出明显的结合；而AECs及UMR-106细胞与FITC-TKPDKGY组则在荧光显微镜下仅见微弱的荧光,且基本为背景荧光,细胞无特异性结合。
流式细胞仪检测结果显示,FITC-TKPDKGY与UMR-106和AECs共孵育时,荧光标记的阳性细胞数百分比分别为0.27%和0.22%,而FITC-TKPDKGY与OAVECs共孵育时,荧光标记的阳性细胞数百分比为89.54%,荧光强度明显强于对照组；单纯FITC与三种细胞共孵育时,荧光标记的阳性细胞数百分比均小于1%。说明短肽TKPDKGY与OAVECs细胞具有较强的特异结合能力。
3.短肽靶向性的体内验证
小动物整体荧光成像实验显示：荷瘤裸鼠注射FITC-TKPDKGY 10min后,肿瘤处即可见荧光富集,但较为微弱,30min时显示明显并逐渐增强,lhr后达最强,4hr后逐渐消退,至24hr基本无荧光残留,而荷瘤裸鼠注射单纯FITC未见明显的肿瘤富集效应。LSCM扫描显示：注射短肽荧光复合物4hr后,肿瘤部位冰冻切片激光共聚焦扫描可见肿瘤血管和血管丛荧光强度较强；血管周围的肿瘤组织也可见少量短肽结合,但显示并不明显,可提示有一定的结合性。心、脑、肺和肝组织显示并不明显。肾脏大部荧光强度较低,镜下可见肾血管并无荧光富集,但肾小球和肾小管部可见荧光富集,且与周围组织对比明显,提示短肽在此因代谢而富集。FITC注射组各部位未见特异性结合。
结论
本研究在已通过噬菌体体内展示技术获得骨肉瘤血管内皮细胞特异性结合肽的基础上,从体外和体内两个角度,对此短肽针对骨肉瘤血管内皮的靶向特异性进行了评价。结果显示在无论在体内还是体外试验中,短肽均显示可很好的靶向结合骨肉瘤血管内皮细胞,同时并不特异性结合其他机体内正常组织,为该特异性短肽进一步结合效应分子应用于骨肉瘤的诊断和治疗奠定了一定的实验基础。通过以上研究,我们得出以下几点结论：
1.成功复制了骨肉瘤荷瘤裸鼠模型；
2.血管内壁贴壁消化法为一种较简易,成功率较高的血管内皮细胞原代培养方法;
3.通过荧光示踪技术和流式细胞技术检测了短肽针对体外培养的骨肉瘤血管内皮细胞的特异结合性,证实骨肉瘤血管内皮细胞是此短肽的靶向细胞；
4.通过小动物整体成像仪和LSCM对短肽针对荷瘤裸鼠模型内骨肉瘤血管内皮组织的靶向性进行评价,发现短肽很好的保持了生物学活性,显示与骨肉瘤血管内皮组织有着良好的结合特异性。
Background
Osteosarcoma is a malignant bone tumor originated from mesenchymal tissue, mainly in the children and adolescents. At present, High dose chemotherapy is the main treatment for osteosarcoma, which is effective but non-selective. Chemotherapy is also of serious toxic effect for the normal tissue. Therefore, to enhance the targeting effect of the chemotherapy for osteosarcoma has become an urgent problem. Tumor angiogenesis is an important aspect of tumor development, because tumor needs the new vascular system to provide adequate nutrition to support its expansion. The researchers have been paying more and more attention on some specific molecules on the tumor endothelium, which are expected to play as the new drug targets. In order to get the above-mentioned specific binding ligands, an effective screening tool is needed. Phage display technology is one of the most effective one.
Phage-display technology is an effective molecular biologic tool that has been developed and widely used since the 1990s. It has been used to create a directly physical linkage between a vast library of random peptide sequences to the DNA encoding each sequence, allowing rapid identification of peptide ligands for a variety of target molecules (antibodies, enzymes, cell-surface receptors, etc.) by an in vitro selection process, which is called panning.
The in vivo phage display technique combining the phage display technology with animal models, is a more effective way to looking for the organ-specific binding peptides. This method can help us to find the target and confirm the domain in the natural environment-tissues or organs, by using the different character of antigen of the phage. In 2007, our team inoculated murine osteosarcoma cells UMR-106 in nude mice, and had established the tumor-bearing animal models successfully. We have screened the endothelial cell of tumor vasculatures of nude mouse for four rounds by in vivo phage display technique without knowing the target. According to the result of in vivo experiment and immunohistochemical staining, we could get conclusion that the TKPDKGY phage can effectively target to the endothelial cell of tumor vasculture of nude mice. The TKPDKGY peptide may became the targeting peptide of vascular targeting therapy, which has laid a good foundation for the following study.
In order to provide a new method and clue for the therapy and mechanism of osteosarcoma, we will identify the binding specification of the short peptide (TKPDKGY) to osteosarcoma-associated vascular endothelial cells.
Objective
To identify the binding specification of the short peptide (TKPDKGY) to osteosarcoma-associated vascular endothelial cells cultured in vitro and to vascular endothelial of osteosarcoma model in nude mice in vivo.
Method
1. Primary culture and identification of AECs and OVAECs
The digestion-adherent method is used in Primary culture of AECs and OVAECs. After serial subcultivation, AECs and OVAECs are identified by immunohistochemical method with anti-Ⅷfactor antibody.
2. The in-vitro functional identification of short peptide binding specifically
The peptide (TKPDKGY) was synthesized with the N-teminal marked with fluorescein isothiocyanate (FITC). Fluorescence microscopy and flow cytometry were used to detect the in-vitro binding activity.
3. The in-vivo functional identification of short peptide binding specifically
Whole-body optical imaging system for Small Animals and Confocal Microscope were used to detect the in-vivo binding activity.
Result
1. Primary culture and identification of AECs and OVAECs
OAVECs and AECs were derived from osteosarcoma by primary culture and characterization was confirmed based on the immunocytochemical technology.
2. The in-vitro functional identification of short peptide binding specifically
Fluorescein-conjugated TKPDKGY peptide selectively bound to OAVECs, whereas only negligible binding to AECs and UMR-106 were observed by Fluorescence microscopy. Flow cytometry also showed that the fluorescence positive cell rate was 89.54% in the OAVECs co-incubated with TKPDKGY-FITC, and were 0.27% and 0.22% in the UMR-106 and AECs incubated with TKPDKGY-FITC respectively.
3. The in-vivo functional identification of short peptide binding specifically
Whole-body optical imaging system showed that tumor was hypofluorescence enrichment in injection 10 min, fluorescence was increased in 30 min gradually, the strongest fluorescence appeared in 1 hr and fluorescence was subsidised 4hrs later, no residual fluorescence in 24hrs. Confocal microscope also showed that the short peptide homed to vascular endothelium in osteosarcoma but was not detectable in heart, brain, lung and liver.
Conclusion
The TKPDKGY peptide has specifically binding activity to vascular endothelium in osteosarcoma. It may be useful as a targeting moiety for selective delivery of therapeutics and as a diagnostic probe for the detection of osteosarcoma. Taken together, we draw the following conclusions:
1. We had successfully established the tumor-bearing animal models of osteosarcoma;
2. The method established in the present study is simple and easy to handle, and can obtain a certain number of high purity AECs and OAVECs;
3. The specifically binding activity to vascular endothelium in osteosarcoma was examined with fluorescent tracer technique and flow cytometer method. The research confirmed that the vascular endothelial cell in osteosarcoma is the target of TKPDKGY.
4. Whole-body optical imaging system and laser scanning confocal microscope is used to evaluate the binding specificity of TKPDKGY. In the study, the biological activity of short peptide was stable in vivo. TKPDKGY shows a good specifically binding activity to OAVECs.
骨肉瘤是起源于间叶组织的恶性骨肿瘤,多发于儿童和青少年。现阶段大剂量的化疗是主要的治疗方法,该方法虽有效但无选择性,当化疗药物进入机体后,对正常的细胞亦有杀伤作用,引起了很严重的副作用。因此,增强化疗药物针对骨肉瘤组织的靶向性,成为目前急待解决的一个课题。肿瘤的血管生成是肿瘤发展的重要环节,肿瘤必须通过形成新的血管系统来提供足够的营养,以支持其继续生长,因此将肿瘤新生血管内皮上的某些特异性分子作为药物作用的新靶点,日益受到研究者的密切关注。为了获取能与上述靶点特异性结合的配体,必须具备有效的筛选手段。噬菌体展示技术的应用为实现此目的提供了一个全新的工具。
噬菌体展示技术是20世纪90年代发展起来并得到广泛应用的新技术。采用该技术,可以在欲筛选的靶标分子结构不明确的情况下,无需事先知道它们之间相互作用的区域及相互作用的性质来进行筛选,从而筛选出一些能与靶分子结合的特异性短肽。近几年兴起的噬菌体体内展示技术更是创造性地将常规的噬菌体展示技术与动物模型相结合,是寻找组织、器官特异性结合多肽的有效手段。此方法可在受体分子尚不清楚的情况下,以受体天然存在的环境——组织器官为配基,利用噬菌体短肽的抗原特异性,寻找未知的靶分子,确定其结构域。
本课题组管明强于2009年利用鼠骨肉瘤细胞UMR-106制作骨肉瘤裸鼠模型,然后利用噬菌体7肽肽库进行体内筛选,得到一条能特异性结合UMR-106骨肉瘤血管内皮细胞的七肽(序列为：TKPDKGY),将表达该序列的噬菌体克隆进行体内回输验证发现,该克隆在鼠骨肉瘤组织的聚集显著高于其它正常组织,免疫组化亦显示该克隆在骨肉瘤血管内皮表面大量聚积,在心脏、肺脏、肾脏及脑组织内仅有少量的聚积,具有明显的鼠骨肉瘤血管内皮细胞特异亲合性,TKPDKGY有可能成为骨肉瘤血管靶向治疗的特异性短肽,为本研究的顺利进行奠定了良好的基础。
目的
验证骨肉瘤血管内皮细胞特异性结合肽(TKPDKGY)对体外培养的骨肉瘤血管内皮细胞和荷瘤裸鼠模型体内的血管内皮细胞的亲和性。
方法
1.血管内皮细胞的原代培养与鉴定采取血管内壁贴壁消化法,分别进行裸鼠主动脉血管内皮细胞和骨肉痈血管内皮细胞的原代培养,同时进行Ⅷ因子免疫组化鉴定。
2.短肽靶向性的体外验证
合成骨肉瘤血管内皮细胞特异性结合肽TKPDKGY,并在其N端加载荧光基团FITC。将短肽荧光复合物与OAVECs结合后于荧光显微镜和流式细胞仪下观察,同时以AECs和UMR-106细胞为对照组,以评估短肽针对OAVECs的靶向性。
3.短肽靶向性的体内验证
将前述合成的短肽荧光复合物注入动物血管内,通过小动物整体荧光成像来观察短肽在活体动物内的分布,同时定时处死动物,切取重要器官和肿瘤部位使用LSCM来评价其动物体内与OAVECs的亲和度。
结果
1.血管内皮细胞的原代培养与鉴定
正常裸鼠于原代培养后的6d后可见明显内皮细胞集落,肿瘤血管内皮细胞则4d后即可见细胞集落。正常血管内皮细胞初期呈团块状排列,细胞较小但形态规则,呈短梭形、多角形或不规则形,核清晰,呈圆或椭圆形,细胞生长迅速。约7-9d后细胞汇合,胞体呈梭形或多角形,互相嵌合,细胞间界限不清。此时细胞生长缓慢,传3-4代时,细胞开始变性、变形、脱落,不再继续生长。OAVECs和AECs形态上无明显差别。Ⅷ因子免疫组化鉴定显示：两种细胞胞质均呈黄褐色着色(阳性细胞率：AECs98%; OAVECs 99%),而不加单抗的对照组无特殊显色。
2.短肽靶向性的体外验证
荧光显微镜示OAVECs与FITC-TKPDKGY组荧光显微镜下细胞表面显示较强且清晰的荧光,表现出明显的结合；而AECs及UMR-106细胞与FITC-TKPDKGY组则在荧光显微镜下仅见微弱的荧光,且基本为背景荧光,细胞无特异性结合。
流式细胞仪检测结果显示,FITC-TKPDKGY与UMR-106和AECs共孵育时,荧光标记的阳性细胞数百分比分别为0.27%和0.22%,而FITC-TKPDKGY与OAVECs共孵育时,荧光标记的阳性细胞数百分比为89.54%,荧光强度明显强于对照组；单纯FITC与三种细胞共孵育时,荧光标记的阳性细胞数百分比均小于1%。说明短肽TKPDKGY与OAVECs细胞具有较强的特异结合能力。
3.短肽靶向性的体内验证
小动物整体荧光成像实验显示：荷瘤裸鼠注射FITC-TKPDKGY 10min后,肿瘤处即可见荧光富集,但较为微弱,30min时显示明显并逐渐增强,lhr后达最强,4hr后逐渐消退,至24hr基本无荧光残留,而荷瘤裸鼠注射单纯FITC未见明显的肿瘤富集效应。LSCM扫描显示：注射短肽荧光复合物4hr后,肿瘤部位冰冻切片激光共聚焦扫描可见肿瘤血管和血管丛荧光强度较强；血管周围的肿瘤组织也可见少量短肽结合,但显示并不明显,可提示有一定的结合性。心、脑、肺和肝组织显示并不明显。肾脏大部荧光强度较低,镜下可见肾血管并无荧光富集,但肾小球和肾小管部可见荧光富集,且与周围组织对比明显,提示短肽在此因代谢而富集。FITC注射组各部位未见特异性结合。
结论
本研究在已通过噬菌体体内展示技术获得骨肉瘤血管内皮细胞特异性结合肽的基础上,从体外和体内两个角度,对此短肽针对骨肉瘤血管内皮的靶向特异性进行了评价。结果显示在无论在体内还是体外试验中,短肽均显示可很好的靶向结合骨肉瘤血管内皮细胞,同时并不特异性结合其他机体内正常组织,为该特异性短肽进一步结合效应分子应用于骨肉瘤的诊断和治疗奠定了一定的实验基础。通过以上研究,我们得出以下几点结论：
1.成功复制了骨肉瘤荷瘤裸鼠模型；
2.血管内壁贴壁消化法为一种较简易,成功率较高的血管内皮细胞原代培养方法;
3.通过荧光示踪技术和流式细胞技术检测了短肽针对体外培养的骨肉瘤血管内皮细胞的特异结合性,证实骨肉瘤血管内皮细胞是此短肽的靶向细胞；
4.通过小动物整体成像仪和LSCM对短肽针对荷瘤裸鼠模型内骨肉瘤血管内皮组织的靶向性进行评价,发现短肽很好的保持了生物学活性,显示与骨肉瘤血管内皮组织有着良好的结合特异性。
Background
Osteosarcoma is a malignant bone tumor originated from mesenchymal tissue, mainly in the children and adolescents. At present, High dose chemotherapy is the main treatment for osteosarcoma, which is effective but non-selective. Chemotherapy is also of serious toxic effect for the normal tissue. Therefore, to enhance the targeting effect of the chemotherapy for osteosarcoma has become an urgent problem. Tumor angiogenesis is an important aspect of tumor development, because tumor needs the new vascular system to provide adequate nutrition to support its expansion. The researchers have been paying more and more attention on some specific molecules on the tumor endothelium, which are expected to play as the new drug targets. In order to get the above-mentioned specific binding ligands, an effective screening tool is needed. Phage display technology is one of the most effective one.
Phage-display technology is an effective molecular biologic tool that has been developed and widely used since the 1990s. It has been used to create a directly physical linkage between a vast library of random peptide sequences to the DNA encoding each sequence, allowing rapid identification of peptide ligands for a variety of target molecules (antibodies, enzymes, cell-surface receptors, etc.) by an in vitro selection process, which is called panning.
The in vivo phage display technique combining the phage display technology with animal models, is a more effective way to looking for the organ-specific binding peptides. This method can help us to find the target and confirm the domain in the natural environment-tissues or organs, by using the different character of antigen of the phage. In 2007, our team inoculated murine osteosarcoma cells UMR-106 in nude mice, and had established the tumor-bearing animal models successfully. We have screened the endothelial cell of tumor vasculatures of nude mouse for four rounds by in vivo phage display technique without knowing the target. According to the result of in vivo experiment and immunohistochemical staining, we could get conclusion that the TKPDKGY phage can effectively target to the endothelial cell of tumor vasculture of nude mice. The TKPDKGY peptide may became the targeting peptide of vascular targeting therapy, which has laid a good foundation for the following study.
In order to provide a new method and clue for the therapy and mechanism of osteosarcoma, we will identify the binding specification of the short peptide (TKPDKGY) to osteosarcoma-associated vascular endothelial cells.
Objective
To identify the binding specification of the short peptide (TKPDKGY) to osteosarcoma-associated vascular endothelial cells cultured in vitro and to vascular endothelial of osteosarcoma model in nude mice in vivo.
Method
1. Primary culture and identification of AECs and OVAECs
The digestion-adherent method is used in Primary culture of AECs and OVAECs. After serial subcultivation, AECs and OVAECs are identified by immunohistochemical method with anti-Ⅷfactor antibody.
2. The in-vitro functional identification of short peptide binding specifically
The peptide (TKPDKGY) was synthesized with the N-teminal marked with fluorescein isothiocyanate (FITC). Fluorescence microscopy and flow cytometry were used to detect the in-vitro binding activity.
3. The in-vivo functional identification of short peptide binding specifically
Whole-body optical imaging system for Small Animals and Confocal Microscope were used to detect the in-vivo binding activity.
Result
1. Primary culture and identification of AECs and OVAECs
OAVECs and AECs were derived from osteosarcoma by primary culture and characterization was confirmed based on the immunocytochemical technology.
2. The in-vitro functional identification of short peptide binding specifically
Fluorescein-conjugated TKPDKGY peptide selectively bound to OAVECs, whereas only negligible binding to AECs and UMR-106 were observed by Fluorescence microscopy. Flow cytometry also showed that the fluorescence positive cell rate was 89.54% in the OAVECs co-incubated with TKPDKGY-FITC, and were 0.27% and 0.22% in the UMR-106 and AECs incubated with TKPDKGY-FITC respectively.
3. The in-vivo functional identification of short peptide binding specifically
Whole-body optical imaging system showed that tumor was hypofluorescence enrichment in injection 10 min, fluorescence was increased in 30 min gradually, the strongest fluorescence appeared in 1 hr and fluorescence was subsidised 4hrs later, no residual fluorescence in 24hrs. Confocal microscope also showed that the short peptide homed to vascular endothelium in osteosarcoma but was not detectable in heart, brain, lung and liver.
Conclusion
The TKPDKGY peptide has specifically binding activity to vascular endothelium in osteosarcoma. It may be useful as a targeting moiety for selective delivery of therapeutics and as a diagnostic probe for the detection of osteosarcoma. Taken together, we draw the following conclusions:
1. We had successfully established the tumor-bearing animal models of osteosarcoma;
2. The method established in the present study is simple and easy to handle, and can obtain a certain number of high purity AECs and OAVECs;
3. The specifically binding activity to vascular endothelium in osteosarcoma was examined with fluorescent tracer technique and flow cytometer method. The research confirmed that the vascular endothelial cell in osteosarcoma is the target of TKPDKGY.
4. Whole-body optical imaging system and laser scanning confocal microscope is used to evaluate the binding specificity of TKPDKGY. In the study, the biological activity of short peptide was stable in vivo. TKPDKGY shows a good specifically binding activity to OAVECs.
引文
[1]Folkman J. Angiogenesis and apoptosis[J]. Semin Cancer Biol,2003, 13(2):159-67.
[2]Denekamp J. Review article:angiogenesis, neovascular proliferation and vascular pathophysiology as targets for cancer therapy. Br J Radiol,1993, 66(783):181-96.
[3]Folkman J. Seminars in medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis. N Engl J Med,1995, 333(26):1757-63.
[4]Kanthou C, Greco O, Stratford A, et al. The tubulin-binding agent combretastatin A-4-phosphate arrests endothelial cells in mitosis and induces mitotic cell death. Am J Pathol,2004,165(4):1401-11.
[5]Burrows FJ, Watanabe Y, Thorpe PE. A murine model for antibody-directed targeting of vascular endothelial cells in solid. Cancer Res, 1992,52(21):5954-62.
[6]Siemann DW, Chaplin DJ, Horsman MR. Vascular-targeting therapies for treatment of malignant disease. Cancer,2004,100(12):2491-99.
[7]Keresztessy Z, Csosz E, Harsfalvi J, et al. Phage display selection of efficient glutamine-donor substrate peptides for transglutaminase[J]. Protein Sci,2006,15(11):2466-80.
[8]Nixon AE. Phage display as a tool for protease ligand discovery[J]. Curr Pharm Biotechnol,2002,3(1):1-12.
[9]Fredericks ZL, Forte C, Capuano IV, et al. Identification of potent human anti-IL-1RI antagonist antibodies[J]. Protein Eng Des Sel,2004,17(1): 95-106.
[10]Venkatesh N, Zaltsman Y, Somjen D, et al. A synthetic peptide with estrogen-like activity derived from a phage-display peptide librar[J]y. Peptides,2002,23(3):573-80.
[11]Lohse PA, Wright MC. In vitro protein display in drug discovery[J]. Curr Opin Drug Discov Devel,2001,4(2):198-204.
[12]Arap W, Pasqualini R, Ruoslahti E.Cancer treatment by targeted drug [J].Science,1998,279(5349):377-80.
[13]Xue Y, Bi F, Zhang X, et al. Inhibition of endothelial cell proliferationby targeting Racl GTPase with small interference RNA in tumor cells[J]. Biochem Biophys Res Commun,2004; 320(4):1309-15.
[14]Folkman J. Angiogenesis and apoptosis[J]. Semin Cancer Biol,2003; 13(2): 159-67.
[15]Folkman J. Seminars in medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis[J]. N Engl J Med,1995, 333(26):1757-63.
[16]Bussolati B, Grange C, Tei L, et al. Targeting of human renal tumor-derived endothelial cells with peptides obtained by phage display[J].J Mol Med, 2007,85(8):897-906.
[17]管明强,史占军,姜勇,等.体内筛选骨肉瘤血管内皮特异性结合肽[J].中华实验外科杂志,2009,26(12)：1763.
[18]MarelliBerg FM, Peek E, Lidington EA, et al. Isolation of endothelial cells from murine tissue. J Immunol Methods,2000,244 (122):205-15.
[19]Bowden RA, Ding ZM, Donnachie EM, et al. Role of alpha4 integrin and VCAM-1 in CD182 independent neutrophil migration across mouse cardiac endothelium. Circ Res,2002,90(5):562-69.
[20]Lim YC, Garcia-Cardena G, Allport JR, et al. Heterogeneity of endothelial cells from different organ sites in T-cell subset recruitment. Am J Pathol, 2003,162(5):1591-1601.
[21]Ulger H, Karabulut AK, Pratten MK. Labelling of rat endothelial cells with antibodies to vWF, RECA21, PECAM21, ICAM21,OX243 and ZO21. Anat Histol Embryol,2002,31(1):31-35.
[22]Ausprunk DH, Folkman J. Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc Res,1977,14(1):53-65.
[1]Ek E, Dass C, Choonq P.Commonly used mouse models of osteosarcoma [J].Crit Rev Oncol Hematol,2006,60(1):1-8
[2]Egenvall A,Nodtvedt A, Euler H.Bone tumors in a population of 400 000 insured Swedish dogs up to 10 y of age:incidence and survival [J].Can J Vet Res,2007,71(4):292-299.
[3]Kondo H, Ishikawa M, Maeda H, et al Spontaneous osteosarcoma in a rabbit [J].Vet Pathol,2007,44(5):691-694.
[4]Dernell W. Tumours of the skeletal system [M]//Dobson J,Lascelles B eds. BSAVA manual of canine and feline oncology.2nd ed. British Small Animal Veterinary Association, 2003:179-195.
[5]Lascelles B, Dernell S, Correa T,et al. Improved survival associated with postoperative wound infection in dogs treated with limb-salvage surgery for osteosarcoma [J]. Ann Surg Oncol,2005,12(12):1073-1083.
[6]Hillers R, Dernell S, Lafferty H, et al. Incidence and prognostic importance of lymph node metastases in dogs with appendicular osteosarcoma:228 cases (1986-2003) [J].J Am Vet Med Assoc,2005,226(8):1364-1367.
[7]Dow S, Elmslie R, Kurzman I, et al. Phase I study of liposome-DNA complexes encoding the interleukin-2 gene in dogs with osteosarcoma lung metastases [J]. Hum Gene Ther,2005 16(8):937-946.
[8]Hosoya K, Poulson M, Azuma C. Osteoradionecrosis and radiation induced bone tumors following orthovoltage radiation therapy in dogs [J].Vet Radiol Ultrasound,2008,49(2):189-195.
[9]王玉民,尤占云,韦志康,等.物理、化学因子诱癌模型及其剂量-效应相关模式[J].职业卫生与应急救援,2000,18(1)：1-3.
[10]Ory B,Heymann F,Kamijo A,et al. Zoledronic acid suppresses lung metastases prolongs overall survival of osteosarcoma-bearing mice [J]. Cancer,2005,104(11):2522-2529.
[11]姚明,孔韩卫,李江,等.SCI-OS人骨肉瘤裸小鼠皮下移植模型的建立[J].肿瘤,2000,20(5)：351-353.
[12]于哲,范清宇,张婷,等.大鼠骨肉瘤模型的建立[J].动物医学进展,2005,26(2)：58-60.
[13]薛华明,梅炯,蔡宣松,等.腺病毒介导干扰RNA对荷人骨肉瘤裸鼠VEGF表达的影响[J].中国肿瘤临床,2008,35(3)：169-172.
[14]Miretti S, Roato I, Taulli,et al. A mouse model of pulmonary metastasis from spontaneous osteosarcoma monitored in vivo by luciferase imaging [J]. PLoS ONE,2008,3(3):e1828
[15]王忠良,李旭良,张德文,等.大鼠骨肉瘤肺转移模型的建立[J].重庆医科大学学报,2007,32(9)：935-939.
[16]罗旭耀,沈茜,苗积生,等.153钐-氯屈膦酸二钠在原位人骨肉瘤裸鼠模型中的疗效[J].第二军医大学学报,2007,28(10)：1094-1097.
[17]师长宏,施新猷,朱德生.LJH-OS人骨肉瘤裸鼠原位移植模型的建立及其生物学特性[J].中国实验动物学报,1999,7(2)：102-106.
[18]屈小鹏,吴道澄,陈军,等.MDP-阿霉素脂质体对裸鼠SOSP-M成骨肉瘤肺转移的治疗[J].第四军医大学学报,2007,28(24)：2232-2234.
[19]祝文涛,郭风劲,许涛,等.不同转移潜能MG63骨肉瘤细胞亚株裸鼠肺转移模型的建立[J].中德临床肿瘤学杂志,2007,6(5)：484-486.
[20]Koto K, Horie N, Kimura S,et al. Clinically relevant dose of zoledronic acid inhibits spontaneous lung metastasis in a murine osteosarcoma model [J]. Cancer Lett, 2009,274(2):271-278
[21]Kaplan B, Moore T, Schreiber K,et al. A new murine tumor model for studying HLA-A2-restricted anti-tumor immunity [J]. Cancer Lett,2005,224(1):153-166.
[22]Berman S, Calo E, Landman A,et al. Metastatic osteosarcoma induced by inactivation of Rb and p53 in the osteoblast lineage [J]. Proc Natl Acad Sci USA,2008,105(33):11851-11856.
[23]Walkley C, Qudsi R, Sankaran V, et al. Conditional mouse osteosarcoma, dependent on p53 loss and potentiated by loss of Rb, mimics the human disease [J]. Genes Dev,2008, 22(12):1662-1676.
[24]Yin D, Jia T,Gong W,et al. VEGF blockade decelerates the growth of a murine experimental osteosarcoma [J]. Int J Oncol,2008,33(2):253-259.
[25]龙晓燕,温敏.转基因动物在人类疾病动物模型中的应用[J].云南中医中药杂志,2007,28(6)：43-45.
[2]Denekamp J. Review article:angiogenesis, neovascular proliferation and vascular pathophysiology as targets for cancer therapy. Br J Radiol,1993, 66(783):181-96.
[3]Folkman J. Seminars in medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis. N Engl J Med,1995, 333(26):1757-63.
[4]Kanthou C, Greco O, Stratford A, et al. The tubulin-binding agent combretastatin A-4-phosphate arrests endothelial cells in mitosis and induces mitotic cell death. Am J Pathol,2004,165(4):1401-11.
[5]Burrows FJ, Watanabe Y, Thorpe PE. A murine model for antibody-directed targeting of vascular endothelial cells in solid. Cancer Res, 1992,52(21):5954-62.
[6]Siemann DW, Chaplin DJ, Horsman MR. Vascular-targeting therapies for treatment of malignant disease. Cancer,2004,100(12):2491-99.
[7]Keresztessy Z, Csosz E, Harsfalvi J, et al. Phage display selection of efficient glutamine-donor substrate peptides for transglutaminase[J]. Protein Sci,2006,15(11):2466-80.
[8]Nixon AE. Phage display as a tool for protease ligand discovery[J]. Curr Pharm Biotechnol,2002,3(1):1-12.
[9]Fredericks ZL, Forte C, Capuano IV, et al. Identification of potent human anti-IL-1RI antagonist antibodies[J]. Protein Eng Des Sel,2004,17(1): 95-106.
[10]Venkatesh N, Zaltsman Y, Somjen D, et al. A synthetic peptide with estrogen-like activity derived from a phage-display peptide librar[J]y. Peptides,2002,23(3):573-80.
[11]Lohse PA, Wright MC. In vitro protein display in drug discovery[J]. Curr Opin Drug Discov Devel,2001,4(2):198-204.
[12]Arap W, Pasqualini R, Ruoslahti E.Cancer treatment by targeted drug [J].Science,1998,279(5349):377-80.
[13]Xue Y, Bi F, Zhang X, et al. Inhibition of endothelial cell proliferationby targeting Racl GTPase with small interference RNA in tumor cells[J]. Biochem Biophys Res Commun,2004; 320(4):1309-15.
[14]Folkman J. Angiogenesis and apoptosis[J]. Semin Cancer Biol,2003; 13(2): 159-67.
[15]Folkman J. Seminars in medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis[J]. N Engl J Med,1995, 333(26):1757-63.
[16]Bussolati B, Grange C, Tei L, et al. Targeting of human renal tumor-derived endothelial cells with peptides obtained by phage display[J].J Mol Med, 2007,85(8):897-906.
[17]管明强,史占军,姜勇,等.体内筛选骨肉瘤血管内皮特异性结合肽[J].中华实验外科杂志,2009,26(12)：1763.
[18]MarelliBerg FM, Peek E, Lidington EA, et al. Isolation of endothelial cells from murine tissue. J Immunol Methods,2000,244 (122):205-15.
[19]Bowden RA, Ding ZM, Donnachie EM, et al. Role of alpha4 integrin and VCAM-1 in CD182 independent neutrophil migration across mouse cardiac endothelium. Circ Res,2002,90(5):562-69.
[20]Lim YC, Garcia-Cardena G, Allport JR, et al. Heterogeneity of endothelial cells from different organ sites in T-cell subset recruitment. Am J Pathol, 2003,162(5):1591-1601.
[21]Ulger H, Karabulut AK, Pratten MK. Labelling of rat endothelial cells with antibodies to vWF, RECA21, PECAM21, ICAM21,OX243 and ZO21. Anat Histol Embryol,2002,31(1):31-35.
[22]Ausprunk DH, Folkman J. Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc Res,1977,14(1):53-65.
[1]Ek E, Dass C, Choonq P.Commonly used mouse models of osteosarcoma [J].Crit Rev Oncol Hematol,2006,60(1):1-8
[2]Egenvall A,Nodtvedt A, Euler H.Bone tumors in a population of 400 000 insured Swedish dogs up to 10 y of age:incidence and survival [J].Can J Vet Res,2007,71(4):292-299.
[3]Kondo H, Ishikawa M, Maeda H, et al Spontaneous osteosarcoma in a rabbit [J].Vet Pathol,2007,44(5):691-694.
[4]Dernell W. Tumours of the skeletal system [M]//Dobson J,Lascelles B eds. BSAVA manual of canine and feline oncology.2nd ed. British Small Animal Veterinary Association, 2003:179-195.
[5]Lascelles B, Dernell S, Correa T,et al. Improved survival associated with postoperative wound infection in dogs treated with limb-salvage surgery for osteosarcoma [J]. Ann Surg Oncol,2005,12(12):1073-1083.
[6]Hillers R, Dernell S, Lafferty H, et al. Incidence and prognostic importance of lymph node metastases in dogs with appendicular osteosarcoma:228 cases (1986-2003) [J].J Am Vet Med Assoc,2005,226(8):1364-1367.
[7]Dow S, Elmslie R, Kurzman I, et al. Phase I study of liposome-DNA complexes encoding the interleukin-2 gene in dogs with osteosarcoma lung metastases [J]. Hum Gene Ther,2005 16(8):937-946.
[8]Hosoya K, Poulson M, Azuma C. Osteoradionecrosis and radiation induced bone tumors following orthovoltage radiation therapy in dogs [J].Vet Radiol Ultrasound,2008,49(2):189-195.
[9]王玉民,尤占云,韦志康,等.物理、化学因子诱癌模型及其剂量-效应相关模式[J].职业卫生与应急救援,2000,18(1)：1-3.
[10]Ory B,Heymann F,Kamijo A,et al. Zoledronic acid suppresses lung metastases prolongs overall survival of osteosarcoma-bearing mice [J]. Cancer,2005,104(11):2522-2529.
[11]姚明,孔韩卫,李江,等.SCI-OS人骨肉瘤裸小鼠皮下移植模型的建立[J].肿瘤,2000,20(5)：351-353.
[12]于哲,范清宇,张婷,等.大鼠骨肉瘤模型的建立[J].动物医学进展,2005,26(2)：58-60.
[13]薛华明,梅炯,蔡宣松,等.腺病毒介导干扰RNA对荷人骨肉瘤裸鼠VEGF表达的影响[J].中国肿瘤临床,2008,35(3)：169-172.
[14]Miretti S, Roato I, Taulli,et al. A mouse model of pulmonary metastasis from spontaneous osteosarcoma monitored in vivo by luciferase imaging [J]. PLoS ONE,2008,3(3):e1828
[15]王忠良,李旭良,张德文,等.大鼠骨肉瘤肺转移模型的建立[J].重庆医科大学学报,2007,32(9)：935-939.
[16]罗旭耀,沈茜,苗积生,等.153钐-氯屈膦酸二钠在原位人骨肉瘤裸鼠模型中的疗效[J].第二军医大学学报,2007,28(10)：1094-1097.
[17]师长宏,施新猷,朱德生.LJH-OS人骨肉瘤裸鼠原位移植模型的建立及其生物学特性[J].中国实验动物学报,1999,7(2)：102-106.
[18]屈小鹏,吴道澄,陈军,等.MDP-阿霉素脂质体对裸鼠SOSP-M成骨肉瘤肺转移的治疗[J].第四军医大学学报,2007,28(24)：2232-2234.
[19]祝文涛,郭风劲,许涛,等.不同转移潜能MG63骨肉瘤细胞亚株裸鼠肺转移模型的建立[J].中德临床肿瘤学杂志,2007,6(5)：484-486.
[20]Koto K, Horie N, Kimura S,et al. Clinically relevant dose of zoledronic acid inhibits spontaneous lung metastasis in a murine osteosarcoma model [J]. Cancer Lett, 2009,274(2):271-278
[21]Kaplan B, Moore T, Schreiber K,et al. A new murine tumor model for studying HLA-A2-restricted anti-tumor immunity [J]. Cancer Lett,2005,224(1):153-166.
[22]Berman S, Calo E, Landman A,et al. Metastatic osteosarcoma induced by inactivation of Rb and p53 in the osteoblast lineage [J]. Proc Natl Acad Sci USA,2008,105(33):11851-11856.
[23]Walkley C, Qudsi R, Sankaran V, et al. Conditional mouse osteosarcoma, dependent on p53 loss and potentiated by loss of Rb, mimics the human disease [J]. Genes Dev,2008, 22(12):1662-1676.
[24]Yin D, Jia T,Gong W,et al. VEGF blockade decelerates the growth of a murine experimental osteosarcoma [J]. Int J Oncol,2008,33(2):253-259.
[25]龙晓燕,温敏.转基因动物在人类疾病动物模型中的应用[J].云南中医中药杂志,2007,28(6)：43-45.