人前列腺癌PC-3细胞的荧光标记及其脊椎转移动物模型的建立
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摘要
第一部分慢病毒介导的绿色荧光蛋白报告基因对人前列腺癌PC-3细胞的标记
[目的]目前仍没有合适的方法标记人前列腺癌PC-3细胞,追踪观察肿瘤的转移情况。本实验旨在探讨慢病毒介导的绿色荧光蛋白(GFP)标记人前列腺癌PC-3细胞是否影响其细胞生物学特性,以及GFP基因能否持久稳定表达,为下一步实验奠定基础。
[方法]常规肿瘤细胞培养、传代,在细胞状态最佳时以不同病毒感染复数(MOI)实行GFP慢病毒对PC-3的感染,7天后在荧光显微镜下观察GFP在PC-3的表达情况。选取感染GFP阳性表达率最高的孔继续培养传代至第三代后,分别用镜下形态观、MTT测生长曲线、划痕实验来对比两种细胞的形态、活性、生长速度和生长状态,以评价GFP基因在PC-3细胞表达的稳定性。
[结果]置荧光显微镜下,转染72h后,部分PC-3细胞可见绿色荧光,转染一周时,绿色荧光表达最强。其中以MOI=20感染率最高,GFP阳性表达率为(92.3±1.2)%,GFP/PC-3在体外持续培养2、4、8周,GFP阳性率没有明显变化。镜下形态观、MTT测生长曲线、划痕实验等结果显示,与转染前相比,慢病毒感染PC-3后,对细胞活力、增殖、凋亡和周期均没有影响(P>0.05)。
[结论]GFP慢病毒能够高效标记PC-3,并且不影响其生物学特性,GFP基因在PC-3细胞中能够持久稳定表达,可以用于下一步的细胞示踪研究。
第二部分前列腺癌脊椎转移模型的建立
[目的]将转染成功后稳定表达绿色荧光蛋白的前列腺癌PC-3细胞(文中称GFP/PC-3细胞)采用悬液注射于小鼠的下腔静脉,建立可在动物活体荧光成像系统下直接观察肿瘤生长及转移的简便、可靠的前列腺癌脊柱转移模型。
[方法]1、3只4周-6周龄的雄性裸鼠,用细胞浓度为1×107个/ml的GFP/PC-3细胞悬液0.15ml行腋窝皮下注射。观察GFP/PC-3的活性和侵袭力。2、16只4周-6周龄的雄性裸鼠,在手术显微镜下暴露下腔静脉,用40ul的胰岛素注射针头抽取细胞浓度为1×107个/ml的GFP/PC-3细胞悬液0.15ml行下腔静脉注射。观察下腔静脉注射肿瘤细胞后裸鼠的存活率及3个月后肿瘤细胞的转移情况。
[结果]1、皮下注射三只裸鼠一周后均可见有成瘤灶并能在活体荧光成像系统下观察到绿色荧光,两周后肿瘤生长迅速,8周后裸鼠呈恶病质状态,12周后死亡。2、下腔静脉注射裸鼠脊柱转移模型成活率为100%(16/16)；一周时活体荧光成像系统下可见3只腰背部有绿色荧光显像,四周时可见表达绿色荧光的肿瘤增大,肿瘤骨转移率为19%。(3/16)。3、三个月后脱颈处死荷瘤鼠,解剖后行大体观察,可见3只裸鼠腰椎有肿瘤浸润,与动物活体荧光成像系统结果一致,转移率约为19%(3/16)。4、病理学观察：大体病理：大体解剖发现腰椎有肿瘤转移,转移肿瘤块呈灰白色团块状隆起于腰椎表面。显微镜下观察：皮下种植肿瘤细胞排列紊乱,形态不规则,细胞分化差,核大深染；椎体转移瘤有反应性成骨显像。
[结论]用绿色荧光蛋白标记的人前列腺癌PC-3细胞成功的建立了裸鼠脊柱骨转移模型,为进一步研究前列腺癌骨转移提供了一种简便、可行的新方法,对前列腺肿瘤治疗药物开发、肿瘤转移机制的干预、基因治疗等许多与临床密切联系的基础实验研究提供了帮助。
Part one Green fluorescent protein reporter gene labeling humans prostate cancer PC-3cells mediated by Lentivirus
OBJECTIVE:there is still no suitable marker of humans prostate cancer PC-3cells tracing observing for tumor metastasis. In order to explore the effect of green fluorescence protein(GFP)labeling mediated by lentivirus on the biophysical properties of humans prostate cancer PC-3,and whether the GFP gene expression is permanent and stable, which can lay the foundation for further experiments.
METHODS:Conventional tumor cell culture,passage. multiplicity of infection (MOI) of different viruses in the cell state is the best implementation of GFP lentivirus infection of PC-3,and7days under a fluorescence microscope to observe GFP expression in PC-3.Select infected with GFP-positive expression rate of the highest hole to continue to foster the passage to the third generation,respectively,with the microscope, Morphology and MTT measured growth curve, the scratch test to compare the two cell morphology, activity, growth rate and growth of state, to evaluate the GFP the stability of gene expression in PC-3cells.
RESULT:Under the fluorescence microscope,72hours later, green fluorescent was found and became strongest at7days. Among them with MOI=20highest infection rate, the GFP positive expression rate was (92.3±1.2)%.GFP/PC-3to sustain the culture in vitro,2,4,8weeks of GFP-positive rate did not change significantly. Microscopically form view, MTT measured growth curve, scratches experimental results show that compared with before transfection, lentiviral infection after PC-3cell viability, proliferation, apoptosis and cell cycle did not affect (P>0.05).
CONCLUSION:GFP lentivirus can efficiently label PC-3cells and has no significant effect on the biophysical properties of PC-3cells. GFP gene expression in PC-3cells is permanent and stable. GFP/PC-3call be used for further cell tracing research.
Part two the vertebral metastasis of prostate cancer model
OBJECTIVE:Successfully transfected prostate cancer PC-3cells (this text said GFP/PC-3cells) suspensions was injected in inferior vena cava of the nude mouse, and the establishment of direct observation of tumor growth and metastasis in animal in vivo fluorescence imaging system simple, reliable prostate cancer spinal metastasis model.
METHODS:1、Total3male nude mice model of4weeks to6-week-old,0.15ml (1.5×106cells) of suspension of GFP/PC-3cells were injected into axillary subcutaneous, and the cells concentration were1x107cells/ml. Observed the GFP/PC-3cells activity and invasiveness.2、Total16male nude mice model of4weeks to6-week-old, under the operating microscope exposure the vena cava, use the40ul insulin needle to extract the cell concentration of1×107cells/ml of GFP/PC-3cells suspension0.15ml and then injected into the inferior vena. Observation of the inferior vena cava after injection of tumor cells in nude mice survival and3months after tumor cell metastasis.
RESULT:1、Tumor was visible in the three nude mice after one week of subcutaneous injection and in vivo fluorescence imaging system observed the green fluorescence. The tumors increased rapidly after two weeks, eight weeks the nude mice was cachexia state, and died in12weeks.2、Inferior vena cava injection in nude mice spinal metastasis model survival rate was100%(16/16); after one week in vivo fluorescence imaging system can be seen three lower back green fluorescence imaging, four weeks later the visible expression of green fluorescence in the tumor increased, tumor bone metastases was19%.(3/16).3、After three months the tumor-bearing mice were sacrificed, anatomy and general observation saw3nude lumbar tumor infiltration, the results are consistent with the animal in vivo fluorescence imaging system, the transfer rate is about19%(3/16).4、Pathological observation:gross pathology:Gross anatomy of the lumbar tumor metastasis, metastatic tumor was gray bulge in the lumbar surface lumps. Observed under the microscope:subcutaneous implantation of tumor cells arranged disorderly, irregular shape, cell differentiation, a large nucleus deeply stained; Vertebral metastases reactive osteogenesis imaging.
CONCLUSION:Labeled with green fluorescent protein in human prostate cancer tumors PC-3cells established a nude mouse spinal bone metastasis model, provides a simple, feasible method for the further study of prostate cancer bone metastasis of prostate cancer treatment drug development, the intervention of the mechanism of tumor metastasis, gene therapy, and many other close contact with basic experimental and clinical research to provide help.
[目的]目前仍没有合适的方法标记人前列腺癌PC-3细胞,追踪观察肿瘤的转移情况。本实验旨在探讨慢病毒介导的绿色荧光蛋白(GFP)标记人前列腺癌PC-3细胞是否影响其细胞生物学特性,以及GFP基因能否持久稳定表达,为下一步实验奠定基础。
[方法]常规肿瘤细胞培养、传代,在细胞状态最佳时以不同病毒感染复数(MOI)实行GFP慢病毒对PC-3的感染,7天后在荧光显微镜下观察GFP在PC-3的表达情况。选取感染GFP阳性表达率最高的孔继续培养传代至第三代后,分别用镜下形态观、MTT测生长曲线、划痕实验来对比两种细胞的形态、活性、生长速度和生长状态,以评价GFP基因在PC-3细胞表达的稳定性。
[结果]置荧光显微镜下,转染72h后,部分PC-3细胞可见绿色荧光,转染一周时,绿色荧光表达最强。其中以MOI=20感染率最高,GFP阳性表达率为(92.3±1.2)%,GFP/PC-3在体外持续培养2、4、8周,GFP阳性率没有明显变化。镜下形态观、MTT测生长曲线、划痕实验等结果显示,与转染前相比,慢病毒感染PC-3后,对细胞活力、增殖、凋亡和周期均没有影响(P>0.05)。
[结论]GFP慢病毒能够高效标记PC-3,并且不影响其生物学特性,GFP基因在PC-3细胞中能够持久稳定表达,可以用于下一步的细胞示踪研究。
第二部分前列腺癌脊椎转移模型的建立
[目的]将转染成功后稳定表达绿色荧光蛋白的前列腺癌PC-3细胞(文中称GFP/PC-3细胞)采用悬液注射于小鼠的下腔静脉,建立可在动物活体荧光成像系统下直接观察肿瘤生长及转移的简便、可靠的前列腺癌脊柱转移模型。
[方法]1、3只4周-6周龄的雄性裸鼠,用细胞浓度为1×107个/ml的GFP/PC-3细胞悬液0.15ml行腋窝皮下注射。观察GFP/PC-3的活性和侵袭力。2、16只4周-6周龄的雄性裸鼠,在手术显微镜下暴露下腔静脉,用40ul的胰岛素注射针头抽取细胞浓度为1×107个/ml的GFP/PC-3细胞悬液0.15ml行下腔静脉注射。观察下腔静脉注射肿瘤细胞后裸鼠的存活率及3个月后肿瘤细胞的转移情况。
[结果]1、皮下注射三只裸鼠一周后均可见有成瘤灶并能在活体荧光成像系统下观察到绿色荧光,两周后肿瘤生长迅速,8周后裸鼠呈恶病质状态,12周后死亡。2、下腔静脉注射裸鼠脊柱转移模型成活率为100%(16/16)；一周时活体荧光成像系统下可见3只腰背部有绿色荧光显像,四周时可见表达绿色荧光的肿瘤增大,肿瘤骨转移率为19%。(3/16)。3、三个月后脱颈处死荷瘤鼠,解剖后行大体观察,可见3只裸鼠腰椎有肿瘤浸润,与动物活体荧光成像系统结果一致,转移率约为19%(3/16)。4、病理学观察：大体病理：大体解剖发现腰椎有肿瘤转移,转移肿瘤块呈灰白色团块状隆起于腰椎表面。显微镜下观察：皮下种植肿瘤细胞排列紊乱,形态不规则,细胞分化差,核大深染；椎体转移瘤有反应性成骨显像。
[结论]用绿色荧光蛋白标记的人前列腺癌PC-3细胞成功的建立了裸鼠脊柱骨转移模型,为进一步研究前列腺癌骨转移提供了一种简便、可行的新方法,对前列腺肿瘤治疗药物开发、肿瘤转移机制的干预、基因治疗等许多与临床密切联系的基础实验研究提供了帮助。
Part one Green fluorescent protein reporter gene labeling humans prostate cancer PC-3cells mediated by Lentivirus
OBJECTIVE:there is still no suitable marker of humans prostate cancer PC-3cells tracing observing for tumor metastasis. In order to explore the effect of green fluorescence protein(GFP)labeling mediated by lentivirus on the biophysical properties of humans prostate cancer PC-3,and whether the GFP gene expression is permanent and stable, which can lay the foundation for further experiments.
METHODS:Conventional tumor cell culture,passage. multiplicity of infection (MOI) of different viruses in the cell state is the best implementation of GFP lentivirus infection of PC-3,and7days under a fluorescence microscope to observe GFP expression in PC-3.Select infected with GFP-positive expression rate of the highest hole to continue to foster the passage to the third generation,respectively,with the microscope, Morphology and MTT measured growth curve, the scratch test to compare the two cell morphology, activity, growth rate and growth of state, to evaluate the GFP the stability of gene expression in PC-3cells.
RESULT:Under the fluorescence microscope,72hours later, green fluorescent was found and became strongest at7days. Among them with MOI=20highest infection rate, the GFP positive expression rate was (92.3±1.2)%.GFP/PC-3to sustain the culture in vitro,2,4,8weeks of GFP-positive rate did not change significantly. Microscopically form view, MTT measured growth curve, scratches experimental results show that compared with before transfection, lentiviral infection after PC-3cell viability, proliferation, apoptosis and cell cycle did not affect (P>0.05).
CONCLUSION:GFP lentivirus can efficiently label PC-3cells and has no significant effect on the biophysical properties of PC-3cells. GFP gene expression in PC-3cells is permanent and stable. GFP/PC-3call be used for further cell tracing research.
Part two the vertebral metastasis of prostate cancer model
OBJECTIVE:Successfully transfected prostate cancer PC-3cells (this text said GFP/PC-3cells) suspensions was injected in inferior vena cava of the nude mouse, and the establishment of direct observation of tumor growth and metastasis in animal in vivo fluorescence imaging system simple, reliable prostate cancer spinal metastasis model.
METHODS:1、Total3male nude mice model of4weeks to6-week-old,0.15ml (1.5×106cells) of suspension of GFP/PC-3cells were injected into axillary subcutaneous, and the cells concentration were1x107cells/ml. Observed the GFP/PC-3cells activity and invasiveness.2、Total16male nude mice model of4weeks to6-week-old, under the operating microscope exposure the vena cava, use the40ul insulin needle to extract the cell concentration of1×107cells/ml of GFP/PC-3cells suspension0.15ml and then injected into the inferior vena. Observation of the inferior vena cava after injection of tumor cells in nude mice survival and3months after tumor cell metastasis.
RESULT:1、Tumor was visible in the three nude mice after one week of subcutaneous injection and in vivo fluorescence imaging system observed the green fluorescence. The tumors increased rapidly after two weeks, eight weeks the nude mice was cachexia state, and died in12weeks.2、Inferior vena cava injection in nude mice spinal metastasis model survival rate was100%(16/16); after one week in vivo fluorescence imaging system can be seen three lower back green fluorescence imaging, four weeks later the visible expression of green fluorescence in the tumor increased, tumor bone metastases was19%.(3/16).3、After three months the tumor-bearing mice were sacrificed, anatomy and general observation saw3nude lumbar tumor infiltration, the results are consistent with the animal in vivo fluorescence imaging system, the transfer rate is about19%(3/16).4、Pathological observation:gross pathology:Gross anatomy of the lumbar tumor metastasis, metastatic tumor was gray bulge in the lumbar surface lumps. Observed under the microscope:subcutaneous implantation of tumor cells arranged disorderly, irregular shape, cell differentiation, a large nucleus deeply stained; Vertebral metastases reactive osteogenesis imaging.
CONCLUSION:Labeled with green fluorescent protein in human prostate cancer tumors PC-3cells established a nude mouse spinal bone metastasis model, provides a simple, feasible method for the further study of prostate cancer bone metastasis of prostate cancer treatment drug development, the intervention of the mechanism of tumor metastasis, gene therapy, and many other close contact with basic experimental and clinical research to provide help.
引文
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[7]Batson O V. THE FUNCTION OF THE VERTEBRAL VEINS AND THEIR ROLE IN THE SPREAD OF METASTASES.[J]. Ann Surg,1940,112(1):138-149.
[8]Chen N, Ye X C, Chu K, etc. A secreted isoform of ErbB3 promotes osteonectin expression in bone and enhances the invasiveness of prostate cancer cells.[J]. Cancer Res,2007,67(14):6544-6548.
[9]Taichman R S, Cooper C, Keller E T, et al. Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone.[J]. Cancer Res,2002,62(6):1832-1837.
[10]张林琳,罗勇,贺大林,等.前列腺癌骨转移动物模型研究进展[J].现代肿瘤医学,2004,12(1)：68-70.
[11]Pollard M, Wolter W R, Sun L. Prostate-seminal vesicle cancers induced in noble rats.[J]. Prostate,2000,43(1):71-74.
[12]Winter S F, Cooper A B, Greenberg N M. Models of metastatic prostate cancer:a transgenic perspective. [J]. Prostate Cancer Prostatic Dis,2003, 6(3):204-211.
[13]Angelucci A, Gravina G L, Rucci N, et al. Evaluation of metastatic potential in prostate carcinoma:an in vivo model.[J]. Int J Oncol, 2004,25(6):1713-1720.
[14]Corey E, Quinn J E, Bladou F, etc. Establishment and characterization of osseous prostate cancer models:intra-tibial injection of human prostate cancer cells.[J]. Prostate,2002,52(1):20-33.
[15]廖晖,陈安民,郭风劲,等.人前列腺癌骨转移动物模型的建立[J].华中科技大学学报：医学版,2007,36(1)：63-66.
[16]Shevrin D H, Kukreja S C, Ghosh L, etc. Development of skeletal metastasis by human prostate cancer in athymic nude mice.[J]. Clin Exp Metastasis,1988,6(5):401-409.
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[18]Zhou J H, Rosser C J, Tanaka M, etc. Visualizing superficial human bladder cancer cell growth in vivo by green fluorescent protein expression. [J]. Cancer Gene Ther,2002,9(8):681-686.
[19]Yang M, Jiang P, Sun F X, etc. A fluorescent orthotopic bone metastasis model of human prostate cancer.[J]. Cancer Res,1999,59(4):781-786.
[1]Jemal A, Siegel R, Xu J, et al. Cancer statistics,2010.[J]. CA Cancer J Clin,2010,60(5):277-300.
[2]Bubendorf L, Schopfer A, Wagner U, et al. Metastatic patterns of prostate cancer:an autopsy study of 1,589 patients.[J]. Hum Pathol,2000, 31(5):578-583.
[3]Coleman R E. Metastatic bone disease:clinical features, pathophysiology and treatment strategies.[J]. Cancer Treat Rev,2001,27(3):165-176.
[4]Bussard K M, Gay C V, Mastro A M. The bone microenvironment in metastasis; what is special about bone?[J]. Cancer Metastasis Rev, 2008,27(1):41-55.
[5]Klein C A. The systemic progression of human cancer:a focus on the individual disseminated cancer cell--the unit of selection.[J]. Adv Cancer Res,2003,89:35-67.
[6]Scott L J, Clarke N W, George N J, et al. Interactions of human prostatic epithelial cells with bone marrow endothelium:binding and invasion.[J]. Br J Cancer,2001,84(10):1417-1423.
[7]Jacobsen K, Kravitz J, Kincade P W, et al. Adhesion receptors on bone marrow stromal cells:in vivo expression of vascular cell adhesion molecule-1 by reticular cells and sinusoidal endothelium in normal and gamma-irradiated mice.[J]. Blood,1996,87(1):73-82.
[8]Sasaki A, Boyce B F, Story B, et al. Bisphosphonate risedronate reduces metastatic human breast cancer burden in bone in nude mice.[J]. Cancer Res,1995,55(16):3551-3557.
[9]Fournier P G, Stresing V, Ebetino F H, et al. How do bisphosphonates inhibit bone metastasis in vivo[J]. Neoplasia,2010,12(7):571-578.
[10]Sun M, Iqbal J, Singh S, et al. The crossover of bisphosphonates to cancer therapy. [J]. Ann N Y Acad Sci,2010,1211:107-112.
[11]Fournier P G, Chirgwin J M, Guise T A. New insights into the role of T cells in the vicious cycle of bone metastases.[J]. Curr Opin Rheu matol,2006,18(4):396-404.
[12]Mundy G R. Metastasis to bone:causes, consequences and therapeutic opportunities.[J]. Nat Rev Cancer,2002,2(8):584-593.
[13]Koeneman K S, Yeung F, Chung L W. Osteomimetic properties of prostate cancer cells:a hypothesis supporting the predilection of prostate cancer metastasis and growth in the bone environment.[J]. Prostate,1999, 39(4):246-261.
[14]Fili S, Karalaki M, Schaller B. Mechanism of bone metastasis:the role of osteoprotegerin and of the host-tissue microenvironment-related survival factors.[J]. Cancer Lett,2009,283(1):10-19.
[15]邬喻,曾甫清,(审校).前列腺癌与骨转移[J].国际肿瘤学杂志,2006,33(4)：293-296.
[16]Batson O V. THE FUNCTION OF THE VERTEBRAL VEINS AND THEIR ROLE IN THE SPREAD OF METASTASES.[J]. Ann Surg,1940,112(1):138-149.
[17]Chen N, Ye X C, Chu K, et al. A secreted isoform of ErbB3 promotes osteonectin expression in bone and enhances the invasiveness of prostate cancer cells.[J]. Cancer Res,2007,67(14):6544-6548.
[18]Taichman R S, Cooper C, Keller E T, et al. Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone.[J]. Cancer Res,2002,62(6):1832-1837.
[19]de S, Chen J, Narizhneva N V, et al. Molecular pathway for cancer metastasis to bone.[J]. J Biol Chem,2003,278(40):39044-39050.
[20]王兆朋,张维东,(审校).前列腺癌骨转移的分子生物学研究进展[J].实用癌症杂志,2005,20(3)：322-324.
[21]Bigler S A, Deering R E, Brawer M K. Comparison of microscopic vascularity in benign and malignant prostate tissue.[J]. Hum Pathol, 1993,24(2):220-226.
[22]Weidner N, Carroll P R, Flax J, et al. Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma.[J]. Am J Pathol, 1993,143(2):401-409.
[23]Nemeth J A, Yousif R, Herzog M, et al. Matrix metalloproteinase activity, bone matrix turnover, and tumor cell proliferation in prostate cancer bone metastasis.[J]. J Natl Cancer Inst,2002,94(1):17-25.
[24]Sweeney P, Karashima T, Kim S J, et al. Anti-vascular endothelial growth factor receptor 2 antibody reduces tumorigenicity and metastasis in orthotopic prostate cancer xenografts via induction of endothelial cell apoptosis and reduction of endothelial cell matrix metalloproteinase type 9 production.[J]. Clin Cancer Res,2002,8(8):2714-2724.
[25]Karashima T, Sweeney P, Slaton J W, et al. Inhibition of angiogenesis by the antiepidermal growth factor receptor antibody ImClone C225 in androgen-independent prostate cancer growing orthotopically in nude mice.[J]. Clin Cancer Res,2002,8(5):1253-1264.
[26]Pollard M, Wolter W R, Sun L. Prostate-seminal vesicle cancers induced in noble rats.[J]. Prostate,2000,43(1):71-74.
[27]Huss W J, Maddison L A, Greenberg N M. Autochthonous mouse models for prostate cancer:past, present and future.[J]. Semin Cancer Biol,2001, 11(3):245-260.
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[1]Bubendorf L, Schopfer A, Wagner U, etc. Metastatic patterns of prostate cancer:an autopsy study of 1,589 patients.[J]. Hum Pathol,2000, 31(5):578-583.
[2]邬喻,曾甫清,(审校).前列腺癌与骨转移[J].国际肿瘤学杂志,2006,33(4)：293-296.
[3]Bussard K M, Gay C V, Mastro A M, etc. The bone microenvironment in metastasis; what is special about bone?[J]. Cancer Metastasis Rev,2008, 27(1):41-55.
[4]Scott L J, Clarke N W, George N J, etc. Interactions of human prostatic epithelial cells with bone marrow endothelium:binding and invasion.[J]. Br J Cancer,2001,84(10):1417-1423.
[5]Jacobsen K, Kravitz J, Kincade P W, etc Adhesion receptors on bone marrow stromal cells:in vivo expression of vascular cell adhesion molecule-1 by reticular cells and sinusoidal endothelium in normal and gamma-irradiated mice.[J]. Blood,1996,87(1):73-82.
[6]Sasaki A, Boyce B F, Story B, etc. Bisphosphonate risedronate reduces metastatic human breast cancer burden in bone in nude mice.[J]. Cancer Res,1995,55(16):3551-3557.
[7]Batson O V. THE FUNCTION OF THE VERTEBRAL VEINS AND THEIR ROLE IN THE SPREAD OF METASTASES.[J]. Ann Surg,1940,112(1):138-149.
[8]Chen N, Ye X C, Chu K, etc. A secreted isoform of ErbB3 promotes osteonectin expression in bone and enhances the invasiveness of prostate cancer cells.[J]. Cancer Res,2007,67(14):6544-6548.
[9]Taichman R S, Cooper C, Keller E T, et al. Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone.[J]. Cancer Res,2002,62(6):1832-1837.
[10]张林琳,罗勇,贺大林,等.前列腺癌骨转移动物模型研究进展[J].现代肿瘤医学,2004,12(1)：68-70.
[11]Pollard M, Wolter W R, Sun L. Prostate-seminal vesicle cancers induced in noble rats.[J]. Prostate,2000,43(1):71-74.
[12]Winter S F, Cooper A B, Greenberg N M. Models of metastatic prostate cancer:a transgenic perspective. [J]. Prostate Cancer Prostatic Dis,2003, 6(3):204-211.
[13]Angelucci A, Gravina G L, Rucci N, et al. Evaluation of metastatic potential in prostate carcinoma:an in vivo model.[J]. Int J Oncol, 2004,25(6):1713-1720.
[14]Corey E, Quinn J E, Bladou F, etc. Establishment and characterization of osseous prostate cancer models:intra-tibial injection of human prostate cancer cells.[J]. Prostate,2002,52(1):20-33.
[15]廖晖,陈安民,郭风劲,等.人前列腺癌骨转移动物模型的建立[J].华中科技大学学报：医学版,2007,36(1)：63-66.
[16]Shevrin D H, Kukreja S C, Ghosh L, etc. Development of skeletal metastasis by human prostate cancer in athymic nude mice.[J]. Clin Exp Metastasis,1988,6(5):401-409.
[17]Ware J L, Paulson D F, Mickey G H, etc. Spontaneous metastasis of cells of the human prostate carcinoma cell line PC-3 in athymic nude mice.[J]. J Urol,1982,128(5):1064-1067.
[18]Zhou J H, Rosser C J, Tanaka M, etc. Visualizing superficial human bladder cancer cell growth in vivo by green fluorescent protein expression. [J]. Cancer Gene Ther,2002,9(8):681-686.
[19]Yang M, Jiang P, Sun F X, etc. A fluorescent orthotopic bone metastasis model of human prostate cancer.[J]. Cancer Res,1999,59(4):781-786.
[1]Jemal A, Siegel R, Xu J, et al. Cancer statistics,2010.[J]. CA Cancer J Clin,2010,60(5):277-300.
[2]Bubendorf L, Schopfer A, Wagner U, et al. Metastatic patterns of prostate cancer:an autopsy study of 1,589 patients.[J]. Hum Pathol,2000, 31(5):578-583.
[3]Coleman R E. Metastatic bone disease:clinical features, pathophysiology and treatment strategies.[J]. Cancer Treat Rev,2001,27(3):165-176.
[4]Bussard K M, Gay C V, Mastro A M. The bone microenvironment in metastasis; what is special about bone?[J]. Cancer Metastasis Rev, 2008,27(1):41-55.
[5]Klein C A. The systemic progression of human cancer:a focus on the individual disseminated cancer cell--the unit of selection.[J]. Adv Cancer Res,2003,89:35-67.
[6]Scott L J, Clarke N W, George N J, et al. Interactions of human prostatic epithelial cells with bone marrow endothelium:binding and invasion.[J]. Br J Cancer,2001,84(10):1417-1423.
[7]Jacobsen K, Kravitz J, Kincade P W, et al. Adhesion receptors on bone marrow stromal cells:in vivo expression of vascular cell adhesion molecule-1 by reticular cells and sinusoidal endothelium in normal and gamma-irradiated mice.[J]. Blood,1996,87(1):73-82.
[8]Sasaki A, Boyce B F, Story B, et al. Bisphosphonate risedronate reduces metastatic human breast cancer burden in bone in nude mice.[J]. Cancer Res,1995,55(16):3551-3557.
[9]Fournier P G, Stresing V, Ebetino F H, et al. How do bisphosphonates inhibit bone metastasis in vivo[J]. Neoplasia,2010,12(7):571-578.
[10]Sun M, Iqbal J, Singh S, et al. The crossover of bisphosphonates to cancer therapy. [J]. Ann N Y Acad Sci,2010,1211:107-112.
[11]Fournier P G, Chirgwin J M, Guise T A. New insights into the role of T cells in the vicious cycle of bone metastases.[J]. Curr Opin Rheu matol,2006,18(4):396-404.
[12]Mundy G R. Metastasis to bone:causes, consequences and therapeutic opportunities.[J]. Nat Rev Cancer,2002,2(8):584-593.
[13]Koeneman K S, Yeung F, Chung L W. Osteomimetic properties of prostate cancer cells:a hypothesis supporting the predilection of prostate cancer metastasis and growth in the bone environment.[J]. Prostate,1999, 39(4):246-261.
[14]Fili S, Karalaki M, Schaller B. Mechanism of bone metastasis:the role of osteoprotegerin and of the host-tissue microenvironment-related survival factors.[J]. Cancer Lett,2009,283(1):10-19.
[15]邬喻,曾甫清,(审校).前列腺癌与骨转移[J].国际肿瘤学杂志,2006,33(4)：293-296.
[16]Batson O V. THE FUNCTION OF THE VERTEBRAL VEINS AND THEIR ROLE IN THE SPREAD OF METASTASES.[J]. Ann Surg,1940,112(1):138-149.
[17]Chen N, Ye X C, Chu K, et al. A secreted isoform of ErbB3 promotes osteonectin expression in bone and enhances the invasiveness of prostate cancer cells.[J]. Cancer Res,2007,67(14):6544-6548.
[18]Taichman R S, Cooper C, Keller E T, et al. Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone.[J]. Cancer Res,2002,62(6):1832-1837.
[19]de S, Chen J, Narizhneva N V, et al. Molecular pathway for cancer metastasis to bone.[J]. J Biol Chem,2003,278(40):39044-39050.
[20]王兆朋,张维东,(审校).前列腺癌骨转移的分子生物学研究进展[J].实用癌症杂志,2005,20(3)：322-324.
[21]Bigler S A, Deering R E, Brawer M K. Comparison of microscopic vascularity in benign and malignant prostate tissue.[J]. Hum Pathol, 1993,24(2):220-226.
[22]Weidner N, Carroll P R, Flax J, et al. Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma.[J]. Am J Pathol, 1993,143(2):401-409.
[23]Nemeth J A, Yousif R, Herzog M, et al. Matrix metalloproteinase activity, bone matrix turnover, and tumor cell proliferation in prostate cancer bone metastasis.[J]. J Natl Cancer Inst,2002,94(1):17-25.
[24]Sweeney P, Karashima T, Kim S J, et al. Anti-vascular endothelial growth factor receptor 2 antibody reduces tumorigenicity and metastasis in orthotopic prostate cancer xenografts via induction of endothelial cell apoptosis and reduction of endothelial cell matrix metalloproteinase type 9 production.[J]. Clin Cancer Res,2002,8(8):2714-2724.
[25]Karashima T, Sweeney P, Slaton J W, et al. Inhibition of angiogenesis by the antiepidermal growth factor receptor antibody ImClone C225 in androgen-independent prostate cancer growing orthotopically in nude mice.[J]. Clin Cancer Res,2002,8(5):1253-1264.
[26]Pollard M, Wolter W R, Sun L. Prostate-seminal vesicle cancers induced in noble rats.[J]. Prostate,2000,43(1):71-74.
[27]Huss W J, Maddison L A, Greenberg N M. Autochthonous mouse models for prostate cancer:past, present and future.[J]. Semin Cancer Biol,2001, 11(3):245-260.
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