存活素反义寡核苷酸对角质形成细胞增殖和凋亡的影响

目的探讨存活素反义寡核苷酸对角质形成细胞增殖和凋亡的影响。方法以脂质体介导存活素反义寡核苷酸转染体外培养的角质形成细胞。采用MTT方法观察存活素反义寡核苷酸对角质形成细胞生长曲线的影响;采用RT-PCR方法观察存活素反义寡核苷酸对角质形成细胞存活素表达水平的影响;采用流式细胞仪检测存活素反义寡核苷酸对角质形成细胞细胞周期和凋亡的影响。结果存活素反义寡核苷酸作用于角质形成细胞后,细胞增殖受到明显抑制,存活素mRNA表达水平明显下降,细胞出现明显的凋亡现象。结论存活素反义寡核苷核酸能够抑制角质形成细胞增殖,促进角质形成细胞凋亡。提示存活素可能会成为一个新的治疗银屑病的靶分子。     

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，     

内源性大麻素(endocannabinoids)调节源于人类外分泌汗腺表皮细胞的生长和存活

有研究证据显示,内源性大麻素系统(endocannabinoid system,ECS)对皮肤稳态的控制具有重要作用.内源性大麻素,如安南得迈(N-花生四烯基乙醇胺,AEA)和2-花生四烯甘油(2-AG),目前参与这些脂质介质的合成及代谢的酶类、以及大麻素受体(cannabinoid receptors,CB)均在各种皮肤细胞群中被识别.不仅如此,皮肤ECS可调节皮肤细胞生长和分化之间的平衡.换言之,AEA可抑制培养的人表皮角质形成细胞的分化,也有关于AEA抑制这些细胞的生长和诱导其凋亡的报道.同样,还报道了局部产生的AEA抑制体外毛干的延伸和诱导凋亡,使毛发提前进入退行期.另外,培养源于人类皮脂腺的皮脂细胞产生的AEA和2-AG使脂质的生成量增加,并诱导细胞凋亡.尽管有报道显示,CB1和CB2在人类外分泌汗腺的表皮细胞上均存在原位表达,但仍缺乏关于内源性大麻素对哺乳动物皮肤最小附属器生物学调节作用的有效资料.因此,该研究旨在明确被广泛研究的AEA和2-AG对人类外分泌汗腺细胞生长和存活的影响.     

生存素与皮肤肿瘤相关性的研究进展

生存素基因是凋亡抑制蛋白家族新成员,因其结构较为独特,且能选择性地在肿瘤中表达,正常终末分化组织中不表达,是皮肤鳞癌、恶性黑素瘤的预后指标;抑制其表达能显著促进常见皮肤恶性肿瘤细胞发生凋亡,对正常组织无明显影响,有望成为肿瘤标记物和肿瘤治疗的新靶点。     

RNA干扰神经突起导向因子 Netrin-1表达下调对鳞状细胞癌SCL-1细胞的影响

目的 探讨神经突起导向因子 Netrin-1表达下调对皮肤鳞状细胞癌（鳞癌）SCL-1细胞增殖、凋亡和迁移的影响。方法 脂质体2000将Netrin-1 siRNA和对照siRNA转染皮肤鳞癌SCL-1细胞。将细胞分为对照1组（未处理组）、对照2组（siRNA对照组）、实验组（Netrin-1 siRNA组）。Western印迹检测转染Netrin-1 siRNA后SCL-1细胞Netrin-1蛋白的表达。人胆囊收缩素/缩胆囊素8肽（CCK-8）ELISA试剂盒分析细胞增殖的变化,流式细胞仪检测细胞凋亡情况。Western 印迹检测caspase-3、caspase-9及MMP-2的表达。结果 Netrin-1 siRNA明显下调SCL-1细胞中Netrin-1蛋白的表达。Netrin-1表达下调明显抑制SCL-1细胞的增殖和诱导细胞凋亡,增加caspase-3和caspase-9的表达,同时可显著抑制MMP-2的表达。结论 Netrin-1表达下调可介导鳞癌细胞增殖抑制、增高细胞凋亡率和降低细胞迁移能力。     

生存素、bcl-2在皮肤鳞状细胞癌皮损及细胞系的表达

目的 探讨凋亡抑制基因生存素、bcl-2在皮肤鳞状细胞癌（简称鳞癌）皮损及皮肤鳞癌细胞系（SCL-1）细胞中的表达意义。方法 细胞免疫组化观察60例鳞癌患者皮损中生存素、bcl－2蛋白的表达并进行统计分析。Western印迹检测HaCaT细胞系和皮肤鳞癌细胞系（SCL-1）细胞中生存素bcl－2蛋白的表达。结果 免疫组化示正常皮肤组织中未见bcl-2与生存素表达;生存素、bcl-2在鳞癌皮损中呈阳性表达,bcl-2、生存素蛋白表达阳性率分别为70%、 60%,两者表达无相关性（P > 0.05）。生存素的表达与年龄、性别、发病部位、病理分级无明显关系,与有无淋巴结转移密切相关。bcl-2蛋白的表达与年龄、性别、发病部位,淋巴结转移无明显关系,但随着鳞癌肿瘤病理分级的升高而降低。Western印迹分析示生存素、bcl-2蛋白在SCL-1细胞中表达明显高于HaCaT细胞。结论 生存素、bcl-2在鳞癌的发生发展中,可能通过不同的抗凋亡路径发挥作用。     

存活素在尖锐湿疣皮损中的表达

目的：探讨凋亡抑制基因存活素在男性包皮尖锐湿疣组织中的表达.方法：采用免疫组化染色法检测30例男性包皮尖锐湿疣皮损组织及30例正常男性包皮组织中存活素表达水平.结果：尖锐湿疣组中存活素蛋白表达水平显著高于对照组（P〈0.01）,两组表达阳性率差异有统计学意义.结论：存活素在尖锐湿疣组织中呈高表达状态, 可能在尖锐湿疣的发病中起重要作用.     

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。     

靶向生存素的小干扰RNA抑制人黑素瘤M14细胞系生长的研究

目的 探讨小干扰RNA（siRNA）对人黑素瘤M14细胞系生存素基因表达的抑制作用及对细胞凋亡、增殖和侵袭的影响。方法 构建靶向生存素的siRNA表达质粒,用脂质体法转染M14人黑素瘤细胞。RT-PCR检测M14细胞生存素mRNA的表达,Western印迹检测生存素蛋白的表达,流式细胞仪检测AnexinV标记的凋亡细胞,噻唑蓝（MTT）法分析细胞增殖,Transwell法分析侵袭能力。结果 与M14细胞和转染空载体的M14细胞比较,转染siRNA的表达质粒可以明显抑制生存素基因在转录和翻译上的表达。M14组和空载体组几乎无凋亡细胞,siRNA表达质粒组的凋亡率明显增加（χ2 ＝ 31.55,P < 0.01）。细胞增殖抑制率（63.6% ± 1.6%）也明显增加,同时Transwell细胞侵袭实验显示,siRNA表达质粒转染组抑制作用显著。结论 靶向生存素的siRNA表达质粒可以特异性抑制生存素的表达,显著抑制肿瘤细胞增殖和侵袭并诱导细胞凋亡。     

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。     

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.     

Proliferation, apoptosis, and survivin expression in keratinocytic neoplasms and hyperplasias

The dysregulation of apoptosis occurs in many cutaneous disease states. Several apoptosis inhibitors have been shown elevated in neoplasms and in some inflammatory conditions, but their relation to proliferative and apoptotic states has not been defined. We examined the expression of the apoptosis inhibitor survivin in a panel of keratinocytic neoplasms and hyperproliferative skin lesions using both immunohistochemistry and a newly developed in situ hybridization technique. Proliferation and apoptotic indices were also assessed by immunohistochemical staining for proliferating cell nuclear antigen and TUNEL, respectively. We found the highest rate of proliferation in verrucae and psoriasis followed by actinic keratosis, squamous and basal cell carcinoma, lichen simplex chronicus, and seborrheic keratosis; all were significantly (P < 0.05) higher than normal skin. Apoptotic rate was increased in squamous (P = 0.05) and basal cell carcinoma (P = 0.03), but not significantly different from normal skin in the other lesions tested. Survivin expression was seen in most neoplasms and hyperproliferative lesions, but not normal skin. Survivin expression was often restricted to the upper third of the epidermis in psoriasis and lichen simplex chronicus, whereas all the other lesions stained diffusely. Survivin expression appears to be a consistent feature of keratinocytic neoplasms and hyperproliferative lesions and may contribute to the formation of epidermal hyperplasia seen in all of these disease states.     

Proliferation, apoptosis, and survivin expression in a spectrum of melanocytic nevi

BACKGROUND: Apoptosis is important for maintenance of tissue homeostasis and often dysregulated in cutaneous neoplasms. The apoptosis inhibitor survivin is expressed in melanoma and non-melanoma skin cancers and benign keratinocytic lesions. Its expression has not been studied in melanocytic nevi. OBJECTIVE: We determined the expression pattern of survivin in benign melanocytic nevi in comparison to markers of proliferation and apoptosis. METHODS: Six cases of each of the following melanocytic nevi were retrieved from a dermatopathology archive: compound dysplastic nevus, intradermal nevus, compound nevus, neurotized intradermal nevus, and Spitz nevus. Survivin expression was evaluated by in situ hybridization. Apoptotic and proliferation indices were calculated by counting immunoreactive cells in terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling and proliferating cell nuclear antigen immunostained sections, respectively. RESULTS: All nevi, regardless of histologic type, expressed survivin. Compound melanocytic lesions expressed survivin in both epidermal and dermal compartments. The apoptotic rate was low for dysplastic, compound, and Spitz nevi, and apoptotic cells were not identified in any neurotized nevus. The proliferative index was highest for Spitz nevi, while all other nevi demonstrated rare positive cells. CONCLUSIONS: Survivin is consistently expressed in benign melanocytic lesions, while apoptotic cells are rarely identified, suggesting the dysregulation of apoptotic pathways with the accumulation of cells in these neoplasms.     

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.     

生存素反义寡核苷酸对人恶性黑素瘤A375细胞生物学行为的影响

目的研究生存素反义寡核苷酸(AS-ODN)经脂质体介导转染对A375细胞增殖、凋亡的影响。方法合成生存素硫代反义寡核苷酸(AS-ODN),脂质体携带转染人恶性黑素瘤A375细胞。采用台盼蓝计数、软琼脂克隆形成试验、电镜、流式细胞仪检测AS-ODN对A375细胞增殖及凋亡的影响。通过裸鼠致瘤实验观察AS-ODN对A375细胞动物体内增殖的抑制作用。结果生存素反义寡核苷酸能降低G2/M期细胞百分比,诱导细胞凋亡发生,明显抑制A375细胞的生长和克隆形成,裸鼠体内A375细胞恶性增殖潜力下降。结论脂质体介导生存素AS-ODN能有效抑制A375细胞增殖、诱导肿瘤细胞凋亡。     

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.     

Mononuclear cell patterns in the skin. An immunohistological and morphometrical analysis

A large variety of skin diseases is characterized by the presence of mononuclear cell infiltrates in the dermis and to some extent in the epidermis. We have investigated frozen material of 566 lesions of benign and malignant skin diseases by immunohistological methods with a panel of 20 monoclonal antibodies; quantitative studies using computer-assisted image analysis were additionally performed in 80 specimens. In all reactive lesions, mononuclear cells were arranged in distinct compartments simulating the architecture of normal lymphatic tissue. A qualitatively uniform T cell compartment (mainly helper-inducer T lymphocytes, suppressor-cytotoxic T lymphocytes, Langerhans' cells) with slight quantitative differences was found in all inflammatory skin diseases, in peritumoral infiltrates, and also in normal skin. The B cell compartment (B lymphocytes, few T lymphocytes and monocytes) is only rarely seen (some B cell lymphomas and pseudolymphomas). The monocyte compartment (monocytes, few T lymphocytes) associated with a T cell compartment is typical for granulomatous skin lesions. The epidermis forms a separate functional region, which might evolve into a lymphoepithelial compartment in diseased skin. Low-grade malignant lymphomas of the skin display similar architectural patterns as reactive lesions, whereas high-grade malignant lymphomas do not. Obviously the mononuclear cells in the skin are usually arranged in compartments and form a limited number of distinct patterns. As similar patterns are found in diseases that are completely different with respect to clinical appearance, histological features, cause, and outcome, the patterns are not disease specific but reflect general anatomical-functional relationships of inflammatory cells and the skin.     

恶性黑素瘤Survivin和C-erbB-2的检测及其意义

目的检测恶性黑素瘤的Survivin和CerbB2及其与临床病理特征的关系。方法应用免疫组化SP法检测72例恶性黑素瘤、61例瘤旁组织、22例黑色素细胞痣和20例正常皮肤组织中Survivin和CerbB2蛋白。结果恶性黑素瘤Survivin蛋白阳性率为59.7%,明显高于瘤旁组织3.3%,黑色素细胞痣4.5%和正常皮肤组织0%(P均<0.01)。Survivin阳性与转移状况和组织学分级有显著相关性。恶性黑素瘤无转移组和Ⅰ级分别明显低于转移组和Ⅱ,Ⅲ,Ⅳ级(P均<0.05)。恶性黑素瘤CerbB2蛋白阳性率为68.1%,明显高于瘤旁组织8.3%,黑色素细胞痣5.6%和正常皮肤组织2.8%(P均<0.01),CerbB2阳性与临床病理特征无明显相关性。Survivin在CerbB2阳性和阴性患者中阳性率分别为69.4%和39.1%,两者间有显著性差异(P<0.05)。结论Survivin和CerbB2蛋白在恶性黑素瘤组织中水平上调,提示两者对恶性黑素瘤发生发展起重要作用。恶性黑素瘤中Survivin蛋白水平与肿瘤转移和组织学分级有关,Survivin可能为临床判断恶性黑素瘤转移和评估预后的有用指标。