【作者】 林云；

【导师】 涂亚庭；

【作者基本信息】 华中科技大学 ， 皮肤病与性病学， 2006， 博士

【摘要】 第一部分Survivin、PEDF在皮肤恶性黑色素瘤中的表达及临床意义目的探讨生存素(Survivin)及色素上皮源性因子(PEDF)在皮肤恶性黑色素瘤中的表达及其临床意义。方法SP免疫组化法检测Survivin、PEDF蛋白在48例原发性皮肤恶性黑色素瘤中的表达水平，以23例色素痣作为对照。结果(1)Survivin在皮肤恶性黑色素瘤中的表达明显强于其在色素痣中的表达，PEDF在二者中的表达无明显差异，(2)Survivin和PEDF蛋白表达均与恶性黑色素瘤病理学类型、淋巴结转移、浸润深度及预后相关，(3)Survivin在黑色素瘤中的表达水平与PEDF呈明显负相关。结论Survivin与皮肤恶性黑色素瘤的发生发展密切相关，对皮肤恶性黑色素瘤的辅助诊断及预后评估具有重要的临床意义。PEDF在皮肤恶性黑色素瘤发展过程中可能起负性调节作用。第二部分EGCG对恶性黑色素瘤A375细胞生物学行为的影响目的探讨EGCG对黑色素瘤A375细胞生物学行为的影响。方法对人恶性黑色素瘤细胞株A375进行培养传代；活细胞计数法、MTT试验检测EGCG不同浓度和不同时间对A375细胞生长的抑制作用；用相差显微镜、电镜和Hochest核染色观察细胞凋亡的形态学改变；TUNEL法定量检测细胞凋亡；用Annexin V-FITC、PI双染分析细胞凋亡的变化；同时用DNA电泳观察凋亡的生化学改变。结果EGCG可明显抑制A375细胞的增殖，其增殖抑制率呈浓度和时间依赖性；细胞凋亡形态学观察到明显凋亡发生；TUNEL染色也观察到凋亡的发生，且凋亡发生和药物浓度、作用时间呈依赖关系；流式细胞技术证实EGCG诱导A375细胞的早期凋亡显示出明显的时间和剂量依赖关系；DNA电泳亦可观察到细胞发生凋亡的典型DNA ladder。结论EGCG对黑色素瘤A375细胞的抑制作用与诱导肿瘤细胞的凋亡有关，为进一步研究EGCG的诱导肿瘤细胞凋亡机制奠定了基础。第三部分Survivin、PEDF在EGCG诱导A375细胞凋亡中的作用目的体外观察EGCG诱导黑色素瘤细胞凋亡过程中凋亡相关蛋白Survivin、PEDF的表达变化及其与EGCG的增殖抑制、凋亡诱导的关系，为更深入地研究EGCG的凋亡调控机制奠定基础。方法对人黑色素瘤细胞株A375进行培养传代；运用细胞免疫组化法检测不同浓度EGCG作用肿瘤细胞后Survivin、PEDF蛋白的表达情况；以western-blot法检测不同作用时间EGCG对A375细胞survivin、PEDF蛋白表达的影响；以RT-PCR技术检测不同浓度EGCG对A375细胞survivin、PEDFmRNA表达的影响；同时采用Caspase-3活性检测试剂盒检测不同浓度EGCG作用肿瘤细胞不同时间后的Caspase-3活性。结果免疫组化法观测EGCG作用A375细胞后，Survivin蛋白的表达明显减弱，PEDF蛋白的表达则明显增强，且药物浓度越高，变化越明显；免疫印迹法检测显示Survivin、PEDF蛋白表达的变化与药物作用时间呈一定的依赖关系；RT-PCR检测发现Survivin mRNA表达水平随着EGCG作用浓度的增加而下降，PEDF mRNA表达水平则随着EGCG作用浓度的增加而增加，Survivin cDNA／β-actin cDNA比值与细胞增殖抑制率和细胞凋亡率呈负相关(P＜0.01)，PEDF／β-actin cDNA比值与细胞增殖抑制率和细胞凋亡率呈正相关(P＜0.01)；Caspase-3活性检测显示Caspase-3的活性变化和药物浓度、作用时间呈依赖关系，且与Survivin的表达下调、PEDF的表达上调呈平行关系。结论EGCG可通过线粒体途径(caspase-3)来诱导A375细胞凋亡，Survivin、PEDF可通过与caspase-3的相互作用发挥其抑制／促进凋亡功能，EGCG通过线粒体途径诱导A375细胞的凋亡可能是通过调节凋亡相关基因Survivin、PEDF的表达来实现。更多还原

【Abstract】 Part I Expression of Survivin and PEDF in Cutaneous MalignantMelanoma and the Relationship between them.Objective To investigate the expression of survivin and PEDF (pigment epithelium derived factor ) and their clinical significance in cutaneous malignant melanoma(CMM).Methods The expression of survivin and PEDF was measured by streptavidin-peroxidase complex immunohistochemical technique in 48 cases of CMM and 23 cases of nevus.Result The positive expression percentage of survivin in CMM was significantly higer than that of nevus, while the positive expression percentage of PEDF in that was similar. The expression of survivin and PEDF in CMM was positively related to pathological histology、 Clark’s grade、 the metastasis to lymph nodes and clinical outcome. The expression of PEDF was inversely correlated with the high expression of survivin in CMM.Conclusions Survivin might play important roles in the carcinogenesis process and progression of CMM and have important implications in the diagnosis and prognostic assessment. PEDF might play negative modulation roles in the progression of CMM. Part II Effect of EGCG on the Biological Behavior of Melanoma A375 cellsObjective TO study the effects of EGCG on the biological behavior of Melanoma A375 cells.Method The melanoma cell line A375 was chosen in this experiment. The inhibitoryeffects of chemotherapeutical drugs on A375 cell line were assayed with MTT test.The EGCG group was divided into different groups by concentration and time.Changes of apoptosis in morphology and chemistry were detected by Phasemicroscope, Electron microscopy and Hochest nuclear staining respectively.Apoptosis was quantitatively determined by TUNEL and detected by Annexin Vstaining. Changes of apoptosis in chemistry were detected by DNA electrophoresis aswell.Results EGCG obviously inhibited the A375 cell growth with dose-andtime-dependent. By Phase microscope, Electron microscopy, Hochest nuclear staining,DNA electrophoresis, Annexin V and TUNEL staining, we all detected the occurrenceof apoptosis. Annexin V and TUNEL staining was in a dose-and time-dependentmanner.Conclusion EGCG can effectively induce apoptosis in A375 cells. Part III The role of Survivin and PEDF in the apoptosis of Melanoma A375 cells induced by EGCGObjective To study the role of Survivin and PEDF in the process of apoptosis, so as to provide the basis for validating the mechanisms of EGCG induce apoptosis. Methods The EGCG group was divided into different groups by concentration and time. The survivin and PEDF protein expression was detected with streptavidin-peroxidase complex immunohistochemical technique and westen blot. Expression of Survivin and PEDF mRNA was detected by RT-PCR. The caspase 3 activities was detected by caspase kit. Results The Survivin protein expression was decreased with the increasing of EGCGconcentration while the expression of PEDF protein was increased by immunohistochemical detection. Western blot showed that the increased expression of Survivin protein and the decreased expression of PEDF in melanoma A375 cells induced by EGCG which is time-dependent. Survivin mRNA was down-regulated and PEDF mRNA was up-regulated with the increase of dose of EGCG The ratio of Survivin and β-actin was negatively associated with the inhibitory rate of cell proliferation and the rate of apoptosis (p<0.01). The ratio of PEDF and β-actin was positively associated with the inhibitory rate of cell proliferation and the rate of apoptosis (p<0.01). The change of caspase-3 activity was in a dose-and time-dependent manner. The increase of of caspase-3 activity positively associated with the decreased expression of Survivin and the decreased expression of PEDF. Conclusion Survivin and PEDF gene expression may play a critical role in the apoptosis of melanoma A375 cells induced by EGCG which throught modulation the caspase-3 activity.更多还原