LyP-1修饰磁纳米颗粒介导的肿瘤磁感应靶向热疗
|
摘要
研究目的
本课题旨在研究主动靶向磁纳米粒子(LyP-1-MNPs)的制备方法,并在细胞水平上完成磁感应靶向热疗的生物相容性研究和靶向性验证,在动物水平上研究LyP-1-MNPs磁感应靶向热疗的安全性与有效性。
研究方法
1.LyP-1-MNPs的制备：采用一步法制备PEG修饰的Fe3O4磁纳米粒子,经活化后在其表面偶联细胞穿膜肽LyP-1。利用傅立叶红外光谱仪(FTIR)、热重分析(TGA)、透射电镜(TEM)、zeta电位与粒度分析仪等分析方法对所制备的PEG修饰的Fe304磁纳米粒子的粒径、形态、蛋白偶联质量比及zeta电位分析等进行表征检测,并将制备样品放置于交变磁场中检测其升温性能,并在室温条件下观察其稳定性和分散性。
2.细胞实验：采用RT-PCR和Western Blot的研究方法对高低表达P32蛋白的肿瘤细胞P32蛋白的表达量进行验证；采用CCK-8法研究靶向性磁纳米粒子的细胞毒性；甲苯胺蓝染色和透射电镜(TEM)观察磁纳米粒子在不同细胞内的聚集状态；激光共聚焦(LCSM)检测磁纳米粒子在细胞内的具体定位情况；电感耦合等离子发射光谱仪(ICP-MS)检测单个细胞对磁纳米粒子的摄取量。
3.动物实验：制作荷瘤鼠模型；施行实验动物分组,随机分为3组：阴性对照组、热疗组、靶向热疗组。实验组肿瘤原位注射磁流体,热疗后称量体重,绘制体重变化曲线；记录肿瘤体积,绘制肿瘤体积变化曲线。热疗30天后对荷瘤鼠进行安乐死,取心、肝、脾、肺、肾、肿瘤,进行组织切片,HE染色观察靶向磁纳米粒子的体内生物相容性；普鲁士蓝染色观察靶向磁纳米粒子在体内的生物分布情况。
研究结果
1.制备的磁纳米粒子稳定性和磁响应性良好,经细胞穿膜肽LyP-1修饰后呈规整的球形,粒径有所增加,约15nm,分散性更好；交变磁场下具有良好的升温能力；一步法制备的PEG修饰的磁纳米粒子表面带负电荷,经LyP-1修饰后表面带正电荷。
2. RT-PCR和Western-Blot验证了人乳腺癌细胞株MCF-7为一种高表达P32蛋白的细胞株,小鼠结直肠癌细胞株CT-26为一种相对低表达P32蛋白的细胞株。
3.所制备的靶向磁纳米粒子无显著细胞毒性,毒性分级为0级或1级,生物相容性好。靶向磁纳米粒子进入细胞的数目高于PEG修饰的非靶向磁纳米粒子进入相同细胞的数目,且高表达P32蛋白的癌细胞MCF-7对靶向磁纳米粒子的摄取量明显高于相对低表达P32蛋白的细胞株CT-26对靶向磁纳米粒子的摄取量。靶向磁纳米粒子在细胞内定位有两种：一部分存在于细胞质中,聚集程度高,推测是吞饮作用或吞噬作用介导的胞吞途径而进入胞内；一部分分散的存在于胞质中或细胞核中,分散性强,猜测是P32蛋白受体介导的内吞途径进入胞内。
4.动物实验结果显示,实验组荷瘤鼠的肿瘤体积均得到有效抑制,热疗组荷瘤鼠的治愈率为30%,靶向热疗组荷瘤鼠治愈率为60%,治愈率提高了一倍。病理切片观察显示,磁纳米粒子在生物体内的生物组织相容性良好,未见明显的免疫反应或炎症反应,普鲁士蓝染色观察磁纳米粒子在体内的分布与代谢情况表明磁纳米粒子在治愈后期主要出现在肿瘤部位或肿瘤消退后的原肿瘤部位,在脏器中也有聚集,主要存在于脾、肺、肝中,而又以脾的铁含量最高。
研究结论
LyP-1修饰的靶向磁纳米粒子具有良好的分散性、磁响应性和升温性能,且生物相容性好,并能有效提高高表达P32蛋白的细胞对靶向纳米粒子的摄取能力。细胞实验和动物实验结果均表明靶向磁性纳米粒用于磁感应靶向热疗是安全的、可行的,磁感应靶向热疗效果是显著的。
Objective
The aims of the present study are to investigate the preparation of LyP-1modified magnetic nanoparticles (LyP-1-MNPs) which could home to the cancer overexpressing protein P32, to study the biocompatibility and targeting properties of LyP-1-MNPs in vitro, and to test the targeting therapy of LyP-1-MNPs in vivo.
Methods
1. Preparation and characterization ofLyP-1modified MNPs
Firstly, Polyethylene glycol(PEG)-coated magnetic nanoparticals (PEG-MNPs) were synthesized by "single-step method", which is chemical co-precipitation method substantially, but it is more simple and cost less time. PEG-MNPs, modified by special agent, were conjugated with cell-penetrating peptide LyP-1. The size, zeta potential, and coating characterizations of LyP-1-MNPs were analyzed by transmission electron microscope (TEM), zetaplus zeta potential measurement, and fourier transform infrared spectroscopy (FTIR).
2. In vitro study
The expressions of P32of cell lines were detected by RT-PCR and western-blot. Cytotoxicity of functional magnetic nanoparticles was evaluated by CCK-8assay, and the aggregation state of LyP-1-MNPs in cells was studied by Prussian blue staining and transmission electron microscopy (TEM). Subcellular localization of LyP-1-MNPs in different cell lines was observed by Laser Scanning Confocal Microscope(LCSM).The iron content of cellular uptake was measured by Inductively coupled plasma mass spectrometry (ICP-MS).
3. In vivo study
The tumor-bearing mice model was built by transplanting human breast cancer cell line MCF-7cells into the subcutaneous tissue of the right hind limb of BALB/c nude mouse. And then all animals were divided into three groups randomly:negative control group, hyperthermia group, and targeting therapy group. The experimental groups were injected with magnetic fluid, after magenetic hyperthermia treatment, the body weight of animals and the tumor volume were measured, then the body weight curves and the tumor volume curves were plotted. When30days had passed after therapy, the mice were euthanized with an intraperitoneal injection of pentobarbital sodium(120mg/kg), the important organs, such as heart, liver, spleen, lungs, kidneys and tumor,were obtained and prepared for histological analysis, then the samples were cut into about5μ m thick section and the sections were stained with either hematoxylin and eosin (H&E) or Prussian blue. Biocompatibility of MNPs was analysed by H&E staining, and biodistrbution of MNPs was observred by Prussian blue staining.
Results
1. Magnetic nanoparticles had good stability, well magnetism response, and great dispersibility. When exposed to alternating magnetic field, MNPs had good warming performance. The morphology of particals modified with LyP-1was approximately spherical and the mean size of the magnetic nanoparticles was about15nm.After being coated by Polyethylene glycol (PEG), the surface of MNPs displayed the negative charge properties, and when LyP-1was conjugated with the PEG-MNPs, the zeta potential was changed into positive charge from negative charge.
2. We found that human breast cancer MCF-7is a P32-high-expressing cell line and relatively,colorectal cancer cell line CT-26is a P32-low-expressing cell line using RT-PCR and western-blot assay.
3. The result of CCK-8assay showed that PEG-MNPs modified with LyP-1had no significant cytotoxicity at a concentration of less than60μg/mL, cytotoxicity grade had a narrow range between0grade and1grade. The cellular uptake of targeting nanoparticals was higher than that of Polyethylene glycol (PEG)-coated magnetic nanoparticals (PEG-MNPs) which has no targeting properties, and the cellular uptake of LyP-1-MNPs in overexpressing P32cell MCF-7was much higher than that in low-expressing P32cell CT-26. The targeting nanoparticals mainly localized in the cytoplasm, and some were found in the nucleus, we speculated that the distribution of LyP-1-MNPs maybe effected by different mechanisms of uptake routines, because of the size of LyP-1-MNPs, the most MNPs aggregated and were uptaked through cells swallowed way into the cytoplasm, while some that had good dispersibility and small size could be transported across the cell wall and nucleus wall through the receptor-mediated swallowed way.
4. The result of in vivo experiment demonstrated that the tumor volume of the experimental groups was suppressed effectively,and the curative ratio of hyperthermia group was30%(n=10), and the curative ratio of targeting therapy group was60%(n=10),which was the twice of that of hyperthermia group. Histological analysis indicated that the biocompatibility of magnetic nanoparticals was very well, because there was no any inflammation and immunoreaction. Prussian blue staining of sections of samples illustrated that the biodistribution of MNPs in body mainly accumulated in the tumors and also showed in the spleen and lungs.
Conclusions
Magnetic nanoparticles had good stability, well magnetism response, great dispersibility, good warming performance, nice biocompatibility. After modified with LyP-1, the LyP-1modified magnetic nanoparticles could home to the overexpressing P32cell lines MCF-7, and the cellular uptake of magnetic nanoparticals was much higher than the non-targeting nanoparticals significantly. We also found that targeting magnetic nanoparticals had a greater inhibitive effect on tumor growth than hyperthermia group or control group.
本课题旨在研究主动靶向磁纳米粒子(LyP-1-MNPs)的制备方法,并在细胞水平上完成磁感应靶向热疗的生物相容性研究和靶向性验证,在动物水平上研究LyP-1-MNPs磁感应靶向热疗的安全性与有效性。
研究方法
1.LyP-1-MNPs的制备：采用一步法制备PEG修饰的Fe3O4磁纳米粒子,经活化后在其表面偶联细胞穿膜肽LyP-1。利用傅立叶红外光谱仪(FTIR)、热重分析(TGA)、透射电镜(TEM)、zeta电位与粒度分析仪等分析方法对所制备的PEG修饰的Fe304磁纳米粒子的粒径、形态、蛋白偶联质量比及zeta电位分析等进行表征检测,并将制备样品放置于交变磁场中检测其升温性能,并在室温条件下观察其稳定性和分散性。
2.细胞实验：采用RT-PCR和Western Blot的研究方法对高低表达P32蛋白的肿瘤细胞P32蛋白的表达量进行验证；采用CCK-8法研究靶向性磁纳米粒子的细胞毒性；甲苯胺蓝染色和透射电镜(TEM)观察磁纳米粒子在不同细胞内的聚集状态；激光共聚焦(LCSM)检测磁纳米粒子在细胞内的具体定位情况；电感耦合等离子发射光谱仪(ICP-MS)检测单个细胞对磁纳米粒子的摄取量。
3.动物实验：制作荷瘤鼠模型；施行实验动物分组,随机分为3组：阴性对照组、热疗组、靶向热疗组。实验组肿瘤原位注射磁流体,热疗后称量体重,绘制体重变化曲线；记录肿瘤体积,绘制肿瘤体积变化曲线。热疗30天后对荷瘤鼠进行安乐死,取心、肝、脾、肺、肾、肿瘤,进行组织切片,HE染色观察靶向磁纳米粒子的体内生物相容性；普鲁士蓝染色观察靶向磁纳米粒子在体内的生物分布情况。
研究结果
1.制备的磁纳米粒子稳定性和磁响应性良好,经细胞穿膜肽LyP-1修饰后呈规整的球形,粒径有所增加,约15nm,分散性更好；交变磁场下具有良好的升温能力；一步法制备的PEG修饰的磁纳米粒子表面带负电荷,经LyP-1修饰后表面带正电荷。
2. RT-PCR和Western-Blot验证了人乳腺癌细胞株MCF-7为一种高表达P32蛋白的细胞株,小鼠结直肠癌细胞株CT-26为一种相对低表达P32蛋白的细胞株。
3.所制备的靶向磁纳米粒子无显著细胞毒性,毒性分级为0级或1级,生物相容性好。靶向磁纳米粒子进入细胞的数目高于PEG修饰的非靶向磁纳米粒子进入相同细胞的数目,且高表达P32蛋白的癌细胞MCF-7对靶向磁纳米粒子的摄取量明显高于相对低表达P32蛋白的细胞株CT-26对靶向磁纳米粒子的摄取量。靶向磁纳米粒子在细胞内定位有两种：一部分存在于细胞质中,聚集程度高,推测是吞饮作用或吞噬作用介导的胞吞途径而进入胞内；一部分分散的存在于胞质中或细胞核中,分散性强,猜测是P32蛋白受体介导的内吞途径进入胞内。
4.动物实验结果显示,实验组荷瘤鼠的肿瘤体积均得到有效抑制,热疗组荷瘤鼠的治愈率为30%,靶向热疗组荷瘤鼠治愈率为60%,治愈率提高了一倍。病理切片观察显示,磁纳米粒子在生物体内的生物组织相容性良好,未见明显的免疫反应或炎症反应,普鲁士蓝染色观察磁纳米粒子在体内的分布与代谢情况表明磁纳米粒子在治愈后期主要出现在肿瘤部位或肿瘤消退后的原肿瘤部位,在脏器中也有聚集,主要存在于脾、肺、肝中,而又以脾的铁含量最高。
研究结论
LyP-1修饰的靶向磁纳米粒子具有良好的分散性、磁响应性和升温性能,且生物相容性好,并能有效提高高表达P32蛋白的细胞对靶向纳米粒子的摄取能力。细胞实验和动物实验结果均表明靶向磁性纳米粒用于磁感应靶向热疗是安全的、可行的,磁感应靶向热疗效果是显著的。
Objective
The aims of the present study are to investigate the preparation of LyP-1modified magnetic nanoparticles (LyP-1-MNPs) which could home to the cancer overexpressing protein P32, to study the biocompatibility and targeting properties of LyP-1-MNPs in vitro, and to test the targeting therapy of LyP-1-MNPs in vivo.
Methods
1. Preparation and characterization ofLyP-1modified MNPs
Firstly, Polyethylene glycol(PEG)-coated magnetic nanoparticals (PEG-MNPs) were synthesized by "single-step method", which is chemical co-precipitation method substantially, but it is more simple and cost less time. PEG-MNPs, modified by special agent, were conjugated with cell-penetrating peptide LyP-1. The size, zeta potential, and coating characterizations of LyP-1-MNPs were analyzed by transmission electron microscope (TEM), zetaplus zeta potential measurement, and fourier transform infrared spectroscopy (FTIR).
2. In vitro study
The expressions of P32of cell lines were detected by RT-PCR and western-blot. Cytotoxicity of functional magnetic nanoparticles was evaluated by CCK-8assay, and the aggregation state of LyP-1-MNPs in cells was studied by Prussian blue staining and transmission electron microscopy (TEM). Subcellular localization of LyP-1-MNPs in different cell lines was observed by Laser Scanning Confocal Microscope(LCSM).The iron content of cellular uptake was measured by Inductively coupled plasma mass spectrometry (ICP-MS).
3. In vivo study
The tumor-bearing mice model was built by transplanting human breast cancer cell line MCF-7cells into the subcutaneous tissue of the right hind limb of BALB/c nude mouse. And then all animals were divided into three groups randomly:negative control group, hyperthermia group, and targeting therapy group. The experimental groups were injected with magnetic fluid, after magenetic hyperthermia treatment, the body weight of animals and the tumor volume were measured, then the body weight curves and the tumor volume curves were plotted. When30days had passed after therapy, the mice were euthanized with an intraperitoneal injection of pentobarbital sodium(120mg/kg), the important organs, such as heart, liver, spleen, lungs, kidneys and tumor,were obtained and prepared for histological analysis, then the samples were cut into about5μ m thick section and the sections were stained with either hematoxylin and eosin (H&E) or Prussian blue. Biocompatibility of MNPs was analysed by H&E staining, and biodistrbution of MNPs was observred by Prussian blue staining.
Results
1. Magnetic nanoparticles had good stability, well magnetism response, and great dispersibility. When exposed to alternating magnetic field, MNPs had good warming performance. The morphology of particals modified with LyP-1was approximately spherical and the mean size of the magnetic nanoparticles was about15nm.After being coated by Polyethylene glycol (PEG), the surface of MNPs displayed the negative charge properties, and when LyP-1was conjugated with the PEG-MNPs, the zeta potential was changed into positive charge from negative charge.
2. We found that human breast cancer MCF-7is a P32-high-expressing cell line and relatively,colorectal cancer cell line CT-26is a P32-low-expressing cell line using RT-PCR and western-blot assay.
3. The result of CCK-8assay showed that PEG-MNPs modified with LyP-1had no significant cytotoxicity at a concentration of less than60μg/mL, cytotoxicity grade had a narrow range between0grade and1grade. The cellular uptake of targeting nanoparticals was higher than that of Polyethylene glycol (PEG)-coated magnetic nanoparticals (PEG-MNPs) which has no targeting properties, and the cellular uptake of LyP-1-MNPs in overexpressing P32cell MCF-7was much higher than that in low-expressing P32cell CT-26. The targeting nanoparticals mainly localized in the cytoplasm, and some were found in the nucleus, we speculated that the distribution of LyP-1-MNPs maybe effected by different mechanisms of uptake routines, because of the size of LyP-1-MNPs, the most MNPs aggregated and were uptaked through cells swallowed way into the cytoplasm, while some that had good dispersibility and small size could be transported across the cell wall and nucleus wall through the receptor-mediated swallowed way.
4. The result of in vivo experiment demonstrated that the tumor volume of the experimental groups was suppressed effectively,and the curative ratio of hyperthermia group was30%(n=10), and the curative ratio of targeting therapy group was60%(n=10),which was the twice of that of hyperthermia group. Histological analysis indicated that the biocompatibility of magnetic nanoparticals was very well, because there was no any inflammation and immunoreaction. Prussian blue staining of sections of samples illustrated that the biodistribution of MNPs in body mainly accumulated in the tumors and also showed in the spleen and lungs.
Conclusions
Magnetic nanoparticles had good stability, well magnetism response, great dispersibility, good warming performance, nice biocompatibility. After modified with LyP-1, the LyP-1modified magnetic nanoparticles could home to the overexpressing P32cell lines MCF-7, and the cellular uptake of magnetic nanoparticals was much higher than the non-targeting nanoparticals significantly. We also found that targeting magnetic nanoparticals had a greater inhibitive effect on tumor growth than hyperthermia group or control group.
引文
1. Alexiou C, Jurgons R, Seliger C, Iro H, Medical application of magnetic nanoparticles, J Nanosci Nanotechnol.2006; 6(9-10):2762-2768.
2. Gazeau F, Levy M, Wilhelm C. Optimizing magnetic nanoparticle design for nanothermotherapy. Nanomed.2008,3(6):831-844.
3. Thiesen B, Jordan A. Clinical applications of magnetic nanoparticles for hyperthermia. Int J Hyperthermia.2008,24(6):467-474.
4. Ito A, Shinkai M, Honda H,et al. Heat-inducible TNF-alpha gene therapy combined with hyperthermia using magnetic nanoparticles as a novel tumor-targeted therapy. Cancer Gene Ther 2001,8(9):649-654.
5. Shinkai M, Ito A. Functional magnetic particles for medical application. Adv Biochem Eng Biotechnol.2004,91:191-220.
6. Chang HL, Eun YK, Kilsoo J, et al. Simple, Efficient, and Reproducible Gene Transfection of Mouse Embryonic Cells by Magnetofectin. Stem Cells Dev,2008, 17:133-141.
7.王骏,阳离子脂质体吸附FLT3L基因肿瘤原位注射联合顺铂治疗小鼠结肠癌的实验研究.四川大学硕士论文,2006年02期
8.陈金玲,郭瑞强,周青,王涛.携重组人促红细胞生成素水凝胶复合物原位注射对兔心肌梗死模型局部心功能的影响.2010中国医学影像技术国际论坛暨《中国医学影像技术》编委换届会论文集
9. Ting-Jung Chen, Tsan-Hwang Cheng, Yu-Chin Hung, et al. Targeted folic acid-PEG nanoparticles for noninvasive imaging of folate receptor by MRI. Journal of Biomedical Materials Research.2008,87(1):165-175
10. Sun C, Sze R, Zhang M. Folic acid-PEG conjugated superparamagnetic nanoparticles for targeted cellular uptake and detection by MRI. J Biomed Mater Res A,2006,78(3):550 7.
11. Kohler N, Sun C, Wang J, et al. Methotrexate-modified superparamagnetic nanoparticles and their intracellular uptake into human cancer cells. Langmuir,2005,21 (19):8858 64.
12. Joy L. Kovar, William Volcheck, Eva Sevick-Muraca et al. Characterization and Performance of a Near-Infrared 2-Deoxyglucose Optical Imaging Agent for Mouse Cancer Models. Analytical Biochemistry,2009,384(2):254-262.
13.胡慧.乳腺癌细胞GLUT1分子成像实验研究.江苏大学硕士学位论文.2010.
14. Janic B, Iskander AS, Rad AM, et al. Effects of ferumoxides-protamine sulfate labeling on immunomodulatory characteristics of macrophage-like THP-1 cells [J]. PLoS ONE, 2008,3(6):e2499.
15. Omidkhoda A, Mozdarani H, Movasaghpoor A, et al. Study of apoptosis in labeled mesenchymal stem cells with superparamagnetic iron oxide using neutral comet assay [J]. Toxicol In Vitro,2007,21(6):1191-1196.
16. Arbab AS, Yocum GT, Kalish H, et al. Efficient magnetic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI [J]. Blood,2004,104(4):1217-1223.
17. Takeda S, Terazono B, Mishima F, et al. Novel drug delivery system by surface modified magnetic nanoparticles [J]. J Nanosci Nanotechnol,2006,6(9-10):3269-3276.
18. Orian-Rousseau V. CD44, a therapeutic target for metastasising tumours. Eur J Cancer 2010;46:1271-7.
19. Choi KY, Chung H, Min KH, Yoon HY, Kim K, Park JH, et al. Self-assembled hyaluronic acid nanoparticles for active tumor targeting. Biomaterials 2010;31:106-14.
20. Choi KY, Min KH, Yoon HY, Kim K, Park JH, Kwon IC, et al. PEGylation of hyaluronic acid nanoparticles improves tumor targetability in vivo. Biomaterials 2011;32:1880-9.
21. Saravanakumar G, Choi KY, Yoon HY, Kim K, Park JH, Kwon IC, et al. Hydrotropic hyaluronic acid conjugates:synthesis, characterization, and implications as a carrier of paclitaxel. Int J Pharm 2010;394:154-61.
22. Laakkonen P, Akerman ME, Biliran H, et al. Antitumor activity of a homing peptide that targets tumor lymphatics and tumor cells. Proc Natl Acad Sci U S A. 2004;101(25):9381-6
23. Al-Jamal WT, Al-Zamal KT, Cakebread A, Halket JM, Kostarelos K. Blood circulation and tissue biodistribution of lipid-quantum dot (L-QD) hybrid vesicles intravenously administered in mice. Bioconjug Chem 2009;20:1696-702.
24. Kim JY, Kim JK, Park JS, Byun Y, Kim CK. The use of PEGylated liposomes to prolong circulation lifetimes of tissue plasminogen activator. Biomaterials 2009;30:5751-6.
25. Lim SM, Kim TH, Jiang HH, Park CW, Lee S, Chen X, et al. Improved biological half-life and anti-tumor activity of TNF-related apoptosis-inducing ligand (TRAIL) using PEG-exposed nanoparticles. Biomaterials 2011;32:3538-46.
26.戴晓晨,靶向性磁性纳米颗粒的制备与磁感应升温作用.北京中医药大学硕士论文.2009
27. Laakkonen P, Porkka K, Hoffman JA, Ruoslahti E.A tumor-homing peptide with a targeting specificity related to lymphatic vessels.Nat Med.2002:8(7):751-5.
28. Laakkonen P, Akerman ME, Biliran H, et al. Antitumor activity of a homing peptide that targets tumor lymphatics and tumor cells. Proc Natl Acad Sci U S A. 2004;101(25):9381-6
29. Jiang J, Zhang Y, Krainer AR, Xu RM. Crystal structure of human p32, a doughnut-shaped acidic mitochondrial matrix protein. Proc Natl Acad Sci U S A.1999;96:35727.
30. Krainer AR, Mayeda A, Kozak D, Binns G. Functional expression of cloned human splicing factor SF2:homology to RNA-binding proteins, Ul 70K, Drosophila splicing regulators. Cell 1991;66:383-94.
31. Ghebrehiwet B, Lim BL, Peerschke El, Willis AC, Reid KB. Isolation, cDNA cloning, and overexpression of a 33-kD cell surface glycoprotein that binds to the globular "heads" of Clq. J ExpMed 1994;179:1809-21.
32. Bialucha CU, Ferber EC, Pichaud F, Peak-Chew SY, Fujita Y. p32 is a novel mammalian Lgl binding protein that enhances the activity of protein kinase Cξ and regulates cell polarity. J Cell Biol 2007;178:575-81.
33. Storz P, Hausser A, Link G, et al. Protein kinase Cμ is regulated by the multifunctional chaperon protein p32. J Biol Chem 2000;275:24601-7.
34. Park JH, von Maltzahn G, Xu MJ, Fogal V, Kotamraju VR, et al.Cooperative nanomaterial system to sensitize, target,and treat tumors. Proc Natl Acad Sci U S A. 2010;107(3):981-6.
35. Jaffer FA, Weissleder R Molecular imaging in the clinical arena.JAMA. 2005;293(7):855-862
36. Sharma R, Wendt JA, Rasmussen JC, Adams KE, Marshall MV, Sevick-Muraca EM New horizons for imaging lymphatic function. Ann N Y Acad Sci.2008; 1131:13-36
37. Winnard PT Jr, Pathak AP, Dhara S, Cho SY, Raman V, Pomper MGMolecular imaging of metastatic potential. J Nucl Med.2008; 49(Suppl 2):96S-112S
38. Qian CN, Berghuis B, Tsarfaty G, Bruch M, Kort EJ, Ditlev J, Tsarfaty I, Hudson E, Jackson DG, Petillo D, Chen J, Resau JH, Teh BT. Preparing the "Soil":the primary tumor induces vasculature reorganization in the sentinel lymph node before the arrival of metastatic cancer cells. Cancer Res.2006;66(21):10365-10376
39. Eiber M, Beer AJ, Holzapfel K, Tauber R, Ganter C, Weirich G, Krause BJ, Rummeny EJ, Gaa J. Preliminary results for characterization of pelvic lymph nodes in patients with prostate cancer by diffusion-weighted mr-imaging. Invest Radiol.2010;45(1):15-23
40. Zhang F, Niu G, Lin X, Jacobson O, Ma Y, Eden HS, He Y, Lu G, Chen X.Imaging tumor-induced sentinel lymph node lymphangio genesis with LyP-1 peptide. Amino Acids. 2011 Jul 19.
41. Uchida M, Kosuge H, Terashima M, Willits DA, Liepold LO, Young MJ, McConnell MV, Douglas T. Protein Cage Nanoparticles Bearing the LyP-1 Peptide for Enhanced Imaging of Macrophage-Rich Vascular Lesions. ACS Nano.2011;5(4):2493-502.
42. Valentina Fogal, Lianglin Zhang, Stan Krajewski, and Erkki Ruoslahti.Mitochondrial/Cell-Surface Protein p32/gC1qR as a Molecular Target in Tumor Cells and Tumor Stroma. Cancer Res,2008; 68:(17).
43. Laakkonen P, Akerman ME, Biliran H, et al. Antitumor activity of a homing peptide that targets tumor lymphatics and tumor cells. Proc Natl Acad Sci U S A 2004; 101:9381-6.
44. Park JH, von Maltzahn G, Xu MJ, Fogal V, Kotamraju VR, Ruoslahti E, Bhatia SN, Sailor MJ.Cooperative nanomaterial system to sensitize, target, and treat tumors.Proc Natl Acad Sci U S A.2010;107(3):981-6.
45. Luo G, Yu X, Jin C, Yang F, Fu D, Long J, Xu J, Zhan C, Lu W.LyP-1-conjugated nanoparticles for targeting drug delivery to lymphatic metastatic tumors.Int J Pharm. 2010;385(1-2):150-6.
46.闫志强,王飞,魏晓丽,俸灵林,何丹农,陆伟跃,LyP-1介导的淋巴转移肿瘤靶向脂质体递药系统研究.2011年中国药学大会暨第11届中国药师周论文集
47. Makela AR, Matilainen H, White DJ, Ruoslahti E, Oker-Blom C.Enhanced baculovirus-mediated transduction of human cancer cells by tumor-homing peptides. J Virol.2006;80(13):6603-11.
48. Lee CN, Wang YM, Lai WF, Chen TJ, Yu MC, Fang CL, Yu FL, Tsai YH, Chang WH, Zuo CS, Renshaw PF.Super-paramagnetic iron oxide nanoparticles for use in extrapulmonary tuberculosis diagnosis.Clin Microbiol Infect.2012, doi: 10.;llll/j.1469-0691.2012.03809.x.
49. Neumaier CE, Baio G, Ferrini S, Corte G, Daga A. MR and iron magnetic nanoparticles. Imaging opportunities in preclinical and translational research. Tumori.2008 Mar-Apr;94(2):226-33.
50. Smirnov P. Cellular magnetic resonance imaging using superparamagnetic anionic iron oxide nanoparticles:applications to in vivo trafficking of lymphocytes and cell-based anticancer therapy. Methods Mol Biol.2009;512:333-53.
51. Laurent S, Boutry S, Mahieu I, Vander Elst L, Muller RN.Iron oxide based MR contrast agents:from chemistry to cell labeling. Curr Med Chem.2009; 16(35):4712-27.
52. Peng XH, Qian X, Mao H, Wang AY, Chen ZG, Nie S, Shin DM.Targeted magnetic iron oxide nanoparticles for tumor imaging and therapy.Int J Nanomedicine. 2008;3(3):311-21.
53. Jin XH, Yang L, Duan XJ, Xie B, Li Z, Tan HB. In vivo MR imaging tracking of supermagnetic iron-oxide nanoparticle-labeled bone marrow mesenchymal stem cells injected into intra-articular space of knee joints:experiment with rabbit. Zhonghua Yi Xue Za Zhi.2007 Dec 4;87(45):3213-8.
54. Shen M, Cai H, Wang X, Cao X, Li K, Wang SH, Guo R, Zheng L, Zhang G, Shi X. Facile one-pot preparation, surface functionalization, and toxicity assay of APTS-coated iron oxide nanoparticles. Nanotechnology.2012 Mar 16;23(10):105601. Epub 2012 Feb 21.
55. Wang S, Wen S, Shen M, Guo R, Cao X, Wang J, Shi X. Aminopropyltriethoxysilane-mediated surface functionalization of hydroxyapatite nanoparticles:synthesis, characterization, and in vitro toxicity assay. Int J Nanomedicine. 2011;6:3449-59..
56. Ho D, Sun X, Sun S.Monodisperse magnetic nanoparticles for theranostic applications. Ace Chem Res.2011 (18);44(10):875-82.
57.陈本科.信号肽修饰的磁性纳米粒用于肿瘤磁感应热疗联合基因治疗的研究.北京中医药大学硕士论文.2010
58.何强芳,李国明,陈烁.水基Fe304磁流体的制备.应用化学,2005；22(6)：665-668
59. Laakkonen P, Porkka K, Hoffman JA, Ruoslahti E.A tumor-homing peptide with a targeting specificity related to lymphatic vessels.Nat Med.2002:8(7):751-5.
60. Ghebrehiwet B, Lim BL, Peerschke El, Willis AC, Reid KB. Isolation, cDNA cloning, and overexpression of a 33-kD cell surface glycoprotein that binds to the globular "heads" of C1q. J Exp Med 1994;179(6);179:1809-1821
61. Gupta S, Batchu RB, Datta K. Purification, partial characterization of rat kidney hyaluronic acid binding protein and its localization on the cell surface. Eur J Cell Biol 1991;56(1):58-67
62. Soltys BJ, Kang D, Gupta RS. Localization of P32 protein (gC1q-R) in mitochondria and at specific extramitochondrial locations in normal tissues. Histochem Cell Biol 2000;114(3):245-255
63. Krainer AR, Mayeda A, Kozak D, Binns G. Functional expression of cloned human splicing factor SF2:homology to RNA-binding proteins, U1 70K, Drosophila splicing regulators. Cell 1991;66(2):383-394
64. Majumdar M, Meenakshi J, Goswami SK, Datta K. Hyaluronan binding protein 1 (HABP1)/C1QBP/p32 is an endogenous substrate for MAP kinase and is translocated to the nucleus upon mitogenic stimulation. Biochem Biophys Res Commun 2002;291(4):829-837.
65. Park JH, von Maltzahn G, Xu MJ, Fogal V, Kotamraju VR, Ruoslahti E, Bhatia SN, Sailor MJ.Cooperative nanomaterial system to sensitize, target, and treat tumors.Proc Natl Acad Sci USA.2010;107(3):981-6.
66. Luo G, Yu X, Jin C, Yang F, Fu D, Long J, Xu J, Zhan C, Lu W.LyP-1-conjugated nanoparticles for targeting drug delivery to lymphatic metastatic tumors.Int J Pharm. 2010;385(1-2):150-6.
67. Laakkonen P, Akerman ME, Biliran H, et al. Antitumor activity of a homing peptide that targets tumor lymphatics and tumor cells. Proc Natl Acad Sci U S A. 2004;101(25):9381-6
68.杨欣.磁性微纳米介质的生物安全性研究.北京中医药大学硕士论文.2010
69. Wilhelm C, Gazeau F. Universal cell labelling with anionic magnetic nanoparticles [J]. Biomaterials,2008,29(22):3161-3174.
70. Gazeau F, Levy M, Wilhelm C. Optimizing magnetic nanoparticle design for nanothermotherapy [J]. Nanomed,2008,3(6):831-844.
71. Englert N.Fine parti cles and human health-a review of epidemiological studies.Toxicol Lett,2004,149:235-242.
72. Bedell MA,Largaespada DA,Jenkins NA,Copeland NGMouse models of human disaease.Part:recent progress and future directions GenesDev,1997,11:11-43.
2. Gazeau F, Levy M, Wilhelm C. Optimizing magnetic nanoparticle design for nanothermotherapy. Nanomed.2008,3(6):831-844.
3. Thiesen B, Jordan A. Clinical applications of magnetic nanoparticles for hyperthermia. Int J Hyperthermia.2008,24(6):467-474.
4. Ito A, Shinkai M, Honda H,et al. Heat-inducible TNF-alpha gene therapy combined with hyperthermia using magnetic nanoparticles as a novel tumor-targeted therapy. Cancer Gene Ther 2001,8(9):649-654.
5. Shinkai M, Ito A. Functional magnetic particles for medical application. Adv Biochem Eng Biotechnol.2004,91:191-220.
6. Chang HL, Eun YK, Kilsoo J, et al. Simple, Efficient, and Reproducible Gene Transfection of Mouse Embryonic Cells by Magnetofectin. Stem Cells Dev,2008, 17:133-141.
7.王骏,阳离子脂质体吸附FLT3L基因肿瘤原位注射联合顺铂治疗小鼠结肠癌的实验研究.四川大学硕士论文,2006年02期
8.陈金玲,郭瑞强,周青,王涛.携重组人促红细胞生成素水凝胶复合物原位注射对兔心肌梗死模型局部心功能的影响.2010中国医学影像技术国际论坛暨《中国医学影像技术》编委换届会论文集
9. Ting-Jung Chen, Tsan-Hwang Cheng, Yu-Chin Hung, et al. Targeted folic acid-PEG nanoparticles for noninvasive imaging of folate receptor by MRI. Journal of Biomedical Materials Research.2008,87(1):165-175
10. Sun C, Sze R, Zhang M. Folic acid-PEG conjugated superparamagnetic nanoparticles for targeted cellular uptake and detection by MRI. J Biomed Mater Res A,2006,78(3):550 7.
11. Kohler N, Sun C, Wang J, et al. Methotrexate-modified superparamagnetic nanoparticles and their intracellular uptake into human cancer cells. Langmuir,2005,21 (19):8858 64.
12. Joy L. Kovar, William Volcheck, Eva Sevick-Muraca et al. Characterization and Performance of a Near-Infrared 2-Deoxyglucose Optical Imaging Agent for Mouse Cancer Models. Analytical Biochemistry,2009,384(2):254-262.
13.胡慧.乳腺癌细胞GLUT1分子成像实验研究.江苏大学硕士学位论文.2010.
14. Janic B, Iskander AS, Rad AM, et al. Effects of ferumoxides-protamine sulfate labeling on immunomodulatory characteristics of macrophage-like THP-1 cells [J]. PLoS ONE, 2008,3(6):e2499.
15. Omidkhoda A, Mozdarani H, Movasaghpoor A, et al. Study of apoptosis in labeled mesenchymal stem cells with superparamagnetic iron oxide using neutral comet assay [J]. Toxicol In Vitro,2007,21(6):1191-1196.
16. Arbab AS, Yocum GT, Kalish H, et al. Efficient magnetic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI [J]. Blood,2004,104(4):1217-1223.
17. Takeda S, Terazono B, Mishima F, et al. Novel drug delivery system by surface modified magnetic nanoparticles [J]. J Nanosci Nanotechnol,2006,6(9-10):3269-3276.
18. Orian-Rousseau V. CD44, a therapeutic target for metastasising tumours. Eur J Cancer 2010;46:1271-7.
19. Choi KY, Chung H, Min KH, Yoon HY, Kim K, Park JH, et al. Self-assembled hyaluronic acid nanoparticles for active tumor targeting. Biomaterials 2010;31:106-14.
20. Choi KY, Min KH, Yoon HY, Kim K, Park JH, Kwon IC, et al. PEGylation of hyaluronic acid nanoparticles improves tumor targetability in vivo. Biomaterials 2011;32:1880-9.
21. Saravanakumar G, Choi KY, Yoon HY, Kim K, Park JH, Kwon IC, et al. Hydrotropic hyaluronic acid conjugates:synthesis, characterization, and implications as a carrier of paclitaxel. Int J Pharm 2010;394:154-61.
22. Laakkonen P, Akerman ME, Biliran H, et al. Antitumor activity of a homing peptide that targets tumor lymphatics and tumor cells. Proc Natl Acad Sci U S A. 2004;101(25):9381-6
23. Al-Jamal WT, Al-Zamal KT, Cakebread A, Halket JM, Kostarelos K. Blood circulation and tissue biodistribution of lipid-quantum dot (L-QD) hybrid vesicles intravenously administered in mice. Bioconjug Chem 2009;20:1696-702.
24. Kim JY, Kim JK, Park JS, Byun Y, Kim CK. The use of PEGylated liposomes to prolong circulation lifetimes of tissue plasminogen activator. Biomaterials 2009;30:5751-6.
25. Lim SM, Kim TH, Jiang HH, Park CW, Lee S, Chen X, et al. Improved biological half-life and anti-tumor activity of TNF-related apoptosis-inducing ligand (TRAIL) using PEG-exposed nanoparticles. Biomaterials 2011;32:3538-46.
26.戴晓晨,靶向性磁性纳米颗粒的制备与磁感应升温作用.北京中医药大学硕士论文.2009
27. Laakkonen P, Porkka K, Hoffman JA, Ruoslahti E.A tumor-homing peptide with a targeting specificity related to lymphatic vessels.Nat Med.2002:8(7):751-5.
28. Laakkonen P, Akerman ME, Biliran H, et al. Antitumor activity of a homing peptide that targets tumor lymphatics and tumor cells. Proc Natl Acad Sci U S A. 2004;101(25):9381-6
29. Jiang J, Zhang Y, Krainer AR, Xu RM. Crystal structure of human p32, a doughnut-shaped acidic mitochondrial matrix protein. Proc Natl Acad Sci U S A.1999;96:35727.
30. Krainer AR, Mayeda A, Kozak D, Binns G. Functional expression of cloned human splicing factor SF2:homology to RNA-binding proteins, Ul 70K, Drosophila splicing regulators. Cell 1991;66:383-94.
31. Ghebrehiwet B, Lim BL, Peerschke El, Willis AC, Reid KB. Isolation, cDNA cloning, and overexpression of a 33-kD cell surface glycoprotein that binds to the globular "heads" of Clq. J ExpMed 1994;179:1809-21.
32. Bialucha CU, Ferber EC, Pichaud F, Peak-Chew SY, Fujita Y. p32 is a novel mammalian Lgl binding protein that enhances the activity of protein kinase Cξ and regulates cell polarity. J Cell Biol 2007;178:575-81.
33. Storz P, Hausser A, Link G, et al. Protein kinase Cμ is regulated by the multifunctional chaperon protein p32. J Biol Chem 2000;275:24601-7.
34. Park JH, von Maltzahn G, Xu MJ, Fogal V, Kotamraju VR, et al.Cooperative nanomaterial system to sensitize, target,and treat tumors. Proc Natl Acad Sci U S A. 2010;107(3):981-6.
35. Jaffer FA, Weissleder R Molecular imaging in the clinical arena.JAMA. 2005;293(7):855-862
36. Sharma R, Wendt JA, Rasmussen JC, Adams KE, Marshall MV, Sevick-Muraca EM New horizons for imaging lymphatic function. Ann N Y Acad Sci.2008; 1131:13-36
37. Winnard PT Jr, Pathak AP, Dhara S, Cho SY, Raman V, Pomper MGMolecular imaging of metastatic potential. J Nucl Med.2008; 49(Suppl 2):96S-112S
38. Qian CN, Berghuis B, Tsarfaty G, Bruch M, Kort EJ, Ditlev J, Tsarfaty I, Hudson E, Jackson DG, Petillo D, Chen J, Resau JH, Teh BT. Preparing the "Soil":the primary tumor induces vasculature reorganization in the sentinel lymph node before the arrival of metastatic cancer cells. Cancer Res.2006;66(21):10365-10376
39. Eiber M, Beer AJ, Holzapfel K, Tauber R, Ganter C, Weirich G, Krause BJ, Rummeny EJ, Gaa J. Preliminary results for characterization of pelvic lymph nodes in patients with prostate cancer by diffusion-weighted mr-imaging. Invest Radiol.2010;45(1):15-23
40. Zhang F, Niu G, Lin X, Jacobson O, Ma Y, Eden HS, He Y, Lu G, Chen X.Imaging tumor-induced sentinel lymph node lymphangio genesis with LyP-1 peptide. Amino Acids. 2011 Jul 19.
41. Uchida M, Kosuge H, Terashima M, Willits DA, Liepold LO, Young MJ, McConnell MV, Douglas T. Protein Cage Nanoparticles Bearing the LyP-1 Peptide for Enhanced Imaging of Macrophage-Rich Vascular Lesions. ACS Nano.2011;5(4):2493-502.
42. Valentina Fogal, Lianglin Zhang, Stan Krajewski, and Erkki Ruoslahti.Mitochondrial/Cell-Surface Protein p32/gC1qR as a Molecular Target in Tumor Cells and Tumor Stroma. Cancer Res,2008; 68:(17).
43. Laakkonen P, Akerman ME, Biliran H, et al. Antitumor activity of a homing peptide that targets tumor lymphatics and tumor cells. Proc Natl Acad Sci U S A 2004; 101:9381-6.
44. Park JH, von Maltzahn G, Xu MJ, Fogal V, Kotamraju VR, Ruoslahti E, Bhatia SN, Sailor MJ.Cooperative nanomaterial system to sensitize, target, and treat tumors.Proc Natl Acad Sci U S A.2010;107(3):981-6.
45. Luo G, Yu X, Jin C, Yang F, Fu D, Long J, Xu J, Zhan C, Lu W.LyP-1-conjugated nanoparticles for targeting drug delivery to lymphatic metastatic tumors.Int J Pharm. 2010;385(1-2):150-6.
46.闫志强,王飞,魏晓丽,俸灵林,何丹农,陆伟跃,LyP-1介导的淋巴转移肿瘤靶向脂质体递药系统研究.2011年中国药学大会暨第11届中国药师周论文集
47. Makela AR, Matilainen H, White DJ, Ruoslahti E, Oker-Blom C.Enhanced baculovirus-mediated transduction of human cancer cells by tumor-homing peptides. J Virol.2006;80(13):6603-11.
48. Lee CN, Wang YM, Lai WF, Chen TJ, Yu MC, Fang CL, Yu FL, Tsai YH, Chang WH, Zuo CS, Renshaw PF.Super-paramagnetic iron oxide nanoparticles for use in extrapulmonary tuberculosis diagnosis.Clin Microbiol Infect.2012, doi: 10.;llll/j.1469-0691.2012.03809.x.
49. Neumaier CE, Baio G, Ferrini S, Corte G, Daga A. MR and iron magnetic nanoparticles. Imaging opportunities in preclinical and translational research. Tumori.2008 Mar-Apr;94(2):226-33.
50. Smirnov P. Cellular magnetic resonance imaging using superparamagnetic anionic iron oxide nanoparticles:applications to in vivo trafficking of lymphocytes and cell-based anticancer therapy. Methods Mol Biol.2009;512:333-53.
51. Laurent S, Boutry S, Mahieu I, Vander Elst L, Muller RN.Iron oxide based MR contrast agents:from chemistry to cell labeling. Curr Med Chem.2009; 16(35):4712-27.
52. Peng XH, Qian X, Mao H, Wang AY, Chen ZG, Nie S, Shin DM.Targeted magnetic iron oxide nanoparticles for tumor imaging and therapy.Int J Nanomedicine. 2008;3(3):311-21.
53. Jin XH, Yang L, Duan XJ, Xie B, Li Z, Tan HB. In vivo MR imaging tracking of supermagnetic iron-oxide nanoparticle-labeled bone marrow mesenchymal stem cells injected into intra-articular space of knee joints:experiment with rabbit. Zhonghua Yi Xue Za Zhi.2007 Dec 4;87(45):3213-8.
54. Shen M, Cai H, Wang X, Cao X, Li K, Wang SH, Guo R, Zheng L, Zhang G, Shi X. Facile one-pot preparation, surface functionalization, and toxicity assay of APTS-coated iron oxide nanoparticles. Nanotechnology.2012 Mar 16;23(10):105601. Epub 2012 Feb 21.
55. Wang S, Wen S, Shen M, Guo R, Cao X, Wang J, Shi X. Aminopropyltriethoxysilane-mediated surface functionalization of hydroxyapatite nanoparticles:synthesis, characterization, and in vitro toxicity assay. Int J Nanomedicine. 2011;6:3449-59..
56. Ho D, Sun X, Sun S.Monodisperse magnetic nanoparticles for theranostic applications. Ace Chem Res.2011 (18);44(10):875-82.
57.陈本科.信号肽修饰的磁性纳米粒用于肿瘤磁感应热疗联合基因治疗的研究.北京中医药大学硕士论文.2010
58.何强芳,李国明,陈烁.水基Fe304磁流体的制备.应用化学,2005；22(6)：665-668
59. Laakkonen P, Porkka K, Hoffman JA, Ruoslahti E.A tumor-homing peptide with a targeting specificity related to lymphatic vessels.Nat Med.2002:8(7):751-5.
60. Ghebrehiwet B, Lim BL, Peerschke El, Willis AC, Reid KB. Isolation, cDNA cloning, and overexpression of a 33-kD cell surface glycoprotein that binds to the globular "heads" of C1q. J Exp Med 1994;179(6);179:1809-1821
61. Gupta S, Batchu RB, Datta K. Purification, partial characterization of rat kidney hyaluronic acid binding protein and its localization on the cell surface. Eur J Cell Biol 1991;56(1):58-67
62. Soltys BJ, Kang D, Gupta RS. Localization of P32 protein (gC1q-R) in mitochondria and at specific extramitochondrial locations in normal tissues. Histochem Cell Biol 2000;114(3):245-255
63. Krainer AR, Mayeda A, Kozak D, Binns G. Functional expression of cloned human splicing factor SF2:homology to RNA-binding proteins, U1 70K, Drosophila splicing regulators. Cell 1991;66(2):383-394
64. Majumdar M, Meenakshi J, Goswami SK, Datta K. Hyaluronan binding protein 1 (HABP1)/C1QBP/p32 is an endogenous substrate for MAP kinase and is translocated to the nucleus upon mitogenic stimulation. Biochem Biophys Res Commun 2002;291(4):829-837.
65. Park JH, von Maltzahn G, Xu MJ, Fogal V, Kotamraju VR, Ruoslahti E, Bhatia SN, Sailor MJ.Cooperative nanomaterial system to sensitize, target, and treat tumors.Proc Natl Acad Sci USA.2010;107(3):981-6.
66. Luo G, Yu X, Jin C, Yang F, Fu D, Long J, Xu J, Zhan C, Lu W.LyP-1-conjugated nanoparticles for targeting drug delivery to lymphatic metastatic tumors.Int J Pharm. 2010;385(1-2):150-6.
67. Laakkonen P, Akerman ME, Biliran H, et al. Antitumor activity of a homing peptide that targets tumor lymphatics and tumor cells. Proc Natl Acad Sci U S A. 2004;101(25):9381-6
68.杨欣.磁性微纳米介质的生物安全性研究.北京中医药大学硕士论文.2010
69. Wilhelm C, Gazeau F. Universal cell labelling with anionic magnetic nanoparticles [J]. Biomaterials,2008,29(22):3161-3174.
70. Gazeau F, Levy M, Wilhelm C. Optimizing magnetic nanoparticle design for nanothermotherapy [J]. Nanomed,2008,3(6):831-844.
71. Englert N.Fine parti cles and human health-a review of epidemiological studies.Toxicol Lett,2004,149:235-242.
72. Bedell MA,Largaespada DA,Jenkins NA,Copeland NGMouse models of human disaease.Part:recent progress and future directions GenesDev,1997,11:11-43.