【作者】 王寅；

【导师】 殷明； 姜远英； 陈新生；

【作者基本信息】 第二军医大学 ， 药理学， 2002， 博士

【摘要】 黑色素瘤是起源于皮肤色素细胞的一种恶性肿瘤，易发生转移，目前尚无特异有效药物治疗其扩散性病变。流行病学研究表明，饮食中多不饱和脂肪酸含量与黑色素瘤的发生密切相关。花生四烯酸是多不饱和脂肪酸的主要成分，其脂氧合酶代谢产物在黑色素瘤的转移过程中起重要作用。本研究通过考察5-脂氧合酶抑制剂对黑色素瘤细胞a375转移过程中相关因素的影响，阐明5-脂氧合酶抑制剂影响黑色素瘤转移的作用机制，为研制开发抗黑色素瘤转移药物提供思路。 实验采用光密度法测定黑色素瘤细胞a375与细胞外基质蛋白的粘附率：以流式细胞仪测定a375细胞表面整合素的表达；以酶联免疫吸附法、流式细胞仪、蛋白质印迹分析测定a375细胞ICAM-1的表达，以Northern blot测定a375细胞ICAM-1的mRNA的表达水平；以双抗体夹心法检测a375细胞NF-κB的活化水平。 以滤膜涂布Matrigel基质的Transwell Chamber测定a375细胞的侵袭能力；以明胶酶谱法测定a375细胞分泌MMP-2和MMP-9的酶活性；以蛋白质印迹分析测定a375细胞产生MMP-2、MMP-9和TIMP-1的蛋白量；以Northern blot测定a375细胞中MMP-2、MMP-9和TIMP-1的mRNA表达水平。 实验结果表明：作为对照的环氧合酶抑制剂吲哚美辛作用后，对黑色素瘤细胞a375与细胞外基质的粘附率没有影响。在5-20μM浓度范围内，非选择性脂氧合酶抑制剂NDGA、5-脂氧合酶抑制剂AA861和5-脂氧合酶激动蛋白抑制剂MK886剂量依赖性地抑制a375细胞对Ⅳ型胶原的粘附，对细胞粘附至纤维粘连蛋白及层粘连蛋白没有影响。抑制剂对a375细胞粘附的抑制作用能被5-脂氧合酶代谢产物5-HETE所逆转，白三烯类对a375细胞粘附性改变无明显作用。 作用浓度为20μM时，NDGA、AA861及MK886能明显抑制细胞内[Ca²⁺]升高导致的a375细胞对Ⅳ型胶原粘附率的增加，对a375细胞PKC活化导致对Ⅳ型胶原粘附增加只有部分抑制作用。 相同浓度的NDGA、AA861及MK886作用后，明显降低a375细胞表面的整合素β₁表达，对细胞整合素α_Ⅱbβ₃的表达基本没有影响。 吲哚美辛对a375细胞ICAM-1表达没有明显影响，在5～20μM浓度范围内，NDGA、AA861及MK886剂量依赖性地降低a375细胞ICAM-1蛋白表达量及mRNA表达水平。NDGA、AA861及MK886作用后，剂量依赖性地抑制a375细胞NF aB活化。 叼噪美辛对a375细胞侵袭重组基底膜能力没有明显影响，NDGA、AA861 和MK886剂量依赖性地抑制a375细胞对Matytael膜的侵袭。这种抑制作用能被5－HETE逆转，白三烯类对0乃细胞的侵袭性改变没有明显影响。 作用浓度为20 p M时，NDGA、AA861及MK886显著抑制a375细胞产生MMP－2和 MMP－9蛋白量及其InRNA表达水平，对a375细胞产生 TIMPI的蛋白量及mRNA表达水平没有明显影响。 这些结果表明，5．脂氧合酶代谢产物的其中一种，5－HETE，在黑色素瘤转移过程中起重要的作用。证实5－脂氧合酶抑制剂通过抑制a375细胞核转录因子活化，在基因转录水平抑制a3 75细胞粘附分子的表达，进而抑制a375细胞对细胞外基质的粘附。5－脂氧合酶抑制剂通过抑制 a375细胞产生、分泌IV型胶原酶，降低 a375细胞对重组基底膜的侵袭性。 本研究结果提示5．脂氧合酶抑制剂作为可能的抗黑色素瘤转移药物，值得深入研究。更多还原

【Abstract】 Melanoma is a kind of malignant tumor originating from pigment cells of skin andprone to metastasis. So far no specific drug has been aPplied to control the diffusedpathological process. Epidemiological studies show that the content of polyunsaturatedfatty acids in diet were closely associated with incidence of malignant melanoma.Arachidonic acid is the main ingredients of polyunsaturated fatty acids and itslipoxygenase metabolites play a key role in the metastasis process of melanoma cancercells. We conducted experiments trying to stlldy the effects and mechanism of 5-lipoxygenase inhibitors on metastasis of human melanoma cell a375 and to obtain theclue of 5-lipoxygenase inhibitor as poteniial anti-metastatic agents for melanoma.Adhesion of a375 cell to extracellular matrix was determined by optical densityassay. The expression of integrins on cancer cell surface was determined by fiowcytometry ELlSA, flow cytometry and Western blot was used to detect the production oflCAM-l in a375 cell. Northern blot was used to measure the expressing level of ICAM-1mRNA in a375 cell. The 1evels of NF- K B in a375 cell was measured by sandwichELISA.Invasiveness of a375 cells was assayed by using Transwell chamber wth filtermembrane coated with Matrigel. The secretion of active matrix metalloproteinases(MMP-2 and MMP-9) was measured by gelatin zymogram. Western blot was used todetect the production of MMP-2, MMP-9 and tissues inhibitors of metalloproteinase(TIMP-l) and the expressing leveI of MMP-2, MMP-9 and TIMP-l mKNA wasmeasured by Northern blot.The results showed that in control grouP treated with indomethacin, acyclooxygenase inhibitors, adhesion of a375 cells to extracellular matrix was not altered.In the range from 5~20 ll M, lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA),5-lipoxygenase inhibitors AA861 and specific 5-lipoxygenase-activating-prote inhibitorMK886 could all inhibit adhesion of a375 cell to collagen IVin a dose-dependent mannerand had no effect on adhesion to fibronectin or vitronectin. 5-lipoxygenase metabolites(5-HETE) reversed the adhesion behavior of inhibitor-treat cancer cell and leukotrieneshad no effect.Pretreated with 20 ll M of NDGA, AA861 and MK886 remarkably inhibited theA23l87-induced enhancement of a375 cell adhesion to collagen IV respectively andonly partly inhibited the TPA-induced enhancement.A375 cells tfeated with NDGA, AA861 and MK886 in the same concentfationexhibited reduction of the expression of integrin 6 l on the surface of melanoma cell, butnot of integrin Q lIb fl 3’Indomechacin had no effect on the production of ICAM-l protein in a375 cell.NDGA, AA861 and MK886 could reduce the production of ICAM-l protein and itsmRNA of a375 cell and suPpress NF- K B activation in a dose-dependeni manner in therange from 5~20 ll M.No significant inhibition of invasiveness to recombinant basement membrane wasseen when a375 ceIls were treated with indomethacin. In the range from 5~20ll MNDGA, AA861 and MK886 significantly inhibited invasiveness of a375 cell throughMatrigel membrane. 5-HETE could reverse this inhibitory effect and leukotrienes had noeffect.NDGA, AA86l and MK886 could significantly inhibit production of MMP-2 andMMP-9 proteins as well as their mRNA expression level. The inhibition of 5-lipoxygenase activity with the shese inhibitors had no significant effect on production ofTIMP-l protein and mRNA level in a375 ceII.These results suggest that one of the 5-lipoxygenase metabolites, 5-HETE, plays animportant role in the metastatic progress of melanoma. 5-lipoxygenase inhibitors caninhibit the process by suPpressing the expression of adhesion molecule through inhibitingthe nuclear factor activation of a375 cell so as to inhibit a375 cell adhesion toextracel1u1ar matrix and by suPPressing the production of MMP-2 and MMP-9 in a375cell so as to decrease the invasiveness to through recombinant basement membrane.That更多还原