發(fā)布時間：2018-07-11 10:16

  本文選題：Hedgehog通路 + 銀屑病�。� 參考：《浙江大學(xué)》2015年博士論文

【摘要】：背景： 銀屑病是一種常見的慢性炎癥性皮膚病,在全世界范圍內(nèi)累及人數(shù)大于125,000,000人,其發(fā)病機制復(fù)雜,包括角質(zhì)形成細(xì)胞的增生和異常分化,顯著的炎癥浸潤等。其中表皮角質(zhì)形成細(xì)胞的過度增生由一系列細(xì)胞因子及信號通路激活,這些刺激因素又來源于免疫細(xì)胞以及角質(zhì)形成細(xì)胞本身。 Hedgehog (Hh)信號通路不僅在胚胎發(fā)育以及成人的內(nèi)穩(wěn)態(tài)維持中發(fā)揮重要作用,其異�；罨矊�(dǎo)致了一系列疾病的產(chǎn)生,包括基底細(xì)胞癌、胰腺癌、成視網(wǎng)膜細(xì)胞瘤以及髓母細(xì)胞瘤等。 在生理性Hh信號通路傳導(dǎo)過程中,Hh配體結(jié)合細(xì)胞表面受體Patched1(PTCH1),從而解除對Smoothened (SMO)的抑制作用。SMO的活化導(dǎo)致一系列細(xì)胞內(nèi)事件的發(fā)生,通過轉(zhuǎn)錄因子GLI家族(GLI1、GLI2、GLI3)導(dǎo)致靶向基因表達(dá)的變化。在缺少Hh配體時,PTCH1抑制SMO的活動,從而使得轉(zhuǎn)錄因子GLI在纖毛內(nèi)降解為抑制體的形式。其降解過程需要suppressor of fused (Sufu)和kinesin family member7(Kif7)的參與。哺乳動物的Hh配體包括三種,分別為Sonic hedgehog (Shh)、Indian hedgehog (Ihh)和Desert hedgehog (Dhh),其中Shh是皮膚中主要的Hh配體。 以往的研究發(fā)現(xiàn)Hh信號通路在銀屑病皮損區(qū)是被激活的,使用Hh通路的抑制劑環(huán)巴胺后能導(dǎo)致皮損的快速消退。但是,又有研究表明在銀屑病皮損區(qū)中并沒有Hh轉(zhuǎn)錄因子mRNA水平上的升高。因此,Hh信號通路是否在銀屑病中過度激活以及其在銀屑病發(fā)病機制中的作用仍需要進(jìn)一步的研究。 目的： 明確Hh信號通路中的各個分子在銀屑病皮膚組織皮損區(qū)角質(zhì)形成細(xì)胞中的表達(dá)情況,并在體外培養(yǎng)的原代人表皮角質(zhì)形成細(xì)胞中使用重組人細(xì)胞因子Sonic Hedgehog激活以及SMO的抑制劑環(huán)巴胺抑制Hh信號通路后觀察細(xì)胞的增殖、凋亡、細(xì)胞周期變化、衰老、活性氧族的產(chǎn)生、遷移以及分化等功能變化；以及環(huán)巴胺在體外對咪喹莫特誘導(dǎo)的銀屑病小鼠模型的作用。 方法： 第一部分：切取正常人以及銀屑病病人皮損區(qū)皮膚,并分離出表皮,分離、培養(yǎng)正常人表皮角質(zhì)形成細(xì)胞,提取總RNA和蛋白質(zhì)。以逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(Reverse transcript polymerase chain reaction, RT-PCR)檢測正常人以及銀屑病病人皮損區(qū)皮膚組織全皮、表皮以及表皮角質(zhì)形成細(xì)胞Hh信號通路中各個分子的mRNA表達(dá)水平；以Western-blot法檢測正常人以及銀屑病病人皮損區(qū)表皮角質(zhì)形成細(xì)胞Hh信號通路中各個分子的蛋白表達(dá)水平；以間接免疫熒光法對Hh信號通路中各個分子在正常人以及銀屑病病人表皮中的表達(dá)進(jìn)行定位；用酶聯(lián)免疫吸附測定(Enzyme Linked Immunosorbent Assay, ELISA)檢測正常人以及銀屑病病人外周血血清中Hh通路的三種配體Shh、Ihh以及Dhh的濃度水平。 第二部分：使用外源性Sonic Hedgehog細(xì)胞因子以及Hh通路的抑制劑環(huán)巴胺處理體外培養(yǎng)的人表皮角質(zhì)形成細(xì)胞,觀察細(xì)胞增殖、凋亡、細(xì)胞周期變化、衰老、活性氧族的產(chǎn)生、遷移以及分化等功能變化。另外使用慢病毒感染人角質(zhì)形成細(xì)胞使其過表達(dá)Hh通路的抑制因子Sufu后觀察Hh通路GLI轉(zhuǎn)錄因子在nRNA、蛋白水平以及亞細(xì)胞定位中的變化,并進(jìn)一步觀察與角質(zhì)形成細(xì)胞分化相關(guān)的蛋白表達(dá)水平變化。 第三部分：體外用咪喹莫特誘導(dǎo)C57BL/6小鼠形成銀屑病模型,并用不同濃度的環(huán)巴胺腹腔注射小鼠,通過PASI評分以及HE染色觀察其對銀屑病皮損形成的抑制作用。 結(jié)果： 一、銀屑病中Hh通路在mRNA水平上的激活： 1.銀屑病病人皮損區(qū)表皮中GLl3的mRNA表達(dá)與正常人相比顯著降低； 2.銀屑病病人皮損區(qū)全皮中Sufu的nRNA表達(dá)與正常人相比顯著降低； 3.體外培養(yǎng)的銀屑病病人皮損區(qū)表皮角質(zhì)形成細(xì)胞中GLl3的mRNA表達(dá)與正常相比顯著降低,而Shh、GLI1、Sufu的mRNA表達(dá)無顯著差異。 二、銀屑病中Hh通路在蛋白水平上的激活： 1.銀屑病病人外周血血清中Shh、Ihh和Dhh濃度與正常人相比顯著升高； 2.體外培養(yǎng)的銀屑病病人皮損區(qū)角質(zhì)形成細(xì)胞中GLI3、SUFU、PTCH1和PTCH2蛋白表達(dá)與正常相比顯著降低,GLl2略有升高,但差異不顯著,Shh和GLl1無顯著差異； 3.免疫熒光檢測結(jié)果發(fā)現(xiàn),銀屑病病人皮損區(qū)以及正常人表皮各層均有GLI1、GLI2和GLl3的表達(dá),其中GLl3在銀屑病皮損區(qū)表皮和正常人中的分布明顯不同,在銀屑病表皮中GLl3主要位于胞漿中,而正常表皮中GLl3主要位于胞核內(nèi)。體外培養(yǎng)的銀屑病病人皮損區(qū)角質(zhì)形成細(xì)胞中GLl3主要位于胞漿中,而在正常人角質(zhì)形成細(xì)胞中GLl3主要位于胞核內(nèi)。 三、重組人Shh刺激體外培養(yǎng)人表皮角質(zhì)形成細(xì)胞引起的功能變化： 1.重組人Shh劑量依賴性地促進(jìn)角質(zhì)形成細(xì)胞中GLl1的mRNA轉(zhuǎn)錄,但對Sufu及GLl3的mRNA轉(zhuǎn)錄無明顯作用； 2.重組人Shh劑量依賴性地促進(jìn)角質(zhì)形成細(xì)胞的增殖； 3.重組人Shh促進(jìn)角質(zhì)形成細(xì)胞中活性氧族的產(chǎn)生。 四、環(huán)巴胺作用于體外培養(yǎng)人表皮角質(zhì)形成細(xì)胞引起的功能變化： 1.環(huán)巴胺劑量依賴性地抑制角質(zhì)形成細(xì)胞的增殖； 2.環(huán)巴胺劑量依賴性地促進(jìn)角質(zhì)形成細(xì)胞的凋亡,而重組人Shh顯著抑制角質(zhì)形成細(xì)胞的凋亡； 3.環(huán)巴胺導(dǎo)致角質(zhì)形成細(xì)胞G1期的阻滯,而TNF-α能逆轉(zhuǎn)環(huán)巴胺的G1期阻滯作用； 4.體外培養(yǎng)的銀屑病病人皮損區(qū)角質(zhì)形成細(xì)胞衰老程度顯著低于正常人；環(huán)巴胺能顯著促進(jìn)細(xì)胞衰老,丙酸氯貝他索能顯著減少細(xì)胞衰老；TNF-α對細(xì)胞衰老無明顯作用,而環(huán)巴胺能顯著逆轉(zhuǎn)丙酸氯貝他索導(dǎo)致的抗衰老作用； 5.環(huán)巴胺能顯著降低角質(zhì)形成細(xì)胞的活性氧族產(chǎn)生,丙酸氯貝他索亦能顯著降低細(xì)胞活性氧族產(chǎn)生；TNF-a能顯著增加細(xì)胞活性氧族的產(chǎn)生,環(huán)巴胺能顯著抑制TNF-a對活性氧族的促進(jìn)作用； 6.環(huán)巴胺能顯著抑制角質(zhì)形成細(xì)胞的遷移,Shh能顯著促進(jìn)細(xì)胞遷移,丙酸氯倍他索能顯著抑制細(xì)胞遷移,TNF-a對細(xì)胞遷移無明顯作用。 五、體外培養(yǎng)人表皮角質(zhì)形成細(xì)胞Sufu過表達(dá)引起的功能變化： 1.人表皮角質(zhì)形成細(xì)胞中mRNA及蛋白水平均驗證了Sufu過表達(dá)的效率； 2.人表皮角質(zhì)形成細(xì)胞中Sufu過表達(dá)后GLI1和GLI2的蛋白表達(dá)均降低了； 3.銀屑病病人皮損區(qū)表皮角質(zhì)形成細(xì)胞中Sufu過表達(dá)后與分化相關(guān)的蛋白表達(dá)升高了； 4.銀屑病病人皮損區(qū)表皮角質(zhì)形成細(xì)胞中Sufu 過表達(dá)后GLI3由胞漿轉(zhuǎn)移至胞核內(nèi)。 六、環(huán)巴胺減輕了小鼠模型中銀屑病皮損的形成： 1.環(huán)巴胺降低了小鼠模型中銀屑病皮損的PASI評分； 2.HE染色表明環(huán)巴胺減輕了咪喹莫特誘導(dǎo)的小鼠銀屑病皮損的嚴(yán)重程度。 結(jié)論： 一、銀屑病中Hh通路在mRNA及蛋白水平上均被激活了； 二、Shh能促進(jìn)角質(zhì)形成細(xì)胞的增殖,抑制角質(zhì)形成細(xì)胞凋亡,顯著增加角質(zhì)形成細(xì)胞活性氧族的產(chǎn)生,促進(jìn)細(xì)胞遷移,從而證明Hh通路的激活在銀屑病的發(fā)病中的作用； 三、環(huán)巴胺能抑制角質(zhì)形成細(xì)胞的增殖,可能與其促進(jìn)細(xì)胞凋亡及阻滯細(xì)胞周期相關(guān)；環(huán)巴胺能促進(jìn)細(xì)胞衰老,抑制活性氧族的產(chǎn)生并抑制細(xì)胞遷移能力；從而證明了阻斷Hh通路對抑制銀屑病發(fā)病的分子機制； 四、Sufu過表達(dá)能抑制角質(zhì)形成細(xì)胞中Hh通路的激活,進(jìn)而促進(jìn)銀屑病表皮角質(zhì)形成細(xì)胞的分化； 五、環(huán)巴胺能抑制咪喹莫特誘導(dǎo)的小鼠模型中銀屑病皮損的形成,從而為應(yīng)用Hh通路抑制劑臨床治療銀屑病提供了可能。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R758.63

【共引文獻(xiàn)】

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