本文關(guān)鍵詞：影響角質(zhì)形成細(xì)胞表面表達(dá)橋粒芯糖蛋白的因素初探　出處：《北京協(xié)和醫(yī)學(xué)院》2013年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 角質(zhì)形成細(xì)胞 橋粒芯糖蛋白1 橋粒芯糖蛋白3 天皰瘡 乙酰膽堿受體

【摘要】：天皰瘡是以抗鈣黏素為自身抗原的自身免疫性皮膚病,該病的發(fā)生與機(jī)體產(chǎn)生致病性抗橋粒芯糖蛋白(DSG)1及抗DSG3為代表的抗橋粒成分自身抗體有關(guān)。近年隨著分子生物學(xué)的飛速發(fā)展,對(duì)天皰瘡的發(fā)病機(jī)制也進(jìn)行了深入研究,但在研究過程中卻出現(xiàn)諸多限制,這些限制主要來源于研究對(duì)象的選擇。以臨床患者作為研究對(duì)象涉及倫理道德問題及人道主義問題；缺乏足夠的動(dòng)物模型,已有動(dòng)物模型維護(hù)價(jià)格高昂；離體細(xì)胞模型資料少且混亂。在上述限制下,本研究擬通過尋找合適的角質(zhì)形成細(xì)胞(KC)及合適的條件,為天皰瘡的離體研究提供一種良好的可重復(fù)、簡(jiǎn)單的實(shí)驗(yàn)對(duì)象。 作為皮膚科慢性、重癥疾病,天皰瘡發(fā)病中自身抗體引起KC出現(xiàn)棘層松解的病理生理學(xué)機(jī)制目前并未完全闡明,DSG補(bǔ)償理論、空間效應(yīng)理論、信號(hào)轉(zhuǎn)導(dǎo)理論這三大主流理論各有利弊,而我們前期研究結(jié)果顯示患者乙酰膽堿受體(AchR)自身抗體的存在及部分DSG3自身抗體陽(yáng)性天皰瘡患者KC AchR特別是m3毒蕈堿型乙酰膽堿受體(mAchR)的變化,這一結(jié)果提示抗DSG3自身抗體與AchR特別是m3mAchR之存在一定的相互作用或影響。因此本研究擬通過更改KC培養(yǎng)條件闡明常見不同亞型天皰瘡患者血清對(duì)KC m3mAchR影響。 第1章橋粒芯糖蛋白1、橋粒芯糖蛋白3在不同角質(zhì)形成細(xì)胞的表達(dá) 目的測(cè)定不同類型KC (HaCat細(xì)胞、A431細(xì)胞及原代KC) DSG1、DSG3及DSG1信使核糖核酸(mRNA)、DSG3mRNA表達(dá)；方法不同KC培養(yǎng)后,直接免疫熒光(DIF)觀察細(xì)胞表而DSG1、DSG3的分布,定量多聚酶鏈反應(yīng)(qPCR)測(cè)定DSG1mRNA、DSG3mRNA相對(duì)表達(dá)水平；結(jié)果三種KC表而均有DSG1與DSG3連續(xù)性表達(dá)；蛋白熒光強(qiáng)度示A431細(xì)胞表而DSG1、DSG3表達(dá)高于HaCat細(xì)胞,而HaCat細(xì)胞又高于原代KC；成纖維細(xì)胞(FB)不表達(dá)DSG1與DSG3；原代KC表達(dá)DSG1與DSG3mRNA的量高于HaCat細(xì)胞,A431細(xì)胞DSG1與DSG3mRNA相對(duì)表達(dá)量低于HaCat細(xì)胞；結(jié)論三種KC均可用于對(duì)DSG1、DSG3進(jìn)行相關(guān)研究；但相對(duì)而言,A431細(xì)胞更適合于單獨(dú)進(jìn)行DSG1、DSG3翻譯水平研究,原代KC更合適于單獨(dú)進(jìn)行轉(zhuǎn)錄水平研究,HaCat細(xì)胞更適用于需同時(shí)進(jìn)行轉(zhuǎn)錄水平與翻譯水平研究。 第2章不同融合狀態(tài)影響角質(zhì)形成細(xì)胞橋粒芯糖蛋白1、橋粒芯糖蛋白3表達(dá) 目的測(cè)定不同融合狀態(tài)下HaCat細(xì)胞DSG1、DSG3及DSG1mRNA、DSG3mRNA表達(dá)差異；方法培養(yǎng)HaCat細(xì)胞后以不同細(xì)胞數(shù)量倍增接種培養(yǎng),DIF結(jié)合激光共聚焦三維成像觀察細(xì)胞表而DSG1、DSG3的表達(dá),qPCR測(cè)定DSG1mRNA、 DSG3mRNA相對(duì)表達(dá)水平；結(jié)果細(xì)胞分裂時(shí),細(xì)胞膜交界處DSG1熒光增加強(qiáng)度較DSG3更為明顯；隨著細(xì)胞數(shù)目增加及接觸增多,DSG1、DSG3熒光在交界處均明顯增強(qiáng)；HaCat細(xì)胞融合數(shù)在10%至40%區(qū)間內(nèi)DSG1mRNA表達(dá)量呈上升趨勢(shì),而在80%至100%區(qū)間呈下降趨勢(shì)；DSG3mRNA則在細(xì)胞融合數(shù)為20%至80%區(qū)間表達(dá)量呈上升趨勢(shì)；細(xì)胞100%融合后,融合初始DSG1mRNA、DSG3mRNA呈現(xiàn)明顯的低表達(dá),完全融合后DSG1mRNA表達(dá)增加,DSG3mRNA無明顯變化；結(jié)論觀察DSG1、DSG3蛋白需要在細(xì)胞融合一定程度后進(jìn)行；DSG1mRNA、DSG3mRNA的測(cè)定在10%至40%區(qū)間或80%至100%區(qū)間受細(xì)胞融合狀態(tài)的影響更��；DSG3mRNA的測(cè)定在20%至80%區(qū)間受細(xì)胞融合狀態(tài)的影響更小。 第3章常見不同亞型天皰瘡患者血清影響角質(zhì)形成細(xì)胞橋粒芯糖蛋白3表達(dá) 目的測(cè)定常見不同臨床亞型天皰瘡患者血清對(duì)HaCat細(xì)胞DSG3及DSG3mRNA表達(dá)的影響：方法采集部分常見臨床亞型天皰瘡患者血清,酶聯(lián)免疫吸附測(cè)定(ELISA)測(cè)定抗DSG1自身抗體、抗DSG3自身抗體滴度后,與HaCat細(xì)胞共同培養(yǎng),DIF觀察細(xì)胞表而DSG3的熒光強(qiáng)度變化,qPCR測(cè)定DSG3mRNA表達(dá)變化；結(jié)果入選的尋常型天皰瘡患者血清抗DSG1抗體、抗DSG3抗體均陽(yáng)性,副腫瘤型天皰瘡患者血清抗DSG3抗體陽(yáng)性,落葉型天皰瘡患者血清抗DSG1抗體陽(yáng)性；三種患者血清均可使離體培養(yǎng)HaCat細(xì)胞集落“收縮”,而均對(duì)集落內(nèi)細(xì)胞形態(tài)影響不明顯；三種患者血清均能降低HaCat釧胞表而DSG3熒光強(qiáng)度；抗DSG1抗體、抗DSG3抗體雙陽(yáng)性的尋常型天皰瘡患者血清及抗DSG3P目性的副腫瘤天皰瘡患者血清作用增加HaCat細(xì)胞DSG3mRNA表達(dá)；抗DSG1抗體陽(yáng)性的落葉型天皰瘡患者血清顯著降低HaCat細(xì)胞DSG3mRNA表達(dá)；結(jié)論尋常型、副腫瘤、落葉型天皰瘡患者血清均對(duì)單個(gè)HaCat細(xì)胞形態(tài)影響不明顯但可影響HaCat釧胞集落的生物學(xué)行為；若不考慮除血清中其他物質(zhì)的影響,發(fā)現(xiàn)抗DSG3抗體陽(yáng)性血清可以降低HaCat細(xì)胞表而DSG3熒光強(qiáng)度,但增加HaCat細(xì)胞DSG3mRNA表達(dá)；單抗DSG1抗體血清降低HaCat細(xì)胞表而DSG3熒光強(qiáng)度及DSG3mRNA表達(dá)。 第4章毒蕈堿型乙酰膽堿受體通路影響角質(zhì)形成細(xì)胞橋粒芯糖蛋白3表達(dá) 目的測(cè)定1mAchR通路對(duì)HaCat釧胞DSG3及L)SG3mRNA表達(dá)的影響；方法細(xì)胞培養(yǎng)后以噻唑藍(lán)(MTT)法測(cè)定不同時(shí)間、不同濃度氯化乙酰膽堿(Ach)及硫酸阿托品(Atr)對(duì)HaCat細(xì)胞活力的影響；選擇不影響HaCat細(xì)胞活力的試劑濃度作用細(xì)胞后,DIF觀察細(xì)胞表而DSG3熒光強(qiáng)度變化,qPCR測(cè)定LDSG3mRNA表達(dá)變化；結(jié)果高濃度(10-2M) Atr顯著殺傷HaCat細(xì)胞,次高濃度(10-3M、10-4M)Atr顯著降低HaCat細(xì)胞活力；高濃度(10-2M)Ach在6h、12h增加HaCat細(xì)胞的活力,10-5～10-6M Atr及10-3M Ach不影響HaCat細(xì)胞活力：1O-5M Atr作用6h、12h后HaCat細(xì)胞表而及細(xì)胞連接處DSG3熒光強(qiáng)度降低,10-3M Ach作用6h、12h后分別增加及降低HaCat釧胞表而及釧胞連接處DSG3熒光強(qiáng)度；10-5M Atr作用2h、12h顯著降低HaCat細(xì)胞DSG3mRNA表達(dá)；10-3M Ach作用6h增加HaCat細(xì)胞DSG3mRNA表達(dá),而作用2h、12h均明顯抑制HaCat細(xì)胞DSG3mRNA表達(dá)；結(jié)論高濃度Atr與Ach分別降低與增加HaCat細(xì)胞活力。mAchR激動(dòng)劑及mAchR拮抗劑短期(12h以內(nèi))對(duì)DSG3的表達(dá)影響不一,長(zhǎng)期(12h以上)存在均可在轉(zhuǎn)錄與翻譯水平,使HaCat細(xì)胞DSG3表達(dá)降低。 第5章常見不同亞型天皰瘡患者血清影響角質(zhì)形成細(xì)胞m3毒草堿型乙酰膽堿受體mRNA表達(dá) 目的測(cè)定常見不同臨床亞型天皰瘡患者血清對(duì)]HaCat細(xì)胞1m3mAchR表達(dá)的影響；方法采集部分常見臨床亞型天皰瘡患者血清,ELISA測(cè)定抗DSG1自身抗體、抗DSG3自身抗體滴度后,與HaCat細(xì)胞共同培養(yǎng),DIF觀察細(xì)胞表面m3mAchR的熒光強(qiáng)度變化,qPCR測(cè)定CHRM3mRNA表達(dá)變化；結(jié)果三種患者血清作用后m3mAchR熒光強(qiáng)度未出現(xiàn)明顯變化；抗DSG1抗體、抗DSG3抗體雙陽(yáng)性的尋常型天皰瘡患者血清及抗DSG3陽(yáng)性的副腫瘤天皰瘡患者血清作用增加HaCat細(xì)胞CHRM3mRNA表達(dá)；抗DSG1抗體陽(yáng)性的落葉型天皰瘡患者血清顯著降低HaCat細(xì)胞CHRM3mRNA表達(dá)；結(jié)論天皰瘡患者血清對(duì)HaCat細(xì)胞m3mAchR熒光強(qiáng)度影響不明顯；若不考慮除血清中其他物質(zhì)的影響,抗DSG3抗體陽(yáng)性血清可以增加HaCat細(xì)胞CHRM3mRNA表達(dá)。
[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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R758.66

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本文編號(hào)：1416933