【作者】 黄巍；

【导师】 涂亚庭；

【作者基本信息】 华中科技大学 ， 皮肤性病学， 2008， 博士

【摘要】 第一部分内皮素对黑素瘤细胞TGF-β1表达及体外生长活性的调节目的研究内皮素1、3(Endothelin1,3 ET-1、ET-3)对恶性黑素瘤细胞系A375、SK-Mel-1体外生长活性的影响,及对TGF-β1蛋白和mRNA水平的调节,探讨ET-1、ET-3对不同细胞系生长的影响因素以及与TGF-β1表达的关系。方法不同浓度的ET对两种MM细胞株进行刺激,MTT法检测体外生长活性,RT-PCR及ELISA技术检测ET-1、ET-3、BQ123、BQ788及LY294002分组干预下,TGF-β1的表达变化。结果ET-3对两种MM细胞系均表现促生长效应,且有浓度依赖关系;ET-1对A375细胞表现为浓度依赖性的生长抑制,对SK-Mel-1细胞生长抑制作用不明显;在蛋白和mRNA水平ET-1明显上调MM细胞TGF-β1的表达,ET-3表现为对TGF-β1表达的抑制;BQ123、BQ788、LY294002均可在不同程度上阻断ET-1、ET-3的上述效应。结论ET-1通过上调TGF-β1的表达抑制MM细胞的体外生长;ET-3对TGF-β1表达的抑制可能是得益于激活了PI3K/Akt信号通路; TGF-β1对MM细胞生长抑制的作用存在细胞系之间的差异。第二部分内皮素诱导Akt蛋白磷酸化对抗细胞凋亡及机制的研究目的研究内皮素1,3(ET-1,ET-3)对恶性黑素瘤细胞PI3K/Akt通路的激活效应,以及ETRA、ETRB拮抗剂BQ123、BQ788,PI3K抑制剂LY294002对通路激活效应的不同影响;探讨ET-1,ET-3对顺铂(Cisplatin)诱导的MM细胞凋亡的抵抗作用。方法Western Blot(免疫印迹法)检测ET-1、ET-3及联合BQ123,BQ788,LY294002刺激下磷酸化AKT蛋白表达的变化,流式细胞术测定内皮素1,3以及与顺铂联合干预下MM细胞的凋亡率。结果ET-1与ET-3均明显上调MM细胞磷酸化Akt蛋白(P-Akt)的表达;ET-3上调P-Akt较ET-1显著;BQ123,BQ788,LY294002可阻断ET-1、ET-3对P-Akt表达的上调;Cisplatin可明显增加MM细胞的凋亡率;ET-1、ET-3可降低MM细胞的基础凋亡;与ET-1比较,ET-3明显抵抗Cisplatin诱导的凋亡。结论ET通过激活PI3K/Akt通路介导了MM细胞抵抗Cisplatin凋亡的效应。第三部分内皮素1诱导黑素瘤细胞磷酸化Smad3蛋白的高表达目的研究内皮素1、内皮素3在在黑素瘤细胞中诱导磷酸化Smad3蛋白表达中的作用,以及内皮素受体拮抗剂对其诱导效应的影响。方法免疫印迹技术检测不同干预条件下(ET-1、ET-3,或联合应用BQ123、BQ788),黑素瘤细胞磷酸化Smad3蛋白的表达变化。结果ET-1明显上调MM细胞磷酸化Smad3的表达,而ET-3对其表达有显著的抑制效应,ETRA拮抗剂BQ123明显阻断ET-1上调磷酸化Smad3的效应,而ETRB拮抗剂BQ788对ET-1的这一效应无明显影响。结论ET-1通过ETRA途径诱导磷酸化Smad3蛋白的表达;ET-3对磷酸化Smad3蛋白的表达有抑制作用。更多还原

【Abstract】 PartⅠThe role of endothelins in TGF-β1 expression and growth effect in malignant melanoma in vitroObject To explore the role of endothelin1,3 in TGF-β1 expresssion on protein secretion and mRNA level and the relationship to MM cell growth effect in cell lines A375 and SK-Mel-1.Methods MM cells stimulated by endothelin1,3 on different concentration, MTT method measure the growth effect in two MM cell lines;RT-PCR and ELISA technique was used to detect the TGF-β1 expression simulated by ET-1,ET-3, or combination with BQ123,BQ788,and LY294002.Results ET-3 promoted cell growth both in A375 and SK-Mel-1 cell lines and show the concentration-dependent. ET-1 inhibits cell growth in A375 but not in SK-Mel-1; ET-1 significantly up-regulates the TGF-β1 expression both on protein and mRNA, ET-3 show oppositely effects. Combination treated with BQ123, BQ788, and LY294002 show block effect of ET stimulation to MM cellsConclusion melanoma cells growth was suppress by TGF-β1 induced by ET-1; ET-3 inhibited TGF-β1 expression through activation of PI3K/Akt pathway; the effect of cell growth inhibition induced by TGF-β1 showed a diversity phenomenon between melanoma cell lines. Endothelins; phosphor-Akt; PI3K/Akt pathway; apoptosis; Cisplatin PartⅢEndothelin1 promotes the over-expression of phospho-Smad3 through ETRA pathway in melanomaObject To investigate the roles of ET1, ET-3 and combination treatment with BQ123, BQ788 in overepression of phospho-Smad3 protein in melanoma cells.Methods Westrn blot technique was applied to detect the expression of phosphor-Smad3 under the ET-1, ET-3stimulation or conjoined treatment with BQ123 and BQ788 .Result ET-1 induced phosphor-Smad3 over-expression significantly, and BQ123 but not BQ788 inhibited this phenomenon. Oppositely, ET-3 suppress the expression of it comparing with control.Conclusion ETRA but not ETRB mediated the over-expression of phosphor-Smad3 induced by endothelin in MM A375 cell line.更多还原