SV40TAg和Cre/loxP系统诱导的正常人可逆性转化黑素细胞在豚鼠体内的存活能力和复色实验

目的 研究SV40TAg基因和Cre/loxP系统在体外诱导正常人黑素细胞的可逆性转化以及转化细胞在豚鼠体内的存活能力和复色效果。方法 用Cre-ERT2病毒上清感染经SV40TAg基因转化的正常人黑素细胞（MCT）,诱导Cre重组酶表达（MCTC）;过氧化氢法建立黄褐色雄性豚鼠白癜风动物模型,按黑素细胞移植方法进行原代黑素细胞和MCTC移植,观察MCTC在豚鼠体内的存活能力及复色效果。结果 MCTC细胞移植实验结果显示,1个月左右移植区即可见色素沉着,连续观察3个月,可见0.5 ～ 1 cm的黑色斑片或褐色斑片形成,移植成功率为82.5%,原代黑素细胞移植成功率为76.7%。病理观察移植区有明显黑素沉积,部分毛囊内也可见黑素沉积。结论 联合应用SV40TAg基因和Cre/loxP系统可以成功地诱导具有良好生物学安全性的人源性可逆性永生化黑素细胞,具有与原代黑素细胞相似的在体存活率,具备在体黑素合成功能,可以达到良好的复色效果。     

InnVit基因沉默对黑素细胞B10BR黑素合成和细胞凋亡的影响

目的观察siRNA抑制InnVit基因后对小鼠永生化B10BR黑素细胞黑素合成和细胞凋亡的影响,进一步探讨InnVit基因的生物学功能。方法将InnVit基因的三对siRNA转染B10BR黑素细胞,半定量RT-PCR检测InnVit基因抑制后对黑素细胞InnVit基因mRNA水平的变化;采用MTT法测定InnVit基因抑制后对细胞增殖率的影响;采用流式细胞仪检测InnVit基因抑制后对黑素细胞凋亡的影响。结果siRNA1转染后,InnVit基因在黑素细胞中的表达明显下降,试验组细胞的早期凋亡率(35.80%)较对照组(6.47%)明显增加。结论通过RNA干扰技术可抑制InnVit在黑素细胞中的表达,可诱导B10BR细胞的凋亡增加。     

SV40T抗原基因转染正常人黑素细胞的研究

目的 研究SV40T抗原基因对体外培养的人正常黑素细胞的转化作用。方法 用阳离子聚合物Sofast^TM介导基因转染,将含有SV40T抗原基因的真核表达载体SV40T-pEGFP导入原代培养的正常人黑素细胞进行稳定表达,G418筛选出阳性克隆,挑独立的单克隆扩大培养,检测SV40T抗原基因及SV40T蛋白在黑素细胞内的表达。结果 分离获得转化细胞阳性克隆。提取DNA及RNA后用PCR法成功扩增出288bp的片段,免疫组化及蛋白质印迹结果证实,转染黑素细胞核内有SV40T蛋白表达。结论 SV40T抗原基因可以成功诱导人黑素细胞的转化。     

慢病毒介导shRNA沉默Nicastrin基因的人永生化角质形成细胞模型构建

目的 构建Nicastrin(NCT)基因的RNA干扰慢病毒表达载体,并在人永生化角质形成细胞(HaCaT细胞)上鉴定其沉默效率,构建NCT基因稳定下调的人永生化角质形成细胞模型,为后续研究NCT基因下调对角质形成细胞的生物学行为影响奠定实验基础.方法 设计3个靶向NCT基因特异性短发卡RNA(shRNA)表达序列并连接到慢病毒表达质粒pGLV3/H 1/GFP+ Puro中,成功构建慢病毒重组表达质粒,并通过测序鉴定.将构建的重组表达质粒与包装质粒共转染293T细胞,产生慢病毒颗粒,并测其滴度.将HaCaT细胞分为空白组(未感染病毒)、阴性对照组(NC组,感染空载病毒LV3-shNC)和干扰组(感染NCT-shRNA1、2、3慢病毒).流式细胞仪检测慢病毒转染效率,实时荧光定量PCR(qRT-PCR)、Western印迹检测靶基因的沉默效率,筛选出沉默效果最佳的干扰序列.结果 测序表明,NCT-shRNA慢病毒重组表达质粒构建成功.重组表达质粒与包装质粒共转染293T细胞后产生的慢病毒颗粒滴度为109 TU/ml.慢病毒感染HaCaT细胞后,流式细胞仪检测,慢病毒转染效率大于95％.qRT-PCR结果,与NC组相比,干扰组NCT mRNA表达量明显下降,其中NCT-shRNA1组NCT基因沉默效果最佳,干扰效率达到75％.Western印迹结果,与NC组相比,shRNA1组NCT蛋白表达抑制率为71.7％.结论 成功筛选出高效NCT-shRNA干扰序列,构建NCT-shRNA慢病毒重组表达载体,并构建NCT基因稳定下调的人永生化角质形成细胞模型.     

InnVit基因对永生化黑素细胞B10BR迁移的影响

目的分析InnVit基因对永生化黑素细胞B10BR迁移的影响,探讨InnVit基因对黑素细胞生物学功能的调控情况。方法化学合成InnVit基因特异性SiRNA,构建InnVit基因表达载体P3XF-P120,lipofectamineTM2000转染B10BR细胞。半定量RT-PCR鉴定抑制和过表达效率;Transwell观察InnVit基因抑制和过表达后黑素细胞的迁移能力,明胶酶谱分析MMP2和MMP9的活性。结果成功在永生化黑素细胞B10BR中抑制和过表达InnVit基因。细胞处理20h后,抑制和过表达组Transwell滤膜下层黏附细胞数与对照组相比无显著性差异(P0.05),MMP2和MMP9活性较对照组也无显著性差异(P0.05)。结论InnVit基因对黑素细胞的迁移无明显调控作用。     

早老蛋白增强子2基因沉默对HaCaT细胞增殖及γ分泌酶表达的影响

目的:构建早老蛋白增强子2（PSENEN）基因沉默的人永生化角质形成细胞（HaCaT）模型,探究PSENEN基因表达下调对HaCaT细胞增殖以及γ分泌酶表达的影响。方法:设计3组靶向PSENEN基因的shRNA序列,与线性化的LV3-pGLV-h1-GFP-puro载体构建慢病毒重组表达质粒,经酶切及测序鉴定后,进行慢...     

hTERT基因永生化人毛乳头细胞系的生物学特征

目的研究hTERT基因永生化人毛乳头细胞(DPC)系的生物学特征。方法采用脂质体转染法,将hTERT基因导入体外培养的正常人DPC,经G418筛选得到阳性克隆,连续传代培养。检测转染细胞内hTERT mRNA的表达,并分析转染细胞的生物学特征。结果转染后获得1个阳性细胞克隆,扩大培养后细胞生长良好,现已连续传至65代。检测证实转染细胞稳定表达hTERT mRNA,且此转染细胞具有体外培养正常DPC的生物学特征。结论hTERT基因永生化人DPC系保持了正常DPC的生物学特征。     

hTERT基因转染激活人毛乳头细胞端粒酶的实验研究

目的:明确外源性人端粒酶反转录酶(hTERT)基因转染能否激活体外培养人毛乳头细胞(DPC)的端粒酶.方法:采用脂质体转染法,将空载体质粒pIRES2-EGFP和含有hTERT基因的质粒pIRES2-EGFP-hTERT分别导入体外培养的正常人DPC,经G418筛选得到阳性克隆,连续传代培养.应用反转录(RT)-PCR法检测hTERT mRNA的表达,端粒重复序列扩增(TRAP)-ELISA法检测细胞端粒酶活性.结果:未转染DPC和空载体转染细胞(DPC-EGFP)无hTERT mRNA表达,端粒酶活性为阴性;而外源性hTERT基因转染细胞(DPC-hTERT)稳定表达hTERT mRNA,同时端粒酶活性转为阳性.结论:外源性hTERT基因转染能够激活体外培养人DPC的端粒酶,为建立永生化细胞系奠定基础.     

槲皮素对鼠黑素细胞株B10BR细胞的抗氧化保护效应及对其生物学活性的影响

目的 探讨槲皮素保护鼠永生化黑素细胞（B10BR）对抗氧化应激的有效性及其对B10BR细胞生物学活性影响。方法 MTT法测定B10BR细胞存活率,流式细胞仪检测细胞凋亡率,倒置显微镜下观察细胞形态改变,并检测槲皮素对酪氨酸酶活性及黑素合成的影响。结果 经33.33 μmol/L槲皮素预处理24 h后细胞活性增高至（94.22 ± 3.36）%,此外黑素细胞的酪氨酸酶活性及黑素含量可分别增高至（107.15 ± 10.96）%和（111.85 ± 9.49）%,与过氧化氢处理组相比,差异均有统计学意义（P < 0.01）。流式细胞仪检测结果表明,槲皮素可抑制过氧化氢诱导的黑素细胞凋亡。结论 槲皮素抑制过氧化氢诱导黑素细胞凋亡的保护效应为其治疗白癜风提供一定的依据。     

Immunohistochemical expression of matrix metalloproteinase-2 and -9 in melanocytic nevi is altered by ultraviolet B

BACKGROUND: Ultraviolet B (UVB) radiation can cause morphological and biological alterations similar to those of melanoma in situ in irradiated melanocytic nevi. matrix metalloproteinase (MMP-2) and -9 appear to be involved with tumour invasion, the formation of metastases and neoangiogenesis in melanomas. The effects of UVB on the immunohistochemical expression of gelatinases in different cell types in melanocytic nevi have not been completely studied. PURPOSE: Evaluate the effects of UVB on the immunohistochemical expression of MMP-2 and -9 in different cell types in melanocytic nevi. METHODS: Forty-two melanocytic nevi had one half irradiated with 2 MEDs of UVB and were excised 1 week later. Three different observers compared the intensity of the immunohistochemical expression of gelatinases in keratinocytes, melanocytes, endothelial cells and fibroblasts on the irradiated (IS) and non-irradiated sides (NIS). RESULTS: All the cell types showed an increase in the expression of MMP-2 on the IS, especially the epidermal melanocytes. No significant increase in the expression of MMP-9 in keratinocytes was detected on the IS; significant increase was observed in all the remaining cells. CONCLUSIONS: One single irradiation of UVB increases the immunohistochemical expression of gelatinases in almost all evaluated cell lines, with the exception of MMP-9 in keratinocytes.     

Differences of bcl-2 protein expression between Merkel cells and Merkel cell carcinomas

The bcl-2 gene, originally identified in B-cell lymphomas, encodes for proteins which may assume oncogenic functions by blocking apoptosis. Bcl-2 proteins are broadly distributed among various tissues, including epithelial ones. Within the skin, bcl-2 is strongly expressed in melanocytes, but its further distribution is yet unclear. The Merkel cells, neuroendocrine-epithelial cells of the skin, are present within the epidermis and hair follicles, mostly nerve-associated, and are believed to be postmitotic and long lived. Possibly they give rise to the malignant Merkel cell carcinomas. In the present study we investigated the bcl-2 expression on the protein level by means of immunohistochemical techniques including double confocal laser scanning microscopy, as well as on the RNA level by RT-PCR techniques, in Merkel cells, Merkel cell carcinomas, and cell lines. Merkel cells were identified by double staining for cytokeratins 20 or 8/18. We demonstrate that fetal epidermal and dermal Merkel cells are immunostained for bcl-2 protein, most of them clearly weaker than melanocytes. Adult Merkel cells also express bcl-2 protein very heterogeneously, mostly weak. In contrast, Merkel cell carcinomas are visually strongly positive for bcl-2 protein with some degree of heterogeneity. This is different from malignant melanomas in which bcl-2 expression is reduced as compared to normal melanocytes. Bcl-2 gene expression was also shown for Merkel cell carcinoma cell lines on both the mRNA and the protein level. Possibly bcl-2 protein expression is downregulated during the life span of Merkel cells, arguing that they may succumb to a certain cell turnover. The comparably high bcl-2 protein level in Merkel cell carcinomas may reflect peculiar biological and clinical characteristics.     

UBA3在黑素瘤细胞的表达及其影响黑素瘤细胞生物学行为的研究

【摘要】 目的 探讨UBA3在黑素瘤细胞中的表达,初步观察干扰UBA3对黑素瘤细胞生物学行为的影响。 方法 用RT-PCR技术观察3株黑素瘤细胞中UBA3的表达,并利用siRNA技术对M14细胞中UBA3的表达进行干扰,检测转染后细胞内UBA3、连接态NEDD8以及p53的表达,并观察转染前后细胞的凋亡及迁移能力的改变。 结果 在A375、M14及MV3中UBA3的表达均较正常黑素细胞上调,其中M14细胞上调最明显,成功干扰M14中UBA3的表达后,细胞内连接态NEDD8的表达下降,p53的表达明显增加,同时M14细胞凋亡增加、迁移能力下降。 结论 构建的质粒可干扰UBA3的表达,影响细胞的Nedd化进程,从而影响细胞的生物学行为。     

Transient expression of a transfected gene in cultured epidermal keratinocytes: implications for future studies

We examine the effect of keratinocyte differentiation upon transient expression of a nonepithelial gene following DNA-mediated transfer. Cultures of primary epidermal keratinocytes were transfected with the reporter gene, chloramphenicol acetyltransferase (CAT). The CAT gene was linked at the 5' end to the long terminal repeat (LTR) regulatory sequences from Rous sarcoma virus, and gene transfer was accomplished by the calcium phosphate coprecipitation method. Transfected cells were fractionated on Ficoll 400 density gradients. The major finding of this study was that the larger, more differentiated cells displayed five- to seven-fold higher levels of CAT activity per cell than the smaller, less differentiated cells. The higher levels of CAT activity did not result from greater uptake of DNA because cells of all gradient fractions contained one to two copies of plasmid DNA per cell. Furthermore, the CAT gene linked to the regulatory sequences from another virus, SV40, gave the same result. We conclude that the CAT gene, when controlled by these viral regulatory sequences, is expressed more efficiently in differentiated keratinocytes. These results have important implications for the interpretation of future studies of gene expression in transfected keratinocytes.     

Human tyrosinase related protein-1 (TRP-1) does not function as a DHICA oxidase activity in contrast to murine TRP-1

Abstract: Tyrosinase related protein-1 is a melanocyte specific protein and a member of the tyrosinase gene family which also includes tyrosinase and TRP-2 (DOPAchrome tautomerase). In murine melanocytes, TRP-1 functions as a 5,6-dihydroxyindole-2-carboxylic acid [DHICA] oxidase during the biosynthetic conversion of tyrosine to eumelanin and mutations affecting TRP-1 result in the synthesis of brown rather than black pelage coloration. In this study, we examined the putative DHICA oxidase activity of TRP-1 in human melanocytes using several approaches. We first utilized a line of cultured melanocytes established from a patient with a form of oculocutaneous albinism completely lacking expression of TRP-1 (OCA3). This line of melanocytes endogenously exhibited the same amount of DHICA oxidase activity as control melanocytes expressing TRP-1. In other experiments, cultured human fibroblasts were transfected with a cDNA for TRP-1, in either the sense or antisense direction, or with the retroviral vector alone. TRP-1 expression was induced in fibroblasts transfected with the TRP-1 cDNA in the sense direction only. Although TRP-1 was expressed by sense-transfected cells, there was no significant DHICA oxidase activity above controls. These results demonstrate that human TRP-1 does not use DHICA as a substrate for oxidation.