【摘要】：目的：銀屑病(psoriasis)是嚴(yán)重影響人類身心健康的自身免疫性皮膚病。導(dǎo)致銀屑病表皮細(xì)胞過度增殖和異常分化的確切原因尚未確定。miRNAs是內(nèi)源性非蛋白編碼單鏈小分子RNAs，主要功能為轉(zhuǎn)錄后水平抑制靶基因的表達(dá)。miRNAs對(duì)其靶基因的調(diào)節(jié)障礙可導(dǎo)致不同疾病的發(fā)生。miR-203具有高度皮膚特異性，在銀屑病皮損中異常表達(dá)，，有報(bào)道推測(cè)p63和SOCS3是其進(jìn)化保守的靶基因。miR-203對(duì)p63和SOCS3的調(diào)節(jié)及其在角質(zhì)形成細(xì)胞增殖、分化和銀屑病發(fā)病機(jī)制中的作用尚不明確。STAT3和Notch信號(hào)通路在銀屑病的發(fā)病中起著關(guān)鍵的作用，而p63和SOCS3是上述信號(hào)通路的2個(gè)重要調(diào)節(jié)子。 本研究旨在通過將miR-203的模擬物和其抑制物轉(zhuǎn)染人永生化角質(zhì)形成細(xì)胞HaCaT細(xì)胞系，確定其對(duì)p63和SOCS3的調(diào)控機(jī)制、明確其與STAT3和Notch信號(hào)通路的關(guān)系及其在銀屑病發(fā)病機(jī)制中的作用，確定它們的調(diào)控方式和相互關(guān)系，為其作為銀屑病新的治療靶標(biāo)提供理論依據(jù)。 方法：用miR-203的模擬物和抑制物(miR-203mimic和miR-203inhibitor)定量轉(zhuǎn)染人角質(zhì)形成細(xì)胞系HaCaT細(xì)胞。通過qRT-PCR、Western blot、流式細(xì)胞術(shù)方法檢測(cè)p63、SOCS3、p-STAT3和Notch1基因及蛋白的表達(dá)水平；并用含TNF-α (20ng/ml)和IL-6(20ng/ml)的培養(yǎng)液處理HaCaT細(xì)胞，并以qRT-PCR法對(duì)miR-203的表達(dá)水平進(jìn)行檢測(cè)，觀察細(xì)胞因子TNF-α和IL-6對(duì)miR-203表達(dá)水平的影響。 結(jié)果：1. qRT-PCR檢測(cè)p63、SOCS3、p-STAT3、Notch1的mRNA表達(dá)水平，其中轉(zhuǎn)染miR-203mimic組與對(duì)照組相比SOCS3、Notch1、p-STAT3的mRNA表達(dá)上調(diào)，Notch1組間差異具有顯著性(P 0.05)，p63mRNA表達(dá)水平無明顯變化；轉(zhuǎn)染miR-203inhibitor組與對(duì)照組相比SOCS3、Notch1、p-STAT3的mRNA表達(dá)下調(diào)，SOCS3和Notch1組間差異有顯著性(P 0.05)，p63mRNA表達(dá)亦無明顯變化。2. Western blot和流式細(xì)胞術(shù)方法檢測(cè)蛋白表達(dá)，轉(zhuǎn)染miR-203mimic的細(xì)胞，p63和SOCS3表達(dá)下降，p-STAT3和Notch1表達(dá)升高;轉(zhuǎn)染miR-203inhibitor的細(xì)胞結(jié)果與之相反。3. HaCaT細(xì)胞經(jīng)TNF-α和IL-6處理后，經(jīng)qRT-PCR方法檢測(cè)miR-203表達(dá)升高，差異具有顯著性(P 0.05)。 結(jié)論：miR-203的過表達(dá)，可以抑制p63和SOCS3表達(dá)，從而使STAT3持續(xù)激活，STAT3的磷酸化水平增加，導(dǎo)致角質(zhì)形成細(xì)胞過度增殖。本研究為探討miR-203對(duì)p63、SOCS3的調(diào)控機(jī)制及其與STAT3信號(hào)轉(zhuǎn)導(dǎo)通路的關(guān)系提供了初步的實(shí)驗(yàn)基礎(chǔ)，有助于了解miR-203在銀屑病發(fā)病機(jī)制中的作用。
[Abstract]:Objective: psoriatic (psoriasis) is an autoimmune dermatosis that seriously affects human physical and mental health. The exact cause of excessive proliferation and abnormal differentiation of psoriatic epidermal cells has not been determined. MiRNAs is an endogenous non-protein encoded single-stranded small molecule RNAs,. The main function of miR-203 is to inhibit the expression of target gene at post-transcriptional level. The regulation of target gene by miRNAs may lead to the occurrence of different diseases. MiR-203 has high skin specificity and abnormal expression in psoriatic lesions. It has been reported that p63 and SOCS3 are the conserved target genes of p63 and SOCS3. MiR-203 regulates p63 and SOCS3 and proliferates in keratinocytes. The role of differentiation and psoriasis pathogenesis is unclear. STAT3 and Notch signaling pathway play a key role in psoriasis pathogenesis, while p63 and SOCS3 are two important regulators of these signaling pathways. The aim of this study was to determine the regulatory mechanism of miR-203 on p63 and SOCS3 by transfection of immortalized human keratinocytes HaCaT cell line with miR-203 mimic and its inhibitor. To clarify its relationship with STAT3 and Notch signaling pathway and its role in the pathogenesis of psoriasis, to determine their regulatory mode and mutual relationship, to provide a theoretical basis for its new therapeutic target for psoriasis. Methods: human keratinocytes HaCaT cells were quantitatively transfected with miR-203 mimics and inhibitors (miR-203mimic and miR-203inhibitor). The expression of p-STAT3 and Notch1 genes and proteins were detected by qRT-PCR,Western blot, flow cytometry. HaCaT cells were treated with TNF- 偽 (20ng/ml) and IL-6 (20ng/ml), and the expression of miR-203 was detected by qRT-PCR method. The effects of cytokines TNF- 偽 and IL-6 on miR-203 expression were observed. Results: 1. QRT-PCR was used to detect the mRNA expression of p63- SOCS3pSTAT3pSTAT3ntch1. The expression of SOCS3,Notch1,p-STAT3 mRNA was up-regulated in the transfected miR-203mimic group compared with the control group. There was significant difference between the Notch1 group and the Notch1 group (P 0.05), but the p63mRNA expression level did not change significantly. Compared with the control group, the mRNA expression of SOCS3,Notch1,p-STAT3 in miR-203inhibitor transfected group was down-regulated, the difference between SOCS3 and Notch1 group was significant (P 0.05), and the expression of p63mRNA was not changed significantly. 2. Western blot and flow cytometry were used to detect the protein expression. The expression of p63 and SOCS3 decreased and the expression of p-STAT3 and Notch1 increased in the cells transfected with miR-203mimic, whereas the expression of p-STAT3 and Notch1 was increased in the transfected miR-203inhibitor cells. After HaCaT cells were treated with TNF- 偽 and IL-6, the expression of miR-203 was detected by qRT-PCR method, and the difference was significant (P 0.05). Conclusion: overexpression of miR-203 can inhibit the expression of p63 and SOCS3, which leads to the sustained activation of STAT3 and the increase of phosphorylation of STAT3, resulting in excessive proliferation of keratinocytes. This study provides a preliminary experimental basis for the study of the regulatory mechanism of miR-203 on p63 SOCS3 and its relationship with STAT3 signal transduction pathway, and helps to understand the role of miR-203 in the pathogenesis of psoriasis.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R758.63

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 湯仲明;;2011年美國(guó)FDA批準(zhǔn)藥物簡(jiǎn)介[J];國(guó)際藥學(xué)研究雜志;2012年01期

2 楊建營(yíng);徐翔峰;向珍蛹;;NF-κB的研究進(jìn)展[J];淮海醫(yī)藥;2011年01期

3 萬永山;張開明;;角質(zhì)形成細(xì)胞在銀屑病發(fā)病中的再認(rèn)識(shí)[J];中國(guó)麻風(fēng)皮膚病雜志;2008年01期

4 邵長(zhǎng)庚;我國(guó)銀屑病的流行和防治現(xiàn)狀[J];中華皮膚科雜志;1996年02期

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