胆碱能受体途径在HaCaT细胞粘附及天疱疮发病中的作用及机制研究

发布时间：2018-05-25 15:17

  本文选题：天疱疮 + 桥粒芯蛋白　； 参考：《北京协和医学院》2015年博士论文

【摘要】：天疱疮是一种以血清中存在抗表皮蛋白抗体为特征的自身免疫性皮肤病,桥粒芯蛋白抗体在本病的发生中具有重要作用。近年的研究发现,除了桥粒芯蛋白途径之外,还有其它因素在该病发生中发挥一定作用,其中胆碱能受体与天疱疮关系较为密切。本研究将以胆碱能受体为研究对象,利用HaCaT细胞构建天疱疮细胞模型,研究胆碱能受体途径在细胞粘附和天疱疮发病中的作用,从而进一步完善天疱疮的发病机制理论。第1部分胆碱能途径药物对HaCaT细胞粘附功能的影响及机制研究目的研究胆碱能途径药物对HaCaT细胞粘附功能的影响及其机制。方法培养HaCaT细胞至融合状态后,分别加入适当浓度的胆碱能途径药物,通过细胞解离实验检测细胞粘附的变化情况,结果以细胞碎片数表示：分别用RIPA和Triton X-100裂解细胞,得到总蛋白和胞浆蛋白,用Western Blot测定细胞表面与粘附相关的蛋白Dsg1、Dsg3、PG、E-钙粘素的变化；用qPCR检测上述细胞表面蛋白在mRNA水平的变化。结果对照组、阿托品、筒箭毒碱、卡巴胆碱作用后细胞碎片数目分别为6.33±1.15、35.00±2.65、6.33±1.53、6.00±1.00；阿托品在蛋白质和mRNA水平减少Dsg3表达,并使Dsg3从胞膜脱离,卡巴胆碱可增加Dsg1、Dsg3、PG表达,并促使Dsg1、PG粘附于胞膜。筒箭毒碱对各种细胞表面蛋白均无明显影响。结论胆碱能途径药物可以调节细胞间粘附分子,尤其是桥粒分子的表达,并影响这些分子在细胞膜上的稳定性,从而介导细胞间粘附。第2部分利用HaCaT细胞构建天疱疮细胞模型目的利用HaCaT细胞与PV-IgG共培养构建天疱疮细胞模型。方法收集典型寻常型天疱疮患者,从血清中纯化IgG,以1mg/ml的浓度与HaCaT细胞共培养,通过细胞解离实验观察PV-IgG对HaCaT细胞粘附功能的影响；通过免疫荧光观察桥粒蛋白的染色模式；通过Western Blot观察桥粒分子蛋白质水平的变化；通过qPCR观察桥粒分子mRNA水平的变化；通过免疫共沉淀研究Dsg3与PG的相互作用情况；通过磷酸化测定观察p38 MAPK、EGFR磷酸化水平。结果PV-IgG与HaCaT细胞共培养后,可以使HaCaT细胞碎片数目增多,桥粒分子内化降解,桥粒稳定性下降,Dsg3与PG相互作用减弱,但对桥粒分子mRNA水平的表达没有影响；也可出现p38 MAPK与EGFR的磷酸化,且p38 MAPK的磷酸化发生于EGFR磷酸化之前。结论利用HaCaT细胞和PV-IgG可以构建天疱疮的细胞模型,桥粒分子出现了与体内棘层松解过程一致的变化。第3部分 胆碱能受体激动剂卡巴胆碱缓解天疱疮棘层松解的机制研究目的研究胆碱能受体激动剂对天疱疮棘层松解的缓解作用及其机制。方法将HaCaT细胞与PV-IgG和胆碱能受体激动剂卡巴胆碱共培养,以PV-IgG诱导的天疱疮细胞模型作为对照,通过细胞解离实验定量分析卡巴胆碱对棘层松解的缓解情况,用免疫荧光方法定性观察桥粒蛋白变化；分别用RIPA和Triton X-100裂解细胞,得到总蛋白和胞浆蛋白,用Western Blot观察细胞表面与粘附相关的Dsg3、PG的变化,以及不同时间点p38 MAPK, EGFR的磷酸化水平；用qPCR检测上述细胞表面蛋白在mRNA水平的变化；通过免疫共沉淀方法定性分析Dsg3与PG相互作用的变化情况。结果在HaCaT细胞与PV-IgG、卡巴胆碱共培养,发现与PV-IgG组相比细胞碎片数明显减少(18.67±2.52 vs 46.67±2.03, t=11.22, p0.01);免疫荧光实验发现卡巴胆碱可以缓解PV-IgG所致的桥粒分子内化。在天疱疮细胞模型中,细胞总的Dsg3和PG含量下降,非桥粒部分的Dsg3下降,非桥粒PG含量增加,且Dsg3与PG的相互作用减弱,加入卡巴胆碱后可缓解上述变化。卡巴胆碱也可使Dsg3 mRNA的相对表达量由1.428±0.215增加至4.974±0.948(t=3.65,p=0.01),PG mRNA的相对表达量由1.563±0.247增加至13.420±1.715(t=6.85,p0.01)。磷酸化实验中,卡巴胆碱可以抑制EGFR磷酸化,而对p38 MAPK磷酸化无明显影响。结论胆碱能受体激动剂卡巴胆碱具有缓解棘层松解的作用,这种缓解作用的机制可能包括：抑制Dsg3和PG内化并增加其表达,增强Dsg3与PG的相互作用,抑制棘层松解关键信号EGFR的磷酸化。第4部分干扰m3、α9 AChR对HaCaT细胞粘附功能的影响目的研究m3、α9 AChR干扰后HaCaT细胞粘附功能和桥粒分子的变化情况。方法利用RNA干扰技术分别干扰m3、α9 AChR的表达,检测干扰效率及最佳的干扰时间；利用细胞解离实验观察m3或α9 AChR表达抑制后HaCaT粘附功能的变化情况：利用Western Blot观察m3或α9 AChR表达抑制后骨架相关和非骨架相关桥粒连接蛋白的变化；利用qPCR观察桥粒蛋白mRNA水平的变化。结果在HaCaT细胞中加入m3或α9AChR干扰序列后,在48h对相应基因的表达均有较好的抑制效果；m3 AChR表达抑制后细胞粘附力下降而α9 AChR的低表达对细胞粘附功能没有明显影响；n3 AChR表达抑制后使桥粒稳定性下降,α9 AChR表达抑制使桥粒稳定性提高；m3 AChR表达抑制后,Dsg1 mRNA表达水平下降、PG表达增多,α9 AChR表达抑制后,Dsg1、PG mRNA的表达增多,Dsg3表达下降。结论干扰m3 AChR的表达后HaCaT细胞的粘附力下降,桥粒稳定性下降；干扰α9 AChR并不影响细胞粘附功能,但可使桥粒稳定性上升。第5部分干扰m3、a9 AChR对天疱疮细胞模型棘层松解的影响目的探索m3或a9 AChR低表达时HaCaT细胞在PV-IgG作用下棘层松解的变化情况。方法通过RNA干扰技术抑制HaCaT细胞m3或a9 AChR的表达,在此基础上与PV-IgG共培养,通过细胞解离实验观察细胞粘附功能的变化；通过免疫荧光观察桥粒分子染色模式的变化；利用Western Blot观察m3或a9 AChR表达抑制后骨架相关和非骨架相关桥粒连接蛋白的变化；通过免疫共沉淀研究Dsg3与PG的相互作用情况；通过磷酸化测定观察p38 MAPK、EGFR磷酸化水平。结果在m3 AChR干扰时,HaCaT在PV-IgG作用下更容易发生棘层松解,骨架相关的Dsg3和PG明显下降,且EGFR的活化程度增强,而a9 AChR干扰对棘层松解有保护作用,在PV-IgG作用下,桥粒分子不发生内化,骨架相关Dsg3和PG不减少,Dsg3与PG的相互作用更紧密,p38 MAPK和EGFR均不发生磷酸化。结论m3和α9AChR在天疱疮的发病中发挥一定作用,且二者在棘层松解某些环节上的作用是相反的。
[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.
【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】：2015
【分类号】：R758.66

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