本文選題：銀屑病　切入點：角質形成細胞　出處：《中南大學》2012年博士論文　論文類型：學位論文

【摘要】：目的 CD147是一種屬于免疫球蛋白超家族的細胞膜表面高糖基化蛋白,其在表皮中的表達對銀屑病的發(fā)生發(fā)展具有促進作用。ABCG2是ATP結合轉運蛋白超家族成員之一,主要行使將多種化療藥物泵出細胞外的功能,進而降低細胞對藥物的敏感性。近年已有文章報道ABCG2與角質形成細胞的增殖相關,但其在銀屑病角質形成細胞中的分布及功能目前尚不清楚。本文旨在探討CD147與ABCG2在銀屑病表皮和HaCaT細胞中的表達、分布及相互調控,為進一步揭示銀屑病的致病機制提供理論依據。 方法 (1)免疫組化分析CD147與ABCG2在銀屑病皮損表皮中的表達及分布； (2)冰凍切片激光共聚焦方法分析CD147與ABCG2在銀屑病皮損表皮的位置關系； (3)構建穩(wěn)定轉染CD147的HaCaT細胞株(HaCaT-C)及米托葸醌(mitoxantrone, MX)誘導高表達ABCG2的HaCaT細胞株(HaCaT-A), Western blot方法鑒定穩(wěn)定株的構建； (4)免疫沉淀(Immunoprecipitation, IP)檢測CD147與ABCG2在HaCaT-A中是否形成復合物； (5)利用HEK293FT細胞共轉染高表達ABCG2質粒及不同CD147片段質粒,IP分析ABCG2與CD147相互結合位點； (6) Western blot檢測MX誘導的ABCG2在HaCaT-C及對照組HaCaT細胞株(HaCaT-N)的表達差異； (7)采用Alarm blue染色方法分析HaCaT-C及HaCaT-N對MX的敏感性(IC50)； (8) Western blot分析TNF-α及對照組PBS誘導HaCaT細胞株中CD147與ABCG2表達水平的差異。 結果 (1)銀屑病皮損與正常皮膚相比,CD147及ABCG2在銀屑病角質形成細胞中的表達全層上調,并且主要表達在細胞膜； (2)激光共聚焦發(fā)現(xiàn)銀屑病皮損角質形成細胞中CD147與ABCG2存在共定位現(xiàn)象； (3)成功構建高表達CD147的HaCaT-C及高表達ABCG2的HaCaT-A細胞株； (4)IP顯示CD147與ABCG2在HaCaT-A細胞中以復合物形式存在； (5)成功建立共同轉染ABCG2質粒及不同CD147片段質粒的8組293FT細胞,IP揭示CD147與ABCG2結合位點位于CD147跨膜區(qū)； (6)MX誘導HaCaT-C細胞株中ABCG2的表達為對照組HaCaT-N細胞株的1.56±0.16倍。(P0.05)； (7) HaCaT-C細胞株對MX的敏感性顯著低于對照組HaCaT-N細胞株,IC50分別為6.83+0.14nM,3.20+0.17nM(P0.05),提示CD147對MX誘導的ABCG2(?)勺蛋白水平及藥物轉運功能均有調控作用； (8)TNF-α誘導HaCaT細胞中ABCG2及CD147表達水平升高。 結論 (1)銀屑病皮損角質形成細胞中CD147與ABCG2的表達增高并存在共定位現(xiàn)象； (2)CD147和ABCG2相結合,結合域位于CD147跨膜區(qū)； (3)CD147對ABCG2蛋白表達及功能具有調控作用。
[Abstract]:Purpose. CD147 is a highly glycosylated protein on the membrane surface of immunoglobulin superfamily. Its expression in epidermis promotes the development of psoriasis. ABCG2 is a member of ATP binding transporter superfamily. In recent years, it has been reported that ABCG2 is related to the proliferation of keratinocytes. The distribution and function of CD147 and ABCG2 in psoriatic keratinocytes are not clear. The purpose of this study is to investigate the expression, distribution and mutual regulation of CD147 and ABCG2 in psoriatic epidermis and HaCaT cells. To further reveal the pathogenesis of psoriasis to provide a theoretical basis. Method. The expression and distribution of CD147 and ABCG2 in the epidermis of psoriatic lesions were analyzed by immunohistochemistry. (2) the relationship between CD147 and ABCG2 in the epidermis of psoriasis was analyzed by laser confocal method. Construction of stable HaCaT cell line transfected with CD147 and mitoxantrone induced by mitoxantrone (MXX) induced by high ABCG2 expression in HaCaT cell line HaCaT-An. The construction of stable cell line was identified by Western blot method. (4) Immunoprecipitation (IP) of CD147 and ABCG2 in HaCaT-A; (5) HEK293FT cells were cotransfected with high expression ABCG2 plasmids and different CD147 fragment plasmids IP to analyze the interaction sites between ABCG2 and CD147. Western blot was used to detect the expression of MX-induced ABCG2 in HaCaT-C and control HaCaT cell line HaCaT-N. The sensitivity of HaCaT-C and HaCaT-N to MX was analyzed by Alarm blue staining. Western blot was used to analyze the expression of CD147 and ABCG2 in HaCaT cells induced by TNF- 偽 and PBS. Results. 1) the expression of CD147 and ABCG2 in psoriatic keratinocytes was up-regulated in the whole layer compared with normal skin, and mainly in the cell membrane of psoriatic keratinocytes. (2) CD147 and ABCG2 in keratinocytes of psoriatic lesions were found to be co-localized by laser confocal focusing. (3) HaCaT-C with high expression of CD147 and HaCaT-A cell line with high expression of ABCG2 were successfully constructed. The expression of CD147 and ABCG2 in HaCaT-A cells was found in the form of complex. 5) eight groups of 293FT cells cotransfected with ABCG2 plasmids and different CD147 fragments were successfully constructed to reveal that the binding sites of CD147 and ABCG2 were located in the transmembrane region of CD147. The expression of ABCG2 was 1.56 鹵0.16 times higher than that of the control HaCaT-N cell line, and the expression of ABCG2 was 1.56 鹵0.16 times higher than that of the control HaCaT-N cell line. The sensitivity of HaCaT-C cell line to MX was significantly lower than that of control HaCaT-N cell line (6.83 0.14nMN) 3.20 0.17nMN P0.05A, which suggested that CD147 was sensitive to MX-induced ABCG2? ) Dipper protein level and drug transport function can be regulated. TNF- 偽 induced increased expression of ABCG2 and CD147 in HaCaT cells. Conclusion. 1) the expression of CD147 and ABCG2 in keratinocytes of psoriatic lesions increased and co-located. The binding domain of CD147 and ABCG2 is located in the transmembrane region of CD147. CD147 can regulate the expression and function of ABCG2 protein.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R758.63

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