Survivin与皮肤恶性肿瘤

生存素（survivin）是凋亡抑制蛋白（IAP）基因家族新成员。具有抑制细胞凋亡和调节细胞分裂及参与肿瘤血管形成等多重功能，于多种肿瘤组织中高表达而在正常成人组织中一般不表达。Survivin在皮肤恶性肿瘤中高表达，与皮肤恶性肿瘤的演主莹、复发、预后、耐药及病人生存率等有关，可作为皮肤恶性肿瘤诊断及治疗新的切入点，     

Survivin的生物学特性及其在皮肤病中表达的意义

Survivin作为凋亡抑制蛋白IAP家族的成员之一，不仅具有抑制由线粒体诱导的凋亡的作用，还协同单一的p53基因抑制凋亡的发生，同时在血管生成过程中抑制血管内皮细胞的凋亡，保护新生血管。Survivin在正常表皮不表达，但在很多皮肤疾病中有充分表达。本文综述了Survivin的分子结构、抑制凋亡的机制以及在各种皮肤组织中，Survivin的表达与诊断、预后、治疗的意义。     

凋亡抑制蛋白livin在人皮肤鳞状细胞癌中表达的初步研究

目的:研究凋亡抑制蛋白livin在人皮肤鳞状细胞癌(简称鳞癌)组织及细胞中的表达情况.方法:①采用免疫组化染色方法检测18例鳞癌组织中livin的表达情况;②利用反转录(RT)-PCR及Western blot的方法检测皮肤鳞癌细胞系A431细胞中livin mRNA及蛋白的表达情况.结果:①livin蛋白在正常皮肤组织中不表达,鳞癌组织中livin蛋白的阳性表达率为72.2%;②livin基因mRNA及蛋白在皮肤鳞癌细胞系A431中表达较高.结论:凋亡抑制基因livin在人皮肤鳞癌组织及细胞中高表达,提示livin基因在皮肤鳞癌发生发展中起一定作用,为皮肤鳞癌的诊断及治疗研究提供实验基础.     

Survivin与皮肤病的研究进展

Survivin是凋亡抑制蛋白家族新成员.其在皮肤恶性肿瘤、癌前期皮肤病、某些良性角质形成细胞过度增生性皮肤病中高表达.Survivin通过抑制细胞凋亡、调节细胞分裂、参与肿瘤血管形成等多重功能参与皮肤疾病的发生.抑癌基因p53通过对抗Survivin蛋白的抗凋亡活性诱导肿瘤细胞凋亡.检测癌组织中Survivin和p53基因的表达可以为肿瘤患者的诊断和判断预后提供参考指标,并且针对Survivin基因及Survivin联合p53基因的靶向治疗将为皮肤癌的治疗开辟新的途径.     

尖锐湿疣皮损中Caspase 8与p-Akt的表达

目的 探讨Caspase 8与p-Akt在尖锐湿疣皮损中的表达及意义.方法 收集尖锐湿疣30例、宫颈癌20例、正常皮肤(包皮)20例,采用免疫组化检测以上组织中Caspase 8及p-Akt的表达及分布.结果 Caspase 8在尖锐湿疣皮损、宫颈癌、正常皮肤中阳性率分别为23.33%、80%、90%.Caspase 8在尖锐湿疣中的表达强度显著低于正常皮肤(P<0.01)和宫颈癌组织(P<0.001).p-Akt在尖锐湿疣皮损、宫颈癌组织、正常皮肤组织中的阳性率分别为93.33%、95%、90%.p-Akt在尖锐湿疣皮损的表达强度显著高于正常皮肤组织(P<0.001),低于宫颈癌组织(P<0.001).在尖锐湿疣皮损和正常皮肤组织中,Caspase 8与p-Akt的表达无相关性.在宫颈癌中,Caspase 8与p-Akt的表达存在显著的正相关(r=0.369,P<0.05).结论 Caspase 8的凋亡启动受抑制及p-Akt的抗凋亡作用可能参与了尖锐湿疣的发生发展.     

Survivin和COX-2在鲍温病和皮肤鳞状细胞癌中的表达及意义

目的探讨鲍温病及皮肤鳞状细胞癌组织中凋亡抑制蛋白生存素(Survivin)和环氧合酶-2(COX-2)的表达及意义。方法用免疫组化SP法分别检测Survivin蛋白和COX-2蛋白在19例鲍温病组织、25例皮肤鳞状细胞癌组织及17例正常皮肤组织中的表达。结果 Survivin蛋白和COX-2蛋白在鲍温病组织及鳞癌组织中的阳性表达率明显高于正常组,二者在鳞癌组织中的阳性率与鲍温病组织中阳性率差异有统计学意义(P0.01);Survivin和COX-2的阳性表达呈正相关(r=0.764,P0.05)。结论 Survivin蛋白和COX-2蛋白参与了鲍温病及皮肤鳞状细胞癌的发生、发展,二者在抑制细胞凋亡方面可能存在协同作用。     

存活素和皮肤病

存活素是凋亡抑制蛋白家族的新成员,不仅具有抑制细胞凋亡的能力,而且参与细胞有丝分裂和胞质分离的调节。存活素仅表达于各种皮肤良恶性病变,而不表达于正常皮肤。综述存活素分子结构、抑制凋亡的机制、在细胞分裂中的作用、与各种良恶性增生性皮肤病的关系以及靶向治疗。     

RPL34基因敲低对人皮肤鳞状细胞癌SCL-1细胞的影响

【摘要】目的 研究ribosomal protein L34 (RPL34)基因敲低后对皮肤SCC细胞增殖、凋亡的影响。方法 首先通过免疫组化对14例皮肤cSCC患者和16例正常对照的组织进行RPL34表达分析。然后利用慢病毒感染皮肤鳞癌SCL-1 细胞RPL34表达进行基因敲低后,流式细胞仪检测细胞周期分布及凋亡,MTT检测细胞增殖,qPCR检测目的基因敲减效率,Western blot分析RPL34表达水平。计数资料用卡方检验进行比较,计量资料用均数±标准差表示,t检验比较2组间差异。α设定为0.05,P<0.05认为有统计学差异。结果免疫组化实验中,cSCC肿瘤组织中RPL34在细胞浆的表达明显高于正常对照皮肤组织（cSCC组细胞核表达评分为2.929±1.542,细胞浆表达评分为2.143±1.956,正常对照皮肤组织中细胞核表达评分为2.563 ±1.153,细胞浆表达评分为0.500±0.516。）。流式细胞仪分析检测细胞凋亡情况,在空白对照组凋亡率为4.58 %,而RPL34-shRNA组的凋亡率为9.42%（P<0.05）。细胞周期分布情况实验组（shRPL34）处于S期的细胞增多(P<0.05),处于G1期的细胞减少(P<0.05),处于G2/M期的细胞无显著变化(P＞0.05)。MTT检测细胞增殖,shRPL34组的SCL-1细胞OD490吸光率及吸收率变化倍数明显低于空白对照组(P<0.05),提示具有活力的细胞数量明显低于对照组。qPCR结果中,经shRNA慢病毒感染后,实验组SCL-1细胞中RPL34基因在mRNA水平的表达量受到抑制,敲减效率达到85.1%（p<0.05）。结论 RPL34在cSCC中可以调节细胞增殖、凋亡,干扰细胞周期。     

紫外线和热休克蛋白研究进展

紫外线辐射通过多条信号传导途径和热休克转录因子诱导皮肤细胞热休克蛋白基因表达，紫外线也通过诱导热休克蛋白磷酸化、转位以发挥其适应性保护功能。热休克蛋白能抑制紫外线诱导的细胞凋亡和皮肤炎症反应。热休克蛋白在紫外线诱导的皮肤肿瘤中的不同表达表明其和皮肤肿瘤的发生、发展和预后密切相关。     

沉默单核细胞趋化蛋白3对人增生性瘢痕成纤维细胞增殖、迁移及凋亡的影响北大核心CSCD

目的:探讨沉默单核细胞趋化蛋白3(MCP-3)对人增生性瘢痕成纤维细胞增殖、迁移及凋亡等功能的影响。方法:取增生性瘢痕组织和正常皮肤组织,qRT-PCR检测正常皮肤组织和增生性瘢痕组织中MCP-3 mRNA表达水平;分离培养增生性瘢痕组织成纤维细胞(HSF)和正常皮肤组织成纤维细胞(NSF),qRT-PCR和Western blot检测两种成纤维细胞中MCP-3 mRNA和蛋白表达水平。采用MCP-3干扰慢病毒(shRNA-MCP-3)和其阴性对照病毒(shRNA-NC)感染HSF,MTT检测各组细胞增殖活性,流式细胞术检测各组细胞凋亡情况,Transwell实验检测细胞的迁移能力,qRT-PCR检测各组细胞中MCP-3、collagenⅠ和collagenⅢmRNA水平,Western blot检测细胞中凋亡相关蛋白MCP-3、Bax、Bcl-2、Cleaved-caspase-3及collagenⅠ和collagenⅢ蛋白表达水平。结果:MCP-3在增生性瘢痕组织及HSF中高表达。与blank组和shRNA-NC组比较,shRNA-MCP-3组细胞中MCP-3 mRNA和蛋白表达水平显著下降(P<0.01),细胞增殖活性、迁移能力及细胞中collagenⅠ、collagenⅢ和Bcl-2表达水平均显著降低(P<0.01),同时细胞凋亡率及凋亡蛋白Bax和Cleaved-caspase-3表达水平显著上升(P<0.01)。结论:干扰MCP-3表达可能通过抑制细胞增殖和诱导细胞凋亡,减少细胞合成胶原,从而发挥抑制瘢痕形成与发展的作用。     

Expression and targeting of the apoptosis inhibitor, survivin, in human melanoma

The newly described apoptosis inhibitor survivin is expressed in many human cancers and appears to play a critical part in both apoptosis regulation and cell cycle progression. Its potential role in malignant melanoma is unknown. In a panel of 30 malignant melanomas, survivin was strongly expressed in all cases (15 of 15) of metastatic malignant melanomas and 13 of 15 cases of invasive malignant melanomas by immunohistochemistry. In invasive malignant melanomas, survivin was also expressed in the in-situ component of the lesion. Survivin expression was found in all cases (11 of 11) of nevi, but not in melanocytes in sections of normal skin. The apoptosis inhibitor bcl-2 was expressed in 26 of 30 cases, but generally at lower levels than that of infiltrating lymphocytes. The mitotic index, as assessed by MIB-1 staining, was consistently higher in metastatic than invasive malignant melanomas. Assessment of apoptotic index by in situ end-labeling revealed extremely low rates of apoptosis in most malignant melanomas. Survivin expression by western blotting was detected in four human metastatic malignant melanoma cell lines but not in cultured normal human melanocytes. Transfection of both YUSAC-2 and LOX malignant melanoma cells with green fluorescence protein-conjugated survivin anti-sense or green fluorescence protein-conjugated survivin dominant negative mutant (Cys84Ala) [corrected] resulted in increased apoptosis in the absence of other genotoxic stimuli. Two-color flow cytometry confirmed that YUSAC-2 cells transfected with survivin anti-sense expressed less endogenous survivin and exhibited an increased fraction of cells with sub-G1 DNA content. These data demonstrate that apoptosis inhibition by survivin may participate in the onset and progression of malignant melanomas, and suggest that therapeutic targeting of survivin may be beneficial in patients with recurrent or metastatic disease.     

Survivin: a dual player in healthy and diseased skin

Survivin belongs to the inhibitor of apoptosis (IAP) protein family, and, in addition to the antiapoptotic functions, it also regulates the cell cycle. The survivin gene generates five major isoforms with diverse and opposite functions. Survivin is highly expressed in cancer and in few normal adult tissues, including skin. It is mostly detected in the nucleus of keratinocyte stem cells (KSCs), but it is also expressed in melanocytes and fibroblasts. Survivin isoforms are differentially detected in subpopulations of human keratinocytes, exerting contrasting activities. Survivin has an important role in the regulation of cell cycle in keratinocytes, and it protects these cells from anoikis and UV-induced apoptosis. In melanoma, survivin is abundantly expressed, and its subcellular localization varies depending upon tumor thickness and invasiveness. Survivin overexpression has been shown in squamous cell carcinoma (SCC), and it is also involved in UVB-induced carcinogenesis. The presence of survivin both in the nucleus and in the cytoplasm throughout the epidermal layers of psoriatic lesions suggests the involvement of this protein in the keratinocyte alterations typical of this disease. Additional studies on the expression of survivin isoforms and their subcellular localization in relation to function will confirm the key role of survivin in the skin and will open the field to new therapeutic strategies for many cutaneous conditions.     

Communication: expression of the novel inhibitor of apoptosis survivin in normal and neoplastic skin.

Apoptosis plays a fundamental part in epidermal homeostasis, and apoptotic cells have been detected in normal and diseased skin. Little is known, however, on the inhibitory mechanisms of apoptosis at the skin level. In addition to bcl-2, a novel inhibitor of apoptosis designated survivin and structurally analogous to IAP apoptosis inhibitors has been recently identified. The expression of survivin in normal and pathologic skin was investigated. Immunohistochemical studies revealed that survivin is expressed in basal keratinocytes, but not in suprabasal epidermal layers, with a pattern similar to bcl-2. In western blots, the anti-survivin antibody recognized a single band of 16.5 kDa in protein extracts from normal human keratinocytes in culture, in agreement with the predicted size of survivin. In addition, survivin immunoreactivity was detected in benign and malignant melanocytic lesions, with strong expression in invasive lesions of melanomas. Whereas survivin staining was undetectable in benign epithelial tumors, such as seborrheic keratoses, it was observed in all epidermal layers in Bowen's disease. Interestingly, at variance with bcl-2, survivin was markedly expressed in squamous cell carcinoma, but virtually lacking in basal cell carcinoma, suggesting that these two apoptosis inhibitors may act through different anti-apoptotic pathways. Deregulation of survivin may influence both epidermal homeostasis and the development of melanoma and nonmelanoma skin cancer.     

Cytoplasmic and nuclear expression of survivin in melanocytic skin lesions

Survivin, a member of the inhibitors of apoptosis protein family, regulates both cellular proliferation and apoptotic cell death. While the human survivin gene is highly expressed in the developing fetus, in adults its expression is restricted to highly proliferating normal tissues and neoplastic tumors tissues. In the present study, we compared the expression of survivin in melanoma and benign melanocytic lesions such as junctional, compound, dermal, congenital, blue and spitz nevi. This analysis reveals a heterogeneous expression of survivin with respect to both the intensity, frequency and cellular localization. In junctional, compound and blue nevi, survivin was present in nuclear localization, whereas in spitz nevi survivin was detectable in the cytoplasm. In dermal and congenital nevi, survivin was present in both localizations with predominance of the nuclear compartment. Interestingly, this distribution was similar to that observed in primary melanoma; whereas in metastatic melanoma the predominance of the nuclear localization of survivin was lost. Our data demonstrate that although survivin is expressed in a large number of benign nevi, the balance between its cytoplasmic and nuclear expression was immensely heterogeneous between lesions with suspected different developmental origins.     

生存素在恶性黑素瘤中的表达及与VEGF和PCNA的关系

目的 探讨人恶性黑素瘤组织中凋亡抑制蛋白生存素的表达,及其与恶性黑素瘤临床特征、血管内皮生长因子(VEGF)和增殖细胞核抗原(PCNA)表达的关系.方法 免疫组化方法检测36例人恶性黑素瘤和30例色素痣组织中生存素、VEGF和PCNA蛋白表达.结果 ①生存素在恶性黑素瘤和色素痣中均有表达,但在恶性黑素瘤中表达明显高于色素痣(P<0.01).②在恶性黑素瘤组织中有30例表达VEGF(阳性率83.3%),36例表达PCNA(阳性率100%),而色素痣组织不表达VEGF和PCNA.③生存素的阳性表达与恶性黑素瘤患者的发病年龄、性别、淋巴结转移均无相关性(P>0.05),但其表达与VEGF和PCNA的表达密切相关(P<0.01).结论 生存素可能参与恶性黑素瘤的增殖和与VEGF相关的肿瘤血管生成.     

几种IAP蛋白及其相关因子在蕈样肉芽肿中的表达及NB-UVB的作用

目的 探讨生存素（Survivin）和Livin两种凋亡抑制蛋白（IAP）及其相关因子Bcl-xl和Caspase-3在蕈样肉芽肿（MF）中的表达情况和窄谱中波紫外线（NB-UVB）的干预作用。方法 采用免疫组化SABC法测定生存素、Livin、Bcl-xl和Caspase-3在MF皮损中的表达，用TUNEL法和原位杂交技术检测NB-UVB治疗前后四种因子mRNA的转录水平和细胞凋亡指数的变化。结果 在MF红斑期、斑块期和肿瘤期，生存素的表达阳性率分别为40.00%、75.00%、77.78%；Bcl-xl为60.00%、68.75%、88.89%，二者在正常皮肤均无表达。Livin在MF三期中阳性率依次为40.00%、25.00%和44.44%；Caspase-3为60.00%、43.75％和22.22％，二者均与正常皮肤相似。NB-UVB治疗后，MF中凋亡细胞显著增多（t = 6.49，P < 0.001），生存素mRNA和Bcl-xl mRNA的表达较照射治疗前有显著降低（P < 0.10），而Caspase-3 mRNA的表达增加（P < 0.10），Livin mRNA的表达在照射治疗前后无显著变化（P > 0.10）。结论 生存素、Bcl-xl和Caspase-3可能参与MF中T淋巴细胞凋亡障碍的调节过程，与MF的发病有关。NB-UVB对MF的治疗作用可能与其抑制生存素mRNA和Bcl-xl mRNA的转录，降低IAP蛋白水平，增加Caspase-3的转录，进而增强MF肿瘤细胞的凋亡有关。     

siRNA抑制生存素基因对恶性黑素瘤细胞A375凋亡的影响

目的 探讨RNA干扰技术抑制恶性黑素瘤细胞株A375生存素基因表达后,对A375细胞凋亡的影响。方法 构建针对凋亡抑制基因生存素的siRNA真核表达载体pU-生存素-siRNA,用电穿孔法转染A375细胞,采用蛋白质印迹技术检测生存素的表达,并用流式细胞仪检测细胞凋亡的变化。结果 转染pU-生存素-siRNA后,生存素在A375细胞中的表达(0.24±0.02)较对照组(0.98±0.21)明显下降,试验组细胞的凋亡率(83%)较对照组(28%)明显增加。结论 通过RNA干扰技术可抑制生存素的表达,诱导A375细胞的凋亡增加。     

Radiosensitization of human melanoma cells by ribozyme-mediated inhibition of survivin expression

Survivin is a structurally unique member of the inhibitors of apoptosis protein family and is involved in the control of cell division and inhibition of apoptosis. The notion that survivin is overexpressed in most human tumors but absent in normal adult tissues with only a few exceptions has led to the proposal of survivin as a promising therapeutic target for novel anticancer therapies. In this context, we generated a hammerhead ribozyme targeting the 3' end of the CUA110 triplet in the survivin mRNA. Two human melanoma cell lines (JR8 and M14) overexpressing survivin were stably transfected with the pRc/CMV vector carrying the ribozyme sequence. Two polyclonal cell populations proven to endogenously express ribozyme and characterized by a markedly lower survivin protein level (-60% and -50%, respectively) than JR8 and M14 parental cells were selected for the study. Ribozyme-expressing cells showed a significantly (p<0.01) increased sensitivity to gamma-irradiation (as detected by clonogenic cell survival) compared to JR8 and M14 cells. Moreover, in the JR8 cell line, the extent of radiation-induced apoptosis (in terms of percentage of apoptotic nuclei in cells stained with propidium iodide and level of caspase-3 catalytic activity) was markedly greater in ribozyme-expressing cells than in parental cells. These results demonstrate for the first time that attenuation of survivin expression renders human melanoma cells more susceptible to gamma-irradiation.     

Endogenous survivin modulates survival and proliferation in UVB-treated human keratinocytes

Abstract:  Survivin is a bi-functional member of inhibitor of apoptosis protein family, as it is able to both inhibit apoptosis and to regulate cell cycle. We investigated the role of survivin in human keratinocytes under normal conditions and during UVB irradiation. Survivin siRNA decreases proliferation and induces apoptosis in human keratinocytes, in a mode consistent with the mitotic catastrophe. Low doses UVB increase survivin expression at earlier times, while high doses down-regulate survivin level. Low doses UVB induce cell cycle arrest in G2/M, while high doses UVB cause apoptosis. Moreover, overexpression of survivin protects keratinocytes from UVB-induced apoptosis, and silencing of survivin renders keratinocytes more susceptible to UVB-induced cell death. Finally, survivin siRNA increases UVB-induced reduction of cell proliferation. Taken together, these results indicate that survivin plays a critical role in epidermal homeostasis in normal conditions and during UVB exposure, with possible implication in skin carcinogenesis.