趋化性细胞因子CCL18对黑素瘤细胞系生物学行为影响的研究
发布时间：2018-07-15 09:39
【摘要】：皮肤恶性黑素瘤是一种高度恶性肿瘤,目前研究认为任何细胞都能分泌细胞因子并表达其受体,而细胞因子在肿瘤细胞的表达已引起广泛的关注和研究,它们是肿瘤细胞侵袭转移的路标。部分黑素瘤细胞系能产生细胞因子并表达其受体,无论阻断配体还是受体都能抑制肿瘤细胞的生长。我们的前期研究,采用全人类基因组芯片对中国人肢端黑素瘤组织的研究发现,趋化性细胞因子CCL18在肿瘤组织的表达明显增高。本研究利用IL-4诱导人单核细胞产生的CCL18、重组CCL18细胞因子与A375黑素瘤细胞株共培养,探讨其在黑素瘤生物学行为方面的作用。 第一部分免疫组化研究CCL18在皮肤恶性黑素瘤的表达 使用免疫组化对67例黑素细胞来源的良、恶性肿瘤进行分析,结果显示良性痣、Spitz痣及发育不良性痣不表达CCL18,而黑素瘤表达CCL18,且CCL18的表达与肿瘤浸润深度呈正比。 第二部分人单核细胞的分离、诱导及CCL18表达的检测 使用密度梯度离心法成功分离人单核细胞,并使用IL-4成功诱导单核细胞,经ELISA和Western-blotting验证,诱导的单核细胞可产生CCL18,非诱导的单核细胞不能产生CCL18。 第三部分CCL18细胞因子对A375黑素瘤细胞株增殖、转移及血管增殖的影响 诱导人单核细胞产生CCL18,与重组人CCL18细胞因子,分别与A375黑素瘤细胞株共培养,通过MTT法检测CCL18对A375增殖的影响；通过趋化试验及重组基底膜侵袭实验,测定CCL18对肿瘤细胞的趋化能力及侵袭作用；通过将A375黑素瘤细胞株接种于鸡胚尿囊膜,测定不同情况下CCL18对肿瘤血管生成的影响。结果提示无论是诱导还是重组CCL18细胞因子都不是影响肿瘤增殖的主要细胞因子,IL-4诱导产生CCL18的单核细胞组及CCL18重组细胞因子组与对照组比较均对肿瘤细胞存在趋化作用,并促进肿瘤细胞的转移；IL-4诱导组及重组CCL18组在鸡胚尿囊膜试验中可促进肿瘤血管的生成作用。 第四部分CCL18细胞因子对A375黑素瘤细胞裸鼠内成瘤作用的影响 裸鼠成瘤的模型显示,CCL18细胞因子可能以某种机制抑制A375移植瘤体积的增加。肿瘤组织切片经Ki-67、Bcl-2免疫组化染色及TUNEL原位凋亡染色后分析,发现诱导及重组CCL18在A375黑素瘤细胞系体内试验中可促进肿瘤的增殖,抑制肿瘤细胞的凋亡。 第五部分小结 CCL18可能与黑素瘤的进展有关,并调节肿瘤的生物学行为。但需要更多研究以明确其作用机制及功能。
[Abstract]:Cutaneous malignant melanoma is a highly malignant tumor. At present, it is believed that any cell can secrete cytokines and express their receptors. However, the expression of cytokines in tumor cells has attracted extensive attention and research. They are landmarks for the invasion and metastasis of tumor cells. Some melanoma cell lines can produce cytokines and express their receptors, both blocking ligands and receptors can inhibit the growth of tumor cells. In our previous study, the expression of chemokine CCL18 in Chinese human limb melanoma tissues was significantly increased by using human genome microarray. In this study, we used IL-4 to induce human monocytes to produce CCL18. The recombinant CCL18 cytokines were co-cultured with A375 melanoma cell line to investigate its role in the biological behavior of melanoma. The expression of CCL18 in malignant melanoma of skin was analyzed by immunohistochemistry in 67 cases of malignant and benign melanocytes. The results showed that CCL18 was not expressed in benign nevus Spitz nevus and dysplastic nevus, but CCL18 was expressed in melanoma, and the expression of CCL18 was proportional to the depth of tumor invasion. The second part of human monocyte isolation, induction and detection of CCL18 expression using density gradient centrifugation method successfully isolated human monocytes, and IL-4 successfully induced monocytes, which were confirmed by Elisa and Western-blotting. CCL18 can be produced by induced monocytes, but not by uninduced monocytes. The effect of CCL18 cytokines on the proliferation, metastasis and vascular proliferation of A375 melanoma cell line induced the production of CCL18 by human monocytes, co-cultured with recombinant human CCL18 cytokines and A375 melanoma cell line, respectively. The effect of CCL18 on A375 proliferation was detected by MTT assay. The chemotaxis and invasion of tumor cells were determined by chemotaxis test and recombinant basement membrane invasion assay. The A375 melanoma cell line was inoculated into chicken embryo allantoic membrane, and A375 melanoma cell line was inoculated into chicken embryo allantoic membrane. The effect of CCL 18 on tumor angiogenesis was measured. The results suggested that neither the induced or recombinant CCL18 cytokines were chemotactic to the tumor cells induced by IL-4, the monocytes that produced CCL18 and the CCL18 recombinant cytokines had chemotactic effects on tumor cells compared with the control group. In addition, IL-4 induction of tumor cells and recombinant CCL18 could promote tumor angiogenesis in chicken embryo allantoic membrane test. The effect of CCL18 cytokines on the tumorigenesis of A375 melanoma cells in nude mice showed that CCL18 cytokines might inhibit the growth of A375 xenografts by some mechanism. By immunohistochemical staining of Ki-67Bcl 2 and Tunel in situ apoptosis staining, it was found that the induction and recombination of CCL18 in A375 melanoma cell line could promote the proliferation of tumor and inhibit the apoptosis of tumor cells. The fifth part concludes that CCL 18 may be related to the progression of melanoma and regulate the biological behavior of the tumor. But more research is needed to clarify its mechanism and function.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.5
本文编号：2123643
[Abstract]:Cutaneous malignant melanoma is a highly malignant tumor. At present, it is believed that any cell can secrete cytokines and express their receptors. However, the expression of cytokines in tumor cells has attracted extensive attention and research. They are landmarks for the invasion and metastasis of tumor cells. Some melanoma cell lines can produce cytokines and express their receptors, both blocking ligands and receptors can inhibit the growth of tumor cells. In our previous study, the expression of chemokine CCL18 in Chinese human limb melanoma tissues was significantly increased by using human genome microarray. In this study, we used IL-4 to induce human monocytes to produce CCL18. The recombinant CCL18 cytokines were co-cultured with A375 melanoma cell line to investigate its role in the biological behavior of melanoma. The expression of CCL18 in malignant melanoma of skin was analyzed by immunohistochemistry in 67 cases of malignant and benign melanocytes. The results showed that CCL18 was not expressed in benign nevus Spitz nevus and dysplastic nevus, but CCL18 was expressed in melanoma, and the expression of CCL18 was proportional to the depth of tumor invasion. The second part of human monocyte isolation, induction and detection of CCL18 expression using density gradient centrifugation method successfully isolated human monocytes, and IL-4 successfully induced monocytes, which were confirmed by Elisa and Western-blotting. CCL18 can be produced by induced monocytes, but not by uninduced monocytes. The effect of CCL18 cytokines on the proliferation, metastasis and vascular proliferation of A375 melanoma cell line induced the production of CCL18 by human monocytes, co-cultured with recombinant human CCL18 cytokines and A375 melanoma cell line, respectively. The effect of CCL18 on A375 proliferation was detected by MTT assay. The chemotaxis and invasion of tumor cells were determined by chemotaxis test and recombinant basement membrane invasion assay. The A375 melanoma cell line was inoculated into chicken embryo allantoic membrane, and A375 melanoma cell line was inoculated into chicken embryo allantoic membrane. The effect of CCL 18 on tumor angiogenesis was measured. The results suggested that neither the induced or recombinant CCL18 cytokines were chemotactic to the tumor cells induced by IL-4, the monocytes that produced CCL18 and the CCL18 recombinant cytokines had chemotactic effects on tumor cells compared with the control group. In addition, IL-4 induction of tumor cells and recombinant CCL18 could promote tumor angiogenesis in chicken embryo allantoic membrane test. The effect of CCL18 cytokines on the tumorigenesis of A375 melanoma cells in nude mice showed that CCL18 cytokines might inhibit the growth of A375 xenografts by some mechanism. By immunohistochemical staining of Ki-67Bcl 2 and Tunel in situ apoptosis staining, it was found that the induction and recombination of CCL18 in A375 melanoma cell line could promote the proliferation of tumor and inhibit the apoptosis of tumor cells. The fifth part concludes that CCL 18 may be related to the progression of melanoma and regulate the biological behavior of the tumor. But more research is needed to clarify its mechanism and function.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.5
【参考文献】
相关期刊论文 前2条
1 郭巨江;苏逢锡;姚和瑞;陈积圣;;替代激活的单核细胞促进人乳腺癌细胞SKBR3的生长侵袭[J];南方医科大学学报;2007年04期
2 叶燕丽,周昕熙,王莲桂;裸小鼠的繁殖及在肿瘤学中的应用[J];实验动物科学与管理;2005年01期
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