本文選題：天皰瘡 + 橋粒芯蛋白��； 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：天皰瘡是一種以血清中存在抗表皮蛋白抗體為特征的自身免疫性皮膚病,橋粒芯蛋白抗體在本病的發(fā)生中具有重要作用。近年的研究發(fā)現(xiàn),除了橋粒芯蛋白途徑之外,還有其它因素在該病發(fā)生中發(fā)揮一定作用,其中膽堿能受體與天皰瘡關(guān)系較為密切。本研究將以膽堿能受體為研究對象,利用HaCaT細(xì)胞構(gòu)建天皰瘡細(xì)胞模型,研究膽堿能受體途徑在細(xì)胞粘附和天皰瘡發(fā)病中的作用,從而進(jìn)一步完善天皰瘡的發(fā)病機(jī)制理論。第1部分膽堿能途徑藥物對HaCaT細(xì)胞粘附功能的影響及機(jī)制研究目的研究膽堿能途徑藥物對HaCaT細(xì)胞粘附功能的影響及其機(jī)制。方法培養(yǎng)HaCaT細(xì)胞至融合狀態(tài)后,分別加入適當(dāng)濃度的膽堿能途徑藥物,通過細(xì)胞解離實(shí)驗(yàn)檢測細(xì)胞粘附的變化情況,結(jié)果以細(xì)胞碎片數(shù)表示：分別用RIPA和Triton X-100裂解細(xì)胞,得到總蛋白和胞漿蛋白,用Western Blot測定細(xì)胞表面與粘附相關(guān)的蛋白Dsg1、Dsg3、PG、E-鈣粘素的變化；用qPCR檢測上述細(xì)胞表面蛋白在mRNA水平的變化。結(jié)果對照組、阿托品、筒箭毒堿、卡巴膽堿作用后細(xì)胞碎片數(shù)目分別為6.33±1.15、35.00±2.65、6.33±1.53、6.00±1.00；阿托品在蛋白質(zhì)和mRNA水平減少Dsg3表達(dá),并使Dsg3從胞膜脫離,卡巴膽堿可增加Dsg1、Dsg3、PG表達(dá),并促使Dsg1、PG粘附于胞膜。筒箭毒堿對各種細(xì)胞表面蛋白均無明顯影響。結(jié)論膽堿能途徑藥物可以調(diào)節(jié)細(xì)胞間粘附分子,尤其是橋粒分子的表達(dá),并影響這些分子在細(xì)胞膜上的穩(wěn)定性,從而介導(dǎo)細(xì)胞間粘附。第2部分利用HaCaT細(xì)胞構(gòu)建天皰瘡細(xì)胞模型目的利用HaCaT細(xì)胞與PV-IgG共培養(yǎng)構(gòu)建天皰瘡細(xì)胞模型。方法收集典型尋常型天皰瘡患者,從血清中純化IgG,以1mg/ml的濃度與HaCaT細(xì)胞共培養(yǎng),通過細(xì)胞解離實(shí)驗(yàn)觀察PV-IgG對HaCaT細(xì)胞粘附功能的影響；通過免疫熒光觀察橋粒蛋白的染色模式；通過Western Blot觀察橋粒分子蛋白質(zhì)水平的變化；通過qPCR觀察橋粒分子mRNA水平的變化；通過免疫共沉淀研究Dsg3與PG的相互作用情況；通過磷酸化測定觀察p38 MAPK、EGFR磷酸化水平。結(jié)果PV-IgG與HaCaT細(xì)胞共培養(yǎng)后,可以使HaCaT細(xì)胞碎片數(shù)目增多,橋粒分子內(nèi)化降解,橋粒穩(wěn)定性下降,Dsg3與PG相互作用減弱,但對橋粒分子mRNA水平的表達(dá)沒有影響；也可出現(xiàn)p38 MAPK與EGFR的磷酸化,且p38 MAPK的磷酸化發(fā)生于EGFR磷酸化之前。結(jié)論利用HaCaT細(xì)胞和PV-IgG可以構(gòu)建天皰瘡的細(xì)胞模型,橋粒分子出現(xiàn)了與體內(nèi)棘層松解過程一致的變化。第3部分 膽堿能受體激動劑卡巴膽堿緩解天皰瘡棘層松解的機(jī)制研究目的研究膽堿能受體激動劑對天皰瘡棘層松解的緩解作用及其機(jī)制。方法將HaCaT細(xì)胞與PV-IgG和膽堿能受體激動劑卡巴膽堿共培養(yǎng),以PV-IgG誘導(dǎo)的天皰瘡細(xì)胞模型作為對照,通過細(xì)胞解離實(shí)驗(yàn)定量分析卡巴膽堿對棘層松解的緩解情況,用免疫熒光方法定性觀察橋粒蛋白變化；分別用RIPA和Triton X-100裂解細(xì)胞,得到總蛋白和胞漿蛋白,用Western Blot觀察細(xì)胞表面與粘附相關(guān)的Dsg3、PG的變化,以及不同時間點(diǎn)p38 MAPK, EGFR的磷酸化水平；用qPCR檢測上述細(xì)胞表面蛋白在mRNA水平的變化；通過免疫共沉淀方法定性分析Dsg3與PG相互作用的變化情況。結(jié)果在HaCaT細(xì)胞與PV-IgG、卡巴膽堿共培養(yǎng),發(fā)現(xiàn)與PV-IgG組相比細(xì)胞碎片數(shù)明顯減少(18.67±2.52 vs 46.67±2.03, t=11.22, p0.01);免疫熒光實(shí)驗(yàn)發(fā)現(xiàn)卡巴膽堿可以緩解PV-IgG所致的橋粒分子內(nèi)化。在天皰瘡細(xì)胞模型中,細(xì)胞總的Dsg3和PG含量下降,非橋粒部分的Dsg3下降,非橋粒PG含量增加,且Dsg3與PG的相互作用減弱,加入卡巴膽堿后可緩解上述變化�？ò湍憠A也可使Dsg3 mRNA的相對表達(dá)量由1.428±0.215增加至4.974±0.948(t=3.65,p=0.01),PG mRNA的相對表達(dá)量由1.563±0.247增加至13.420±1.715(t=6.85,p0.01)。磷酸化實(shí)驗(yàn)中,卡巴膽堿可以抑制EGFR磷酸化,而對p38 MAPK磷酸化無明顯影響。結(jié)論膽堿能受體激動劑卡巴膽堿具有緩解棘層松解的作用,這種緩解作用的機(jī)制可能包括：抑制Dsg3和PG內(nèi)化并增加其表達(dá),增強(qiáng)Dsg3與PG的相互作用,抑制棘層松解關(guān)鍵信號EGFR的磷酸化。第4部分干擾m3、α9 AChR對HaCaT細(xì)胞粘附功能的影響目的研究m3、α9 AChR干擾后HaCaT細(xì)胞粘附功能和橋粒分子的變化情況。方法利用RNA干擾技術(shù)分別干擾m3、α9 AChR的表達(dá),檢測干擾效率及最佳的干擾時間；利用細(xì)胞解離實(shí)驗(yàn)觀察m3或α9 AChR表達(dá)抑制后HaCaT粘附功能的變化情況：利用Western Blot觀察m3或α9 AChR表達(dá)抑制后骨架相關(guān)和非骨架相關(guān)橋粒連接蛋白的變化；利用qPCR觀察橋粒蛋白mRNA水平的變化。結(jié)果在HaCaT細(xì)胞中加入m3或α9AChR干擾序列后,在48h對相應(yīng)基因的表達(dá)均有較好的抑制效果；m3 AChR表達(dá)抑制后細(xì)胞粘附力下降而α9 AChR的低表達(dá)對細(xì)胞粘附功能沒有明顯影響；n3 AChR表達(dá)抑制后使橋粒穩(wěn)定性下降,α9 AChR表達(dá)抑制使橋粒穩(wěn)定性提高；m3 AChR表達(dá)抑制后,Dsg1 mRNA表達(dá)水平下降、PG表達(dá)增多,α9 AChR表達(dá)抑制后,Dsg1、PG mRNA的表達(dá)增多,Dsg3表達(dá)下降。結(jié)論干擾m3 AChR的表達(dá)后HaCaT細(xì)胞的粘附力下降,橋粒穩(wěn)定性下降；干擾α9 AChR并不影響細(xì)胞粘附功能,但可使橋粒穩(wěn)定性上升。第5部分干擾m3、a9 AChR對天皰瘡細(xì)胞模型棘層松解的影響目的探索m3或a9 AChR低表達(dá)時HaCaT細(xì)胞在PV-IgG作用下棘層松解的變化情況。方法通過RNA干擾技術(shù)抑制HaCaT細(xì)胞m3或a9 AChR的表達(dá),在此基礎(chǔ)上與PV-IgG共培養(yǎng),通過細(xì)胞解離實(shí)驗(yàn)觀察細(xì)胞粘附功能的變化；通過免疫熒光觀察橋粒分子染色模式的變化；利用Western Blot觀察m3或a9 AChR表達(dá)抑制后骨架相關(guān)和非骨架相關(guān)橋粒連接蛋白的變化；通過免疫共沉淀研究Dsg3與PG的相互作用情況；通過磷酸化測定觀察p38 MAPK、EGFR磷酸化水平。結(jié)果在m3 AChR干擾時,HaCaT在PV-IgG作用下更容易發(fā)生棘層松解,骨架相關(guān)的Dsg3和PG明顯下降,且EGFR的活化程度增強(qiáng),而a9 AChR干擾對棘層松解有保護(hù)作用,在PV-IgG作用下,橋粒分子不發(fā)生內(nèi)化,骨架相關(guān)Dsg3和PG不減少,Dsg3與PG的相互作用更緊密,p38 MAPK和EGFR均不發(fā)生磷酸化。結(jié)論m3和α9AChR在天皰瘡的發(fā)病中發(fā)揮一定作用,且二者在棘層松解某些環(huán)節(jié)上的作用是相反的。
[Abstract]:Pemphigus is an autoimmune dermatosis characterized by the presence of anti epidermal protein antibodies in the serum. The serotonin antibody plays an important role in the occurrence of this disease. In recent years, other factors have been found to play a role in the pathogenesis of the disease in addition to the bridging kernel protein pathway, including cholinergic receptor and pemphigus. The study will take the cholinergic receptor as the research object, and use HaCaT cells to construct pemphigus cell model, study the role of cholinergic receptor pathway in cell adhesion and pemphigus, and further improve the pathogenesis theory of pemphigus. First parts of the cholinergic pathway drugs on the adhesion function of HaCaT cells The effects of cholinergic pathway drugs on the adhesion function of HaCaT cells and its mechanism were studied. Methods the appropriate concentration of cholinergic pathway was added to HaCaT cells and the cell dissociation test was used to detect the changes of cell adhesion. The results were indicated by the number of cell fragments: RIPA and Tr, respectively. Iton X-100 cracked cells, obtained total protein and cytoplasmic protein, and measured the changes of cell surface and adhesion related protein Dsg1, Dsg3, PG, E- cadherin with Western Blot, and detected the change of the protein on the mRNA level by qPCR. Results of the control group, atropine, canistromine, and Kaba choline, the number of cell fragments was 6.33 1.15,35.00 + 2.65,6.33 + 1.53,6.00 + 1; atropine reduced Dsg3 expression at the level of protein and mRNA, and separated Dsg3 from the membrane. Kaba choline could increase Dsg1, Dsg3, PG expression, and promote Dsg1, PG adherence to the membrane. The expression of adhesion molecules, especially the molecular weight of pemphigus, affects the stability of these molecules on the cell membrane and mediates the intercellular adhesion. The second part uses HaCaT cells to construct pemphigus cell models to construct pemphigus cell models by co culture of HaCaT cells with PV-IgG. IgG was purified with the concentration of 1mg/ml and co cultured with HaCaT cells. The effect of PV-IgG on the adhesion function of HaCaT cells was observed through the cell dissociation test. The staining pattern of the grained protein was observed by immunofluorescence; the change of the molecular protein level of the bridge particles was observed by Western Blot; the variation of the mRNA level of the bridge particles was observed through qPCR; The interaction between Dsg3 and PG was studied by immunoprecipitation, and the phosphorylation of p38 MAPK and EGFR was observed by phosphorylation. Results after co culture of PV-IgG and HaCaT cells, the number of HaCaT cell fragments increased, the molecular degradation of the bridging particles, the stability of the grained grain and the interaction of Dsg3 and PG weakened, but the mRNA level of the molecular weight of the grained particles could be reduced. There is no influence; phosphorylation of p38 MAPK and EGFR may also occur, and the phosphorylation of p38 MAPK occurs before EGFR phosphorylation. Conclusion using HaCaT cells and PV-IgG to construct the cell model of pemphigus, the bridging molecules appear to be consistent with the process of acantholysis in the body. Third division of cholinergic agonist Kaba choline relieves the day Study on the mechanism of peminysis in pemphigus objective to study the effect and mechanism of cholinergic receptor agonist on pemphigus pemphigus. Methods HaCaT cells were co cultured with PV-IgG and cholinergic receptor agonist Kaba choline, and the PV-IgG induced pemphigus cell model was used as the control, and the quantitative analysis of Kaba choline was carried out through cell dissociation test. The changes of PK were observed by immunofluorescence. The total protein and cytoplasmic protein were obtained by RIPA and Triton X-100, respectively. The changes of Dsg3, PG, p38 MAPK and EGFR were observed by Western Blot, and the phosphorylation level of p38 MAPK and EGFR at different time points was detected by qPCR detection. The changes in the level of cell surface protein at mRNA level; qualitative analysis of the interaction between Dsg3 and PG by immunoprecipitation. Results in co culture of HaCaT cells with PV-IgG and Kaba choline, it was found that the number of cell fragments decreased significantly compared with the PV-IgG group (18.67 + 2.52 vs 46.67 + 2.03, t=11.22, P0.01); immunofluorescence experimental discovery card In the pemphigus cell model, the total number of Dsg3 and PG in the pemphigus cell model decreased, the Dsg3 decreased in the pemphigoid part, the non bridging PG content increased, and the interaction between the Dsg3 and PG weakened, and the addition of Kaba choline could alleviate the above-mentioned changes. The relative expression of Dsg3 mRNA by Kaba choline could also be 1 of the relative expression of Dsg3 mRNA by 1 .428 + 0.215 increased to 4.974 + 0.948 (t=3.65, p=0.01), and the relative expression of PG mRNA increased from 1.563 + 0.247 to 13.420 + 1.715 (t=6.85, P0.01). In the phosphorylation experiment, Kaba choline could inhibit the phosphorylation of EGFR, but had no obvious effect on the phosphorylation of p38 MAPK. Conclusion cholinergic agonist Kaba choline has the effect of relieving spinous release. The mechanisms of this remission may include inhibiting the internalization of Dsg3 and PG and increasing their expression, enhancing the interaction between Dsg3 and PG, and inhibiting the phosphorylation of EGFR, the key signal of the spinous release of EGFR. The fourth part interferes with M3, and the effect of alpha 9 AChR on HaCaT cell adhesion function is to study the adherence function of HaCaT cells and the change of the pbridge molecules after the interference of alpha 9 AChR. Methods RNA interference technique was used to interfere with the expression of M3, alpha 9 AChR, to detect interference efficiency and the best interference time, and to observe the changes of HaCaT adhesion function after the inhibition of M3 or alpha 9 AChR expression by cell dissociation experiment: using Western Blot to observe the cytoskeleton related and non skeleton related bridging connection after the expression of M3 or alpha 9 AChR expression The changes in the protein mRNA were observed by qPCR. The results showed that 48h had a better inhibitory effect on the expression of the corresponding genes after the addition of M3 or alpha 9AChR interference in HaCaT cells; the adhesion function of the cell adhesion decreased and the low expression of alpha 9 AChR had no obvious effect on the cell adhesion function after the expression of M3 AChR; N3 AC. After inhibition of expression of hR, the stability of bridging grain decreased and the expression of alpha 9 AChR inhibited the stability of the grain. After the expression of M3 AChR was inhibited, the expression level of Dsg1 mRNA decreased, the expression of PG increased, the expression of Dsg1, PG mRNA increased and Dsg3 expression decreased after the inhibition of the expression of alpha 9 AChR. Conclusion the adhesion of the cells decreased and the grain stability was stable after the expression of interference. Decrease in sex; interfering with alpha 9 AChR does not affect cell adhesion, but it can increase the stability of PON. The fifth part interferes with the effect of M3, A9 AChR on the spinous release of pemphigus cell models. Objective to explore the changes of the acanthosis of HaCaT cells under the action of PV-IgG under the low expression of M3 or A9 AChR. Methods the HaCaT cell m3 is inhibited by RNA interference technique. Or the expression of A9 AChR, on this basis, co culture with PV-IgG, observe the changes in cell adhesion function through cell dissociation test, observe the changes in the staining pattern of the bridge particles by immunofluorescence, and observe the changes of the skeleton related and non skeleton related grained connexin after the inhibition of the expression of M3 or A9 AChR by Western Blot; The interaction between Dsg3 and PG was studied by coprecipitation, and the phosphorylation of p38 MAPK and EGFR was observed by phosphorylation. The results showed that when m3 AChR interfered, HaCaT was more likely to occur acanthosis under PV-IgG, and the Dsg3 and PG of skeleton decreased obviously, and the activation degree of EGFR was enhanced. Under the action of -IgG, the molecules of pons do not internalize, the cytoskeleton related Dsg3 and PG do not decrease, the interaction between Dsg3 and PG is more closely, p38 MAPK and EGFR do not produce phosphorylation. Conclusion m3 and alpha 9AChR play a role in the pathogenesis of pemphigus, and the role of the two in some stages of acanthosis is opposite.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R758.66

【共引文獻(xiàn)】

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