单核细胞衍生的巨噬细胞应用IFN-γ和Lps处理的抗肿瘤实验研究
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摘要
单核／巨噬细胞是一类重要的免疫效应细胞，在人体抗肿瘤免疫中发挥重要作用。目前国外已有单核／巨噬细胞Ⅰ期临床试验的报道，而国内关于人体单核／巨噬细胞基础临床及动物实验方面的资料尚系空白。本实验以外周血为原料，首次观察了人体单核／巨噬细胞的体外分离、纯化、增殖、激活及体内体外抗肿瘤效应，比较了单核／巨噬细胞与LAK细胞的体内抗肿瘤活性差异，检测了单核／巨噬细胞培养上清中及细胞中TNF-α及IL-12的含量，并观察了单核／巨噬细胞培养上清对荷瘤小鼠的体内抗肿瘤效应。旨在对单核／巨噬细胞的抗肿瘤功能作一基础性研究，为今后单核／巨噬细胞的临床应用打下一定的理论基础。
实验方法：我们采用CS-3000 Plus血细胞分离机采集了7位肿瘤病人经化疗及G-CSF动员后的外周血单个核细胞，以密度梯度离心及贴壁法分离及纯化外周血单核细胞，以GM-CSF1000U／ml作单核／巨噬细胞对比增殖实验；以台盼蓝染色法检测细胞活率；以非特异性酯酶染色法鉴定单核细胞；以流式细胞技术检测单核／巨噬细胞表型以判断细胞分化程度；以透射电镜对培养8天单核／巨噬细胞进行形态学观察：以[’H]TdR掺合法检测单核／巨噬细胞对K562靶细胞的体外静止细胞毒活性；采用皮下预先注射的方法以观察激活巨噬细胞对接种K562靶细胞的生长抑制作用；采用瘤内注射法观察单核／巨噬细胞对菏瘤小鼠的体内抗肿瘤活性并与LAK细胞比较；采用Elisa法检测单核／巨噬细胞培养上清中TNF-α及IL-12的浓度；以Western-blot法检测单核／巨噬细胞中TNF-α及IL-12的含量；以激活巨噬细胞培养上清对菏瘤小鼠行腹腔注射观察细胞培养上清的体内抗肿瘤活性。
结果：1、采用CS-3000 Plus血细胞分离机可大量采集外周血中的单核细胞，在采血量为10000-12000ml时，平均每次单核细胞采集量为5.8×108[(0.83-11.75)×108]，其数量可满足实验要求。2、GM-CSF可刺激单核／
第二军医大学 博士学位论文
巨噬细胞轻度增殖，单核／巨噬细胞经GM（sF刺激后可扩增5乃倍（P＜o．05人
3、台盼蓝染色法证明培养第2天及第8天的单核l巨噬细胞活串均大于90％。
4、非特异性酯酌染色显示外周血贴壁细胞胞浆广泛深染，并有棕褐色颗粒，
证明贴壁细胞为单核细胞。5、流式细胞表型检测显示外周血贴壁细胞高表
达＊D14抗原，培养吕天及经过1＊冲YO00＊偷1）和u9＊咖u联合刺激后
大部分己经分化成熟。6、培养吕天的单核l巨噬细胞透射电镜观察显示胞浆
内丰富的内质网、线粒体及溶酶体，显示细胞活跃的吞噬和分泌功能。7、
单核／巨噬细胞体外静止细胞毒试验证明单核l巨噬细胞具有明显的抗肿瘤活
性，其抗肿庙效应存在剂量一效应依赖关系，在效／靶比＝8：1 时其抗瘤活性
达到最大值：！FN·丁a00＊八n1）和＊＊8山glinlm！）均可活化单核J巨噬细胞，增强
其抗肿瘤活性（P功．05入 其对巨噬细胞的活化作用两者之间无显著差异
（P叩刀5hWNWN．可＋＊PS 联合刺激可进一步提高巨噬细胞抗肿瘤活性
（P＜o．01）。吕、激活巨喧细胞的预防注射可显著减少肿瘤的发生（P＜o．05）。
9、单核／巨噬细胞及LAK细胞均有明显的体内抗肿盾活性，以微活巨噬细胞
及活化LAK细胞瘤内注射的菏瘤小鼠组肿瘤平均面积及瘤重均显著小于对
照组（P＜0刀1及P＜0刀5）：在同一效／M比条件下，激活巨噬纫胞抗肿鹰活性
强于＊＊K细胞（P＜o．05）；病理切片显示巨噬细胞组庙结广泛坏死，残留肿
瘤组织很少，伴弥漫性淋巴细胞浸润；LAK细胞组瘤结有轻到中度坏死，局
部淋巴细胞浸润：而对照组瘤结肿瘤细胞呈旺盛生长态势，伴点状坏死，无
淋巴细胞浸润。10、Elisa法测定细胞培养上清中 TNF a及 IL．12浓度显示
LPS0ug／ml）可显著增强单核／巨噬细胞对WP山及IL12的分泌，LPS组细
胞上清中TNF·Q及IL－12浓度显著高于对照组（P＜0．of及P＜0．05）。IFN．Y
（200U／m）对单核／巨噬细胞的此种刺激作用不明显，但和LPS＊U咖l）有协同
作用，可强烈刺激单核 l巨噬细胞分泌 iF a及 ILd，IFN·Ya00Ulml）＋
LPSOuglml）联合处理组细胞上清中TNF a及IL12浓度显著高于对照组
（P＜0．00）。11、Western．blot法测定单核l巨噬细胞中 hF．口及肌．12含量
4
第二军医大学 博士学位论文
同样显示LPS＊ug／ml）可显著刺激单核／巨噬细胞对该两细胞因子的合成，IFN－
m仅00U／ml）单独刺激作用不明显，但和 LPS有协同作用，可明显提高细胞中
TNF a及 IL－12的含量。12、激活巨噬细胞培养上清有明显抗肿瘤作用，腹
腔注射激活巨噬细胞培养上清组小鼠瘤重显著小于对照组（P＜0刀5），生存时
间显著高于对照组（P＜0刀5）。
结论：l、采用 CS－3000 PISS血细胞分离机可大量采集外周血中的单核
细胞，为单核／巨噬细胞的实验研究提供了一条行之有效的细胞采集方法。二、
GMcSF可刺激单核／巨噬细胞轻度增殖。3、单核／巨噬细胞具有明显的抗肿
瘤效应。4、IFN－mC00U／ml）及 L
Monocyte/macrophage is a kind of immune effect cell, and plays an important
role in anti-tumor immunity. There has already been reported abroad about the phase
I clinical trial of human monocyte/macrophage, however, in our country, the
basically clinical investigation or animal experiments of anti-tumor effect of human
monocyte/macrophage has yet not been reported. In our study, we have observed
mainly the ex vivo isolation, purification, proliferation, activation, and in vitro and in
vivo anti-tumor effect of monocyte/macrophage derived from human peripheral blood,
compared its in vivo anti-tumor activity with LAK cell, detected the content of TNF-
a and IL- 12 in cultured monocyte/macrophage and its supematant, and observed the
in vivo anti-tumor effect of activated monocyte/macrophage culture supematant on
scid mice0 The main aim of this study is to explore further the anti-tumor effect of
monocyte/macrophage through our basic research, and to lay some theoretical
foundation for clinical application of monocyte/macrophage henceforth in our
country.
Method: By using CS-3000 Plus blood cell seperator, mononuclear cells were
isolated from peripheral blood of seven tumor patient who were preliminarily treated
with chemotherapy and G-CSF mobilization, then the isblated mononuclear were
purified further by density centrifugation with Ficoll and by adherence; Proliferation-
comparison study of monocyte/macrophage is carried out under GM-CSF I000U/ml
stimulation; Cell survival rate is assessed by trypan blue dye; Monocyte is identified
by non-specific esterase staining; Differentiation of monocyte/macrophage is assessed
by flow cytometry analysis; Morphological change of eight-day cultured cell is
observed by electron microscope; In vitro static cytotoxic activity to K562 target cells
of monocyte/macrophage is analyzed by 3H-TdR incorporation; The inhibition of
tumor incidence by activated macrophage to inoculated K562 target cells is assessed
by prophylactic subcutaneous injectiom In vivo anti-tumor activity to tumor bearing
scid mice of activated macrophage or LAK is analyzed by direct intratumor injection;
The content of TNF-a and IL- 12 in culture supematant of monocyte/macrophage is
assessed by ELISA assay;The content of TNF-a and IL-12 in cultured
monocyte/macrophage is analyzed by Western-blot. In vivo anti-tumor activity of
activated monocyte/macrophage culture supernatant is detected by intraperineal
injection.
Result: 1 .By using CS-3000 Plus blood cell seperator, large amounts of
peripheral blood monocytes can be collected. When the sampling blood amounts to
10000 to l2000ml, the average number of collected monocyte is about 5.80 X
1O抂(0.83-11.75)X 10抅, which can well satisfies our study demand. 2. GM-CSF can
stimulate gently the proliferation of monocyte/macrophage,after GM-CSF stimulation,
the number of monocyte/macrophage can add to five to six-fold(P<0.05).3. trypan
blue dye demonstrates the survival rate of monocyte/macrophage cultured after the
second day and the eighth day is more than 90%. 4.Non-speciflc esterase staining
shows the cytoplasm of peripheral blood adherent cells is dyed extensively and
profoundly, and has brown granule deposits, demonstrating that these adherent cells
are monocytes. 5. Flow cytometry analysis shows peripheral blood adherent cells
highly express CD 14 antigen, after eight days culture and by a costimulation of IFN-y
(200U/ml) and LPS(lug/ml), majority
实验方法：我们采用CS-3000 Plus血细胞分离机采集了7位肿瘤病人经化疗及G-CSF动员后的外周血单个核细胞，以密度梯度离心及贴壁法分离及纯化外周血单核细胞，以GM-CSF1000U／ml作单核／巨噬细胞对比增殖实验；以台盼蓝染色法检测细胞活率；以非特异性酯酶染色法鉴定单核细胞；以流式细胞技术检测单核／巨噬细胞表型以判断细胞分化程度；以透射电镜对培养8天单核／巨噬细胞进行形态学观察：以[’H]TdR掺合法检测单核／巨噬细胞对K562靶细胞的体外静止细胞毒活性；采用皮下预先注射的方法以观察激活巨噬细胞对接种K562靶细胞的生长抑制作用；采用瘤内注射法观察单核／巨噬细胞对菏瘤小鼠的体内抗肿瘤活性并与LAK细胞比较；采用Elisa法检测单核／巨噬细胞培养上清中TNF-α及IL-12的浓度；以Western-blot法检测单核／巨噬细胞中TNF-α及IL-12的含量；以激活巨噬细胞培养上清对菏瘤小鼠行腹腔注射观察细胞培养上清的体内抗肿瘤活性。
结果：1、采用CS-3000 Plus血细胞分离机可大量采集外周血中的单核细胞，在采血量为10000-12000ml时，平均每次单核细胞采集量为5.8×108[(0.83-11.75)×108]，其数量可满足实验要求。2、GM-CSF可刺激单核／
第二军医大学 博士学位论文
巨噬细胞轻度增殖，单核／巨噬细胞经GM（sF刺激后可扩增5乃倍（P＜o．05人
3、台盼蓝染色法证明培养第2天及第8天的单核l巨噬细胞活串均大于90％。
4、非特异性酯酌染色显示外周血贴壁细胞胞浆广泛深染，并有棕褐色颗粒，
证明贴壁细胞为单核细胞。5、流式细胞表型检测显示外周血贴壁细胞高表
达＊D14抗原，培养吕天及经过1＊冲YO00＊偷1）和u9＊咖u联合刺激后
大部分己经分化成熟。6、培养吕天的单核l巨噬细胞透射电镜观察显示胞浆
内丰富的内质网、线粒体及溶酶体，显示细胞活跃的吞噬和分泌功能。7、
单核／巨噬细胞体外静止细胞毒试验证明单核l巨噬细胞具有明显的抗肿瘤活
性，其抗肿庙效应存在剂量一效应依赖关系，在效／靶比＝8：1 时其抗瘤活性
达到最大值：！FN·丁a00＊八n1）和＊＊8山glinlm！）均可活化单核J巨噬细胞，增强
其抗肿瘤活性（P功．05入 其对巨噬细胞的活化作用两者之间无显著差异
（P叩刀5hWNWN．可＋＊PS 联合刺激可进一步提高巨噬细胞抗肿瘤活性
（P＜o．01）。吕、激活巨喧细胞的预防注射可显著减少肿瘤的发生（P＜o．05）。
9、单核／巨噬细胞及LAK细胞均有明显的体内抗肿盾活性，以微活巨噬细胞
及活化LAK细胞瘤内注射的菏瘤小鼠组肿瘤平均面积及瘤重均显著小于对
照组（P＜0刀1及P＜0刀5）：在同一效／M比条件下，激活巨噬纫胞抗肿鹰活性
强于＊＊K细胞（P＜o．05）；病理切片显示巨噬细胞组庙结广泛坏死，残留肿
瘤组织很少，伴弥漫性淋巴细胞浸润；LAK细胞组瘤结有轻到中度坏死，局
部淋巴细胞浸润：而对照组瘤结肿瘤细胞呈旺盛生长态势，伴点状坏死，无
淋巴细胞浸润。10、Elisa法测定细胞培养上清中 TNF a及 IL．12浓度显示
LPS0ug／ml）可显著增强单核／巨噬细胞对WP山及IL12的分泌，LPS组细
胞上清中TNF·Q及IL－12浓度显著高于对照组（P＜0．of及P＜0．05）。IFN．Y
（200U／m）对单核／巨噬细胞的此种刺激作用不明显，但和LPS＊U咖l）有协同
作用，可强烈刺激单核 l巨噬细胞分泌 iF a及 ILd，IFN·Ya00Ulml）＋
LPSOuglml）联合处理组细胞上清中TNF a及IL12浓度显著高于对照组
（P＜0．00）。11、Western．blot法测定单核l巨噬细胞中 hF．口及肌．12含量
4
第二军医大学 博士学位论文
同样显示LPS＊ug／ml）可显著刺激单核／巨噬细胞对该两细胞因子的合成，IFN－
m仅00U／ml）单独刺激作用不明显，但和 LPS有协同作用，可明显提高细胞中
TNF a及 IL－12的含量。12、激活巨噬细胞培养上清有明显抗肿瘤作用，腹
腔注射激活巨噬细胞培养上清组小鼠瘤重显著小于对照组（P＜0刀5），生存时
间显著高于对照组（P＜0刀5）。
结论：l、采用 CS－3000 PISS血细胞分离机可大量采集外周血中的单核
细胞，为单核／巨噬细胞的实验研究提供了一条行之有效的细胞采集方法。二、
GMcSF可刺激单核／巨噬细胞轻度增殖。3、单核／巨噬细胞具有明显的抗肿
瘤效应。4、IFN－mC00U／ml）及 L
Monocyte/macrophage is a kind of immune effect cell, and plays an important
role in anti-tumor immunity. There has already been reported abroad about the phase
I clinical trial of human monocyte/macrophage, however, in our country, the
basically clinical investigation or animal experiments of anti-tumor effect of human
monocyte/macrophage has yet not been reported. In our study, we have observed
mainly the ex vivo isolation, purification, proliferation, activation, and in vitro and in
vivo anti-tumor effect of monocyte/macrophage derived from human peripheral blood,
compared its in vivo anti-tumor activity with LAK cell, detected the content of TNF-
a and IL- 12 in cultured monocyte/macrophage and its supematant, and observed the
in vivo anti-tumor effect of activated monocyte/macrophage culture supematant on
scid mice0 The main aim of this study is to explore further the anti-tumor effect of
monocyte/macrophage through our basic research, and to lay some theoretical
foundation for clinical application of monocyte/macrophage henceforth in our
country.
Method: By using CS-3000 Plus blood cell seperator, mononuclear cells were
isolated from peripheral blood of seven tumor patient who were preliminarily treated
with chemotherapy and G-CSF mobilization, then the isblated mononuclear were
purified further by density centrifugation with Ficoll and by adherence; Proliferation-
comparison study of monocyte/macrophage is carried out under GM-CSF I000U/ml
stimulation; Cell survival rate is assessed by trypan blue dye; Monocyte is identified
by non-specific esterase staining; Differentiation of monocyte/macrophage is assessed
by flow cytometry analysis; Morphological change of eight-day cultured cell is
observed by electron microscope; In vitro static cytotoxic activity to K562 target cells
of monocyte/macrophage is analyzed by 3H-TdR incorporation; The inhibition of
tumor incidence by activated macrophage to inoculated K562 target cells is assessed
by prophylactic subcutaneous injectiom In vivo anti-tumor activity to tumor bearing
scid mice of activated macrophage or LAK is analyzed by direct intratumor injection;
The content of TNF-a and IL- 12 in culture supematant of monocyte/macrophage is
assessed by ELISA assay;The content of TNF-a and IL-12 in cultured
monocyte/macrophage is analyzed by Western-blot. In vivo anti-tumor activity of
activated monocyte/macrophage culture supernatant is detected by intraperineal
injection.
Result: 1 .By using CS-3000 Plus blood cell seperator, large amounts of
peripheral blood monocytes can be collected. When the sampling blood amounts to
10000 to l2000ml, the average number of collected monocyte is about 5.80 X
1O抂(0.83-11.75)X 10抅, which can well satisfies our study demand. 2. GM-CSF can
stimulate gently the proliferation of monocyte/macrophage,after GM-CSF stimulation,
the number of monocyte/macrophage can add to five to six-fold(P<0.05).3. trypan
blue dye demonstrates the survival rate of monocyte/macrophage cultured after the
second day and the eighth day is more than 90%. 4.Non-speciflc esterase staining
shows the cytoplasm of peripheral blood adherent cells is dyed extensively and
profoundly, and has brown granule deposits, demonstrating that these adherent cells
are monocytes. 5. Flow cytometry analysis shows peripheral blood adherent cells
highly express CD 14 antigen, after eight days culture and by a costimulation of IFN-y
(200U/ml) and LPS(lug/ml), majority
引文
1． Rosenberg SA, Lotze MT, Muul MT, etal. Observation on the administration of autologous, lymphokine activated killer cells and recombinant interleukin-2 to patient with cancer. N Engl J Med 1985; 313:1485-92
2． Fidler, I.J. Inhibition of pulmonary metastasis by intravenous injection of specification activated macrophages. Cancer Res 1974; 34:1074-1078
3． Berdel,W.E., Bausert,W.R., Weltaien,H.U., etal. The influence of alkyl-lysophospholipids and lysophospholipid-activated macrophages on the development of metastasis of 3-lewis lung carcinoma. Eur J Cancer 1980; 16: 1199-1204
4． Figdor.C, Bont.W.S, Vries.J.D, etal. Isolation of large numbers of highly purified lymphocytes and monocytes with a modified centrifugal elutriation technique. J Immunol Methods 1981; 40:275-288
5． Stevenson HC, Keenan AM, Woodhouse C, Ohaw RI, Miller P, Steller EP, Foon KA, etal. Fate of IFN-gamma activated killer blood monocytes adoptively transferred into the abdominal cavity of patients with peritoneal carcinoma. Cancer Res 1987; 47:6100
6． Bartoleyns.J, Romet-lemonne.JL, Chokri.M, Lopez.M. Immune therapy with macrophages: present status and critical requirements for implementation. Immunobiol 1996; 195:550-562
7． B.Hennemann, A.Rehm,A.Kottke, N.Meidenbauer, and R.Andreesen. Adoptive immunotherapy with tumor-cytotoxic macrophages derived from recombinant human granulocyte-macrophage colony-stimulating factor (rhu GM-CSF) mobilized peripheral blood monocytes. J Immunother 1997; 20(5) : 365-371
8． B.Hennemann, G.Beckmann, A.Eichelmann, A.Rehm, and R.Andereesen. Phase I trial of adoptive immunotherapy of cancer patients using momocyte-derived macrophages activated with interferon y and lipopolysaccharide. Cancer Immunol
Immunother 1998; 45: 250-256
9． R.Adreesen, KL.Bross, J.Osterholz, and F.Emmrich. Human macrophage maturation and heterogeneity with a newly generated set of monoclonal antibodies to differentiation antigens. Blood 1986; 67: 1257
10． R.Adreesen, J.Picht, and G.W.Lohr. Primary cultures of human blood-born macrophages grown on hydrophobic teflon membranes. J Immunol Meth 1983; 56: 295
11 .R.Adreesen, C.Scheibenbogen, M.Kreutz, S.Krause, and A.Rehm. Regulation of neopterm secretion during monocyte to macrophage differentiation in vitro. Pteridins 1990; 23:1140
12． C.Scheibenbogen, and R.Andreesen. Developmental regulation of the cytokine repertoire in human macrophages: IL-2, IL-6, TNF-a , and M-CSF. J Leukocyte Biol 1991; 34:1236
13． R.Adreesen, C.Scheibenbogen, H.Moser, S.Krause, and H.Engler. Adoptive transfer of activated monocyte-derived macrophages initiates the coagulation cascade. Proc. Am. Assoc. Cancer Res 1990; 31: 245
14． RB.Herberman, JR.Ortaldo, A.Mantovani, DS.Hobbs, HF.Kung, and S.Pestka. Effect of human recombinant interferon on cytotoxic activity of natural killer (NK) and monocytes. Cell Immunol 1982; 67: 160-167
15． R.Adreesen, S.Gadd, W.Brugger, GW.Lohr, and RC.Atkins. Activation of human monocyte-derived macrophages cultured on teflon: response to interferon-Y during terminal maturation in vitro. Immunobiol 1988; 177: 186-198
16． Meerchaert I.M, B.Furie. Monocytes use either CD11/CD58 or VLA4 to migrate across human erdothelium in vitro. J Immunol 1994; 152: 1915
17． M.Lopez, J.Fechtenbaum, B.David, C.Martinache, M.Chokri, S.Canepa, etal. Adoptive immunorherapy with activated macrophages grown in vitro from monocytes
in cancer patients: a pilot study. J Immunother 1992; 11: 209-217
18． J.C.Eymard, M.Lopez, A.Cattan, Q.Bouche, J.C.Adjizian, and J.Bernard. Phase I /II trial of autologous activated macrophages in advanced colorectal cancer. Euro J Cancer Vol 1996; 11: 1905-1911
19． B.Hennemann, C.Scheibenbogen, C.Schumichen, and R.Andreesen. Intrahepatic adoptive immunotherapy with autologous tumorcytotoxic macrophages in patients with cancer. J Immunother 1995; 18 (1) : 19-27
20． Broxmeyer HE,Cooper S.Williams DE,et al.Growth characteristics of marrow hematopoietic progenitor/precursor cells from patients on a phase I clinical trial with purified recombinant human granulocyte-macrophage colony-stimulating factor. Exp Hematol 1988; 16: 594-602
21． Hermann F,Schulz G,Lindemann A,et al. Hematopoietic responses in patients with advanced malignancy treated with recombinant human granulocyte-macrophage colony-stimulating factor. J Clin Oncol 1989; 7: 159-67
22． Lieschke GL,Maher D.Cebon J,et al. Effects of bacterial synthesized recombinant human granulocyte-macrophage colony-stimulating factor in patients with advanced malignancy. Ann Intern Med 1989; 110:357-64
23． R.Andreesen, J.Osterholz, HH.Bodemann, KJ.Bross, U.Costabel, etal. Expression of transferring receptors and intracellular ferritin during terminal differentiation of human monocytes. Blut 1984; 49: 195
24． Wright.SD. Multiple receptors for endoxin. Curr Opin Immunol 1991; 3: 83-90
25． E.Ferrero, SM.Goyert. Nucleotide of the gene encoding the monocyte differentiation antigen, CD14． Nucleic Acids Res 1988; 16: 4173
26． M.Setoguchi, N.Nasu, S.Yoshida, Y.Higuchi, S.Akizuki, and S.Yamamoto. Mouse and human CD14 (Myeloid Cell-specific Leucine-rich Glycoprotein) primary
structure deduced from CDNA clones. Biochem Biophys Acta 1989; 1008: 213-222
27． Med Miorobiol Immunol 1994; 183: 279-297
28． R.Andreesen, B.Hennemann. Adoptive immunotherapy with autologous macrophages: current status and future perspectives. Pathobiology 1991; 59: 259
29． R.Andreesen, C.Schibenbogen, W.Brugger, S.Krause, HG.Meerpohl, etal. Adoptive transfer of tumorcytotoxic macrophages generated in vitro from circulating blood monocytes: a new approach to cancer immunotherapy. Cancer Res 1990; 50: 7450
30． Evans R, Alexander P. Role of macrophages in tumor immunity. Immunology 1972; 23: 615
31． Hibbs JB, Chapman HA, Weinberg JB. The macrophages as an antineoplastic surveillance cell: biological perspectives. J Reticuloendothel Soc 1978; 24: 549
32． Chakraborty NG, Okino T, Stabach P, etal. Adoptive transfer of activated human autologous macrophages results in regression of transplanted human melanoma cells in scid mice. In Viro 1991; 5: 609-14
33． Bartholeyns J. Lombard Y, Poindron P. Immunotherapy of murine sarcoma by adoptive transfer of resident peritoneal macrophages proliferating in culture. Anticancer Res 1988; 8: 145-52
34． Chokri M, Freudenberg M, Galanos C, etal. Antitumoral effects of lipopolysaccharides, tumor necrosis factor, interferon and activated macrophages: synergism and tissue distribution. Anticancer Res 1989; 9: 1185-90
35． AA.Loosdrecht, GJ.Ossenkoppele, RH.J.Beelen, MG.Broekhoven, and MM.A.C.Langenhuijsen. Role of interferon-Y and tumor necrosis factor a in monocyte-mediated cytostasis and cytotoxicity against a human histiocytic lymphoma cell line. Cancer Immunol Immunother 1992; 34: 393-398
36． Cameron DJ, and WH.Churchill. Cytotoxicity of human macrophages for
tumor cells: enhancement by bacterial lipopolysaccharides (LPS). J Immunol 1980; 124: 708
37． Peri G, N. Polentarutti, C.Sessa, C.Mangiotii, and A.Mantovani. Tumoricidal activity of macrophages isolated from human ascitic and solid ovarian carcinomas:augmentation by interferon, lymphokines and endotoxin. Int. J. Cancer 1981; 28: 143
38． Boraschi D, and A.Tagliabue. Interferon-induced enhancement of macrophage-mediated tumor cytolysis and its difference from activation by lymphokines. Eur. J. Immunol 1981; 11: 110
39． Boraschi D, D.Soldateschi, and A.Tagliabue. Macrophage activation by interferon: dissociation between tumoricidal capacity and suppressive activity. Eur. J. Immunol 1982; 12: 320
40． Mantovani A, J.H.Dean, T.RJerrells, and R.B.Herberman. Augmentation of tumoricidal activity of human monocytes and macrophages by lymphokines. Int. J. Cancer 1980; 25: 691
41． Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, and Mathison JC. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 1990;249:1431-1433
42． Shumann RR,Leong SR, Flaggs GW, Gray PW, Wright SD, Mathison JC, Tobias PS, and Ulevithch RJ. Structure and function of lipopolysaccharide binding protein. Science 1990; 249: 1429-1430．
43． Schwamberger G, Flesch I, Ferber E. Characterisation and purification of a high molecular weight tumoridal activity secreted by murine bone marrow macrophage. Int Immunol 1992; 4: 253
44． 曹雪涛.白细胞介素2的基础与临床,北京科技技术出版社,1990．
45． Kubin M, Chow JM, Trinchieri G. Differential regulation of interleukin-12
(IL-12) ,tumor necrosis factor a ,and IL-1 3 production in human myeloid leukemia cell lines and peripheral blood mononuclear cells. Blood 1994;83:1047-55
46． Gyorgh H, David GK, Jiang FC, MA,.Schwarzschild, EA.Deitch,etal. Adenosine inhibits IL-12 and TNF-a production via adenosine A2a receptor-dependent and independent mechanisms. FASEB 2000;14:2065
47． J.Liu, M.Chen, and X.Wang. Calcitonin gene-related peptide inhibits lipopolysaccharide-induced interleukin-12 release from mouse peritoneal macrophages,mediated by the cAMP pathway. Immunol 2000;101:61-67
48． Munker R, Dipersio J, Koeffler HP. Tumor necrosis factor: receptors on hematopietic cells. Blood 1987;70:1730
49． Pennica D, Nedwin E, Hayflick JS, Seeburg PH, Derynck R,etal. Human tumour necrosis factor: precursor structure, expression and homology to lymphotoxin. Nature 1984;312:724
50． Urban JL, Shepard HM, Rothstein JL, Sugarman J, Schreiber H. Tumor necrosis factor: a potent effector molecule for tumor cell killing by activated macrophages. Proc Natl Acad Sci USA 1986;83:5233
51． Goossens V, Grooten J, Vos KD and Fiers W. Direct evidence for rumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity. 1995;92:8115-8119
52． Leist M, Gantner F, Jilg S, etal. Activation of the 55Kda TNF receptor is necessary and sufficient for TNF-induced liver failure, hepatocyte apoptosis, and nitrite release. J Immunol 1995;54:1307
53． Grell M, Douni E, Wajant H, etal. The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80Kda tumor necrosis factor receptor. Cell 1995;83:793-802
54． Decker T, Lohmann Matthes ML, Gifford GE. Cell-associated tumor necrosis
factor (TNF) as a killing mechanism of activated cytotoxic macrophages. J Immunol 1987;138:957
55． Philip R, Epstein LB. Tumor necrosis factor as immuro-modulator and mediator of monocyte cytotoxicity induced by itself, y-interferon and interleukin-1． Nature 1986;323:86
56． JA.Hendrzak, MJ.Brunda. Interleukin-12: biologic activity, therapeutic utility, and role in disease. Laboratory Investigation 1995;72(6) :619
57． Brunda MJ, Luistro L, Warrier RR, Wright RB, Hubbard BR, Murphy M, etal. Antitumor and antimetastatic activity of interleukin-12 against murine tumors. J Exp Med 1993;178:1223-30
58． Gately MK, Gubler U, Brunda MJ, Nadeau RR, Anderson TA, Lipman JM,etal. Interleukin-12: a cytokine with therapeutic potential in oncology and infectious diseases. Therapeutic Immunol 1994;1: 187-96
59． Nastala CL, Edington HD, Mckinney TG, Tahara H, Naslenik MA, Brunda MJ, etal. Recombinant IL-12 administration induces tumor regression in association with IFN-y production. J Immunol 1994; 153:1697-706
60． Stem LL, Tarby CM, Tamborini B, Truitt GA. Preclinical development of IL-12 as an anticancer drug: comparison to IL-2 (abstract). Proc Am Assoc Cancer Res 1993;34:520
61． Verbik DJ, Stinson WW, Brunda MJ, Kessinger A, Joshi SS. Therapeutic effects of in vivo administration of Interleukin-12 against residual lymphoma (abstract). Proc Am Assoc Cancer Res 1995;36:480
62． Geldhof AB, Moser M, Lespagnard L, etal. Interleukin-12-activated natural killer cells recognize B7 costimulatory molecules on tumor cells and autologous dendritic cells. Blood 1998;91:196
63． Mori S, Jewett A, Murakami MK,etal. The participation of the Fas-mediated
cytotoxic pathway by natural killer cells is tumor-cell-dependent. Cancer Immunol Immunother 1997;44:2o2
64． Nanni P, Rossi I, Giovanni CD, etal. Interleukin-12 gene therapy of MHC-negative murine melanoma metastases. Cancer Res 1998;58:1225
65． Hiscox S, Hallett MB, Puntis MC, etal. Inhibition of cancer cell motility and invasion by interleukin-12． Clin Exp Metastasis 1995; 13:396
66． Tsung K, Meko JB, Peplinski GR, etal. IL-12 induces T helper 1-directed antitumor response. J Immunol 1997; 158:3359
2． Fidler, I.J. Inhibition of pulmonary metastasis by intravenous injection of specification activated macrophages. Cancer Res 1974; 34:1074-1078
3． Berdel,W.E., Bausert,W.R., Weltaien,H.U., etal. The influence of alkyl-lysophospholipids and lysophospholipid-activated macrophages on the development of metastasis of 3-lewis lung carcinoma. Eur J Cancer 1980; 16: 1199-1204
4． Figdor.C, Bont.W.S, Vries.J.D, etal. Isolation of large numbers of highly purified lymphocytes and monocytes with a modified centrifugal elutriation technique. J Immunol Methods 1981; 40:275-288
5． Stevenson HC, Keenan AM, Woodhouse C, Ohaw RI, Miller P, Steller EP, Foon KA, etal. Fate of IFN-gamma activated killer blood monocytes adoptively transferred into the abdominal cavity of patients with peritoneal carcinoma. Cancer Res 1987; 47:6100
6． Bartoleyns.J, Romet-lemonne.JL, Chokri.M, Lopez.M. Immune therapy with macrophages: present status and critical requirements for implementation. Immunobiol 1996; 195:550-562
7． B.Hennemann, A.Rehm,A.Kottke, N.Meidenbauer, and R.Andreesen. Adoptive immunotherapy with tumor-cytotoxic macrophages derived from recombinant human granulocyte-macrophage colony-stimulating factor (rhu GM-CSF) mobilized peripheral blood monocytes. J Immunother 1997; 20(5) : 365-371
8． B.Hennemann, G.Beckmann, A.Eichelmann, A.Rehm, and R.Andereesen. Phase I trial of adoptive immunotherapy of cancer patients using momocyte-derived macrophages activated with interferon y and lipopolysaccharide. Cancer Immunol
Immunother 1998; 45: 250-256
9． R.Adreesen, KL.Bross, J.Osterholz, and F.Emmrich. Human macrophage maturation and heterogeneity with a newly generated set of monoclonal antibodies to differentiation antigens. Blood 1986; 67: 1257
10． R.Adreesen, J.Picht, and G.W.Lohr. Primary cultures of human blood-born macrophages grown on hydrophobic teflon membranes. J Immunol Meth 1983; 56: 295
11 .R.Adreesen, C.Scheibenbogen, M.Kreutz, S.Krause, and A.Rehm. Regulation of neopterm secretion during monocyte to macrophage differentiation in vitro. Pteridins 1990; 23:1140
12． C.Scheibenbogen, and R.Andreesen. Developmental regulation of the cytokine repertoire in human macrophages: IL-2, IL-6, TNF-a , and M-CSF. J Leukocyte Biol 1991; 34:1236
13． R.Adreesen, C.Scheibenbogen, H.Moser, S.Krause, and H.Engler. Adoptive transfer of activated monocyte-derived macrophages initiates the coagulation cascade. Proc. Am. Assoc. Cancer Res 1990; 31: 245
14． RB.Herberman, JR.Ortaldo, A.Mantovani, DS.Hobbs, HF.Kung, and S.Pestka. Effect of human recombinant interferon on cytotoxic activity of natural killer (NK) and monocytes. Cell Immunol 1982; 67: 160-167
15． R.Adreesen, S.Gadd, W.Brugger, GW.Lohr, and RC.Atkins. Activation of human monocyte-derived macrophages cultured on teflon: response to interferon-Y during terminal maturation in vitro. Immunobiol 1988; 177: 186-198
16． Meerchaert I.M, B.Furie. Monocytes use either CD11/CD58 or VLA4 to migrate across human erdothelium in vitro. J Immunol 1994; 152: 1915
17． M.Lopez, J.Fechtenbaum, B.David, C.Martinache, M.Chokri, S.Canepa, etal. Adoptive immunorherapy with activated macrophages grown in vitro from monocytes
in cancer patients: a pilot study. J Immunother 1992; 11: 209-217
18． J.C.Eymard, M.Lopez, A.Cattan, Q.Bouche, J.C.Adjizian, and J.Bernard. Phase I /II trial of autologous activated macrophages in advanced colorectal cancer. Euro J Cancer Vol 1996; 11: 1905-1911
19． B.Hennemann, C.Scheibenbogen, C.Schumichen, and R.Andreesen. Intrahepatic adoptive immunotherapy with autologous tumorcytotoxic macrophages in patients with cancer. J Immunother 1995; 18 (1) : 19-27
20． Broxmeyer HE,Cooper S.Williams DE,et al.Growth characteristics of marrow hematopoietic progenitor/precursor cells from patients on a phase I clinical trial with purified recombinant human granulocyte-macrophage colony-stimulating factor. Exp Hematol 1988; 16: 594-602
21． Hermann F,Schulz G,Lindemann A,et al. Hematopoietic responses in patients with advanced malignancy treated with recombinant human granulocyte-macrophage colony-stimulating factor. J Clin Oncol 1989; 7: 159-67
22． Lieschke GL,Maher D.Cebon J,et al. Effects of bacterial synthesized recombinant human granulocyte-macrophage colony-stimulating factor in patients with advanced malignancy. Ann Intern Med 1989; 110:357-64
23． R.Andreesen, J.Osterholz, HH.Bodemann, KJ.Bross, U.Costabel, etal. Expression of transferring receptors and intracellular ferritin during terminal differentiation of human monocytes. Blut 1984; 49: 195
24． Wright.SD. Multiple receptors for endoxin. Curr Opin Immunol 1991; 3: 83-90
25． E.Ferrero, SM.Goyert. Nucleotide of the gene encoding the monocyte differentiation antigen, CD14． Nucleic Acids Res 1988; 16: 4173
26． M.Setoguchi, N.Nasu, S.Yoshida, Y.Higuchi, S.Akizuki, and S.Yamamoto. Mouse and human CD14 (Myeloid Cell-specific Leucine-rich Glycoprotein) primary
structure deduced from CDNA clones. Biochem Biophys Acta 1989; 1008: 213-222
27． Med Miorobiol Immunol 1994; 183: 279-297
28． R.Andreesen, B.Hennemann. Adoptive immunotherapy with autologous macrophages: current status and future perspectives. Pathobiology 1991; 59: 259
29． R.Andreesen, C.Schibenbogen, W.Brugger, S.Krause, HG.Meerpohl, etal. Adoptive transfer of tumorcytotoxic macrophages generated in vitro from circulating blood monocytes: a new approach to cancer immunotherapy. Cancer Res 1990; 50: 7450
30． Evans R, Alexander P. Role of macrophages in tumor immunity. Immunology 1972; 23: 615
31． Hibbs JB, Chapman HA, Weinberg JB. The macrophages as an antineoplastic surveillance cell: biological perspectives. J Reticuloendothel Soc 1978; 24: 549
32． Chakraborty NG, Okino T, Stabach P, etal. Adoptive transfer of activated human autologous macrophages results in regression of transplanted human melanoma cells in scid mice. In Viro 1991; 5: 609-14
33． Bartholeyns J. Lombard Y, Poindron P. Immunotherapy of murine sarcoma by adoptive transfer of resident peritoneal macrophages proliferating in culture. Anticancer Res 1988; 8: 145-52
34． Chokri M, Freudenberg M, Galanos C, etal. Antitumoral effects of lipopolysaccharides, tumor necrosis factor, interferon and activated macrophages: synergism and tissue distribution. Anticancer Res 1989; 9: 1185-90
35． AA.Loosdrecht, GJ.Ossenkoppele, RH.J.Beelen, MG.Broekhoven, and MM.A.C.Langenhuijsen. Role of interferon-Y and tumor necrosis factor a in monocyte-mediated cytostasis and cytotoxicity against a human histiocytic lymphoma cell line. Cancer Immunol Immunother 1992; 34: 393-398
36． Cameron DJ, and WH.Churchill. Cytotoxicity of human macrophages for
tumor cells: enhancement by bacterial lipopolysaccharides (LPS). J Immunol 1980; 124: 708
37． Peri G, N. Polentarutti, C.Sessa, C.Mangiotii, and A.Mantovani. Tumoricidal activity of macrophages isolated from human ascitic and solid ovarian carcinomas:augmentation by interferon, lymphokines and endotoxin. Int. J. Cancer 1981; 28: 143
38． Boraschi D, and A.Tagliabue. Interferon-induced enhancement of macrophage-mediated tumor cytolysis and its difference from activation by lymphokines. Eur. J. Immunol 1981; 11: 110
39． Boraschi D, D.Soldateschi, and A.Tagliabue. Macrophage activation by interferon: dissociation between tumoricidal capacity and suppressive activity. Eur. J. Immunol 1982; 12: 320
40． Mantovani A, J.H.Dean, T.RJerrells, and R.B.Herberman. Augmentation of tumoricidal activity of human monocytes and macrophages by lymphokines. Int. J. Cancer 1980; 25: 691
41． Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, and Mathison JC. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 1990;249:1431-1433
42． Shumann RR,Leong SR, Flaggs GW, Gray PW, Wright SD, Mathison JC, Tobias PS, and Ulevithch RJ. Structure and function of lipopolysaccharide binding protein. Science 1990; 249: 1429-1430．
43． Schwamberger G, Flesch I, Ferber E. Characterisation and purification of a high molecular weight tumoridal activity secreted by murine bone marrow macrophage. Int Immunol 1992; 4: 253
44． 曹雪涛.白细胞介素2的基础与临床,北京科技技术出版社,1990．
45． Kubin M, Chow JM, Trinchieri G. Differential regulation of interleukin-12
(IL-12) ,tumor necrosis factor a ,and IL-1 3 production in human myeloid leukemia cell lines and peripheral blood mononuclear cells. Blood 1994;83:1047-55
46． Gyorgh H, David GK, Jiang FC, MA,.Schwarzschild, EA.Deitch,etal. Adenosine inhibits IL-12 and TNF-a production via adenosine A2a receptor-dependent and independent mechanisms. FASEB 2000;14:2065
47． J.Liu, M.Chen, and X.Wang. Calcitonin gene-related peptide inhibits lipopolysaccharide-induced interleukin-12 release from mouse peritoneal macrophages,mediated by the cAMP pathway. Immunol 2000;101:61-67
48． Munker R, Dipersio J, Koeffler HP. Tumor necrosis factor: receptors on hematopietic cells. Blood 1987;70:1730
49． Pennica D, Nedwin E, Hayflick JS, Seeburg PH, Derynck R,etal. Human tumour necrosis factor: precursor structure, expression and homology to lymphotoxin. Nature 1984;312:724
50． Urban JL, Shepard HM, Rothstein JL, Sugarman J, Schreiber H. Tumor necrosis factor: a potent effector molecule for tumor cell killing by activated macrophages. Proc Natl Acad Sci USA 1986;83:5233
51． Goossens V, Grooten J, Vos KD and Fiers W. Direct evidence for rumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity. 1995;92:8115-8119
52． Leist M, Gantner F, Jilg S, etal. Activation of the 55Kda TNF receptor is necessary and sufficient for TNF-induced liver failure, hepatocyte apoptosis, and nitrite release. J Immunol 1995;54:1307
53． Grell M, Douni E, Wajant H, etal. The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80Kda tumor necrosis factor receptor. Cell 1995;83:793-802
54． Decker T, Lohmann Matthes ML, Gifford GE. Cell-associated tumor necrosis
factor (TNF) as a killing mechanism of activated cytotoxic macrophages. J Immunol 1987;138:957
55． Philip R, Epstein LB. Tumor necrosis factor as immuro-modulator and mediator of monocyte cytotoxicity induced by itself, y-interferon and interleukin-1． Nature 1986;323:86
56． JA.Hendrzak, MJ.Brunda. Interleukin-12: biologic activity, therapeutic utility, and role in disease. Laboratory Investigation 1995;72(6) :619
57． Brunda MJ, Luistro L, Warrier RR, Wright RB, Hubbard BR, Murphy M, etal. Antitumor and antimetastatic activity of interleukin-12 against murine tumors. J Exp Med 1993;178:1223-30
58． Gately MK, Gubler U, Brunda MJ, Nadeau RR, Anderson TA, Lipman JM,etal. Interleukin-12: a cytokine with therapeutic potential in oncology and infectious diseases. Therapeutic Immunol 1994;1: 187-96
59． Nastala CL, Edington HD, Mckinney TG, Tahara H, Naslenik MA, Brunda MJ, etal. Recombinant IL-12 administration induces tumor regression in association with IFN-y production. J Immunol 1994; 153:1697-706
60． Stem LL, Tarby CM, Tamborini B, Truitt GA. Preclinical development of IL-12 as an anticancer drug: comparison to IL-2 (abstract). Proc Am Assoc Cancer Res 1993;34:520
61． Verbik DJ, Stinson WW, Brunda MJ, Kessinger A, Joshi SS. Therapeutic effects of in vivo administration of Interleukin-12 against residual lymphoma (abstract). Proc Am Assoc Cancer Res 1995;36:480
62． Geldhof AB, Moser M, Lespagnard L, etal. Interleukin-12-activated natural killer cells recognize B7 costimulatory molecules on tumor cells and autologous dendritic cells. Blood 1998;91:196
63． Mori S, Jewett A, Murakami MK,etal. The participation of the Fas-mediated
cytotoxic pathway by natural killer cells is tumor-cell-dependent. Cancer Immunol Immunother 1997;44:2o2
64． Nanni P, Rossi I, Giovanni CD, etal. Interleukin-12 gene therapy of MHC-negative murine melanoma metastases. Cancer Res 1998;58:1225
65． Hiscox S, Hallett MB, Puntis MC, etal. Inhibition of cancer cell motility and invasion by interleukin-12． Clin Exp Metastasis 1995; 13:396
66． Tsung K, Meko JB, Peplinski GR, etal. IL-12 induces T helper 1-directed antitumor response. J Immunol 1997; 158:3359
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