本文選題：糖皮質(zhì)激素 + 糖皮質(zhì)激素受體��； 參考：《浙江大學(xué)》2012年博士論文

【摘要】：【研究背景】 銀屑病是一種常見的多基因遺傳、多環(huán)境因素誘導(dǎo)的慢性炎癥性增殖性皮膚病。目前認(rèn)為銀屑病不僅僅只是一種皮膚疾病,因?yàn)殂y屑病患者往往伴有糖尿病、高血壓、冠心病、肥胖、抑郁和代謝綜合征等系統(tǒng)性疾病,所以,銀屑病現(xiàn)已成為嚴(yán)重影響廣大人民群眾身心健康的復(fù)雜性疾病之一 眾所周知,外用糖皮質(zhì)激素制劑在治療銀屑病中應(yīng)用最廣泛,是銀屑病治療的一線藥物,其對(duì)銀屑病皮損的清除十分迅速有效。糖皮質(zhì)激素(Glucocorticoid,GC)的作用如此強(qiáng)大,主要是通過其受體——糖皮質(zhì)激素受體(Glucocorticoid receptor, GR)來實(shí)現(xiàn)的。GR屬于核受體家族,廣泛分布于幾乎所有細(xì)胞上。 大量研究表明GR能夠在細(xì)胞核和細(xì)胞漿之間穿梭。在沒有激素的情況下,GR主要存在于細(xì)胞胞漿中,與分子伴侶和其他蛋白組成多蛋白復(fù)合物。GC與GR結(jié)合后誘導(dǎo)多蛋白復(fù)合物發(fā)生構(gòu)象變化,促使GR從復(fù)合物中解離,并轉(zhuǎn)位至細(xì)胞核中。根據(jù)細(xì)胞類型的不同,未轉(zhuǎn)化的GR可以位于胞漿或胞核中。一旦與配體結(jié)合,未轉(zhuǎn)化的GR即通過核轉(zhuǎn)錄因子轉(zhuǎn)位進(jìn)入細(xì)胞核,細(xì)胞核中GR與特異性DNA序列糖皮質(zhì)激素反應(yīng)元件結(jié)合,調(diào)節(jié)靶基因的轉(zhuǎn)錄。 我們的初步研究表明,正常人角質(zhì)形成細(xì)胞中GR位于細(xì)胞核中,而銀屑病角質(zhì)形成細(xì)胞中GR則位于細(xì)胞漿中,即銀屑病角質(zhì)形成細(xì)胞中GR發(fā)生核質(zhì)轉(zhuǎn)運(yùn)障礙。哪些因素導(dǎo)致了GR核質(zhì)轉(zhuǎn)運(yùn)障礙的發(fā)生?GR核質(zhì)轉(zhuǎn)運(yùn)障礙的機(jī)制和意義是什么?這些問題促使我們通過體外細(xì)胞培養(yǎng)和建立銀屑病小鼠模型對(duì)銀屑病皮損角質(zhì)形成細(xì)胞中GR的表達(dá)和功能進(jìn)行深入的綜合分析,探討GR在銀屑病發(fā)生發(fā)展過程中的變化及其在表皮角質(zhì)形成細(xì)胞增殖過程中的作用。 [目的] 明確在銀屑病角質(zhì)形成細(xì)胞中,存在糖皮質(zhì)激素受體的核質(zhì)轉(zhuǎn)運(yùn)障礙；闡明銀屑病角質(zhì)形成細(xì)胞糖皮質(zhì)激素受體核質(zhì)轉(zhuǎn)運(yùn)障礙的原因以及可能的信號(hào)傳導(dǎo)通路的作用；明確導(dǎo)致銀屑病角質(zhì)形成細(xì)胞糖皮質(zhì)激素受體核質(zhì)轉(zhuǎn)運(yùn)障礙的機(jī)制,發(fā)現(xiàn)新的與糖皮質(zhì)激素受體核質(zhì)轉(zhuǎn)運(yùn)有關(guān)的分子及其作用；闡明銀屑病角質(zhì)形成細(xì)胞核質(zhì)轉(zhuǎn)運(yùn)障礙的意義,從而通過促進(jìn)糖皮質(zhì)激素受體的核轉(zhuǎn)位,發(fā)現(xiàn)治療銀屑病的新方法。 [方法] 第一部分：間接免疫熒光法檢測(cè)糖皮質(zhì)激素受體在正常表皮和銀屑病皮損的表達(dá)和定位；分離和培養(yǎng)正常人和銀屑病患者表皮角質(zhì)形成細(xì)胞,以間接免疫熒光法檢測(cè)糖皮質(zhì)激素受體在細(xì)胞中的定位；以Western Blot法比較糖皮質(zhì)激素受體在正常人和銀屑病患者表皮角質(zhì)形成細(xì)胞中表達(dá)；ELISA(?)去檢測(cè)銀屑病患者外周血中促腎上腺皮質(zhì)激素和皮質(zhì)醇的水平；將細(xì)胞因子(如VEGF165和IFN-γ等)與體外培養(yǎng)的角質(zhì)形成細(xì)胞共孵育,間接免疫熒光法檢測(cè)對(duì)糖皮質(zhì)激素受體的核質(zhì)分布影響。 第二部分：分離和培養(yǎng)正常人和銀屑病患者表皮角質(zhì)形成細(xì)胞,與GSK-3、p53和微管蛋白抑制劑共培養(yǎng),間接免疫熒光法檢測(cè)上述因素對(duì)糖皮質(zhì)激素受體核轉(zhuǎn)位的影響；以外源性細(xì)胞因子刺激培養(yǎng)的正常人和銀屑病角質(zhì)形成細(xì)胞,間接免疫熒光法檢測(cè)對(duì)糖皮質(zhì)激素受體的影響。 第三部分：外用咪喹莫特誘導(dǎo)Balb/c小鼠產(chǎn)生銀屑病樣皮損；靜脈注射IL-13和微管抑制劑vincristine,觀察對(duì)咪喹莫特誘導(dǎo)銀屑病樣皮損的影響；對(duì)小鼠標(biāo)本進(jìn)行Masson三染,評(píng)價(jià)IL-13和vincristine對(duì)咪喹莫特誘導(dǎo)小鼠的影響。 [結(jié)果] 一、糖皮質(zhì)激素受體在銀屑病表皮和正常表皮的定位和定量表達(dá)： 1.糖皮質(zhì)激素受體強(qiáng)烈表達(dá)于銀屑病表皮和正常表皮除角質(zhì)層外的各層細(xì)胞； 2.糖皮質(zhì)激素受體在銀屑病表皮和正常表皮細(xì)胞內(nèi)的分布是不同的：糖皮質(zhì)激素受體位于銀屑病表皮細(xì)胞的胞漿中,而在正常表皮細(xì)胞中卻位于胞核內(nèi)； 3.體外培養(yǎng)的銀屑病和正常角質(zhì)形成細(xì)胞內(nèi)糖皮質(zhì)激素受體的分布與組織免疫熒光結(jié)果一致：糖皮質(zhì)激素受體位于銀屑病角質(zhì)形成細(xì)胞的胞漿中,而位于正常角質(zhì)形成細(xì)胞的胞核內(nèi)； 4.銀屑病與正常角質(zhì)形成細(xì)胞之間糖皮質(zhì)激素受體的表達(dá)量無差異； 二、銀屑病與正常角質(zhì)形成細(xì)胞糖皮質(zhì)激素受體核轉(zhuǎn)運(yùn)影響因素：1.銀屑病患者與正常對(duì)照之間外周血促腎上腺皮質(zhì)激素和皮質(zhì)醇水平無差異; 2. VEGF165和IFN-γ可誘導(dǎo)正常角質(zhì)形成細(xì)胞糖皮質(zhì)激素受體核轉(zhuǎn)運(yùn)障礙； 3.地塞米松可逆轉(zhuǎn)VEGF165和IFN-γ誘導(dǎo)的糖皮質(zhì)激素受體核轉(zhuǎn)運(yùn)障礙； 4.內(nèi)皮素-1、肝細(xì)胞生長(zhǎng)因子、轉(zhuǎn)化生長(zhǎng)因子、白介素10、白介素13、白介素17、血小板源性生長(zhǎng)因子(?)(?)A/AB/BB對(duì)正常角質(zhì)形成細(xì)胞糖皮質(zhì)激素受體核質(zhì)轉(zhuǎn)運(yùn)無影響； 5.ATP水平對(duì)正常角質(zhì)形成細(xì)胞糖皮質(zhì)激素受體的核質(zhì)轉(zhuǎn)運(yùn)無影響。 三、銀屑病角質(zhì)形成細(xì)胞糖皮質(zhì)激素受體核質(zhì)轉(zhuǎn)運(yùn)障礙機(jī)制： 1.p53抑制劑可抑制VEGF165和IFN-y誘導(dǎo)的糖皮質(zhì)激素受體核質(zhì)轉(zhuǎn)運(yùn)障礙； 2.微管抑制劑vincristine可抑制糖皮質(zhì)激素受體的核攝��； 3.GSK-3抑制劑不能抑制糖皮質(zhì)激素受體的核攝取； 4.地塞米松促進(jìn)銀屑病表皮角質(zhì)形成細(xì)胞胞漿糖皮質(zhì)激素受體進(jìn)入細(xì)胞核內(nèi)； 5. IL-13、PDGF-AA、PDGF-AB和PDGF-BB可促進(jìn)銀屑病表皮角質(zhì)形成細(xì)胞糖皮質(zhì)激素受體進(jìn)入細(xì)胞核內(nèi),而ET-1、HGF、TGF-β1、IL-10和IL-17不能促進(jìn)糖皮質(zhì)激素受體進(jìn)入細(xì)胞核內(nèi)； 6.突然撤去地塞米松可誘導(dǎo)正常角質(zhì)形成細(xì)胞糖皮質(zhì)激素受體聚集在胞漿中。 四、IL-13和微管抑制劑對(duì)咪喹莫特誘導(dǎo)的小鼠銀屑病模型的影響： 1.咪喹莫特誘導(dǎo)的Balb/c小鼠模型具有銀屑病的主要特征,包括表皮過度增殖肥厚、炎癥細(xì)胞浸潤(rùn)和真皮微血管增生； 2.IL-13可減輕咪喹莫特誘導(dǎo)小鼠的紅斑、鱗屑、表皮厚度和浸潤(rùn)炎癥細(xì)胞的數(shù)目； 3.微管抑制劑可加重咪喹莫特誘導(dǎo)小鼠的紅斑、鱗屑和微血管數(shù)目。 【結(jié)論】 一、銀屑病角質(zhì)形成細(xì)胞糖皮質(zhì)激素受體發(fā)生了核質(zhì)轉(zhuǎn)運(yùn)障礙； 二、銀屑病角質(zhì)形成細(xì)胞糖皮質(zhì)激素受體發(fā)生的核質(zhì)轉(zhuǎn)運(yùn)障礙與體內(nèi)促腎上腺皮質(zhì)激素和皮質(zhì)醇水平無關(guān)； 三、VEGF165和IFRN-γ可誘導(dǎo)正常角質(zhì)形成細(xì)胞糖皮質(zhì)激素受體發(fā)生核質(zhì)轉(zhuǎn)運(yùn)障礙,而地塞米松可逆轉(zhuǎn)此誘導(dǎo)效應(yīng)； 四、角質(zhì)形成細(xì)胞糖皮質(zhì)激素受體發(fā)生核質(zhì)轉(zhuǎn)運(yùn)障礙是非APT依賴的； 五、VEGF165和IFN-γ誘導(dǎo)的糖皮質(zhì)激素受體核質(zhì)轉(zhuǎn)運(yùn)障礙需要p53參與； 六、微管介導(dǎo)糖皮質(zhì)激素受體的核攝取,p53介導(dǎo)糖皮質(zhì)激素受體的核輸出,而GSK-3抑制劑不能抑制糖皮質(zhì)激素受體的核轉(zhuǎn)運(yùn)； 七、地塞米松促進(jìn)銀屑病角質(zhì)形成細(xì)胞糖皮質(zhì)激素受體核轉(zhuǎn)運(yùn)需要微管參與； 八、IL-13、PDGF-AA、PDGF-AB和PDGF-BB可促進(jìn)銀屑病表皮角質(zhì)形成細(xì)胞糖皮質(zhì)激素受體進(jìn)入細(xì)胞核內(nèi)； 九、突然撤去地塞米松可誘導(dǎo)正常角質(zhì)形成細(xì)胞糖皮質(zhì)激素受體聚集于胞漿； 十、IL-13可減輕咪喹莫特誘導(dǎo)小鼠的銀屑病樣損害； 十一、微管抑制劑可加重咪喹莫特誘導(dǎo)小鼠的銀屑病樣損害。
[Abstract]:BACKGROUND OF THE STUDY

psoriasis is a common multi - gene , multi - environment - induced chronic inflammatory proliferative skin disease . It is believed that psoriasis is not just a kind of skin disease , as psoriasis is often accompanied by systemic diseases such as diabetes , hypertension , coronary heart disease , obesity , depression and metabolic syndrome , so psoriasis has now become one of the complex diseases which seriously affect the physical and mental health of the general population

It is well known that topical glucocorticoid formulations are most widely used in the treatment of psoriasis and are a first - line drug for psoriasis treatment , which is very rapid and effective in the removal of psoriasis lesions . The effect of glucocorticoid ( GC ) is so powerful that it is mainly achieved by its receptor _ glucocorticoid receptor ( GR ) . GR belongs to the nuclear receptor family and is widely distributed on almost all cells .

In the absence of a hormone , GR is mainly present in the cytoplasm of the cell , and is composed of a polyprotein complex with the molecular partner and other proteins . After the combination of GC and GR , the protein complex is induced to undergo conformational change , which causes the GR to be dissociated from the complex and transferred to the nucleus . Once the GR is combined with the ligand , the untransformed GR can be located in the nucleus and the nucleus is transferred to the nucleus by the nuclear transcription factor , and the GR in the nucleus is combined with the specific DNA sequence glucocorticoid receptor element to regulate the transcription of the target gene .

Our preliminary study shows that GR in keratinocytes of normal human keratinocytes is located in the nucleus , while GR in keratinocytes is located in the cytoplasm , i.e . , GR occurs in the keratinocytes of psoriasis . What are the mechanisms and significance of GR in the pathogenesis and significance of GR nuclear transport disorders ? These problems have prompted us to study the changes of GR in the course of development of psoriasis and to study the role of GR in the formation of keratinocytes in keratinocytes .

Purpose of the project

It is clear that in the keratinocytes of psoriasis , there is a nuclear transport disorder of the glucocorticoid receptor ;
Clarifying the cause of glucocorticoid receptor nuclear translocation in keratinocytes and the possible role of signal transduction pathways .
The mechanism for the formation of glucocorticoid receptor nuclear transport disorders in keratinocytes has been identified , and it has been found that new molecules associated with the nuclear transport of the glucocorticoid receptor and their role are found ;
To clarify the significance of keratinocytes in the formation of nuclear transport disorders , and to find a new method for the treatment of psoriasis by promoting the nuclear translocation of the glucocorticoid receptor .

Methodology

The first part : indirect immunofluorescence assay was used to detect the expression and localization of glucocorticoid receptor in normal epidermis and psoriasis lesions ;
separating and culturing epidermal keratinocytes of normal persons and psoriasis patients , and detecting the position of the glucocorticoid receptor in the cells by indirect immunofluorescence ;
Western Blot was used to compare the expression of glucocorticoid receptor in keratinocytes of normal and psoriasis patients .
The levels of adrenocorticotropic hormone and cortisol in peripheral blood of patients with psoriasis were detected by ELISA ( ? ) ;
cytokines ( such as VEGF165 and IFN - gamma , etc . ) were incubated with keratinocytes cultured in vitro , and the effects of indirect immunofluorescence on the nuclear distribution of the glucocorticoid receptor were detected .

The second part : separating and culturing epidermal keratinocytes of normal and psoriasis patients , co - culturing with gsk - 3 , p53 and microtube protein inhibitors , and indirect immunofluorescence to detect the effect of the above factors on the nuclear translocation of glucocorticoid receptor ;
The effects of exogenous cytokine stimulation on glucocorticoid receptor in healthy volunteers and psoriasis keratinocytes were detected by indirect immunofluorescence assay .

The third part : imiquimod was used to induce psoriasis - like lesions in Balb / c mice .
The effect of imiquimod on psoriasis - like lesions was observed by intravenous injection of IL - 13 and microtube inhibitor vincristine .
The effects of IL - 13 and vincristine on imiquimod - induced mice were evaluated .

The result is not valid .

I . Localization and quantitative expression of glucocorticoid receptor in the epidermis and normal epidermis of psoriasis :

1 . glucocorticoid receptor is strongly expressed in the epidermis of psoriasis and normal epidermis except the stratum corneum ;


2 . The distribution of glucocorticoid receptor in the epidermis of psoriasis and normal epidermal cells is different : glucocorticoid receptor is located in the cytoplasm of psoriasis epidermal cells , while in normal epidermal cells , it is located in the nucleus ;


3 . The distribution of glucocorticoid receptor in cultured psoriasis and normal keratinocytes was consistent with that of tissue immunofluorescence . The glucocorticoid receptor was located in the cytoplasm of keratinocytes in psoriasis , while in the nucleus of normal keratinocytes .


4 . There was no difference in the expression of glucocorticoid receptor between psoriasis and normal keratinocytes .


2 . The effects of psoriasis and normal keratinocytes on the nuclear transport of glucocorticoid receptor : 1 . There was no difference between the levels of adrenocorticotropic hormone and cortisol in peripheral blood between patients with psoriasis and normal controls ;

2 . VEGF165 and IFN - 緯 can induce normal keratinocytes to form glucocorticoid receptor nuclear transport disorder ;


3 . Dexamethasone can reverse the expression of VEGF165 and IFN - 緯 - induced glucocorticoid receptor nuclear transport .


4 . Endothelin - 1 , hepatocyte growth factor , transforming growth factor , interleukin - 10 , interleukin - 13 , interleukin - 17 , platelet - derived growth factor ( ? ) ( ? ) A / AB / BB have no effect on the nuclear transport of glucocorticoid receptor in normal keratinocytes ;


5 . ATP level has no effect on the nuclear transport of glucocorticoid receptor in normal keratinocytes .

III . Mechanism of glucocorticoid receptor nuclear transport barrier in keratinocytes of psoriasis :

1 . p53 inhibitors inhibit glucocorticoid receptor nuclear transport disorders induced by VEGF165 and IFN - y ;


2 . Microtube inhibitor vincristine can inhibit the nuclear uptake of glucocorticoid receptor ;


3 . The gsk - 3 inhibitor does not inhibit the nuclear uptake of the glucocorticoid receptor ;


4 . Dexamethasone promotes psoriasis epidermal keratinocytes to form a cellular plasma glucocorticoid receptor into the nucleus ;


5 . IL - 13 , PDGF - AA , PDGF - AB and PDGF - BB promote the formation of glucocorticoid receptor in keratinocytes of psoriasis , while ET - 1 , HGF , TGF - 尾1 , IL - 10 and IL - 17 do not promote glucocorticoid receptor into nucleus ;


6 . Dexamethasone can induce normal keratinocytes to form the glucocorticoid receptor in the cytoplasm .

IV . Effect of IL - 13 and microtube inhibitors on imiquimod - induced psoriasis model :

1 . imiquimod - induced Balb / c mouse model has the main characteristics of psoriasis , including hyperproliferation of epidermis , infiltration of inflammatory cells and dermal vascular hyperplasia ;


2 . IL - 13 could alleviate the erythema , scale , epidermis thickness and infiltration of inflammatory cells in mice induced by imiquimod .


3 . Microtube inhibitor can aggravate the erythema , scale and microvessels in mice induced by imiquimod .

Conclusion

1 . The glucocorticoid receptor in the keratinocytes of psoriasis has undergone a nuclear transport disorder .


Second , the nuclear transport disturbance of glucocorticoid receptor in the keratinocytes of psoriasis is not related to the level of adrenocorticotropic hormone and cortisol in vivo ;


( 3 ) VEGF165 and IFRN - 緯 can induce nuclear transport disturbance of glucocorticoid receptor in normal keratinocytes , and dexamethasone can reverse this induced effect ;


4 . Keratinocyte - forming cell glucocorticoid receptor is non - APT dependent .


Five , VEGF165 and IFN - 緯 - induced glucocorticoid receptor nuclear transport disorders require p53 involvement ;


( 6 ) Microtubule - mediated nuclear uptake of glucocorticoid receptor , p53 - mediated nuclear output of glucocorticoid receptor , while gsk - 3 inhibitor does not inhibit the nuclear translocation of glucocorticoid receptor ;


7 . Dexamethasone promotes the formation of glucocorticoid receptor nuclear transport in the keratinocytes of psoriasis , which requires micro - tube participation ;


8 , IL - 13 , PDGF - AA , PDGF - AB and PDGF - BB promote the formation of glucocorticoid receptor in keratinocytes of psoriasis .


9 . sudden withdrawal of dexamethasone can induce normal keratinocytes to form the glucocorticoid receptor to accumulate in the cytoplasm ;


X . IL - 13 can reduce the psoriasis - like damage in mice induced by imiquimod ;


XI . Microtube inhibitor can aggravate the psoriasis - like damage in mice induced by imiquimod .
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R758.63

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相關(guān)期刊論文 前2條

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