Hedgehog信号通路在银屑病发病中的机制研究

发布时间：2018-07-11 10:16

本文选题：Hedgehog通路 + 银屑病　；参考：《浙江大学》2015年博士论文

【摘要】：背景：银屑病是一种常见的慢性炎症性皮肤病,在全世界范围内累及人数大于125,000,000人,其发病机制复杂,包括角质形成细胞的增生和异常分化,显著的炎症浸润等。其中表皮角质形成细胞的过度增生由一系列细胞因子及信号通路激活,这些刺激因素又来源于免疫细胞以及角质形成细胞本身。 Hedgehog (Hh)信号通路不仅在胚胎发育以及成人的内稳态维持中发挥重要作用,其异常活化也导致了一系列疾病的产生,包括基底细胞癌、胰腺癌、成视网膜细胞瘤以及髓母细胞瘤等。在生理性Hh信号通路传导过程中,Hh配体结合细胞表面受体Patched1(PTCH1),从而解除对Smoothened (SMO)的抑制作用。SMO的活化导致一系列细胞内事件的发生,通过转录因子GLI家族(GLI1、GLI2、GLI3)导致靶向基因表达的变化。在缺少Hh配体时,PTCH1抑制SMO的活动,从而使得转录因子GLI在纤毛内降解为抑制体的形式。其降解过程需要suppressor of fused (Sufu)和kinesin family member7(Kif7)的参与。哺乳动物的Hh配体包括三种,分别为Sonic hedgehog (Shh)、Indian hedgehog (Ihh)和Desert hedgehog (Dhh),其中Shh是皮肤中主要的Hh配体。以往的研究发现Hh信号通路在银屑病皮损区是被激活的,使用Hh通路的抑制剂环巴胺后能导致皮损的快速消退。但是,又有研究表明在银屑病皮损区中并没有Hh转录因子mRNA水平上的升高。因此,Hh信号通路是否在银屑病中过度激活以及其在银屑病发病机制中的作用仍需要进一步的研究。目的：明确Hh信号通路中的各个分子在银屑病皮肤组织皮损区角质形成细胞中的表达情况,并在体外培养的原代人表皮角质形成细胞中使用重组人细胞因子Sonic Hedgehog激活以及SMO的抑制剂环巴胺抑制Hh信号通路后观察细胞的增殖、凋亡、细胞周期变化、衰老、活性氧族的产生、迁移以及分化等功能变化；以及环巴胺在体外对咪喹莫特诱导的银屑病小鼠模型的作用。方法：第一部分：切取正常人以及银屑病病人皮损区皮肤,并分离出表皮,分离、培养正常人表皮角质形成细胞,提取总RNA和蛋白质。以逆转录-聚合酶链反应(Reverse transcript polymerase chain reaction, RT-PCR)检测正常人以及银屑病病人皮损区皮肤组织全皮、表皮以及表皮角质形成细胞Hh信号通路中各个分子的mRNA表达水平；以Western-blot法检测正常人以及银屑病病人皮损区表皮角质形成细胞Hh信号通路中各个分子的蛋白表达水平；以间接免疫荧光法对Hh信号通路中各个分子在正常人以及银屑病病人表皮中的表达进行定位；用酶联免疫吸附测定(Enzyme Linked Immunosorbent Assay, ELISA)检测正常人以及银屑病病人外周血血清中Hh通路的三种配体Shh、Ihh以及Dhh的浓度水平。第二部分：使用外源性Sonic Hedgehog细胞因子以及Hh通路的抑制剂环巴胺处理体外培养的人表皮角质形成细胞,观察细胞增殖、凋亡、细胞周期变化、衰老、活性氧族的产生、迁移以及分化等功能变化。另外使用慢病毒感染人角质形成细胞使其过表达Hh通路的抑制因子Sufu后观察Hh通路GLI转录因子在nRNA、蛋白水平以及亚细胞定位中的变化,并进一步观察与角质形成细胞分化相关的蛋白表达水平变化。第三部分：体外用咪喹莫特诱导C57BL/6小鼠形成银屑病模型,并用不同浓度的环巴胺腹腔注射小鼠,通过PASI评分以及HE染色观察其对银屑病皮损形成的抑制作用。结果：一、银屑病中Hh通路在mRNA水平上的激活： 1.银屑病病人皮损区表皮中GLl3的mRNA表达与正常人相比显著降低； 2.银屑病病人皮损区全皮中Sufu的nRNA表达与正常人相比显著降低； 3.体外培养的银屑病病人皮损区表皮角质形成细胞中GLl3的mRNA表达与正常相比显著降低,而Shh、GLI1、Sufu的mRNA表达无显著差异。二、银屑病中Hh通路在蛋白水平上的激活： 1.银屑病病人外周血血清中Shh、Ihh和Dhh浓度与正常人相比显著升高； 2.体外培养的银屑病病人皮损区角质形成细胞中GLI3、SUFU、PTCH1和PTCH2蛋白表达与正常相比显著降低,GLl2略有升高,但差异不显著,Shh和GLl1无显著差异； 3.免疫荧光检测结果发现,银屑病病人皮损区以及正常人表皮各层均有GLI1、GLI2和GLl3的表达,其中GLl3在银屑病皮损区表皮和正常人中的分布明显不同,在银屑病表皮中GLl3主要位于胞浆中,而正常表皮中GLl3主要位于胞核内。体外培养的银屑病病人皮损区角质形成细胞中GLl3主要位于胞浆中,而在正常人角质形成细胞中GLl3主要位于胞核内。三、重组人Shh刺激体外培养人表皮角质形成细胞引起的功能变化： 1.重组人Shh剂量依赖性地促进角质形成细胞中GLl1的mRNA转录,但对Sufu及GLl3的mRNA转录无明显作用； 2.重组人Shh剂量依赖性地促进角质形成细胞的增殖； 3.重组人Shh促进角质形成细胞中活性氧族的产生。四、环巴胺作用于体外培养人表皮角质形成细胞引起的功能变化： 1.环巴胺剂量依赖性地抑制角质形成细胞的增殖； 2.环巴胺剂量依赖性地促进角质形成细胞的凋亡,而重组人Shh显著抑制角质形成细胞的凋亡； 3.环巴胺导致角质形成细胞G1期的阻滞,而TNF-α能逆转环巴胺的G1期阻滞作用； 4.体外培养的银屑病病人皮损区角质形成细胞衰老程度显著低于正常人；环巴胺能显著促进细胞衰老,丙酸氯贝他索能显著减少细胞衰老；TNF-α对细胞衰老无明显作用,而环巴胺能显著逆转丙酸氯贝他索导致的抗衰老作用； 5.环巴胺能显著降低角质形成细胞的活性氧族产生,丙酸氯贝他索亦能显著降低细胞活性氧族产生；TNF-a能显著增加细胞活性氧族的产生,环巴胺能显著抑制TNF-a对活性氧族的促进作用； 6.环巴胺能显著抑制角质形成细胞的迁移,Shh能显著促进细胞迁移,丙酸氯倍他索能显著抑制细胞迁移,TNF-a对细胞迁移无明显作用。五、体外培养人表皮角质形成细胞Sufu过表达引起的功能变化： 1.人表皮角质形成细胞中mRNA及蛋白水平均验证了Sufu过表达的效率； 2.人表皮角质形成细胞中Sufu过表达后GLI1和GLI2的蛋白表达均降低了； 3.银屑病病人皮损区表皮角质形成细胞中Sufu过表达后与分化相关的蛋白表达升高了； 4.银屑病病人皮损区表皮角质形成细胞中Sufu 过表达后GLI3由胞浆转移至胞核内。六、环巴胺减轻了小鼠模型中银屑病皮损的形成： 1.环巴胺降低了小鼠模型中银屑病皮损的PASI评分； 2.HE染色表明环巴胺减轻了咪喹莫特诱导的小鼠银屑病皮损的严重程度。结论：一、银屑病中Hh通路在mRNA及蛋白水平上均被激活了；二、Shh能促进角质形成细胞的增殖,抑制角质形成细胞凋亡,显著增加角质形成细胞活性氧族的产生,促进细胞迁移,从而证明Hh通路的激活在银屑病的发病中的作用；三、环巴胺能抑制角质形成细胞的增殖,可能与其促进细胞凋亡及阻滞细胞周期相关；环巴胺能促进细胞衰老,抑制活性氧族的产生并抑制细胞迁移能力；从而证明了阻断Hh通路对抑制银屑病发病的分子机制；四、Sufu过表达能抑制角质形成细胞中Hh通路的激活,进而促进银屑病表皮角质形成细胞的分化；五、环巴胺能抑制咪喹莫特诱导的小鼠模型中银屑病皮损的形成,从而为应用Hh通路抑制剂临床治疗银屑病提供了可能。
[Abstract]:Background:
Psoriasis is a common chronic inflammatory dermatosis, involving more than 125000000 people worldwide. The pathogenesis is complex, including the proliferation and abnormal differentiation of keratinocytes and significant inflammatory infiltration. The hyperproliferation of epidermal keratinocytes is activated by a series of cytokines and signaling pathways. The stimulant also comes from immune cells and keratinocytes themselves.
The Hedgehog (Hh) signaling pathway not only plays an important role in the development of embryo and the homeostasis of adult, but its abnormal activation also leads to the production of a series of diseases, including basal cell carcinoma, pancreatic cancer, retinoblastoma and medulloblastoma.
In the process of physiological Hh signaling pathway, the Hh ligand binding to the cell surface receptor Patched1 (PTCH1), thus relieving the inhibition of Smoothened (SMO), the activation of.SMO leads to the occurrence of a series of intracellular events. The transcriptional factor GLI family (GLI1, GLI2, GLI3) leads to the change of the target gene expression. The activity of MO causes the transcription factor GLI to degrade into the cilium as a form of inhibition. The degradation process requires the participation of suppressor of fused (Sufu) and kinesin family member7 (Kif7). The main Hh ligands.
Previous studies have found that the Hh signaling pathway is activated in the psoriatic lesion area, and the use of the Hh pathway inhibitor, cyclic amine, can lead to rapid degeneration of the skin lesions. However, further studies have shown that there is no increase in the level of Hh transcription factor mRNA in the lesions of the psoriasis. Therefore, whether the Hh signaling pathway is excessively activated in psoriasis and it can be used in psoriasis. The role of psoriasis in the pathogenesis of psoriasis still needs further study.
Objective:
The expression of each molecule in the Hh signaling pathway in the keratinocytes of the skin lesion of psoriasis was identified, and the recombinant human cytokine Sonic Hedgehog was activated in the cultured human epidermal keratinocytes, and the proliferation, apoptosis, and apoptosis of the cells were observed after the inhibition of the Hh signaling pathway by the inhibitor of SMO. The changes in cell cycle, senescence, the generation, migration and differentiation of the active oxygen group, and the effect of cycamines on the model of psoriasis induced by imiquimod in vitro.
Method:
The first part: cutting the skin of normal and psoriatic patients, separating the epidermis, separating and cultivating the normal human epidermal keratinocyte, extracting the total RNA and protein, and using reverse transcription polymerase chain reaction (Reverse transcript polymerase chain reaction, RT-PCR) to detect the skin group of the skin lesions of the patients with psoriasis and the skin lesions of the patients with psoriasis. The mRNA expression level of each molecule in the Hh signaling pathway of the skin, the epidermis and the epidermal keratinocytes; the protein expression level of each molecule in the Hh signaling pathway of the epidermal keratinocytes in the skin lesions of psoriasis patients was detected by Western-blot method; and the molecules in the Hh signaling pathway were in positive effect by indirect immunofluorescence. The expression in the epidermis of the normal and psoriatic patients was located, and the concentrations of the three ligands, Shh, Ihh, and Dhh, were detected by Enzyme Linked Immunosorbent Assay (ELISA) and the Hh pathway in the peripheral blood serum of psoriasis patients.
The second part: using the exogenous Sonic Hedgehog cytokine and the inhibitor of Hh pathway to treat the cultured human epidermal keratinocytes in vitro, observe the cell proliferation, apoptosis, cell cycle change, senescence, the generation, migration and differentiation of the active oxygen group. In addition, the human keratinocytes are infected with lentivirus. After overexpressing the inhibitory factor Sufu of the Hh pathway, the changes in the GLI transcription factors of the Hh pathway in nRNA, protein level and subcellular localization were observed, and the changes in the protein expression level related to the differentiation of keratinocytes were further observed.
The third part: Imiquimod induced C57BL/6 mice to form a psoriasis model in vitro and intraperitoneally injected with different concentrations of cyclic amine. The inhibition of psoriasis on the formation of psoriasis was observed by PASI score and HE staining.
Result:
1. Activation of Hh pathway in psoriasis at mRNA level:
1. the mRNA expression of GLl3 in epidermis of psoriatic patients was significantly lower than that of normal persons.
2. the nRNA expression of Sufu in skin lesions of psoriatic patients was significantly lower than that of normal persons.
3. the mRNA expression of GLl3 in epidermal keratinocytes from the skin lesions of the patients with psoriasis in vitro was significantly lower than that of normal, but there was no significant difference in the expression of mRNA in Shh, GLI1 and Sufu.
Two, activation of Hh pathway in psoriasis at protein level:
1. the concentrations of Shh, Ihh and Dhh in peripheral blood of psoriasis patients were significantly higher than those of normal persons.
2. the expression of GLI3, SUFU, PTCH1 and PTCH2 in the keratinocytes of the skin lesions of the patients with psoriasis in vitro decreased significantly compared with the normal, and the GLl2 increased slightly, but the difference was not significant, and there was no significant difference between Shh and GLl1.
3. the results of immunofluorescence detection showed that GLI1, GLI2 and GLl3 were expressed in the skin lesions and the normal human epidermis of psoriasis, and the distribution of GLl3 in the epidermis of psoriatic lesions and the normal people was distinct. In the epidermis of psoriasis, GLl3 was mainly located in the cytoplasm, and the GLl3 in the normal epidermis was mainly located in the nucleus. In vitro culture, the silver was cultured in the epidermis of the psoriasis. GLl3 is mainly located in cytoplasm of keratinocytes in skin lesions of patients with scraps disease. GLl3 is mainly located in the nucleus of normal human keratinocytes.
Three, recombinant human Shh stimulates human epidermal keratinocytes to induce functional changes in vitro.
1. recombinant human Shh promoted mRNA transcription of GLl1 in keratinocytes dose dependently, but had no significant effect on mRNA transcription of Sufu and GLl3.
2. recombinant human Shh promotes proliferation of keratinocytes in a dose-dependent manner.
3. recombinant human Shh promotes the production of reactive oxygen species in keratinocytes.
Four, the effects of cyclic Amban on human keratinocytes cultured in vitro:
1. cyclic Amban inhibited the proliferation of keratinocytes in a dose-dependent manner.
2. cyclic Amban promoted the apoptosis of keratinocytes in a dose dependent manner, while recombinant human Shh significantly inhibited the apoptosis of keratinocytes.
3. cyclic Amban causes G1 phase arrest in keratinocytes, while TNF- alpha can reverse the G1 arrest effect of cyclic amines.
4. the senescence of keratinocytes in the skin lesions of the patients with psoriasis in vitro was significantly lower than that of the normal people; cyclic amine could significantly promote cell senescence, and chloramalin could significantly reduce cell senescence; TNF- alpha had no obvious effect on cell senescence, while cyclic amine could significantly reverse the antiaging effect of chloropamines.
5. cyclic amine can significantly reduce the production of reactive oxygen species in keratinocytes, and chloramoxen propionate can also significantly reduce the production of reactive oxygen species. TNF-a can significantly increase the production of reactive oxygen species, and cyclic amine can significantly inhibit the promoting effect of TNF-a on the active oxygen group.
6. cyclic amine can significantly inhibit the migration of keratinocytes, Shh can significantly promote cell migration. Chloramatropine propionate can significantly inhibit cell migration, and TNF-a has no significant effect on cell migration.
Five, functional changes induced by overexpression of Sufu in human keratinocytes cultured in vitro:
The efficiency of Sufu overexpression was verified by mRNA and protein levels in 1. keratinocytes.
The expression of GLI1 and GLI2 protein in 2. keratinocytes was decreased after overexpression of Sufu.
3. the expression of Sufu in epidermal keratinocytes of psoriatic patients increased after overexpression.
4. Sufu in epidermis of psoriatic lesions.
After overexpression, the GLI3 was transferred from the cytoplasm to the nucleus.
Six, cyclic Amban alleviated the formation of silver scraps lesions in mice.
1. cyclic Amban reduced the PASI score of silver scraps lesions in mice.
2.HE staining showed that cimetidine alleviated the severity of psoriasis induced by imiquimod in mice.
Conclusion:
First, the Hh pathway in psoriasis was activated at mRNA and protein levels.
Two, Shh can promote the proliferation of keratinocytes, inhibit the apoptosis of keratinocytes, increase the production of reactive oxygen species in keratinocytes, and promote cell migration, thus proving the role of activation of Hh pathway in the pathogenesis of psoriasis.
Three, cyclamamine inhibits the proliferation of keratinocytes, which may be related to the promotion of cell apoptosis and cell cycle arrest; cyclamamine can promote cell senescence, inhibit the production of reactive oxygen species and inhibit cell migration, which proves the molecular mechanism of blocking Hh pathway to inhibit the onset of psoriasis.
Four, Sufu overexpression can inhibit the activation of Hh pathway in keratinocytes and promote the differentiation of epidermal keratinocytes in psoriasis.
Five, cimiquine can inhibit the formation of the skin lesion of the silver chip disease in imiquimod induced mouse models, thus providing a possibility for the clinical treatment of psoriasis using Hh pathway inhibitors.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R758.63

【共引文献】