發(fā)布時間：2018-07-15 09:39

【摘要】：皮膚惡性黑素瘤是一種高度惡性腫瘤,目前研究認(rèn)為任何細(xì)胞都能分泌細(xì)胞因子并表達(dá)其受體,而細(xì)胞因子在腫瘤細(xì)胞的表達(dá)已引起廣泛的關(guān)注和研究,它們是腫瘤細(xì)胞侵襲轉(zhuǎn)移的路標(biāo)。部分黑素瘤細(xì)胞系能產(chǎn)生細(xì)胞因子并表達(dá)其受體,無論阻斷配體還是受體都能抑制腫瘤細(xì)胞的生長。我們的前期研究,采用全人類基因組芯片對中國人肢端黑素瘤組織的研究發(fā)現(xiàn),趨化性細(xì)胞因子CCL18在腫瘤組織的表達(dá)明顯增高。本研究利用IL-4誘導(dǎo)人單核細(xì)胞產(chǎn)生的CCL18、重組CCL18細(xì)胞因子與A375黑素瘤細(xì)胞株共培養(yǎng),探討其在黑素瘤生物學(xué)行為方面的作用。 第一部分免疫組化研究CCL18在皮膚惡性黑素瘤的表達(dá) 使用免疫組化對67例黑素細(xì)胞來源的良、惡性腫瘤進(jìn)行分析,結(jié)果顯示良性痣、Spitz痣及發(fā)育不良性痣不表達(dá)CCL18,而黑素瘤表達(dá)CCL18,且CCL18的表達(dá)與腫瘤浸潤深度呈正比。 第二部分人單核細(xì)胞的分離、誘導(dǎo)及CCL18表達(dá)的檢測 使用密度梯度離心法成功分離人單核細(xì)胞,并使用IL-4成功誘導(dǎo)單核細(xì)胞,經(jīng)ELISA和Western-blotting驗證,誘導(dǎo)的單核細(xì)胞可產(chǎn)生CCL18,非誘導(dǎo)的單核細(xì)胞不能產(chǎn)生CCL18。 第三部分CCL18細(xì)胞因子對A375黑素瘤細(xì)胞株增殖、轉(zhuǎn)移及血管增殖的影響 誘導(dǎo)人單核細(xì)胞產(chǎn)生CCL18,與重組人CCL18細(xì)胞因子,分別與A375黑素瘤細(xì)胞株共培養(yǎng),通過MTT法檢測CCL18對A375增殖的影響；通過趨化試驗及重組基底膜侵襲實驗,測定CCL18對腫瘤細(xì)胞的趨化能力及侵襲作用；通過將A375黑素瘤細(xì)胞株接種于雞胚尿囊膜,測定不同情況下CCL18對腫瘤血管生成的影響。結(jié)果提示無論是誘導(dǎo)還是重組CCL18細(xì)胞因子都不是影響腫瘤增殖的主要細(xì)胞因子,IL-4誘導(dǎo)產(chǎn)生CCL18的單核細(xì)胞組及CCL18重組細(xì)胞因子組與對照組比較均對腫瘤細(xì)胞存在趨化作用,并促進(jìn)腫瘤細(xì)胞的轉(zhuǎn)移；IL-4誘導(dǎo)組及重組CCL18組在雞胚尿囊膜試驗中可促進(jìn)腫瘤血管的生成作用。 第四部分CCL18細(xì)胞因子對A375黑素瘤細(xì)胞裸鼠內(nèi)成瘤作用的影響 裸鼠成瘤的模型顯示,CCL18細(xì)胞因子可能以某種機(jī)制抑制A375移植瘤體積的增加。腫瘤組織切片經(jīng)Ki-67、Bcl-2免疫組化染色及TUNEL原位凋亡染色后分析,發(fā)現(xiàn)誘導(dǎo)及重組CCL18在A375黑素瘤細(xì)胞系體內(nèi)試驗中可促進(jìn)腫瘤的增殖,抑制腫瘤細(xì)胞的凋亡。 第五部分小結(jié) CCL18可能與黑素瘤的進(jìn)展有關(guān),并調(diào)節(jié)腫瘤的生物學(xué)行為。但需要更多研究以明確其作用機(jī)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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R739.5

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本文編號：2123643