影响角质形成细胞表面表达桥粒芯糖蛋白的因素初探

发布时间：2018-01-13 01:38

  本文关键词：影响角质形成细胞表面表达桥粒芯糖蛋白的因素初探　出处：《北京协和医学院》2013年博士论文　论文类型：学位论文

  更多相关文章： 角质形成细胞 桥粒芯糖蛋白1 桥粒芯糖蛋白3 天疱疮 乙酰胆碱受体

【摘要】：天疱疮是以抗钙黏素为自身抗原的自身免疫性皮肤病,该病的发生与机体产生致病性抗桥粒芯糖蛋白(DSG)1及抗DSG3为代表的抗桥粒成分自身抗体有关。近年随着分子生物学的飞速发展,对天疱疮的发病机制也进行了深入研究,但在研究过程中却出现诸多限制,这些限制主要来源于研究对象的选择。以临床患者作为研究对象涉及伦理道德问题及人道主义问题；缺乏足够的动物模型,已有动物模型维护价格高昂；离体细胞模型资料少且混乱。在上述限制下,本研究拟通过寻找合适的角质形成细胞(KC)及合适的条件,为天疱疮的离体研究提供一种良好的可重复、简单的实验对象。 作为皮肤科慢性、重症疾病,天疱疮发病中自身抗体引起KC出现棘层松解的病理生理学机制目前并未完全阐明,DSG补偿理论、空间效应理论、信号转导理论这三大主流理论各有利弊,而我们前期研究结果显示患者乙酰胆碱受体(AchR)自身抗体的存在及部分DSG3自身抗体阳性天疱疮患者KC AchR特别是m3毒蕈碱型乙酰胆碱受体(mAchR)的变化,这一结果提示抗DSG3自身抗体与AchR特别是m3mAchR之存在一定的相互作用或影响。因此本研究拟通过更改KC培养条件阐明常见不同亚型天疱疮患者血清对KC m3mAchR影响。 第1章桥粒芯糖蛋白1、桥粒芯糖蛋白3在不同角质形成细胞的表达 目的测定不同类型KC (HaCat细胞、A431细胞及原代KC) DSG1、DSG3及DSG1信使核糖核酸(mRNA)、DSG3mRNA表达；方法不同KC培养后,直接免疫荧光(DIF)观察细胞表而DSG1、DSG3的分布,定量多聚酶链反应(qPCR)测定DSG1mRNA、DSG3mRNA相对表达水平；结果三种KC表而均有DSG1与DSG3连续性表达；蛋白荧光强度示A431细胞表而DSG1、DSG3表达高于HaCat细胞,而HaCat细胞又高于原代KC；成纤维细胞(FB)不表达DSG1与DSG3；原代KC表达DSG1与DSG3mRNA的量高于HaCat细胞,A431细胞DSG1与DSG3mRNA相对表达量低于HaCat细胞；结论三种KC均可用于对DSG1、DSG3进行相关研究；但相对而言,A431细胞更适合于单独进行DSG1、DSG3翻译水平研究,原代KC更合适于单独进行转录水平研究,HaCat细胞更适用于需同时进行转录水平与翻译水平研究。 第2章不同融合状态影响角质形成细胞桥粒芯糖蛋白1、桥粒芯糖蛋白3表达 目的测定不同融合状态下HaCat细胞DSG1、DSG3及DSG1mRNA、DSG3mRNA表达差异；方法培养HaCat细胞后以不同细胞数量倍增接种培养,DIF结合激光共聚焦三维成像观察细胞表而DSG1、DSG3的表达,qPCR测定DSG1mRNA、 DSG3mRNA相对表达水平；结果细胞分裂时,细胞膜交界处DSG1荧光增加强度较DSG3更为明显；随着细胞数目增加及接触增多,DSG1、DSG3荧光在交界处均明显增强；HaCat细胞融合数在10%至40%区间内DSG1mRNA表达量呈上升趋势,而在80%至100%区间呈下降趋势；DSG3mRNA则在细胞融合数为20%至80%区间表达量呈上升趋势；细胞100%融合后,融合初始DSG1mRNA、DSG3mRNA呈现明显的低表达,完全融合后DSG1mRNA表达增加,DSG3mRNA无明显变化；结论观察DSG1、DSG3蛋白需要在细胞融合一定程度后进行；DSG1mRNA、DSG3mRNA的测定在10%至40%区间或80%至100%区间受细胞融合状态的影响更小；DSG3mRNA的测定在20%至80%区间受细胞融合状态的影响更小。 第3章常见不同亚型天疱疮患者血清影响角质形成细胞桥粒芯糖蛋白3表达 目的测定常见不同临床亚型天疱疮患者血清对HaCat细胞DSG3及DSG3mRNA表达的影响：方法采集部分常见临床亚型天疱疮患者血清,酶联免疫吸附测定(ELISA)测定抗DSG1自身抗体、抗DSG3自身抗体滴度后,与HaCat细胞共同培养,DIF观察细胞表而DSG3的荧光强度变化,qPCR测定DSG3mRNA表达变化；结果入选的寻常型天疱疮患者血清抗DSG1抗体、抗DSG3抗体均阳性,副肿瘤型天疱疮患者血清抗DSG3抗体阳性,落叶型天疱疮患者血清抗DSG1抗体阳性；三种患者血清均可使离体培养HaCat细胞集落“收缩”,而均对集落内细胞形态影响不明显；三种患者血清均能降低HaCat钏胞表而DSG3荧光强度；抗DSG1抗体、抗DSG3抗体双阳性的寻常型天疱疮患者血清及抗DSG3P目性的副肿瘤天疱疮患者血清作用增加HaCat细胞DSG3mRNA表达；抗DSG1抗体阳性的落叶型天疱疮患者血清显著降低HaCat细胞DSG3mRNA表达；结论寻常型、副肿瘤、落叶型天疱疮患者血清均对单个HaCat细胞形态影响不明显但可影响HaCat钏胞集落的生物学行为；若不考虑除血清中其他物质的影响,发现抗DSG3抗体阳性血清可以降低HaCat细胞表而DSG3荧光强度,但增加HaCat细胞DSG3mRNA表达；单抗DSG1抗体血清降低HaCat细胞表而DSG3荧光强度及DSG3mRNA表达。 第4章毒蕈碱型乙酰胆碱受体通路影响角质形成细胞桥粒芯糖蛋白3表达 目的测定1mAchR通路对HaCat钏胞DSG3及L)SG3mRNA表达的影响；方法细胞培养后以噻唑蓝(MTT)法测定不同时间、不同浓度氯化乙酰胆碱(Ach)及硫酸阿托品(Atr)对HaCat细胞活力的影响；选择不影响HaCat细胞活力的试剂浓度作用细胞后,DIF观察细胞表而DSG3荧光强度变化,qPCR测定LDSG3mRNA表达变化；结果高浓度(10-2M) Atr显著杀伤HaCat细胞,次高浓度(10-3M、10-4M)Atr显著降低HaCat细胞活力；高浓度(10-2M)Ach在6h、12h增加HaCat细胞的活力,10-5～10-6M Atr及10-3M Ach不影响HaCat细胞活力：1O-5M Atr作用6h、12h后HaCat细胞表而及细胞连接处DSG3荧光强度降低,10-3M Ach作用6h、12h后分别增加及降低HaCat钏胞表而及钏胞连接处DSG3荧光强度；10-5M Atr作用2h、12h显著降低HaCat细胞DSG3mRNA表达；10-3M Ach作用6h增加HaCat细胞DSG3mRNA表达,而作用2h、12h均明显抑制HaCat细胞DSG3mRNA表达；结论高浓度Atr与Ach分别降低与增加HaCat细胞活力。mAchR激动剂及mAchR拮抗剂短期(12h以内)对DSG3的表达影响不一,长期(12h以上)存在均可在转录与翻译水平,使HaCat细胞DSG3表达降低。 第5章常见不同亚型天疱疮患者血清影响角质形成细胞m3毒草碱型乙酰胆碱受体mRNA表达 目的测定常见不同临床亚型天疱疮患者血清对]HaCat细胞1m3mAchR表达的影响；方法采集部分常见临床亚型天疱疮患者血清,ELISA测定抗DSG1自身抗体、抗DSG3自身抗体滴度后,与HaCat细胞共同培养,DIF观察细胞表面m3mAchR的荧光强度变化,qPCR测定CHRM3mRNA表达变化；结果三种患者血清作用后m3mAchR荧光强度未出现明显变化；抗DSG1抗体、抗DSG3抗体双阳性的寻常型天疱疮患者血清及抗DSG3阳性的副肿瘤天疱疮患者血清作用增加HaCat细胞CHRM3mRNA表达；抗DSG1抗体阳性的落叶型天疱疮患者血清显著降低HaCat细胞CHRM3mRNA表达；结论天疱疮患者血清对HaCat细胞m3mAchR荧光强度影响不明显；若不考虑除血清中其他物质的影响,抗DSG3抗体阳性血清可以增加HaCat细胞CHRM3mRNA表达。
[Abstract]:Pemphigus is anti cadherin to autoantigen in autoimmune skin disease, the disease occurrence and the production of pathogenic anti desmoglein (DSG) 1 and DSG3 as the representative of the anti anti desmosomal components of autoantibodies. In recent years, with the rapid development of molecular biology, the pathogenesis of pemphigus also conducted in-depth research, but in the course of the study there are many restrictions, these restrictions mainly comes from the selection of research object. In clinical patients as the research object to ethical issues and humanitarian issues; the lack of adequate animal models, the animal model of maintenance of high prices; in vitro cell model of the data is few and in confusion. These limitations, this study intends to find suitable keratinocytes (KC) and suitable conditions for pemphigus, in vitro study provides a good repeatability, simple experimental object.
Department of dermatology as a chronic, severe disease, the pathogenesis of pemphigus autoantibody induced KC pathophysiology of acantholysis mechanism is not fully elucidated, DSG compensation theory, spatial effect theory, signaling theory the three mainstream theories have advantages and disadvantages, and our preliminary results of the study showed that patients with acetylcholine receptor (AchR) has its own antibody and DSG3 autoantibodies in patients with pemphigus KC AchR especially M3 muscarinic acetylcholine receptor (mAchR) changes, the results showed that the anti DSG3 antibody and AchR m3mAchR especially there is a certain interaction or mutual influence. The purpose of this study was to clarify the common culture conditions by changing the KC of different subtypes of pemphigus the serum of KC m3mAchR.
The expression of granular core glycoprotein 1 and keratocyte glycoprotein 3 in different keratinocytes in the first chapter
Objective to determine the different types of KC (HaCat cells, A431 cells and primary KC) DSG1, DSG3 and DSG1 messenger RNA (mRNA), the expression of DSG3mRNA after KC culture; different methods, direct immunofluorescence (DIF) to observe cell surface DSG1, DSG3 distribution, quantitative polymerase chain reaction (qPCR) was DSG1mRNA, the relative the expression level of DSG3mRNA and KC; the results of the three tables were DSG1 and DSG3 continuity of expression; the fluorescence intensity of A431 cell surface protein showed higher expression of DSG3 and DSG1, HaCat cells, and HaCat cells was higher than that of primary KC; fibroblast (FB) expression of DSG1 and DSG3; primary KC expression of DSG1 and the amount of DSG3mRNA higher than that of HaCat cells, A431 cells and DSG1 DSG3mRNA expression than HaCat cells; conclusion three KC can be used for DSG1, DSG3 related research; however, the A431 cell is more suitable for DSG1 alone, on the translation level of DSG3, the primary KC more appropriate In a single transcriptional level study, HaCat cells are more suitable for simultaneous transcriptional and translation levels.
In the second chapter, different fusion states affect keratinocyte core glycoprotein 1, and the expression of GP 3
Objective to determine DSG1 HaCat cell fusion state, DSG3 and DSG1mRNA, the difference of DSG3mRNA expression; methods the cultured HaCat cells in different cell after multiplying the number of inoculated DIF combined with confocal laser 3D imaging to observe the cell surface expression of DSG1, DSG3, qPCR were measured by DSG1mRNA, the relative expression level of DSG3mRNA; the results of cell division, cell membrane at the junction of DSG1 fluorescence intensity increase with DSG3 is more obvious; with the increase in cell number and contact increased, DSG1, DSG3 were significantly enhanced fluorescence at the junction; HaCat cell fusion number DSG1mRNA in the 10% to 40% range expression increased, decreased in the 80% to 100% range; DSG3mRNA in the number of cell fusion increased from 20% to 80% interval expression; 100% cell fusion, fusion of initial DSG1mRNA, expression of DSG3mRNA decreased obviously, completely fused DSG1mRNA increased, DSG3mR Conclusion NA has no obvious change; observation of DSG1, DSG3 protein to a certain extent in the fusion cells; DSG1mRNA, DSG3mRNA determination in 10% to 40% less interval or 80% to 100% range from fusion cells; Determination of DSG3mRNA in the 20% to 80% range with less influence by fusion cells.
Serum of patients with different subtype pemphigus affects the expression of keratocyte core glycoprotein 3 in the third chapter
Effects of different clinical subtypes in patients with pemphigus serum on the expression of HaCat cell DSG3 and DSG3mRNA determination method of acquisition part of the common objective: clinical subtypes in patients with pemphigus sera, enzyme-linked immunosorbent assay (ELISA) determination of anti DSG1 antibodies, anti DSG3 antibody titer after co cultured with HaCat cells, DIF cells were observed in table DSG3 and the change of fluorescence intensity, qPCR determination of the expression of DSG3mRNA; the results were in patients with pemphigus vulgaris serum anti DSG1 antibody and anti DSG3 antibody were positive, paraneoplastic pemphigus patients with positive anti DSG3 antibody, pemphigus foliaceus serum anti DSG1 antibody positive; three patients can make the in vitro culture of HaCat cell colony contractions, and influence on colony in cell morphology was not obvious; three patients with HaCat were reduced, and the fluorescence intensity of DSG3 cells; anti DSG1 antibody, anti DSG3 The serum of patients with vulgaris pemphigus and double positive anti DSG3P mesh of paraneoplastic pemphigus patients serum HaCat cells increased the expression of DSG3mRNA in serum of patients with deciduous type; anti DSG1 antibody positive pemphigus significantly decreased HaCat cell DSG3mRNA expression; conclusion vulgaris, paraneoplastic, effects of sera from patients with pemphigus foliaceus were to form a single HaCat the cell is not obvious but it can affect the biological behavior of HaCat cells, set off; if you do not take into account in addition to the impact of other substances in serum, anti DSG3 antibody positive serum can reduce HaCat cell surface and the fluorescence intensity of DSG3, but increased HaCat cell DSG3mRNA expression; monoclonal antibody DSG1 antibody decreased HaCat cell surface expression of DSG3 and fluorescence intensity of DSG3mRNA.
The fourth chapter of the muscarinic acetylcholine receptor pathway affects the expression of keratocyte core glycoprotein 3 in keratinocyte
Objective to determine the 1mAchR pathway on HaCat cell, DSG3 and L) SG3mRNA expression method; cell culture by thiazolyl blue (MTT) method for the determination of different time, different concentrations of acetylcholine (Ach) and atropine sulfate (Atr) effect on HaCat cell viability; reagent concentration has no effect on HaCat cells induced cell viability after DIF and DSG3, observed the change of fluorescence intensity, qPCR determination of the expression of LDSG3mRNA (10-2M); the high concentration of Atr significantly kill HaCat cells. High concentration (10-3M, 10-4M) Atr HaCat significantly decreased cell viability; high concentration (10-2M) of Ach in 6h, 12h increased HaCat cell viability, 10-5 ~ 10-6M Atr 10-3M and Ach did not affect the viability of HaCat cells: 1O-5M Atr 6h, 12h HaCat cells and cell junctions of the fluorescence intensity of DSG3 was decreased, 10-3M Ach 6h, 12h and HaCat were increased after decreased, cell surface and cell junction, DS The fluorescence intensity of G3 10-5M Atr 2h, 12h; HaCat decreased the expression of DSG3mRNA 10-3M Ach 6h; HaCat cells increased the expression of DSG3mRNA and 12h significantly inhibited 2H HaCat cell DSG3mRNA expression; conclusion high concentration of Atr and Ach respectively decreased with the increase of HaCat cell activity with.MAchR agonists and antagonists of mAchR short-term (less than 12h the influence of DSG3 on the expression of) a long-term (more than 12h) can exist in the transcription and translation level, the HaCat cells decreased expression of DSG3.
The fifth chapter is common in different subtypes of patients with pemphigus day affect the expression of keratinocyte M3 muscarinic acetylcholine receptor mRNA
Effects of different clinical subtypes in patients with pemphigus serum on the expression of]HaCat cell 1m3mAchR determination; method of acquisition part of common clinical subtypes in patients with pemphigus sera, determination of anti DSG1 antibodies ELISA, anti DSG3 antibody titer after co cultured with HaCat cells, the fluorescence intensity changes observed on the surface of m3mAchR DIF, qPCR by CHRM3mRNA the expression results of the three patients; serum m3mAchR fluorescence intensity did not change significantly; anti DSG1 antibody, anti DSG3 antibody in serum of patients with vulgaris pemphigus and double positive anti DSG3 positive side of tumor patients with pemphigus serum HaCat increased the expression of CHRM3mRNA in serum of patients with deciduous type; anti DSG1 antibody positive pemphigus HaCat decreased the expression of CHRM3mRNA in serum of patients with pemphigus; conclusion: the effect of m3mAchR on HaCat cell fluorescence intensity is not obvious; if you do not consider the addition The effect of other substances in serum, anti DSG3 antibody positive serum can increase the expression of CHRM3mRNA in HaCat cells.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R758.66

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