【作者】 李美洲；

【导师】 左连富；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 目的:随着社会老龄化,皮肤肿瘤发病率逐渐升高,其中表皮细胞肿瘤如皮肤基底细胞癌和皮肤鳞状细胞癌以及脂溢性角化病等最常见,对人类健康造成威胁。因而,探讨科学有效的治疗方法具有重要的临床意义和社会意义。Survivin是凋亡蛋白抑制因子(inhibitor of apoptosis proteins, IAPs)家族的新成员,特异表达于人和鼠的胚胎发育组织和多数人类肿瘤。Survivin基因定位于染色体17q25,全长125 kb,由四个外显子和三个内含子组成,mRNA1,619nt,编码的蛋白含142个氨基酸残基,分子量为16.5Kda,是IAP基因家族的最小成员。由于Survivin在肿瘤组织中的特异表达及其在抑制细胞凋亡作用和调节细胞分裂中的重要作用,使其成为一个具有潜在应用价值的肿瘤标志物和肿瘤治疗的新靶点。PTEN基因是迄今为止发现的第一个具有双特异性磷酸源性的抑癌基因,在维持细胞的增殖分裂和凋亡的平衡中起重要作用,在多种恶性肿瘤中发生突变或丧失,导致其抑癌功能减弱或丧失,在多种恶性肿瘤的发生、发展中起重要作用。对PTEN基因的研究将为恶性肿瘤的早期诊断、临床监测、基因靶向治疗、预后评估、新药研发和一级预防提供科学的理论依据。Livin基因是最近发现的一个凋亡蛋白抑制因子(inhibitor of apoptosis protein IAP)家族的一个成员,该基因位于人染色体20q13.3,全长4.6kb,包含7个外显子,基因转录产物mRNA长1.4kb。有研究认为Livin既在胞质内呈丝状表达,同时也表达于胞核,并认为LivinC末端的RING结构对介导其在亚细胞水平上的分布有重要作用。有研究证实Livin mRNA在人多种类型的肿瘤细胞系中高表达,可能与人某些类型肿瘤的形成存在密切关系。上世纪中后期,我国学者使用三氧化二砷治疗急性早幼粒白血病取得显著疗效,未出现严重不良反应。研究还表明三氧化二砷可以诱导多种实体瘤细胞生长阻值和凋亡,三氧化二砷抗实体瘤具有如下机制:三氧化二砷可诱导肿瘤细胞分化,改变其恶性特征;三氧化二砷以蛋白巯基为直接作用靶点,改变和影响酶的活性,继而引起一系列的抗癌效应;三氧化二砷通过线粒体途径诱导肿瘤细胞凋亡;活性氧化物在三氧化二砷诱导凋亡中起作用;三氧化二砷可以抑制端粒酶活性,可达到抑制肿瘤的目的。2004年9月,我国食品药品管理监督局批准三氧化二砷注射液可用于晚期原发性肝癌的治疗。但三氧化二砷对实体瘤的治疗作用和抗肿瘤机制仍未明确。在此基础上,本研究拟观察与细胞凋亡密切相关的Survivin基因,PTEN,和Livin等在皮肤基底细胞癌中的表达情况,探讨皮肤基底细胞癌发生、发展的病理机制,并初步探讨三氧化二砷对皮肤基底细胞癌细胞增殖的影响,观察对皮肤基底细胞癌细胞株Survivin表达的抑制或影响,企图为应用三氧化二砷治疗皮肤基底细胞癌提供理论和实验室依据。方法:1 Survivin和PTEN在皮肤基底细胞癌中的表达的研究选取2005年11月-2007年11月我科就疹的皮肤肿瘤患者:皮肤基底细胞癌30例,其中男性18例,女性12例,年龄45-83岁,平均年龄67.40岁;部位:颜面部12例;头皮部:9例;躯干部:6例;四肢3例;病程12个月~15年,平均36.6个月。脂溢性角化病30例,其中棘层肥厚型13例,角化过度型8例,巢状型4例,腺样型3例,刺激型2例;男性16例,女性14,年龄38岁-70岁,平均年龄54.8岁。正常皮肤组织20例,男13例,女7例,年龄17~68岁,均来自我科手术患者,年龄、部位与患者组相匹配。常规组织学技术观察表皮肿瘤的病理特点;并运用免疫组织化学技术、流式细胞术技术观察Survivin和PTEN基因在皮肤基底细胞癌中蛋白水平表达及相关性情况,进一步探讨Survivin和PTEN基因在皮肤基底细胞癌发病及发展中的作用。2 Livin蛋白在基底细胞癌中的表达及其与survivin的相关性分析收集标本同第一部分,运用免疫组化、流式细胞术观察Livin在皮肤基底细胞癌和脂溢性角化等表皮肿瘤中的蛋白水平表达情况。并分析Livin蛋白与皮肤基底细胞癌发生、发展的关系。并在第一部分的基础上,分析Livin蛋白与survivin蛋白表达之间的相关性,了解二者在表皮肿瘤发病过程中的相互关系。分析Livin蛋白与PTEN蛋白表达之间的相关性,了解二者在皮肤基底细胞癌发病过程中的相互关系。并观察Livin蛋白表达与皮肤基底细胞癌临床病理特征之间的关系。3三氧化二砷对人基底细胞癌细胞株A-431增殖及相关基因表达的影响常规培养皮肤基底细胞癌细胞株A-431,加入不同浓度的三氧化二砷,采用MTT法观察不同浓度的As2O3对A431细胞增殖在不同时间点(0h、24h、48h、72h)的影响,应用流式细胞仪检测三氧化二砷对A-431细胞细胞周期、凋亡和Survivin等基因表达的变化。应用Western Blot免疫印迹技术观察三氧化二砷对A-431细胞Survivin基因表达的影响。观察不同浓度的三氧化二砷对培养的皮肤基底细胞癌细胞株增殖的影响,观察对Survivin基因蛋白水平表达的影响。结果:1 Survivin和PTEN在皮肤基底细胞癌中的表达及与临床病理特征之间关系(1)免疫组化结果显示,在30例脂溢性角化病皮损中,19例中Survivin蛋白阳性表达,正常20例皮肤组织中均为阴性表达,差异有显著性(P<0.05);30例脂溢性角化病皮损中,16例PTEN蛋白阳性表达,正常20例皮肤组织中14例有PTEN表达,二者比较差异无显著性(P>0.05)。(2)在基底细胞癌中,Survivin蛋白的表达高于在正常皮肤组织(P<0.05)和脂溢性角化病(P<0.05)中的表达;而PTEN蛋白的表达则低于在正常皮肤组织(P<0.05)和脂溢性角化病(P<0.05)中的表达。(3)相关性分析发现,Survivin和PTEN蛋白在SK、BCC等表皮良恶性肿瘤中的表达呈显著负相关(P<0.05)。随肿瘤恶性度逐渐增高,Survivin表达逐渐升高,而PTEN表达逐渐下降。(4)在基底细胞癌组中,Survivin在年龄>60岁组中的表达高于在年龄≤60岁组中的表达,两组之间表达差异有显著性(P<0.05);PTEN在年龄>60岁组中的表达低于在年龄≤60岁组中的表达,两组之间表达差异有显著性(P<0.05)。Survivin在皮损平均直径>2cm组中的表达高于在皮损平均直径≤2cm组中的表达(P<0.05);PTEN在皮损平均直径>2cm组中的表达低于在皮损平均直径≤2cm组中的表达(P<0.05)。Survivin在病程>平均病程组中的表达高于在病程≤平均病程组中的表达(P<0.05)而PTEN在病程>平均病程组中的表达低于在病程≤平均病程组中的表达(P<0.05)。Survivin和PTEN在基底细胞癌中的表达与患者性别、发病部位、皮损数目无关(P>0.05)。2 Livin蛋白在皮肤基底细胞癌中的表达及其与Survivin的相关性分析(1)免疫组化染色结果Livin蛋白表现为细胞浆着色,在30例基底细胞癌中,26例中有明显的表达(阳性率86.67%),而30例脂溢性角化病组中有16例有阳性表达(阳性率53.33%),20例正常对照组织中仅见1例有弱阳性表达(阳性率5.00%)。Livin蛋白在基底细胞癌组中的表达阳性率明显高于在脂溢性角化病组的表达,两者比较差异有显著性(P<0.05);同时也高于在正常皮肤组中的表达,两者比较差异有显著性(P<0.05)。Livin蛋白在脂溢性角化病组织中的表达也高于在正常皮肤组中的表达,统计结果显示,差异有显著性(P<0.05)。从正常皮肤到脂溢性角化病到BCC,Livin蛋白的表达阳性率逐渐增高。Livin在年龄>60岁组中的表达高于在年龄≤60岁组中的表达,两组之间表达差异有显著性(P<0.05);Livin在皮损平均直径>2cm组中的表达高于在皮损平均直径≤2cm组中的表达(P<0.05);Livin在病程>平均病程组中的表达高于在病程≤平均病程组中的表达(P<0.05)。Livin在皮肤基底细胞癌中的表达与患者性别、发病部位、皮损数目无关(P>0.05)。Spearmen秩相关检验相关性分析发现,在该组病例中,Livin蛋白和survivin蛋白表达之间存在正相关(r=0.557, P<0.05)。而Livin蛋白和PTEN蛋白表达之间存在负相关(r=-0.453, P<0.05)。(2)流式细胞术定量检测Livin蛋白在皮肤基底细胞癌中表达的结果在基底细胞癌组织中,Livin蛋白的免疫荧光均道值(586.75±33.73)明显高于脂溢性角化病(459.49±62.74),差异有显著性(P<0.05);同时也高于在正常组织中的免疫荧光均道值(327.57±98.37),差异有显著性(P<0.05);在脂溢性角化病组织中,Livin蛋白的免疫荧光均道值高于正常皮肤组织,两者比较差异有统计学意义(P<0.05)。从正常皮肤到脂溢性角化病到基底细胞癌,Livin蛋白的表达量的荧光均道值逐渐增高。采用Person相关性分析发现,Livin蛋白和Survivin蛋白之间存在正相关(r=0.547, P<0.05);而Livin蛋白和PTEN蛋白表达之间存在负相关(r=-0.553, P<0.05)。与免疫组化的结果相一致。3三氧化二砷对表皮癌细胞株A-431增殖、凋亡、细胞周期以及Survivin基因表达的影响(1) MTT分析发现从浓度10umol/L到60umol/L,三氧化二砷可以显著地抑制A-431细胞的增殖,随着浓度的增加和时间的延长,OD值逐渐下降(P<0.05),该抑制过程呈时间依赖性和浓度依赖性。(2)流式细胞术检测结果提示,As2O3影响A-431细胞周期的分布,不同浓度的As2O3作用A-431细胞后S期细胞群增多(P<0.05),存在S期阻滞;并且细胞凋亡数目增多(P<0.05)。(3)与对照组比较,As2O3能抑制A431细胞survivin基因的表达,该蛋白表达量下降(P<0.05)。三氧化二砷对体外培养的皮肤基底细胞癌细胞株有显著的增殖抑制作用,有诱导表皮细胞癌凋亡作用;并可能与下调凋亡抑制基因Survivin的表达有关。结论:1本研究首次应用免疫组化和流式细胞术发现表皮肿瘤皮肤基底细胞癌和SK的发生发展可能与Survivin基因和PTEN基因的异常表达可能有关。联合检测这两种基因有助于判断基底细胞癌的预后和恶性程度的可能性。2本研究首次应用流式细胞术和免疫组织化学的方法发现皮肤基底细胞癌的发生发展可能与凋亡抑制基因Livin的异常表达可能有关。Livin基因有可能成为皮肤基底细胞癌免疫治疗的靶基因。研究发现Livin和Survivin在皮肤基底细胞癌中的表达呈正相关,在皮肤基底细胞癌发病过程中二者之间可能存在协同作用。而Livin和PTEN在皮肤基底细胞癌中的表达呈负相关。3本研究首次研究了三氧化二砷对体外培养的皮肤基底细胞癌细胞株增殖和survivin基因表达的影响,结果显示三氧化二砷对皮肤基底细胞癌细胞株有显著的增殖抑制作用,并且有诱导皮肤基底细胞癌细胞凋亡作用和细胞周期阻滞作用;该抑制过程并可能与下调凋亡抑制基因Survivin的表达和影响细胞周期分布有关。更多还原

【Abstract】 Objectives:Epidermal tumors are ones of the most common malignant or benign tumors in the world. The incidence of basal cell carcinoma (BCC) and seborrheic keratosis (SK) is high in China.They are a great threaten to human health. Therefore, it is extremely important to investigate pathogenesis and search new target and effective drugs for BCC and SK therapy to decrease mortality and prolong life span of patients with epidermal tumors.Survivin is a novel member of the apoptosis protein family member. Survivin has correlation with tumor development and progress in many human tumors. Its main biological functions include inhibiting cell apoptosis and enhancing cell proliferation. Survivin selectively expresses in most common hunman noeplasms. More and more researchers study it as an ideal target for cancer treatment. Down-regulation of survivin expression can induce cancer cell apoptosis and enhances radiosensitivity in many human cancers. It can be served as marker of prognosis.PTEN is a dual-specificity lipid phosphase originally identified as a tumor suuressor gene. It frequently mutates and is absent in a number of human cancers, which leads to the weaking or absence of tumor suppressing functions. PTEN plays a critical role in the development and progression of many cancers. PTEN regulates complex signal transduction pathways (such as P13K/AKT pathway, FAK/MAPK cacade, PTEN/P53/MDM2 network), induces G1 cell-cycle arrest and apoptosis, suppresses the vscular formation and inhibits invasion and metastasis of tumors. With further study on the important function of PTEN, an earlier diagnosis, appropriate clinical surveillance, gene-targeted immunotherapy, prognostic evaluation, new drug development and population-oriented first-line prevention of cancers might be possible. Livin, as a new member of IAPs, plays an important role in anti-cancers. Livin regulates complex signal transduction pathways (such as TAK1/JNK1, et al) in antiapoptotic functions. Many studies have discovered that livin protein and livin mRNA are expressed in many malignant human tumors, but not or down-regulatedly expressed in normal tissues and benign tumors. With further sdudy on the important functions of livin, it can be a gene target in immunotherapy for cancers.Clinical trials have demonstrated that arsenic trioxide is effective for acute promyelocytic leukemia; it also has the action of induction of apoptosis and inhibition of growth of some solid cancer cells. It can induce differentiation of human nasopharyngeal carcinoma in BALB/C nude mice xenograft model. But the mechanisms are not clearly understood. Based on these facts, studies have been carried out to study its roles in the treatment of solid cancers. Many studies have discovered that arsenic trioxide can be used in many therapies for solid neoplasm.Therefore, in the present study we examined the expression of survivin, PTEN proteins in the process of carcinogenesis of epidermal tumors and determined the correlation between the levels of these proteins and various clinical and pathological features. Then, we also examined the expression of livin in the process of carcinogenesis of BCC and SK and determined the correlation between the levels of these proteins and various clinical and pathological features;We also analysed the correlation between linvin and survivin, PTEN in BCC and SK. Next, we studied the effects of arsenic trioxide on proliferation, cell cycle distribution, and expression of survivin, PTEN and livin in human epidermal cancer A-431cells.It may deepen our knowledge about epidermal tumors such as basal cell carcinoma and Sk,et al.It might provide theoretical and experimental evidence for utilization of arsenic trioxide in epidermal carcinoma therapy.Methods:1 Expression of survivin, PTEN in epidermal tumors and normal skin tissues and the correlation between the levels of these proteins and clinical-pathological features.Basal cell carcinoma tissues, seborrheic keratosis tissues and normal skin tissues were obtained from resected surgical specimens of epidermal tumors in our dermatology department. There were thirty cases of basal cell carcinoma including 18males and 12 females, who were aged from 45 to 83 years old (averaged 67.40 years old).There were 30 cases of SK including 16 males and 14 females, who were aged from 38 to 70 years old (averaged 54.80 years old).There were 20 cases of normal skin tissues which were from the patients who received surgical treatments in our dermatology department.All the specimens were verified by pathologic diagnosis. Survivin, PTEN protein expression in normal skin tissues and the lesions of basal cell carcinoma and seborrheic keratosis were examined by immunohistochemistry (IHC). Survivin, PTEN protein expression in normal skin tissues and the lesions of basal cell carcinoma and seborrheic keratosis were also examined by flow cytometry (FCM). Relationship between their expressions and clinical pathological features was analyzed.2 Expression of livin protein in epidermal tumors and normal skin tissues and the correlation between the levels of this protein and clinical-pathological features and the correlation with survivin and PTEN based on part one study.In this study, basal cell carcinoma tissues, seborrheic keratosis tissues and normal skin tissues were obtained from resected surgical specimens of epidermal tumors in our dermatology department. All the specimens were verified by pathologic diagnosis. They were the same as in part one study. Livin protein expression in normal skin tissues and the lesions of basal cell carcinoma and seborrheic keratosis were examined by immunohistochemistry (IHC). Livin protein expression in normal skin tissues and the lesions of basal cell carcinoma and seborrheic keratosis were also examined by flow cytometry (FCM). Relationship between its expressions and clinical pathological features was analyzed. Statistical treatment with SPSS was applied to deal with all the experimental data.We also analysed the correlation with survivin and PTEN based on part one study. 3 Effects of arsenic trioxide on cell proliferation, apoptosis, cell cycle distribution and gene expression in human epidermis cancer cell line A-431Human epidermal carcinoma cells (cell line A431) were conventionally cultured. After treatment with arsenic trioxide in different concentrations, inhibition of cellular growth was determined by measuring MTT dye absorption of living cells in 24,48,72h. We calculated the OD values.Then, apoptosis, cell cycle distribution and survivin expression were assessed by flow cytometry and western Blot methods.Results:1 Expression of survivin, PTEN in epidermal tumors and normal skin tissues and the correlation between the levels of these proteins and clinical-pathological features(1) The protein of survivin was positively expressed in 18 cases of the 30 SK specimens while none was expressed in the 20 normal controls (P<0.05). The protein of PTEN was positively expressed in 17 cases of the 30 SK specimens and in 13 cases of the 20 normal controls (P>0.05).(2) The protein of survivin was highly expressed in BCC lesions than in SK lesions and in normal controls (P<0.05), while the protein of PTEN was lowly expressed in BCC than in SK lesions and normal controls (P<0.05).(3) There had been a significant negative correlation between the expressions of survivin and PTEN (r= -0.546, P<0.01) in BCC and SK.(4) The expressions of survivin and PTEN were related with the patients’age, areas of the lesions and the disease duration (P<0.05), but not related with the patients’genders, sites of lesions and numbers of lesions (P>0.05) in patients with BCC.2 Expression of livin protein in epidermal tumors and normal skin tissues and the correlation between the levels of this protein and clinical-pathological features and the correlation with survivin and PTEN.(1) The protein of Livin was highly positively expressed in basal cell carcinoma than in seborrheic keratosis. There was significant difference (P<0.05) and it was also highlier expressed in BCC tissues than in normal controls (P<0.05).(2) The protein of Livin was highlier expressed in SK lesions than in normal controls, there was significant difference (P<0.05).(3) From normal skin to seborrheic keratosis and to basal cell carcinoma, the expressions of Livin were getting gradually highlier.(4) The expressions of livin were related with the patients’age, areas of the lesions and the disease duration (P<0.05), but not related with the patients’gender, sites of lesions and numbers of lesions(P>0.05) in patients with basal cell carcinoma..(5) There had been a significant positive correlation between the expressions of Livin and Survivin (r= 0.557, P<0.05) in basal cell carcinoma and seborrheic keratosis. There had been a significant positive correlation between the expressions of Livin and PTEN (r=- 0.453, P<0.05) in basal cell carcinoma and seborrheic keratosis.3 Effects of arsenic trioxide on cell proliferation, apoptosis, cell cycle distribution and gene expression in human epidermis cancer cell A-431MTT assay showed that with concentrations from 10umol/L to 40umol/L, arsenic trioxide can significantly inhibited the proliferation of A431 cells in a dose- and time-dependent manner (P<0.05). After A-431 cells were dealed with different concentration of arsenic trioxide in 24-72hours, the OD values of the treatment groups decreased to some extent compared with the control group. There were statistically significant differences between them. Survivin expression was significantly downed regulated by arsenic trioxide in A-431 cell line (P<0.05). Arsenic trioxide can inhibit cell proliferation and induce apoptosis in A-431 cells. Arsenic trioxide can also down-regulate the expression of Survivin in A-431 cells. The anti-proliferation effect of arsenic trioxide on human epidermoid carcinoma A-431 maybe is related to down-regulated expression of Survivin gene (P<0.05). Arsenic trioxide can also affect the cell cycle distribution in A-431 cells in our FCM assays. Every group of Arsenic trioxide blocked A-431 cell cycle at S-phase (P<0.05).Cellular morphology was observed under light microscope: cells shrinkage after treatment, the breakdown was irregular in shapes. Pseudo-foot lenders appeared in some cells at both ends. A-431 cells showed morphological changes.Conclusions:(1)Our study found that in the process of carcinogenesis of the basal cell carcinoma, the expression of survivin may play a role in the pathgenesis of BCC and SK while the expression of PTEN may take a part in the protection of BCC and SK. There propably is an interaction between the two genes in BCC and SK. Detecting the two protein levels can provide certain reference value in evaluating the malignant probability and in judging the prognosis of seborrheic keratosis.(2) It was first found that the abnormally high expressions of Livin protein may play a role in the pathogenesis of seborrheic keratosis and basal cell carcinoma through inhibiting cell apoptosis. From normal skin tissues to seberric kerotosis and to basal cell carcinoma, livin expressions are increasing gradually. Our study suggests that Livin can be a gene target in immunotherapy for epidermal tumors such as BCC and SK. There had been a significant positive correlation between the expressions of Livin and Survivin in BCC and SK.There may be a co-operation between the two genes in basal cell carcinoma and seborrheic keratosis. There had been a significant negtive correlation between the expressions of Livin and PTEN in BCC and SK.(3) In our study, it was first found that arsenic trioxide can inhibit cell proliferation and induce apoptosis in A-431 cells. Arsenic trioxide can also down-regulate the expression of survivin in A-431 cells. The anti-proliferation effect of arsenic trioxide on human epidermoid carcinoma A-431 maybe is related to down-regulated expression of survivin gene. Arsenic trioxide can also affect the cell cycle distribution in A-431 cells.更多还原