膜结合型巨噬细胞集落刺激因子对巨噬细胞功能性极化影响的探索

 目的　探讨高表达膜结合型巨噬细胞集落刺激因子（mM-CSF）的血液肿瘤细胞对巨噬细胞的异常活化作用。方法　采用稳定表达mM-CSF的淋巴瘤细胞系Namalwa-M和巨噬细胞系RAW264.7体外共培养体系,以转入空载体的Namalwa-V细胞系共培养体系为对照组。流式细胞术检测RAW264.7的CD206（选择性活化巨噬细胞高表达的表面分子）和细胞内白细胞介素（IL）-6、IL-10、IL-12及TNF-α的表达水平;墨汁吞噬实验检测共培养RAW264.7细胞的功能变化。 结果　与Namalwa-M共培养的RAW264.7 的CD206表达水平明显升高平均均荧光强度为55.12±3.77,提示其巨噬细胞活性增强;IL-10、TNF-α表达上调（201±6）%、（136±6）%;IL-12、IL-6表达下调（990±528）%、（60±26）%;吞噬能力明显提高。结论　高表达mM-CSF的血液肿瘤细胞可异常活化巨噬细胞成为高表达IL-10、TNF-α,低表达IL-12、IL-6的异常活化巨噬细胞。     

转GM-CSF基因瘤苗联合IL-12治疗T细胞淋巴瘤的实验研究

目的　探讨用转粒细胞巨噬细胞 集落刺激因子 (GM CSF)基因瘤苗 ,联合白介素 12 (IL 12 )治疗小鼠T细胞淋巴瘤的方法。方法　将小鼠GM CSF真核表达质粒 ,用电穿孔法导入小鼠T细胞淋巴瘤细胞系RMA ,有限稀释法制备单个细胞克隆 ,经RT PCR、骨髓祖细胞增殖实验和集落形成实验 ,筛选相对高表达GM CSF的RMA克隆 ,该克隆细胞用丝裂霉素灭活后免疫小鼠 ,以诱导产生抵抗RMA肿瘤细胞再攻击的能力 ,并观察其与IL 12联合应用对淋巴瘤的治疗作用。结果　获得了高表达GM CSF的小鼠T细胞淋巴瘤克隆 ,其动物体内致瘤活性降低 ,将其用丝裂霉素灭活后作为瘤苗可使小鼠产生抗肿瘤的免疫保护力 ,与IL 12联合应用增强其抗肿瘤活性。结论　转GM CSF基因瘤苗可作为有效的抗肿瘤瘤苗 ,与IL 12联合应用较其单独应用更为有效     

G-CSF/G-CSFR在实体肿瘤中的研究进展

粒细胞集落刺激因子(G-CSF)是一种多肽链的粒细胞生长因子,其与粒细胞集落刺激因子受体(G-CSFR)相结合,可以促进骨髓细胞的增殖、分化和迁移.G-CSFR主要表达于造血干细胞、髓祖细胞和成熟粒细胞等造血细胞表面.因此,以往关于G-CSF/G-CSFR的研究多集中于造血系统,然而,近年来的肿瘤研究显示G-CSF和G-CSFR在肺癌、胃癌、结肠癌、膀胱癌、食管肉瘤、脑胶质瘤等多种实体瘤中均有一定的表达.G-CSF和G-CSFR可能通过影响炎性浸润的免疫细胞以及骨髓髓系细胞的分化,改变实体肿瘤的微环境,促进肿瘤细胞增殖、抑制肿瘤细胞凋亡或诱导血管形成,参与肿瘤的发生、发展,并且可能对肿瘤细胞的干性特征产生一定的影响.因此,本文主要就G-CSF/G-CSFR调节肿瘤免疫微环境以及肿瘤细胞干性特征方面的研究作一简要综述.     

粒细胞-巨噬细胞集落刺激因子联合放疗研究进展

粒细胞-巨噬细胞集落刺激因子（granulocyte-macrophage colony-stimulating factor，GM-CSF）作为一种造血因子可以有效诱导多种具有抑瘤效应的免疫细胞增殖，从而发挥抗肿瘤免疫反应。放疗作为肿瘤治疗的主要手段之一，不仅可以直接杀伤肿瘤细胞，而且会对抗肿瘤免疫产生影响。多项临床研究提示，放疗联合GM-CSF可以诱导产生旁观者效应并增强对肿瘤的远期控制，从而增强放疗的抗肿瘤效应。本文就GM-CSF及GM-CSF联合放疗的研究进展予以综述。      

粒细胞集落刺激因子及其受体在肺癌中表达与作用的研究进展

粒细胞集落刺激因子(granulocyte colony-stimulating factor,G-CSF)被广泛应用于治疗肿瘤放、化疗所致的白细胞减少.研究发现,多种实体肿瘤中有G-CSF及粒细胞集落刺激因子受体(G-CSF receptor, G-CSFR)的表达,且G-CSF可通过G-CSF/G-CSFR的自分泌/旁分泌通路与环氧化酶通路等影响肿瘤细胞增殖、凋亡和微血管形成,但其确切机制尚需要进一步研究.本文就G-CSF 及G-CSFR在肺癌中的表达及其作用机制的研究进展作综述.     

GM-CSF基因重组腺病毒载体的构建及鉴定

目的：构建人粒细胞-巨噬细胞集落刺激因子（GM-CSF）基因的重组腺病毒载体，为进一步研究GM-CSF基因在肿瘤基因治疗中的应用提供实验基础。方法：采用PCR方法，从重组质粒pcDNA3．1-GM-CSF扩增出GM-CSF基因片段，通过穿梭质粒pShuttle，将带有CMV启动子的目的片段克隆入Adeno-X腺病毒DNA中，获得重组腺病毒DNA，通过脂质体转染HEK293细胞，经包装扩增后，获得重组腺病毒Adeno-X-GM-CSF，PCR鉴定，ELISA法检测表达产物。结果：含GM-CSF基因的重组腺病毒Adeno-X-GM-CSF构建成功，经PCR鉴定和DNA测序等证实了其正确性，重组腺病毒上清液中GM-CSF表达量达26ng／mL。结论：含GM-CSF基因的重组腺病毒构建成功。     

巨噬细胞集落刺激因子与肿瘤

巨噬细胞集落刺激因子(M-CSF)是一具有谱系特异性的细胞因子，刺激巨噬细胞集落形成。最近研究发现许多肿瘤的M-CSF表达异常。M-CSF参与了肿瘤的发生发展过程，对肿瘤的生长、侵袭和转移以及新生血管的形成均有重要的作用，在肿瘤的治疗研究中也取得了一系列进展。     

免疫粘附素:一种新的研究和生物治疗工具——GM-CSF/Fc_(γ2)融合蛋白载体的构建与表达

粒细胞集落刺激因子和粒细胞-巨噬细胞等集落刺激因子是肿瘤病人化疗后提高机体造血和免疫功能所必须的药物,但由于各种集落刺激因子在体内的半衰期极短,因此须使用较大的剂量,而这又将产生明显的毒副作用.延长这些集落刺激因子在体内的半衰期,减少其用量有可能减轻毒副作用.免疫粘附素(Immunoadhesin)由于其特有的生物学活性,是近年来受人瞩目的一种新的研究和生物治疗工具.     

瘤内/瘤周注射巨噬细胞集落刺激因子或(和)干扰素γ基因转染的巨噬细胞对肿瘤的治疗作用

目的观察巨噬细胞集落刺激因子（ＭＣＳＦ）和干扰素γ（ＩＦＮγ）基因单独或联合转染的巨噬细胞（ｍａｃｒｏｐｈａｇｅ，Ｍ）对局部黑色素瘤的治疗效果及相关免疫机理。方法荷瘤小鼠经基因转染的巨噬细胞治疗后，观察其长期存活期并检测其体内抗肿瘤免疫功能。用４小时５１Ｃｒ释放法检测荷瘤小鼠脾细胞自然杀伤细胞（ＮＫ）、细胞毒Ｔ淋巴细胞（ＣＴＬ）活性，间接ＭＴＴ法检测荷瘤小鼠腹腔巨噬细胞的杀伤活性，常规方法检测脾细胞诱导上清中肿瘤坏死因子（ＴＮＦ）、白细胞介素２（ＩＬ２）、γ干扰素（ＩＦＮγ）活性，局部肿瘤经治疗后行常规病理分析。结果经ＩＦＮγ基因及联合基因转染的巨噬细胞治疗后的荷瘤小鼠有２５％能长期存活，体外诱导的ＣＴＬ和小鼠腹腔巨噬细胞杀伤活性显著增强，脾细胞经诱导产生的细胞因子有不同程度的增加，与对照组相比差别显著。常规病理显示，ＭＣＳＦ和ＩＦＮγ联合基因转染的巨噬细胞治疗的小鼠肿瘤局部有大量的淋巴细胞浸润。结论ＭＣＳＦ和ＩＦＮγ基因转染的巨噬细胞瘤内瘤周注射对肿瘤有较好的治疗效果，其机制除了与巨噬细胞在局部直接接触杀伤瘤细胞有关外，宿主抗肿瘤免疫功能的增强亦起了重要的作用     

人白血病细胞系J6-1变异M-CSF的表达及受体结合活性研究

目的：从人白血病细胞系J6－1克隆并表达变异的M－CSF的功能区，研究其受体结合活性。方法：RT－PCR克隆muM-CSF并在大肠杆菌BL21trxB(DE3),pET32c( )系统中表达；镍柱鏊合层析，抗体亲和层析纯化。ELISA法测定其受体结合活性和解离常数，并测定对J6－1细胞增殖刺激的活性。结果；muM-CSF可在本文实验系统呈可溶性表达，纯化产物具有M－CSF受体的结合活性，解离常数为3.7nmol/L低于正常M－CSF，且刺激细胞体外增殖能力大于正常M－CSF。结论：从人白血病细胞系J6－1克隆的muM-CSF能在BL21，pET32c( )系统表达，可得电泳纯产物。     

Granulocyte‐colony‐stimulating factor enhances invasive potential of human head‐and‐neck‐carcinoma cell lines

Granulocyte‐colony‐stimulating factor (G‐CSF), a hematopoietic cytokine, regulates the proliferation and differentiation of granulocytic progenitor cells and functionally activated mature neutrophils. G‐CSF also affects non‐hematopoietic tumor cells by the binding of G‐CSF to its specific receptor (G‐CSFR) on the cells. In this study, we investigated the effect of G‐CSF on the invasive potential of head‐and‐neck carcinoma cells, and explored the intracellular events initiated by the binding of G‐CSF in tumor cells. In vitro treatment of head‐and‐neck‐carcinoma cell lines, IMC‐2, IMC‐3, KB, Ca9‐22, SCCKN and SCCTF, with recombinant G‐CSF (rG‐CSF) significantly augmented their invasive potential in dose‐ and time‐dependent manners. Among these cancer cells, IMC‐2, IMC‐3, KB and Ca9‐22 cells produced little G‐CSF, while large amounts of G‐CSF were produced by SCCKN and SCCTF cell lines. Anti‐G‐CSF antibody (Ab) abrogated the rG‐CSF‐enhanced invasiveness to the control level of that in untreated cancer cell lines. Immunocytochemical staining and Western blotting using anti‐G‐CSFR monoclonal antibody (MAb) revealed the expression of G‐CSFR on head‐and‐neck‐cancer cell lines exhibiting the enhancement of invasive activity by rG‐CSF. IMC‐2 cells, having the highest invasive ability among the cell lines used, showed augmentation of G‐CSFR expression on stimulation with rG‐CSF. Furthermore, stimulation of IMC‐2 cells with rG‐CSF induced rapid activation of tyrosine‐phosphorylated JAK1, suggesting that the G‐CSF signal may be transduced into the cells through G‐CSFR. Moreover, the gelatinolytic activity of IMC‐2 cells was enhanced by stimulation of rG‐CSF, and the enhanced invasiveness was inhibited on addition of the tissue inhibitors of metalloproteinases (TIMPs). These results suggest that exogenous rG‐CSF may increase the risk of metastasis and/or local recurrence in patients with G‐CSFR‐positive head‐and‐neck squamous‐cell carcinoma, via an invasive mechanism. Int. J. Cancer 80:78–84, 1999. © 1999 Wiley‐Liss, Inc.     

Carcinoembryonic antigen‐related cell adhesion molecule 1 negatively regulates granulocyte colony‐stimulating factor production by breast tumor‐associated macrophages that mediate tumor angiogenesis

Carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1), a cell adhesion molecule expressed on epithelial cells and activated immune cells, is downregulated in many cancers and plays a role in inhibition of inflammation in part by inhibition of granulocyte colony‐stimulating factor (G‐CSF) production by myeloid cells. As macrophages are associated with a poor prognosis in breast cancer, but play important roles in normal breast, we hypothesized that CEACAM1 downregulation would lead to tumor promotion under inflammatory conditions. Cocultures of proinflammatory M1 macrophages with CEACAM1 negative MCF7 breast cells produced high levels of G‐CSF (10 ng/mL) compared to CEACAM1‐transfected MCF7/4S cells (1 ng/mL) or anti‐inflammatory M2 macrophage cocultures (0.5 or 0.1 ng/mL, MCF7 or MCF7/4S, respectively). The expression of CEACAM1 on M1s was much greater than for M2s and was observed only in cocultures with either MCF7 or MCF7/4S cells. When M1 macrophages were mixed with MCF7 cells and implanted in murine mammary fat pads of nonobese diabetic/severe combined immunodeficient mice, tumor size and blood vessel density were significantly greater than MCF7 or MCF7/4S only tumors which were hardly detected after 8 weeks of growth. In contrast, M1 cells had a much reduced effect on MCF7/4S tumor growth and blood vessel density, indicating that the tumor inhibitory effect of CEACAM1 is most likely related to its anti‐inflammatory action on inflammatory macrophages. These results support our previous finding that CEACAM1 inhibits both G‐CSF production by myeloid cells and G‐CSF‐stimulated tumor angiogenesis.     

Supplementary granulocyte macrophage colony‐stimulating factor to chemotherapy and programmed death‐ligand 1 blockade decreases local recurrence after surgery in bladder cancer

Despite advances and refinements in surgery and perioperative chemotherapy, there are still unmet medical needs with respect to radical cystectomy for muscle‐invasive bladder cancer (MIBC). We investigated the potential benefit of supplementary granulocyte macrophage colony‐stimulating factor (GM‐CSF) to chemoimmunotherapy with programmed cell death protein‐1 (PD‐1)/programmed death‐ligand 1 (PD‐L1) axis blockade and standard neoadjuvant chemotherapy in bladder cancer. We inoculated 2 × 10⁵ MBT2 cells s.c. in C3H mice to create a syngeneic animal model of local recurrence (LR). When the tumor diameter reached 12 mm, the mice were allocated randomly as follows: (i) non‐treated control (vehicle only); (ii) anti‐mPD‐L1 monotherapy; (iii) mGM‐CSF monotherapy; (iv) anti‐mPD‐L1 plus mGM‐CSF; (v) gemcitabine and cisplatin (GC); (vi) GC plus anti‐mPD‐L1; (vii) GC plus mGM‐CSF; and (viii) GC plus anti‐mPD‐L1 plus mGM‐CSF. After completing 2‐week neoadjuvant therapy, tumors were resected for resection margin evaluation and immunohistochemical staining and blood was collected for flow cytometry and ELISA. Operative wounds were sutured, and the operative site was monitored to detect LR. Addition of anti‐mPD‐L1 and mGM‐CSF to neoadjuvant GC chemotherapy enhanced the antitumor effect and reduced positive resection margins (50% vs 12.5%). Combination of GC, anti‐mPD‐L1, and mGM‐CSF resulted in longer LR‐free survival and cancer‐specific survival compared to those in other groups. These effects involved an immunotherapy‐related decrease in oncological properties such as tumor invasion capacity and epithelial‐mesenchymal transition. mGM‐CSF significantly decreased the accumulation of myeloid‐derived suppressor cells in both the blood and tumor microenvironment and blood interleukin‐6 levels. Supplementary GM‐CSF to neoadjuvant GC plus PD‐L1 blockade could decrease LR after radical surgery by immune modulation in the blood and tumor microenvironment.     

M-CSF可溶性受体在烟草中的表达及其抗M-CSF作用研究

目的:在烟草中高效表达人M-CSF可溶性受体并研究其抗M-CSF活性.方法:采用基因重组技术构建表达C端含组氨酸标签和内质网滞留信号的M-CSF可溶性受体植物表达载体,通过根瘤土壤杆菌转化烟草,获得转基因植株.PCR、蛋白印迹实验鉴定转基因植株,ELISA方法测定重组蛋白表达水平,集落形成实验测定重组蛋白活性.结果:获得50余株转基因烟草植株,PCR证实目的基因已整合到烟草基因组中,蛋白印迹实验证实转基因植株表达目的蛋白;M-CSFsR在烟草中获得高效表达,但不同转基因植株的表达水平存在差异,其中表达水平最高的一株M-CSFsR占叶片可溶性蛋白的1.92%;集落形成实验证明M-CSFsR可以抑制J6-1细胞的集落形成.结论:在烟草中高效表达有生物学活性的重组人M-CSFsR.     

促红细胞生成素、粒细胞及粒-巨噬细胞集落刺激因子对慢性粒细胞白血病急变细胞生长的影响

为了解外源性细胞因子对慢性粒细胞白血病急变细胞增殖的影响，观察了促红细胞生成素、粒细胞集落刺激因子和粒－巨噬细胞集落刺激因子在体外对慢粒急变细胞生长的影响。实验结果显示慢粒急变细胞的增殖不受这些外源性细胞生长因子的影响，提示急变期细胞的生长特性发生了改变，同时这一结果也对细胞生长因子的临床应用有一定的意义     

Expression of granulocyte-colony-stimulating factor and its receptor in human Ewing sarcoma cells and patient tumor specimens: potential consequences of granulocyte-colony-stimulating factor administration

BACKGROUND: Ewing sarcoma (ES) is a highly vascular malignancy. It has been demonstrated that both angiogenesis and vasculogenesis contribute to the growth of ES tumors. Granulocyte-colony-stimulating factor (G-CSF), a cytokine known to stimulate bone marrow (BM) stem cell production and angiogenesis, is routinely administered to ES patients after chemotherapy. Whether ES cells and patient tumor samples express G-CSF and its receptor (G-CSFR) and whether treatment with this factor enhances tumor growth was examined. METHODS: Human ES cell lines were analyzed for expression of G-CSF and G-CSFR in vitro and in vivo. Sixty-eight paraffin-embedded and 15 frozen tumor specimens from patients with ES were also evaluated for the presence of G-CSF and G-CSFR. The in vivo effect of G-CSF on angiogenesis and BM cell migration was determined. Using a TC/7-1 human ES mouse model, the effect of G-CSF administration on ES tumors was investigated. RESULTS: G-CSF and G-CSFR protein and RNA expression was identified in all ES cell lines and patient samples analyzed. In addition, G-CSF was found to stimulate angiogenesis and BM cell migration in vivo. Tumor growth was found to be significantly increased in mice treated with G-CSF. The average tumor volume for the group treated with G-CSF was 1218 mm(3) compared with 577 mm(3) for the control group (P = .006). CONCLUSIONS: The findings that ES cells and patient tumors expressed both G-CSF and its receptor in vitro and in vivo and that the administration of G-CSF promoted tumor growth in vivo suggest that the potential consequences of G-CSF administration should be investigated further.     

ANTITUMOR EFFECT OF INTRATUMORAL INJECTION OF LIPOSOME-ENCAPSULATED G-CSF GENE AND IN SITU BIOLOGICAL CHARACTERISTICS OF THE TREATED TUMOR CELLS

ＡＮＴＩＴＵＭＯＲＥＦＦＥＣＴＯＦＩＮＴＲＡＴＵＭＯＲＡＬＩＮＪＥＣＴＩＯＮＯＦＬＩＰＯＳＯＭＥＥＮＣＡＰＳＵＬＡＴＥＤＧＣＳＦＧＥＮＥＡＮＤＩＮＳＩＴＵＢＩＯＬＯＧＩＣＡＬＣＨＡＲＡＣＴＥＲＩＳＴＩＣＳＯＦＴＨＥＴＲＥＡＴＥＤＴＵＭＯＲＣＥＬＬ...     

Development of macrophage and granulocyte colonies from human peripheral blood

The presence of macrophage and granulocyte progenitor cells in the human peripheral blood enabled the establishment of colonies from this accessible tissue and obviated the need for bone marrow to achieve this task. We have developed a method of obtaining reproducible growth of macrophage/granulocyte colonies from human peripheral blood. Colonies of macrophages and granulocytes were obtained by plating peripheral blood mononuclear cells (PBM) in methylcellulose containing medium in the presence of medium conditioned by nonstimulated PBM (CM). At early stages of colony growth both macrophage and granulocyte colonies were detected while following 20-25 days in culture all colonies tested revealed monocyte-macrophage morphology. To obtain higher numbers of colonies, we tested different cell sources, different CM preparations and the effect of steroid hormones on colony development. We found that the mononuclear cells obtained from cord blood (CB) or from some patients with inflammatory bowel disease yielded much higher numbers of colonies than PBM from normal individuals. Colony development from these two sources did not depend on an external source of colony stimulating factor (CSF) but was augmented as a result of CSF supplementation. CM obtained from CB mononuclear cells as well as supernatants from some human monoblastic cell lines were similar in their CSF activity to CM from normal PBM and made possible the development of macrophage/granulocyte colonies. Higher numbers of colonies were induced by including physiological concentrations of estradiol in the culture medium, in the absence of external sources of CSF. The system described above enabled the analysis of cloned macrophages and their circulating progenitor cells as well as the assay of different preparations of CSF.