树突状细胞在血管内皮生长因子作用下内皮样化现象的初步探讨
摘要
研究背景
树突状细胞(dendritic cell，DC)是目前已知的体内抗原呈递功能最强的，而且是唯一能激活初始T细胞(naive T cell)的专职抗原呈递细胞(antigen presenting cell，APC)，是启动、调控、并维持免疫反应的中心环节。由DC激活的T细胞介导的免疫反应在机体抗肿瘤免疫中起着主导作用，DC的数量或功能的缺陷是肿瘤逃脱机体免疫监视的重要原因，因此DC的生物功能状态与肿瘤的发生发展有着重要的关系。
血管内皮生长因子(vascular endothelial growth factor，VEGF)，是肿瘤细胞分泌的众多因子中的一种强力的血管生成因子，是高效的内皮细胞有丝分裂原，可直接特异地作用于内皮细胞，促进肿瘤血管新生。近年来研究已经证实，VEGF可通过抑制核因子-κB(NF-κB)的活性，使DC分化成熟障碍，并可影响DC的功能及促使其凋亡。VEGF对DC的抑制降低了机体的抗肿瘤免疫功能，从而使肿瘤细胞逃脱机体的免疫监管，促进肿瘤的生长。
最新研究报道，在高分泌VEGF的荷瘤鼠和人卵巢癌肿瘤组织中发现了一种新的细胞群，能够同时表达DC和内皮细胞的标志，且体外培养的小鼠骨髓来源的DC在用高表达VEGF的肿瘤条件培养基培养时也可出现内皮样化改变，这些研究提示我们在高分泌VEGF的肿瘤环境中DC有可能出现内皮样化，但具体机制尚不清楚。这种内皮样化现象使DC在抗肿瘤免疫反应中不能发挥应有的抗原呈递作用，不但使成熟有功能的DC数量进一步减少，而且内皮样化的DC有可能参与肿瘤血管形成，反而促进肿瘤生长。国内外近年来关于DC的研究多集中于如何提高DC的抗原呈递功能及制备有效的DC瘤苗等方面，而关于DC内皮样化现象及其在人类肿瘤血管形成中的作用等则有待于进一步的研究。
目的
本实验拟对体外培养的人外周血单个核细胞来源的DC在VEGF诱导下能否发生内皮样化现象进行初步探讨。
实验方法
采集健康志愿者新鲜外周血，经Ficoll密度梯度离心法获取单个核细胞，用RPMI 1640完全培养液调整细胞浓度为3×10~6／ml，接种于24孔培养板中，每孔1ml，移入二氧化碳孵育箱(5％CO_2，37℃)静置3h，去除悬浮细胞，获取贴壁生长的DC前体细胞，各孔中加入1ml含rhGM-CSF(100ng／ml)和rhIL-4(5ng／ml)的RPMI 1640完全培养液培养。培养过程中观察细胞形态并计数。正常对照组在培养过程中收集第1d、3d、7d、10d、14d的细胞采用流式细胞仪动态检测细胞表型变化。实验组于培养第3d和第7d分别加入rhVEGF(10ng／ml和100ng／ml)进行诱导培养，并于隔天半量换液时半量补充因子量。分别用VEGF诱导培养7d后收集细胞，经荧光素标记抗体后，采用流式细胞技术检测DC特异性标志CD1a和内皮细胞特异性表面分子CD34、CD31的表达情况，并分别用免疫荧光法及免疫细胞化学法检测内皮细胞特异性标志vWF的表达情况。
采用统计学软件包SPSS12.0对结果进行统计分析，结果用(?)±s表示，各组之间的比较采用单因素方差分析(ONE-WAY ANOVA)，组间两两比较用q检验，显著性检验水准为α=0.05。
结果
1．正常对照组细胞在培养的第5d开始出现突起，第7d时突起更加明显，并且大量突起交织，之后细胞逐渐增大变圆，出现悬浮趋势。VEGF诱导的各实验组细胞生长过程明显落后于正常对照组，而且细胞密度较低。
2．正常对照组细胞在培养初期出现少量CD34、CD31表达，随培养时间的延长，其表达量逐渐降低，而DC的特异性标志CD1a的表达量逐渐升高。用VEGF诱导7d后，各实验组细胞CD1a的表达受抑制。各实验组细胞CD1a与内皮细胞表面标志CD34、CD31的共表达率均高于相应正常对照组，且差异具有统计学意义，并且其共表达情况存在如下趋势：3d大剂量组＞7d大剂量组＞3d小剂量组和7d小剂量组。
3．用VEGF诱导的实验组中存在胞浆着色的阳性细胞，提示有vWF的表达，并且各实验组之间的表达趋势和上述流式细胞仪检测到的共表达趋势相似，而且积分光密度(IOD)和阳性区面积百分比(Aera％)均高于其相应正常对照组，且差异具有统计学意义。
结论：
1．在体外培养过程中，VEGF能抑制或减缓DC的生长过程，并使DC的特异性表面标志CD1a表达减少。
2．在VEGF诱导下，DC可以表达一些内皮细胞特异性标志(CD31、CD34、vWF)，出现内皮样化倾向，而且大剂量诱导比小剂量诱导更容易出现内皮样化倾向、早期诱导比晚期诱导更容易出现内皮样化倾向。
Background
Dendritic cells (DC) are the best professional antigen presenting cells (APC) known so far, which are uniquely able to activate a naive T-cell response. They play the central role in initiating, regulating and maintaining immune responses. The T-cell mediated immune reaction actived by dendritic cells play a key role in anti-tumor immune responses. The defection on numbers and functions of DCs in tumor-bearing mice and in cancer patients is the main reason for the tumor cells to escape initial immune recognition. Therefore, the biological function of DCs is closely connected with the specific anti-tumor response and plays a key role in it.
Vascular endothelial growth factor (VEGF) is one of the most potent direct-acting angiogenic molecules secreted by tumor cells, which is a highly effective specific mitogen for endothelial cells. It plays a key role on tumor angiogenesis and metastasis. Reports have proved that VEGF from tumor cells could inhibit the activation of NF-κB , then affected the differentiation development and function of DCs in tumor bearing host. The inhibition could induce abnormality of the host's immune system, and promote the tumor growth.
A recent research reported a novel leukocyte subset within ovarian carcinoma that coexpressed endothelial and dendritic cell markers. It has been proved that murine bone marrow-derived DCs could undergo endothelialisation in vitro after incubation in tumor-conditioned medium containing high levels of VEGF favours. These results suggest that VEGF may contribute to the endothelialisation of DCs in tumor microenvironment. It indicates that by promoting the endothelialisation of DCs, the tumor might not only ensure a sufficient blood supply but also prevent these DCs from initiating an immune response against tumor antigens. In this case, therapeutic targeting of this pathway could both reduce tumour growth and promote immune attack. There are many reports about the use of intratumoural DC inoculation as a means for tumor vaccination in the world, but research on endothelialisation of DCs is still limited. The effects of this phenomenon also need further investigation.
Experiment aim
The purposes of our study is to incubate human monocyte-derived DCs under the induction of VEGF, and observe the degree of endothelialisation.
Methods
Precursors of DC were separated from peripheral blood monouclear cells (PBMC) divided from healthy volunteers' peripheral blood by Ficoll density gradient centrifuge. After 3 hours incubation, remove non-adherent cells. The left precursors were cultured at 3×10~6/ml with RPMI1640 medium containing GM-CSF, IL-4. And different concentration rhVEGF(10ng/ml, 100ng/ml) were added to DCs on the 3_(rd),7_(th) days of culture. Observed the appearance character of DCs with microscope during the culture process. Normal control DCs without induction of VEGF were analysed the phenotype by Flow cytometer at l_(st), 3_(rd), 7_(th), 10_(th), 14_(th) days of culture. The experimental group cells were collected after 7 days induction, then we analysed the expression of CDla,CD34,CD31 by Flow cytometer , and detected the expression of vWF by immunocytochemistry and immunofluorescence . All data were expressed as mean value±S.D., and analyzed by statistical software SPSS12.0. P-value of < 0.05 was considered to be statistically significant.
Results
1. The Morphologic changes of normal control cells were coincide with the typicalDCs' reported. The experimental cells' growth speed was slower than the control cells', and the cell density was lower.
2. The expression of CD34 and CD31 in normal DCs reduced gradually, and the expression of CD1a elevated along with the culture time lengthening. The expression of CD1a was suppressed in experimental cells induced 7days by VEGF. The coexpression of endothelial and dendritic cell markers was higher than normal control cells'.
3. Immunocytochemistry and immunofluorescence results suggested the expression of vWF in experimental cells induced by VEGF, and the Aera% , IOD of experimental group cells were higher than normal control cells'.
Conclusion
DCs derived from human monocyte could appear endothelialisation in vitro under the induction of VEGF, and in certain ranges showing a dose-dependent.
树突状细胞(dendritic cell，DC)是目前已知的体内抗原呈递功能最强的，而且是唯一能激活初始T细胞(naive T cell)的专职抗原呈递细胞(antigen presenting cell，APC)，是启动、调控、并维持免疫反应的中心环节。由DC激活的T细胞介导的免疫反应在机体抗肿瘤免疫中起着主导作用，DC的数量或功能的缺陷是肿瘤逃脱机体免疫监视的重要原因，因此DC的生物功能状态与肿瘤的发生发展有着重要的关系。
血管内皮生长因子(vascular endothelial growth factor，VEGF)，是肿瘤细胞分泌的众多因子中的一种强力的血管生成因子，是高效的内皮细胞有丝分裂原，可直接特异地作用于内皮细胞，促进肿瘤血管新生。近年来研究已经证实，VEGF可通过抑制核因子-κB(NF-κB)的活性，使DC分化成熟障碍，并可影响DC的功能及促使其凋亡。VEGF对DC的抑制降低了机体的抗肿瘤免疫功能，从而使肿瘤细胞逃脱机体的免疫监管，促进肿瘤的生长。
最新研究报道，在高分泌VEGF的荷瘤鼠和人卵巢癌肿瘤组织中发现了一种新的细胞群，能够同时表达DC和内皮细胞的标志，且体外培养的小鼠骨髓来源的DC在用高表达VEGF的肿瘤条件培养基培养时也可出现内皮样化改变，这些研究提示我们在高分泌VEGF的肿瘤环境中DC有可能出现内皮样化，但具体机制尚不清楚。这种内皮样化现象使DC在抗肿瘤免疫反应中不能发挥应有的抗原呈递作用，不但使成熟有功能的DC数量进一步减少，而且内皮样化的DC有可能参与肿瘤血管形成，反而促进肿瘤生长。国内外近年来关于DC的研究多集中于如何提高DC的抗原呈递功能及制备有效的DC瘤苗等方面，而关于DC内皮样化现象及其在人类肿瘤血管形成中的作用等则有待于进一步的研究。
目的
本实验拟对体外培养的人外周血单个核细胞来源的DC在VEGF诱导下能否发生内皮样化现象进行初步探讨。
实验方法
采集健康志愿者新鲜外周血，经Ficoll密度梯度离心法获取单个核细胞，用RPMI 1640完全培养液调整细胞浓度为3×10~6／ml，接种于24孔培养板中，每孔1ml，移入二氧化碳孵育箱(5％CO_2，37℃)静置3h，去除悬浮细胞，获取贴壁生长的DC前体细胞，各孔中加入1ml含rhGM-CSF(100ng／ml)和rhIL-4(5ng／ml)的RPMI 1640完全培养液培养。培养过程中观察细胞形态并计数。正常对照组在培养过程中收集第1d、3d、7d、10d、14d的细胞采用流式细胞仪动态检测细胞表型变化。实验组于培养第3d和第7d分别加入rhVEGF(10ng／ml和100ng／ml)进行诱导培养，并于隔天半量换液时半量补充因子量。分别用VEGF诱导培养7d后收集细胞，经荧光素标记抗体后，采用流式细胞技术检测DC特异性标志CD1a和内皮细胞特异性表面分子CD34、CD31的表达情况，并分别用免疫荧光法及免疫细胞化学法检测内皮细胞特异性标志vWF的表达情况。
采用统计学软件包SPSS12.0对结果进行统计分析，结果用(?)±s表示，各组之间的比较采用单因素方差分析(ONE-WAY ANOVA)，组间两两比较用q检验，显著性检验水准为α=0.05。
结果
1．正常对照组细胞在培养的第5d开始出现突起，第7d时突起更加明显，并且大量突起交织，之后细胞逐渐增大变圆，出现悬浮趋势。VEGF诱导的各实验组细胞生长过程明显落后于正常对照组，而且细胞密度较低。
2．正常对照组细胞在培养初期出现少量CD34、CD31表达，随培养时间的延长，其表达量逐渐降低，而DC的特异性标志CD1a的表达量逐渐升高。用VEGF诱导7d后，各实验组细胞CD1a的表达受抑制。各实验组细胞CD1a与内皮细胞表面标志CD34、CD31的共表达率均高于相应正常对照组，且差异具有统计学意义，并且其共表达情况存在如下趋势：3d大剂量组＞7d大剂量组＞3d小剂量组和7d小剂量组。
3．用VEGF诱导的实验组中存在胞浆着色的阳性细胞，提示有vWF的表达，并且各实验组之间的表达趋势和上述流式细胞仪检测到的共表达趋势相似，而且积分光密度(IOD)和阳性区面积百分比(Aera％)均高于其相应正常对照组，且差异具有统计学意义。
结论：
1．在体外培养过程中，VEGF能抑制或减缓DC的生长过程，并使DC的特异性表面标志CD1a表达减少。
2．在VEGF诱导下，DC可以表达一些内皮细胞特异性标志(CD31、CD34、vWF)，出现内皮样化倾向，而且大剂量诱导比小剂量诱导更容易出现内皮样化倾向、早期诱导比晚期诱导更容易出现内皮样化倾向。
Background
Dendritic cells (DC) are the best professional antigen presenting cells (APC) known so far, which are uniquely able to activate a naive T-cell response. They play the central role in initiating, regulating and maintaining immune responses. The T-cell mediated immune reaction actived by dendritic cells play a key role in anti-tumor immune responses. The defection on numbers and functions of DCs in tumor-bearing mice and in cancer patients is the main reason for the tumor cells to escape initial immune recognition. Therefore, the biological function of DCs is closely connected with the specific anti-tumor response and plays a key role in it.
Vascular endothelial growth factor (VEGF) is one of the most potent direct-acting angiogenic molecules secreted by tumor cells, which is a highly effective specific mitogen for endothelial cells. It plays a key role on tumor angiogenesis and metastasis. Reports have proved that VEGF from tumor cells could inhibit the activation of NF-κB , then affected the differentiation development and function of DCs in tumor bearing host. The inhibition could induce abnormality of the host's immune system, and promote the tumor growth.
A recent research reported a novel leukocyte subset within ovarian carcinoma that coexpressed endothelial and dendritic cell markers. It has been proved that murine bone marrow-derived DCs could undergo endothelialisation in vitro after incubation in tumor-conditioned medium containing high levels of VEGF favours. These results suggest that VEGF may contribute to the endothelialisation of DCs in tumor microenvironment. It indicates that by promoting the endothelialisation of DCs, the tumor might not only ensure a sufficient blood supply but also prevent these DCs from initiating an immune response against tumor antigens. In this case, therapeutic targeting of this pathway could both reduce tumour growth and promote immune attack. There are many reports about the use of intratumoural DC inoculation as a means for tumor vaccination in the world, but research on endothelialisation of DCs is still limited. The effects of this phenomenon also need further investigation.
Experiment aim
The purposes of our study is to incubate human monocyte-derived DCs under the induction of VEGF, and observe the degree of endothelialisation.
Methods
Precursors of DC were separated from peripheral blood monouclear cells (PBMC) divided from healthy volunteers' peripheral blood by Ficoll density gradient centrifuge. After 3 hours incubation, remove non-adherent cells. The left precursors were cultured at 3×10~6/ml with RPMI1640 medium containing GM-CSF, IL-4. And different concentration rhVEGF(10ng/ml, 100ng/ml) were added to DCs on the 3_(rd),7_(th) days of culture. Observed the appearance character of DCs with microscope during the culture process. Normal control DCs without induction of VEGF were analysed the phenotype by Flow cytometer at l_(st), 3_(rd), 7_(th), 10_(th), 14_(th) days of culture. The experimental group cells were collected after 7 days induction, then we analysed the expression of CDla,CD34,CD31 by Flow cytometer , and detected the expression of vWF by immunocytochemistry and immunofluorescence . All data were expressed as mean value±S.D., and analyzed by statistical software SPSS12.0. P-value of < 0.05 was considered to be statistically significant.
Results
1. The Morphologic changes of normal control cells were coincide with the typicalDCs' reported. The experimental cells' growth speed was slower than the control cells', and the cell density was lower.
2. The expression of CD34 and CD31 in normal DCs reduced gradually, and the expression of CD1a elevated along with the culture time lengthening. The expression of CD1a was suppressed in experimental cells induced 7days by VEGF. The coexpression of endothelial and dendritic cell markers was higher than normal control cells'.
3. Immunocytochemistry and immunofluorescence results suggested the expression of vWF in experimental cells induced by VEGF, and the Aera% , IOD of experimental group cells were higher than normal control cells'.
Conclusion
DCs derived from human monocyte could appear endothelialisation in vitro under the induction of VEGF, and in certain ranges showing a dose-dependent.
引文
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34. Jaffe EA. Endothelial cells and the biology of factor Ⅷ. N EngI J Med, 1997, 296: 377.
1 Banchereau J, Briere F, Caux C, et al. Immunobiology of dendritic cells [J]. Annu Rev Immunol, 2000, 18:767-773.
2 Ferralra N, Davis-Smyth T. The biology of vascular endothelial growth factor [J].Endocr Rev, 1997,18:4.
3 Tsunehiro Oyama, Sophia Ran, Tadao Ishida, et al. Vascular endothelial growth factor affects dendritic cell maturation through the inbition of nuclear factor activation in hemopoietic progenitor cells[J]. The Journal of Immunology, 1998, 160: 1224.
4 Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature, 1998, 392: 245-252.
5 Yao V, Platell C, Hall JC. Dendritic cells [J]. ANZ Surgery, 2002,72:501-506.
6 Steinman RM, Cohn ZA. Identification of a novel cell type in peripheral lymphoid organs of mice.I.Morphology, quantitation, tissue distribution. J Exp Med ,1973,137:1142-1162.
7 Bodey B, Siegel SE, Kaiser HE. Antigen presentation by dendritic cells and their significance in antineoplastie immunotherapy. Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA. In Vivo, 2004,18:81-100.
8 Inaba K, Inaba M, Deguchi M, et al. Granulocytes ,macrophages,and dendritic cells arise from a common major histocomatibility complex class Ⅱ—negative progenitor in mouse bone marrow[J].Proc Natl Acad Sci U S A,1993,90:3038-42.
9 Shortman K, Wu L, Suss G, et al. Dendritic cells and T lymphocytes: developmental and functional interactions[J].Ciba Found Symp, 1997,204:130-138.
10 Schwarz FS. Positive and negative regulation of the myeloid dendritic cell lineage[J]. J Leu Bio, 1999,66:209-216.
11 Auffermann GS, Keffe EB, Levy S, et al. Impaired dendritic cell maturation in patients with chronic,but not resolved, hepatitis C virus infection[J]. Blood, 2001,97:3171.
12 Jong C, Vieira L, Kalinski P, et al. Microbial compounds selectively induce Th1 cell-promoting or Th2 cell-promoting dendritic cells in vitro with diverse Th cell-polarizing signals. J Immunology, 2002,168: 1704-1709.
13 Macia F, Im SH, Garcia-Cozar FJ, et al. T-cell anergy. Current Opinion in Immunology, 2004, 16:209-216.
14 Sallusto F, Lanzavecchia A. Understanding dendritic cell and T-lymphocyte traffic through the analysis of chemokine receptor expression. Immunol Rev, 2000, 177:134-140.
15 Weninger W, von Andrian UH. Chemokine regulation of native T cell traffic in health and disease[J]. Semin Immunol ,2003,15:257-270.
16 Steinman RM. The dendritic cell system and its roles in immunogenicity.Ann Rev, 1991,9:271.
17 Hart DNJ. Dendritic cells: unique leukocyte populations which control the primary immune response.Blood, 1997,90:3245.
18 Inaba K, Inaba M, Romani N, et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with GM-CSF. J Exp Med,1992, 176:1693.
19 郑秋红．人外周血及脐血树突状细胞的体外分离培养．基础与临床，1999，4：92-96．
20 Grabbe S , Granstein R. Modulation of antigen-presenting cell function as apotential regulatory mechanism in tumor-host immune reaactions. In Vivo, 1993, 7: 265-270.
21 Becker Y. Anticancer role of dendritic cell in human and experimental cancer. A review. Anticancer Res, 1992,120:511-520.
22 朱学军．树突状细胞与肿瘤免疫治疗研究进展．国外医学．免疫学分册．1998，21：322-325．
23 Lespagnard L,Gancberg D,Rouas G, et al. Tumor-infiltrating dendritic cells in adenocarcinomas of the breast: a study of 143 neoplasms with a correlation to usual prognostic factors and to clinical outcome .Int J Cancer 1999,84:309-314.
24 Chaux P, N Favre, M Martin ,et al. Tumor-infilting dendritic cells are defective in their antigen-presenting function and inducible B7 expression in rats. Int J Cancer 1997,72:619-625.
25 Almand B, Clark JI, Nikitina E, et al. Increased production of immature myeloid cells in cancer patients: A mechanism of immunosuppression in cancer .J Immunol 2001, 166:678-689.
26 王正昕，曹雪涛，张明徽等．人肿瘤浸润性树突状细胞的表型分析及原因探讨．中国免疫学杂志，1999，11．
27 Troy AJ ,Davidson PJ, Atkinson C, et al. Phenotypic characterization of the dendritic cell infiltrate in prostate cancer. J Urol, 1998, 160:214-219.
28 Dmitry I ,Frank C, David P, et al. Dendritic cells in antitumor immune responses defective antigen presentation in tumour bearing hosts. Cell Immunol 1996, 170: 101-103.
29 Lissoni P, Vigore L,Ferranti R ,et al.Circulating and advanced cancer patients: diminished percentdendritic cells in earlyin metastatic desease. J Biol Regal Homeost Agents. 1999, 13:216-219.
30 Tsunehiro Oyama, Sophia Ran ,Tadao Ishida ,et al. Vascular endothelial growth factor affects dendritic cell maturation through the inbition of nuclear factor-κ3 activation in hemopoetic progenitor cells [J].The Journal of Immunology, 1998,160:1224.
31 Khanna A ,Morelli AE,Zhong CP, et al. Effects of liver-derived dendritic cell progenitors on Th1- and Th2- like cytokine responses in vitro and in vivo.[J] ,J Immunol, 2000, 164: 1346.
32 Lin CM, Wang FH, Lee PK,et al. (ALL)Activated human CD4+Tcells induced by dendritic cell stimulation are most sensitive to transforming growth factor-beta: implications for dendritic cell immunization against cancer[J]. Clin Immunol, 2002, 102: 96.
33 Folkman J. Tumor angiogensis: therapeutic implications. N Engl J Med, 1971, 185:1182-1186.
34 Ferrara N, Houck K,Jakeman L, et al. Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocrine Rev 1992, 13:18-32.
35 Ferrara N,Henzel WJ. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun,1998,161:851-855.
36 Tischer E,Mitchell R, Hartman T, et al. The human gene for vascular endothelial growth factor. Multiple protein forms are encode through alternative exon splicing .J Biol Chem 1991,266:11947-11954.
37 Neufeld G, Cohen T, Gengrinovitch S,et al. Vascular endothelial growth factor and its receptors. FASEB J 1999,13:9-22.
38 Leung DW, Cahianes G, Kuang W J, et al.Vascular endothelial growth factor is a secreted angiogenic mitogen. Science, 1989,246:1306-1309.
39 Ikeda N,Adachi M,Taki T,et al. Prognostic significance of angiogenesis in human pancreatic cancer.Br J Cancer. 1999,79: 1553-1563.
40 Tatsuya F, Yu ji T, Suminon A,et al. Expression of thymidine phosphozylase and vascular endothelial cell growth factor in human head and neck squamous cell carcinoma and their different characteristics [J]. Cancer, 1999,85:960-969.
41 Fujimoto J,Sakaguchi R,Hirose R,et al. Expression of vascular endothelial growth factor (VEGF) and its mRNA in uterine cancersBr J Cancer, 1999,80: 827-833.
42 K Jin Kim,Bing LI,Jane W, et al. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo. Nature, 1993, 362: 841.
43 Asano M,Yukita A,Suzuki H.Wide spectrum of antitumor activity of a neutralizing monoclonal antibody to human vascular endothelial growth factor [J].Jpn J Cancer Res, 1999,90:93-100.
44 Harold FD, Lawrence FB, Michael D,et al. Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability ,and angiogenesis. Am J Pathol, 1995,146:1029.
45 Nor JE, Christensen J, Mooney D J, et al. Vascular endothelial growth factor—mediated angiogenesis is associated with enhanced endothelial cell survival and induction of bcl-2 expression. Am J Pathol, 1999,154:375-384.
46 郭仁德，陈剑秋．血管内皮生长因子与肿瘤研究进展．医学综述，2002，8：68-70．
47 Van der Zee R,Murohara T, Lou Z,et al. Vascular endothelial growth factor/vascular permeability factor augments nitric oxide release from quiescent rabbit and human vascular endothelium. Circulation, 1997,95:1030-1037.
48 Pertovaara L, Kaipainen A, Mustonen T, et al.Vascular endothelial growth factor is induced in response to transforming growth factor-beta in fibroblastic and epithelial cells. J Biol Chem,1994,269:6271-6274.
49 Ryuto M, One M, Lzumi H, et al. Induction of vascular endothelial growth factor by tumor necrosis factor α in human glioma cells. J Biol Chem, 1996,271:28220-28228.
50 Saito H, Tsujitani S, Oka S, et al. The expression of transforming growth factor-beta is significantly correlated with the expression of vascular endothelial growth factor and poor prognosis of patients with advanced gastric carcinoma. Cancer, 1999,86:1455-62.
51 Wang D, Huang HJ, Kazlauskas A, et al. Induction of vascular endothelial growth factor expression in endothelial cells by platelet-derived growth factor through activation of phosphatidylinosito13-kinase. Cancer Res, 1999,59:1464-72.
52 Gasparini C.. Tumor angiogenesis and accessibility role of vascular endothelial growth factor.Lancet Oncol ,2001,2:733～740.
53 Shurin MR,Gabrilovich DI. Regulation of dendritic cell system by tumor. Cancer Res Ther Control, 2001, 11:65-78.
54 Oyama T, Ran S, Ishida T, et al.Vascular endothelial growth factor affects dendritic cell maturation through the inhibition of nuclear factor-κB activation in hemopoietic progenitor cells.J Immunol, 1998,160:1224-1232.
55 Dikov MM, Oyarna T, Cheng P, et al. Vascular endothelial growth factor effects on nuclear factor-kappaB activation in hematopoietic progenitor cells.Cancer Res, 2001,61:2015-2021.
56 Ishida T, Oyama T, Carbone DP, et al. Defective function of langerhans cells in tumor-bearing animals is the result of defective maturation from hemopoietic progenitor. J Immunol, 1998,161:4842-4851.
57 Gabrilovich D,Ishida T, Nadaf S,et al.Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function. Clin Cancer Res, 1999,5:2963-2570.
58 Almand B, Resser JR,Lindman B, et al. Clinical significance of defective dendritic cell differentiation in cancer. Clin Cancer Res,2000,6:1755-1766.
59 H Saito,S Tsujutanl,M Ikeguchi, et al. Relationship between the expression of vascular endothelial growth factor and the density of dendritic cells in gastric adenocarcinoma tissue[J]. British Journal of Cancer,1998,78:1573.
60 Takahashi A,Kono K,Itakura J,et al. Correlation of vascular endothelial growth factor-C espression with tumor-infiltrating dendritic cells in gastric cancer[J]. Ontology,2002,62:121.
61 Lissoni P, Malugani F, Bonfanti A ,et al.Abnormally enhanced blood concentrations of vascular endothelial growth factor(VEGF) in metastatic cancer patients and their relation to circulating dendritic cells,IL-12 and endothelin-1[J]. J Biol Regul Homeost Agents,2001,15:140.
62 Isner J M,Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J Clin Invest, 1999, 103: 1231-1236.
63 Holash J,Maisonpierre PC,Compton D,et al. Vessel cooption, regression, and growth in tumors mediated by angiopoietin and VEGF. Science, 1999, 284 : 1994-1998.
64 Conejo-Garcia JR, Benencia F, Coukos G, et al. Tumor-infiltrating dendritic cell precursors recruited by a beta-defensin contribute to vasculogenesis under the influence of Vegf-A. Nat Med ,2004, 10: 950-958.
65 Conejo-Garcia JR, Buckanovich RJ, Coukos G,et al. Vascular leukocytes contribute to tumor vascularization. Blood,2005,105:679-681.
66 G Coukos, F Beneneia, Buckanovich RJ, et al. The role of dendritic cell precursors in tumour vasculogenesis. British Journal of Cancer,2005,92:1182-1187.
2. Gatti E, Pierre P. Understanding the cell biology of antigen presentation: the dendritic cell contribution. Curr Opin Cell Biol ,2003,15(4):468-473.
3. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature, 1998,392:245-252.
4. Vleeschouwer S, Arredouani M, Ade M, et al. Uptake and presentation of malignant glioma tumor cell lysates by monocyte-derived dendritic cells .Cancer Immunol,2005,54(4):372-382.
5. Gunzer M, Janich S, Varga G, et al. Dendritic cells and tumor immunity. Semin Immunol,2001,13(5):291-302.
6. Almand B, Clark JI, Nikitina E, et al. Increased production of immature myeloid cells in cancer patients: A mechanism of immunosuppression in cancer. J Immunol, 2001, 166:678-689.
7. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med, 1971,285 (21) :1182-1186.
8. Ferrara N, Houck K, Jakeman L, et al. Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocrine Rev, 1992,13:18-32.
9. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocrine Rev, 1997,18:4-25.
10. Toi M, Taniguchi T, Yamamoto Y, et al .Clinical significance of the determination of angiogenic factors .Eur J Cancer, 1996,32:2513-2519.
11. Ellis LM, Fidler IJ. Angiogenesis and metatasis .Eur J Cancer ,1996,32:2451-2460.
12. Oyama T, Ran S, Ishida T, et al. Vascular endothelial growth factor affects dendritic cell maturation through the inhibition of nuclear factor-κB activation in hemopoietic progenitor cells. J Immunol, 1998,160:1224-1232.
13. Shurin MR, Gabrilovich DI. Regulation of dendritic cell system by tumor. Cancer Res Ther Control,2001,11:65-78.
14. Conejo-Garcia JR, Benencia F, Coukos G, et al. Tumor-infiltrating dendritic cell precursors recruited by a beta-defensin contribute to vasculogenesis under the influence of Vegf-A. Nat Med 2004, 10: 950-958.
15. Conejo-Garcia JR, Buckanovich RJ, Coukos G ,et al. Vascular leukocytes contribute to tumor vascularization. Blood, 2005,105:679-681.
16. G Coukos, F Benencia, Buckanovich RJ, et al. The role of dendritic cell precursors in tumor vasculogenesis. British Journal of Cancer ,2005,92:1182-1187.
17．潘建平，曹雪涛．树突状细胞与肿瘤免疫治疗研究进展．实用肿瘤杂志，2003，18(6)：427-430．
18. Gan TO, Kagaku, Ryoho. Intratumoral injection of immature dendritic cells (DC) for cancer patients. Hum Immunol, 2005,32(11): 1574-1575.
19. Bodey B, Siegel SE, Kaiser HE. Antigen presentation by dendritic cells and their significance in antineoplastic immunotherapy. Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA. In Vivo.,2004,18(1):81-100.
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23. Oyarna T, Ran S, Ishida T, et al.Vascular endothelial growth factor affects dendritic cell maturation through the inhibition of nuclear factor-κB activation in hemopoietic progenitor cells.J Immunol, 1998,160:1224-1232.
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32. Kuzu I, Bicknell R, Harris AL, et al. Heterogeneity of vascular endothelial ceils with relevance to diagnosis of vascular tumors [J]. J Clin Pathol, 1992, 45(2): 143-148.
33. Denis CV. Molecular and cellular biology of von Willebrand factor. Int J Hematol,2002,75(1):3-8.
34. Jaffe EA. Endothelial cells and the biology of factor Ⅷ. N EngI J Med, 1997, 296: 377.
1 Banchereau J, Briere F, Caux C, et al. Immunobiology of dendritic cells [J]. Annu Rev Immunol, 2000, 18:767-773.
2 Ferralra N, Davis-Smyth T. The biology of vascular endothelial growth factor [J].Endocr Rev, 1997,18:4.
3 Tsunehiro Oyama, Sophia Ran, Tadao Ishida, et al. Vascular endothelial growth factor affects dendritic cell maturation through the inbition of nuclear factor activation in hemopoietic progenitor cells[J]. The Journal of Immunology, 1998, 160: 1224.
4 Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature, 1998, 392: 245-252.
5 Yao V, Platell C, Hall JC. Dendritic cells [J]. ANZ Surgery, 2002,72:501-506.
6 Steinman RM, Cohn ZA. Identification of a novel cell type in peripheral lymphoid organs of mice.I.Morphology, quantitation, tissue distribution. J Exp Med ,1973,137:1142-1162.
7 Bodey B, Siegel SE, Kaiser HE. Antigen presentation by dendritic cells and their significance in antineoplastie immunotherapy. Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA. In Vivo, 2004,18:81-100.
8 Inaba K, Inaba M, Deguchi M, et al. Granulocytes ,macrophages,and dendritic cells arise from a common major histocomatibility complex class Ⅱ—negative progenitor in mouse bone marrow[J].Proc Natl Acad Sci U S A,1993,90:3038-42.
9 Shortman K, Wu L, Suss G, et al. Dendritic cells and T lymphocytes: developmental and functional interactions[J].Ciba Found Symp, 1997,204:130-138.
10 Schwarz FS. Positive and negative regulation of the myeloid dendritic cell lineage[J]. J Leu Bio, 1999,66:209-216.
11 Auffermann GS, Keffe EB, Levy S, et al. Impaired dendritic cell maturation in patients with chronic,but not resolved, hepatitis C virus infection[J]. Blood, 2001,97:3171.
12 Jong C, Vieira L, Kalinski P, et al. Microbial compounds selectively induce Th1 cell-promoting or Th2 cell-promoting dendritic cells in vitro with diverse Th cell-polarizing signals. J Immunology, 2002,168: 1704-1709.
13 Macia F, Im SH, Garcia-Cozar FJ, et al. T-cell anergy. Current Opinion in Immunology, 2004, 16:209-216.
14 Sallusto F, Lanzavecchia A. Understanding dendritic cell and T-lymphocyte traffic through the analysis of chemokine receptor expression. Immunol Rev, 2000, 177:134-140.
15 Weninger W, von Andrian UH. Chemokine regulation of native T cell traffic in health and disease[J]. Semin Immunol ,2003,15:257-270.
16 Steinman RM. The dendritic cell system and its roles in immunogenicity.Ann Rev, 1991,9:271.
17 Hart DNJ. Dendritic cells: unique leukocyte populations which control the primary immune response.Blood, 1997,90:3245.
18 Inaba K, Inaba M, Romani N, et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with GM-CSF. J Exp Med,1992, 176:1693.
19 郑秋红．人外周血及脐血树突状细胞的体外分离培养．基础与临床，1999，4：92-96．
20 Grabbe S , Granstein R. Modulation of antigen-presenting cell function as apotential regulatory mechanism in tumor-host immune reaactions. In Vivo, 1993, 7: 265-270.
21 Becker Y. Anticancer role of dendritic cell in human and experimental cancer. A review. Anticancer Res, 1992,120:511-520.
22 朱学军．树突状细胞与肿瘤免疫治疗研究进展．国外医学．免疫学分册．1998，21：322-325．
23 Lespagnard L,Gancberg D,Rouas G, et al. Tumor-infiltrating dendritic cells in adenocarcinomas of the breast: a study of 143 neoplasms with a correlation to usual prognostic factors and to clinical outcome .Int J Cancer 1999,84:309-314.
24 Chaux P, N Favre, M Martin ,et al. Tumor-infilting dendritic cells are defective in their antigen-presenting function and inducible B7 expression in rats. Int J Cancer 1997,72:619-625.
25 Almand B, Clark JI, Nikitina E, et al. Increased production of immature myeloid cells in cancer patients: A mechanism of immunosuppression in cancer .J Immunol 2001, 166:678-689.
26 王正昕，曹雪涛，张明徽等．人肿瘤浸润性树突状细胞的表型分析及原因探讨．中国免疫学杂志，1999，11．
27 Troy AJ ,Davidson PJ, Atkinson C, et al. Phenotypic characterization of the dendritic cell infiltrate in prostate cancer. J Urol, 1998, 160:214-219.
28 Dmitry I ,Frank C, David P, et al. Dendritic cells in antitumor immune responses defective antigen presentation in tumour bearing hosts. Cell Immunol 1996, 170: 101-103.
29 Lissoni P, Vigore L,Ferranti R ,et al.Circulating and advanced cancer patients: diminished percentdendritic cells in earlyin metastatic desease. J Biol Regal Homeost Agents. 1999, 13:216-219.
30 Tsunehiro Oyama, Sophia Ran ,Tadao Ishida ,et al. Vascular endothelial growth factor affects dendritic cell maturation through the inbition of nuclear factor-κ3 activation in hemopoetic progenitor cells [J].The Journal of Immunology, 1998,160:1224.
31 Khanna A ,Morelli AE,Zhong CP, et al. Effects of liver-derived dendritic cell progenitors on Th1- and Th2- like cytokine responses in vitro and in vivo.[J] ,J Immunol, 2000, 164: 1346.
32 Lin CM, Wang FH, Lee PK,et al. (ALL)Activated human CD4+Tcells induced by dendritic cell stimulation are most sensitive to transforming growth factor-beta: implications for dendritic cell immunization against cancer[J]. Clin Immunol, 2002, 102: 96.
33 Folkman J. Tumor angiogensis: therapeutic implications. N Engl J Med, 1971, 185:1182-1186.
34 Ferrara N, Houck K,Jakeman L, et al. Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocrine Rev 1992, 13:18-32.
35 Ferrara N,Henzel WJ. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun,1998,161:851-855.
36 Tischer E,Mitchell R, Hartman T, et al. The human gene for vascular endothelial growth factor. Multiple protein forms are encode through alternative exon splicing .J Biol Chem 1991,266:11947-11954.
37 Neufeld G, Cohen T, Gengrinovitch S,et al. Vascular endothelial growth factor and its receptors. FASEB J 1999,13:9-22.
38 Leung DW, Cahianes G, Kuang W J, et al.Vascular endothelial growth factor is a secreted angiogenic mitogen. Science, 1989,246:1306-1309.
39 Ikeda N,Adachi M,Taki T,et al. Prognostic significance of angiogenesis in human pancreatic cancer.Br J Cancer. 1999,79: 1553-1563.
40 Tatsuya F, Yu ji T, Suminon A,et al. Expression of thymidine phosphozylase and vascular endothelial cell growth factor in human head and neck squamous cell carcinoma and their different characteristics [J]. Cancer, 1999,85:960-969.
41 Fujimoto J,Sakaguchi R,Hirose R,et al. Expression of vascular endothelial growth factor (VEGF) and its mRNA in uterine cancersBr J Cancer, 1999,80: 827-833.
42 K Jin Kim,Bing LI,Jane W, et al. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo. Nature, 1993, 362: 841.
43 Asano M,Yukita A,Suzuki H.Wide spectrum of antitumor activity of a neutralizing monoclonal antibody to human vascular endothelial growth factor [J].Jpn J Cancer Res, 1999,90:93-100.
44 Harold FD, Lawrence FB, Michael D,et al. Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability ,and angiogenesis. Am J Pathol, 1995,146:1029.
45 Nor JE, Christensen J, Mooney D J, et al. Vascular endothelial growth factor—mediated angiogenesis is associated with enhanced endothelial cell survival and induction of bcl-2 expression. Am J Pathol, 1999,154:375-384.
46 郭仁德，陈剑秋．血管内皮生长因子与肿瘤研究进展．医学综述，2002，8：68-70．
47 Van der Zee R,Murohara T, Lou Z,et al. Vascular endothelial growth factor/vascular permeability factor augments nitric oxide release from quiescent rabbit and human vascular endothelium. Circulation, 1997,95:1030-1037.
48 Pertovaara L, Kaipainen A, Mustonen T, et al.Vascular endothelial growth factor is induced in response to transforming growth factor-beta in fibroblastic and epithelial cells. J Biol Chem,1994,269:6271-6274.
49 Ryuto M, One M, Lzumi H, et al. Induction of vascular endothelial growth factor by tumor necrosis factor α in human glioma cells. J Biol Chem, 1996,271:28220-28228.
50 Saito H, Tsujitani S, Oka S, et al. The expression of transforming growth factor-beta is significantly correlated with the expression of vascular endothelial growth factor and poor prognosis of patients with advanced gastric carcinoma. Cancer, 1999,86:1455-62.
51 Wang D, Huang HJ, Kazlauskas A, et al. Induction of vascular endothelial growth factor expression in endothelial cells by platelet-derived growth factor through activation of phosphatidylinosito13-kinase. Cancer Res, 1999,59:1464-72.
52 Gasparini C.. Tumor angiogenesis and accessibility role of vascular endothelial growth factor.Lancet Oncol ,2001,2:733～740.
53 Shurin MR,Gabrilovich DI. Regulation of dendritic cell system by tumor. Cancer Res Ther Control, 2001, 11:65-78.
54 Oyama T, Ran S, Ishida T, et al.Vascular endothelial growth factor affects dendritic cell maturation through the inhibition of nuclear factor-κB activation in hemopoietic progenitor cells.J Immunol, 1998,160:1224-1232.
55 Dikov MM, Oyarna T, Cheng P, et al. Vascular endothelial growth factor effects on nuclear factor-kappaB activation in hematopoietic progenitor cells.Cancer Res, 2001,61:2015-2021.
56 Ishida T, Oyama T, Carbone DP, et al. Defective function of langerhans cells in tumor-bearing animals is the result of defective maturation from hemopoietic progenitor. J Immunol, 1998,161:4842-4851.
57 Gabrilovich D,Ishida T, Nadaf S,et al.Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function. Clin Cancer Res, 1999,5:2963-2570.
58 Almand B, Resser JR,Lindman B, et al. Clinical significance of defective dendritic cell differentiation in cancer. Clin Cancer Res,2000,6:1755-1766.
59 H Saito,S Tsujutanl,M Ikeguchi, et al. Relationship between the expression of vascular endothelial growth factor and the density of dendritic cells in gastric adenocarcinoma tissue[J]. British Journal of Cancer,1998,78:1573.
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