银屑病表皮角质形成细胞糖皮质激素受体核质转运障碍的机制与功能研究

发布时间：2018-05-27 14:46

本文选题：糖皮质激素 + 糖皮质激素受体　；参考：《浙江大学》2012年博士论文

【摘要】：【研究背景】银屑病是一种常见的多基因遗传、多环境因素诱导的慢性炎症性增殖性皮肤病。目前认为银屑病不仅仅只是一种皮肤疾病,因为银屑病患者往往伴有糖尿病、高血压、冠心病、肥胖、抑郁和代谢综合征等系统性疾病,所以,银屑病现已成为严重影响广大人民群众身心健康的复杂性疾病之一众所周知,外用糖皮质激素制剂在治疗银屑病中应用最广泛,是银屑病治疗的一线药物,其对银屑病皮损的清除十分迅速有效。糖皮质激素(Glucocorticoid,GC)的作用如此强大,主要是通过其受体——糖皮质激素受体(Glucocorticoid receptor, GR)来实现的。GR属于核受体家族,广泛分布于几乎所有细胞上。大量研究表明GR能够在细胞核和细胞浆之间穿梭。在没有激素的情况下,GR主要存在于细胞胞浆中,与分子伴侣和其他蛋白组成多蛋白复合物。GC与GR结合后诱导多蛋白复合物发生构象变化,促使GR从复合物中解离,并转位至细胞核中。根据细胞类型的不同,未转化的GR可以位于胞浆或胞核中。一旦与配体结合,未转化的GR即通过核转录因子转位进入细胞核,细胞核中GR与特异性DNA序列糖皮质激素反应元件结合,调节靶基因的转录。我们的初步研究表明,正常人角质形成细胞中GR位于细胞核中,而银屑病角质形成细胞中GR则位于细胞浆中,即银屑病角质形成细胞中GR发生核质转运障碍。哪些因素导致了GR核质转运障碍的发生?GR核质转运障碍的机制和意义是什么?这些问题促使我们通过体外细胞培养和建立银屑病小鼠模型对银屑病皮损角质形成细胞中GR的表达和功能进行深入的综合分析,探讨GR在银屑病发生发展过程中的变化及其在表皮角质形成细胞增殖过程中的作用。 [目的] 明确在银屑病角质形成细胞中,存在糖皮质激素受体的核质转运障碍；阐明银屑病角质形成细胞糖皮质激素受体核质转运障碍的原因以及可能的信号传导通路的作用；明确导致银屑病角质形成细胞糖皮质激素受体核质转运障碍的机制,发现新的与糖皮质激素受体核质转运有关的分子及其作用；阐明银屑病角质形成细胞核质转运障碍的意义,从而通过促进糖皮质激素受体的核转位,发现治疗银屑病的新方法。 [方法] 第一部分：间接免疫荧光法检测糖皮质激素受体在正常表皮和银屑病皮损的表达和定位；分离和培养正常人和银屑病患者表皮角质形成细胞,以间接免疫荧光法检测糖皮质激素受体在细胞中的定位；以Western Blot法比较糖皮质激素受体在正常人和银屑病患者表皮角质形成细胞中表达；ELISA(?)去检测银屑病患者外周血中促肾上腺皮质激素和皮质醇的水平；将细胞因子(如VEGF165和IFN-γ等)与体外培养的角质形成细胞共孵育,间接免疫荧光法检测对糖皮质激素受体的核质分布影响。第二部分：分离和培养正常人和银屑病患者表皮角质形成细胞,与GSK-3、p53和微管蛋白抑制剂共培养,间接免疫荧光法检测上述因素对糖皮质激素受体核转位的影响；以外源性细胞因子刺激培养的正常人和银屑病角质形成细胞,间接免疫荧光法检测对糖皮质激素受体的影响。第三部分：外用咪喹莫特诱导Balb/c小鼠产生银屑病样皮损；静脉注射IL-13和微管抑制剂vincristine,观察对咪喹莫特诱导银屑病样皮损的影响；对小鼠标本进行Masson三染,评价IL-13和vincristine对咪喹莫特诱导小鼠的影响。 [结果] 一、糖皮质激素受体在银屑病表皮和正常表皮的定位和定量表达： 1.糖皮质激素受体强烈表达于银屑病表皮和正常表皮除角质层外的各层细胞； 2.糖皮质激素受体在银屑病表皮和正常表皮细胞内的分布是不同的：糖皮质激素受体位于银屑病表皮细胞的胞浆中,而在正常表皮细胞中却位于胞核内； 3.体外培养的银屑病和正常角质形成细胞内糖皮质激素受体的分布与组织免疫荧光结果一致：糖皮质激素受体位于银屑病角质形成细胞的胞浆中,而位于正常角质形成细胞的胞核内； 4.银屑病与正常角质形成细胞之间糖皮质激素受体的表达量无差异；二、银屑病与正常角质形成细胞糖皮质激素受体核转运影响因素：1.银屑病患者与正常对照之间外周血促肾上腺皮质激素和皮质醇水平无差异; 2. VEGF165和IFN-γ可诱导正常角质形成细胞糖皮质激素受体核转运障碍； 3.地塞米松可逆转VEGF165和IFN-γ诱导的糖皮质激素受体核转运障碍； 4.内皮素-1、肝细胞生长因子、转化生长因子、白介素10、白介素13、白介素17、血小板源性生长因子(?)(?)A/AB/BB对正常角质形成细胞糖皮质激素受体核质转运无影响； 5.ATP水平对正常角质形成细胞糖皮质激素受体的核质转运无影响。三、银屑病角质形成细胞糖皮质激素受体核质转运障碍机制： 1.p53抑制剂可抑制VEGF165和IFN-y诱导的糖皮质激素受体核质转运障碍； 2.微管抑制剂vincristine可抑制糖皮质激素受体的核摄取； 3.GSK-3抑制剂不能抑制糖皮质激素受体的核摄取； 4.地塞米松促进银屑病表皮角质形成细胞胞浆糖皮质激素受体进入细胞核内； 5. IL-13、PDGF-AA、PDGF-AB和PDGF-BB可促进银屑病表皮角质形成细胞糖皮质激素受体进入细胞核内,而ET-1、HGF、TGF-β1、IL-10和IL-17不能促进糖皮质激素受体进入细胞核内； 6.突然撤去地塞米松可诱导正常角质形成细胞糖皮质激素受体聚集在胞浆中。四、IL-13和微管抑制剂对咪喹莫特诱导的小鼠银屑病模型的影响： 1.咪喹莫特诱导的Balb/c小鼠模型具有银屑病的主要特征,包括表皮过度增殖肥厚、炎症细胞浸润和真皮微血管增生； 2.IL-13可减轻咪喹莫特诱导小鼠的红斑、鳞屑、表皮厚度和浸润炎症细胞的数目； 3.微管抑制剂可加重咪喹莫特诱导小鼠的红斑、鳞屑和微血管数目。【结论】一、银屑病角质形成细胞糖皮质激素受体发生了核质转运障碍；二、银屑病角质形成细胞糖皮质激素受体发生的核质转运障碍与体内促肾上腺皮质激素和皮质醇水平无关；三、VEGF165和IFRN-γ可诱导正常角质形成细胞糖皮质激素受体发生核质转运障碍,而地塞米松可逆转此诱导效应；四、角质形成细胞糖皮质激素受体发生核质转运障碍是非APT依赖的；五、VEGF165和IFN-γ诱导的糖皮质激素受体核质转运障碍需要p53参与；六、微管介导糖皮质激素受体的核摄取,p53介导糖皮质激素受体的核输出,而GSK-3抑制剂不能抑制糖皮质激素受体的核转运；七、地塞米松促进银屑病角质形成细胞糖皮质激素受体核转运需要微管参与；八、IL-13、PDGF-AA、PDGF-AB和PDGF-BB可促进银屑病表皮角质形成细胞糖皮质激素受体进入细胞核内；九、突然撤去地塞米松可诱导正常角质形成细胞糖皮质激素受体聚集于胞浆；十、IL-13可减轻咪喹莫特诱导小鼠的银屑病样损害；十一、微管抑制剂可加重咪喹莫特诱导小鼠的银屑病样损害。
[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 - 纬 can induce normal keratinocytes to form glucocorticoid receptor nuclear transport disorder ;

3 . Dexamethasone can reverse the expression of VEGF165 and IFN - 纬 - 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 - 纬 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 - 纬 - 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 .
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R758.63

【参考文献】