大疱及疱疹性皮肤病

20042967　副肿瘤性天疱疮伴发Castleman肿瘤分泌致病相关抗体的初步研究/王亮春(北京大学第一医院皮肤科)…//中华皮肤科杂志.-2004,37(2).-74～76分离副肿瘤性天疱疮患者的肿瘤淋巴细胞进行培养,将上清液超滤离心获得浓缩的IgG抗体为一抗,分别以鼠膀胱冰冻切片为底物行间接免疫荧光检查,以正常皮肤蛋白提取物为底物进行免疫印迹试验。RT-PCR、克隆测序分析培养肿瘤B细胞免疫球蛋白重链基因并与其他6例该病患者的基因相比较。结果显示,培养细胞上清液中的IgG抗体能在鼠膀胱上皮细胞间沉积;这些抗体能识别皮肤中分子量分别为210000和19…     

抗白念珠菌人鼠嵌合抗体真核表达载体的构建与表达

目的构建抗白念珠菌人鼠嵌合抗体的真核表达载体,并实现真核表达。方法从含有单克隆天然抗白念珠菌抗体3B4可变区基因的载体pMDT-V2H和pUC-VL中PCR克隆单抗3B4的可变区VH和VL基因,依次插入含有人免疫球蛋白κ轻链恒定区基因和γ1重链恒定区基因的真核表达载体pMH-CA。经DNA序列测定正确后,电穿孔转染J558L细胞进行嵌合抗体表达,RT-PCR,ELISA方法对抗体表达进行鉴定。结果成功构建了抗白念珠菌人鼠嵌合抗体,转染真核细胞后RT-PCR显示人鼠嵌合抗体重链和轻链mRNA的转录,ELISA证实了抗白念珠菌人鼠嵌合抗体的表达以及对白念珠菌的识别。结论成功构建抗白念珠菌人鼠嵌合抗体的真核表达载体,并表达出具有结合活性的基因工程抗体。     

伴发Castleman病的副肿瘤性天疱疮发病机制研究

目的研究Castleman病在副肿瘤性天疱疮患者(PNP)发病机制中的作用。方法肿瘤组织常规免疫组化染色。RT-PCR扩增6例患者肿瘤组织中RNA,将PCR产物进行聚丙烯酰胺凝胶电泳及克隆测序;并以原位杂交法与6例无皮肤黏膜损害的Castleman病和3例淋巴结反应性增生患者肿瘤B细胞克隆相比较。以RNA印迹法了解重排基因的表达情况。结果6例PNP患者RT-PCR产物聚丙烯酰胺凝胶电泳皆获得1条约124bp的锐利条带;克隆测序得到2种高度同源的序列,分别为128bp,122bp,其中6例患者皆具有长序列,4例患者同时还有相同短序列。以128bp的重排基因为探针进行原位杂交,阳性信号仅见于PNP患者肿瘤组织切片的淋巴滤泡样结构中;RNA印迹示该探针与6例PNP患者肿瘤组织mRNA皆有较强的杂交信号。结论6例PNP患者肿瘤组织中主要B细胞克隆是相同的。这种B细胞克隆的免疫球蛋白重排基因在肿瘤组织中有丰富表达,所产生的免疫球蛋白可能直接与皮肤中的抗原发生作用,引起皮肤黏膜的免疫损伤。     

副肿瘤性天疱疮患者免疫荧光与免疫印迹检测研究

目的 研究副肿瘤性天疱疮患者的免疫学特点。方法 采用免疫荧光和免疫印迹方法检测4例副肿瘤性天疱疮患者肿瘤切除前、后的血清。结果 以鼠膀胱为底物行间接免疫荧光示IgG和C3棘细胞间沉积，肿瘤切除后抗体滴度较术前下降，且与病情的好转成正比。比正常人皮肤及大鼠舌、食管及猴舌为底物行间接免疫荧光阳性；以大鼠支气管、心肌、肝脏、肾脏等组织为底物行间接免疫荧光阴性。免疫印迹示患者血清可识别人角质形成细胞提取物中的210000和190000抗原。结论 以鼠膀胱为底物的间接免疫荧光可作为副肿瘤性天疱疮的过筛试验，且可通过抗体滴度的改变监测病情的变化。副肿瘤性天疱疮更易累及粘膜部位上皮组织。免疫印迹结果可确诊4例患者为副肿瘤性天疱疮。     

金黄色葡萄球菌肠毒素A基因在C57BL/6小鼠淋巴细胞中的表达

目的 通过复制缺陷型腺病毒载体的介导,将金黄色葡萄球菌肠毒素A(SEA)基因转染入C57BL/6小鼠淋巴细胞中,观察基因表达产物对B16细胞的作用。方法 通过MTT法直接测定重组腺病毒颗粒感染的淋巴细胞的增殖情况,采用双抗体夹心ABC-ELISA法,测定淋巴细胞培养上清液中白介素2的水平。MTT法测定活细胞数目,以了解活化的C57BL/6小鼠淋巴细胞对B16细胞的杀伤效应。结果 加入不同滴度的重组腺病毒颗粒后,C57BL/6小鼠淋巴细胞明显增殖;同时淋巴细胞培养上清液中白介素2水平也明显增加。重组腺病毒颗粒转染的C57BL/6小鼠淋巴细胞可明显杀伤B16细胞,效/靶比越高,杀伤效应越明显。结论 SEA基因可以在C57BL/6小鼠淋巴细胞中表达,并且表达产物能够活化C57BL/6小鼠淋巴细胞,进而杀伤B16细胞。     

特发性系统性红斑狼疮中抑制T细胞的异常

已发现抑制T细胞(Suppressor T-cell)通过抑制B细胞合成免疫球蛋白(Ig)或抗DNA抗体的分泌来调节B细胞。作者将从外周血分离到的T细胞和B细胞在美洲商陆作为刺激原时一起培养,用异硫氰酸荧光素标记的抗Ig血清染胞浆以检测B细胞Ig的合成,用培养细胞的上清与氚标记的天然脱氧核糖核酸(DNA)的结合测定抗DNA抗体的分泌,对9例特发性系统性红斑狼疮(SLE)患者血液     

白介素2、10和干扰素对B细胞分泌天疱疮自身抗体的作用

天疱疮是一种自身抗体介导的具有器官特异性、自身免疫性大疱性皮肤黏膜疾病，其特征为患者血清中出现针对角质形成细胞表面桥粒成分(主要为桥粒芯糖蛋白，Dsg)的特异性IgG抗体引起棘层细胞松解，主要的致病性抗体亚类为IgG4。B细胞产生自身抗体需要主要组织相容性复合体(MHC)-Ⅱ限制性的CD4^ T细胞的辅助，体外实验证实天疱疮患者外周血中的CD4^ T     

自身免疫性大疱性皮肤病患者血清中IgM抗体所识别的皮肤抗原的研究

目的:研究自身免疫性大疱性皮肤病患者血清中IgM抗体所识别的皮肤抗原成分.方法:该研究共纳入10例大疱性类天疱疮患者、15例天疱疮患者(包括7例红斑/落叶型和8例寻常型).首先通过直接免疫荧光法分析受试患者皮肤中所沉积的免疫球蛋白或补体,再通过免疫印迹方法分析患者血清中的IgM抗体所识别的皮肤抗原成分.结果:在10例大疱性类天疱疮患者的皮肤中,C3、IgG、IgM单独沉积的例数分别是4例、2例、1例,IgG和C3共同沉积的2例,IgG、C3和IgA三者共同沉积的1例;在15例天疱疮患者中,C3、IgG单独沉积的例数分别是4例和2例,IgG和C3共同沉积的6例,IgM和C3共同沉积的3例.免疫印迹研究发现9例(9/10)大疱性类天疱疮患者、11例(11/15)天疱疮患者血清中的IgM抗体可以识别皮肤中分子量约80 kD的蛋白质.结论:IgM在自身免疫性大疱性皮肤病患者的皮肤中沉积的几率很低,但大多数患者血清中的IgM抗体都能够识别分子量约80 kD的皮肤抗原.     

In-cell PCR技术扩增白癜风自身抗体基因

目的 从白癜风患者外周血B淋巴细胞中扩增获得原始亲本配对的抗体基因.方法 以正常人的黑素细胞为抗原,用活细胞ELISA法筛选50例白癜风血清,选择其中7例抗黑素细胞膜抗体强阳性的患者外周血分离得到CD19⁺B淋巴细胞,以10%甲醛溶液固定,蛋白酶K溶液通透化处理,进行in-cell PCR.用特异性引物进行in-cell RT-PCR扩增人免疫球蛋白轻链和重链可变区基因,采用Cre/loxP系统在细胞内完成轻重链的连接,然后经巢式PCR扩增获得具有细胞内原始亲本配对特性的单链抗体基因.结果 经in-cell PCR扩增,获得了与预期单链抗体大小一致的单一800bp产物.结论 利用in-cell PCR技术可以扩增白癜风原始亲本配对的自身抗体基因.     

活动性系统性红斑狼疮病人血中免疫球蛋白分泌细胞增加

作者应用逆转溶血空斑法测定了24例活动性SLE 患者和49名正常对照者在外周血中分泌免疫球蛋白活性细胞的数量。此试验能查出所有分泌免疫球蛋白的细胞,且还能测定分泌每一型 Ig(IgG、IgA 和 IgM)细胞的数目。结果:对照组几何平均值为137 IgG 分泌细胞/10~6外周血单一棱细胞±2标准差(33～563);SLE 患者 IgG 分泌细胞的平均值为489/10~6单一核细胞。SLE 患者外周血中 IgG 分泌细胞显著多于对照组(P<0.005)。24例 SLE 中其值超过正常范围的最高线者有11例,某些 SLE 患者甚至超过正常平均值40倍以上.正常 IgA 分泌细胞几何平     

KSHV/HHV8-associated primary cutaneous plasmablastic lymphoma in a patient with Castleman's disease and Kaposi's sarcoma

Abstract:  We report a unique case of Kaposi's sarcoma (KS)-associated herpesvirus (KSHV)/human herpesvirus (HHV)8-associated lymphoma in a 56-year-old man with a history of acquired immune deficiency syndrome, Castleman's disease, KS, and idiopathic thrombocytopenic purpura. Three months following the diagnosis of KS affecting a left cervical lymph node and Castleman's disease with bone marrow involvement, he presented with a subcutaneous, tender lesion on his left arm. A skin biopsy demonstrated a superficial and deep, interstitial-nodular infiltrate of severely atypical lymphoid cells showing plasmacytoid features, numerous mitotic figures, and frequent individual apoptotic tumor cells. The morphologic features were those of plasmablastic lymphoma (PBL). Immunohistochemical study showed that the lymphoma cells strongly expressed CD45, CD30, and KSHV/HHV8 latency-associated nuclear antigen. KSHV/HHV8 was also detected in the biopsy sections of the patient's KS and Castleman's disease. Epstein–Barr virus in situ hybridization was diffusely positive. In situ hybridization demonstrated κ-light chain restriction. Although KSHV/HHV8 has been individually associated with KS, Castleman's disease, and PBL, this appears to be the first reported case in which all three entities were present simultaneously in one person, suggesting a critical role of KSHV/HHV8 as a common denominator in the pathogenesis of these diseases.     

与复发性Castleman病相关的副肿瘤天疱疮1例

报告1例与复发性Castleman病相关的副肿瘤天疱疮。患者男，29岁。主因进行性加重的疼痛性粘膜损害4个月，躯干部多形性皮疹5天入院。7年前患者切除一腹膜后Castleman肿瘤，此次入院检查发现右腹膜后肿物，手术切除并经病理证实为Castleman肿瘤。患者临床、病理及免疫学特点均符合副肿瘤天疱疮。经大剂量静滴丙种球蛋白及口服强的松联合治疗，术后7周皮疹完全消退，留下广泛色素沉着，术后9周粘膜损害基本愈合。     

Induction of manganese superoxide dismutase in human dermal fibroblasts: a UV-B-mediated paracrine mechanism with the release of epidermal interleukin 1 alpha,interleukin 1 beta,and tumor necrosis factor alpha

BACKGROUND: Reactive oxygen species generated in the skin by UV irradiation promote photoaging and photocarcinogenesis. The manganese (Mn) superoxide dismutase (SOD) is a primary antioxidant enzyme that crucially contributes to the homeostasis of oxygen radicals within the mitochondria, and thus critically participates in the control of senescence and tumor generation. OBJECTIVE: To determine whether repetitive UV-B exposure, as practiced for light hardening during phototherapy for various photodermatoses, can enhance the adaptive antioxidant response by up-regulating MnSOD activity in either the epidermal or the dermal skin compartment. DESIGN: In vitro experiments to determine MnSOD activity levels in cultured human dermal fibroblasts and epidermal cells (HaCaT cells and primary keratinocytes) at different times after direct UV-B exposure or after incubation of human dermal fibroblasts with supernatants from UV-B-irradiated epidermal cells. SETTING: Photobiological research laboratory in a university dermatology department. INTERVENTION: Irradiation of cultured human dermal fibroblasts and epidermal cells with UV-B. MAIN OUTCOME MEASURES: Manganese SOD messenger RNA and activity levels in cultured irradiated or mock-treated skin cells. RESULTS: No increase in MnSOD activity could be detected in fibroblasts or epidermal cells until 24 hours after UV-B irradiation. However, fibroblasts incubated with supernatants from UV-B-irradiated epidermal cells showed a marked increase in specific MnSOD messenger RNA and activity. Removal of interleukin 1alpha, interleukin 1beta, and tumor necrosis factor alpha from the supernatants led to a significant reduction of MnSOD mRNA in fibroblasts. CONCLUSION: Irradiation of the epidermal cells with UV-B induced a release of soluble factors that amplified MnSOD activity in fibroblasts via a paracrine mechanism.     

银屑病患者角质形成细胞培养上清液对单核细胞形态、表型的影响

目的　探讨银屑病角质形成细胞 (keratinocytes ,KC)在单核细胞向郎格汉斯细胞 (langerhanscells ,LC)分化过程中的作用。方法　从健康志愿者外周血分离单核细胞 ,分别经银屑病患者及正常人KC培养上清单独培养 5天 ,然后通过Leica显微镜观察其形态 ,通过流式细胞仪分析其表型。结果　健康志愿者外周血单核细胞经银屑病患者KC上清培养后 ,细胞的聚集体明显较正常组减少 ,而与阴性对照组间无显著差异 ;CDla、CD80、CD86的表达均较正常人KC上清组减弱。结论　银屑病患者KC中缺乏某些因子而影响了单核细胞向LC的分化 ,或表达了某些因子而抑制了单核细胞向LC的分化。     

Coexistence of Castleman's disease and Kaposi's sarcoma. Report of a case and a speculation

A case report of multicentric Castleman's disease associated with Kaposi's sarcoma is presented. Histologic examinations of cutaneous lesions and of an axillary lymph node were made. The cutaneous lesions showed Kaposi's sarcoma and a lymph node revealed Castleman's disease and foci of spindle cells characteristic of Kaposi's sarcoma. The association of Castleman's disease and Kaposi's sarcoma appears too frequently to be due to chance alone. We speculate that these two entities may reflect different tissue reactions to the same causative agent.     

Loss of tuberin, the tuberous-sclerosis-complex-2 gene product is associated with angiogenesis

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominantly inherited disorder associated with an alteration of the TSC2 tumor suppressor gene which encodes for the protein product tuberin. The disease is characterized by the development of hamartomas, e.g. cutaneous angiofibromas which consist of vascular cells, interstitial cells, and normal components of the skin. The Eker rat model, an animal model of inherited cancer, has been shown to carry a mutation of TSC2. METHODS: Immunohistochemical analyses of human angiofibromas were performed using antibodies directed against tuberin and angiogenic growth factors. Proliferation of human dermal microvascular endothelial cells (HDMEC) was determined after incubation with the supernatants of TSC2 (+/+) and TSC2 (-/-) rat embryonic fibroblasts (REF) that were derived from the Eker strain. RESULTS: Loss of the expression of tuberin was observed in the interstitial cells of 13 of 39 angiofibromas. The expression of tuberin was retained in the vascular cells. In all analyzed angiofibromas, the angiogenic factors bFGF, PD-ECGF, VEGF and angiogenin were detected in the interstitial cells and/or vascular cells. Expression of PDGF-B and TGF-beta1 was weak. Tissue culture supernatants from TSC2 (-/-) REF stimulated the growth of HDMEC significantly more than supernatants from TSC2 (+/+) REF. CONCLUSION: A functional loss of tuberin may stimulate vascular growth.     

伴限局性Castleman病的副肿瘤性天疱疮临床及实验室研究

目的：报告6例伴限局性Castleman病副肿瘤性天疱疮，从临床、组织病理和免疫学方面进行了研究，从而能早期诊断，早期治疗。方法：采用全面临床检查，包括CT检查，常规组织病理检查，间接免疫荧光检查，免疫印迹及酶联免疫吸附测定（ELISA）。结果：6例患者临床上均有严重的口腔黏膜糜烂或溃疡。工有形态各异及程度不等的皮损，CT检查均示有潜在单发的内脏肿瘤。组织病理检查示表皮内疱，基底细胞液化变性，表皮内坏死角质形成细胞及真皮浅层以淋巴细胞为主的浸润。免疫学检查示患者血清中含抗棘细菌间抗体，它们能与桥粒的envoplakin及periplakin发生特异结合。手术切除后病理检查均为Castleman病，术后皮损逐渐消退。结论：副肿瘤性天疱疮无论在临床，皮损的组织病理学改变，还是免疫学上都具有特征性，对于临床上口腔黏膜有广泛糜烂或溃疡，按天疱疮治疗无效的患者，应考虑副肿瘤性天疱疮的可能性。早期诊断，及时切除肿瘤或作相应治疗，患者的预后是良好。     

Cbl-b基因沉默对小鼠原代淋巴细胞免疫活性影响的研究

目的 体外初步研究Cbl-b基因经特异性siRNA沉默后对小鼠淋巴细胞免疫活性的影响。 方法 摘取C57BL/6小鼠的脾脏,体外无菌分离小鼠脾脏淋巴细胞后进行培养。通过EntransterTM-R4000试剂将Cbl-b siRNA转染入小鼠原代淋巴细胞以沉默细胞内Cbl-b的表达。转染72 h后通过酶联免疫吸附实验（ELISA）检测淋巴细胞培养上清中干扰素γ（INF-γ）及肿瘤坏死因子α（TNF-α）的表达量。通过与B16F10黑素瘤细胞共培养,研究Cbl-b基因沉默后的淋巴细胞对B16F10黑素瘤细胞免疫杀伤活性的影响。 结果 Cbl-b siRNA成功转染进小鼠原代淋巴细胞并能有效沉默细胞内Cbl-b的表达。与阴性对照转染组及空白组相比,转染Cbl-b siRNA的淋巴细胞IFN-γ、TNF-α分泌量增加（P < 0.05）。共培养检测结果显示,Cbl-b siRNA转染组比转染阴性对照组能更高效地杀伤小鼠B16F10细胞。 结论 Cbl-b基因沉默能够促进小鼠淋巴细胞INF-γ、TNF-α分泌能力,并能增强淋巴细胞对B16F10黑素瘤细胞的体外免疫杀伤作用。     

Induction of Intercellular Adhesion Molecule-1 and Adherence of HTLV-1-Infected T-cells to Cultured Keratinocytes

Cutaneous lesions of T-cell proliferative disorders are characterized by epidermotropic infiltration of the neoplastic cells and expression of intercellular adhesion molecule-1 (ICAM-1) and HLA-DR by lesional keratinocytes. Using cloned HTLV-1-infected T-cells obtained from patients with adult T-cell leukemia (ATL), we have studied immunobiological activities of cytokines released from the T-cell lines and their ability to adhere to cultured keratinocytes. Three out of the five CD-4-positive, HTLV-1-infected T-cell clones secreted both IFN-γ and IL-4, similar to murine Th0 clones. The other two clones did not produce such cytokines. ICAM-1 and HLA-DR molecules were induced on cultured normal human keratinocytes and organ-cultured skin specimens by co-cultivation with IFN-γ-producing T-cell clones or their culture supernatants. Induction of both molecules was markedly inhibited by pretreatment of the supernatants with excess amounts of anti-IFN-γ monoclonal antibody. The number of cells adherent to the normal cultured keratinocytes was greater in the IFN-γ-producing clones than in the non-producing ones. These data suggest that some HTLV-1-infected clones produce cytokines, including IFN-γ, which in turn induce ICAM-1 on keratinocytes, thereby enhancing the ability of the T-cell clones to adhere to the keratinocytes.