CD147与ABCG2在银屑病表皮和HaCaT细胞中的表达及相互作用

发布时间：2018-03-17 04:37

本文选题：银屑病　切入点：角质形成细胞　出处：《中南大学》2012年博士论文　论文类型：学位论文

【摘要】：目的 CD147是一种属于免疫球蛋白超家族的细胞膜表面高糖基化蛋白,其在表皮中的表达对银屑病的发生发展具有促进作用。ABCG2是ATP结合转运蛋白超家族成员之一,主要行使将多种化疗药物泵出细胞外的功能,进而降低细胞对药物的敏感性。近年已有文章报道ABCG2与角质形成细胞的增殖相关,但其在银屑病角质形成细胞中的分布及功能目前尚不清楚。本文旨在探讨CD147与ABCG2在银屑病表皮和HaCaT细胞中的表达、分布及相互调控,为进一步揭示银屑病的致病机制提供理论依据。方法 (1)免疫组化分析CD147与ABCG2在银屑病皮损表皮中的表达及分布； (2)冰冻切片激光共聚焦方法分析CD147与ABCG2在银屑病皮损表皮的位置关系； (3)构建稳定转染CD147的HaCaT细胞株(HaCaT-C)及米托葸醌(mitoxantrone, MX)诱导高表达ABCG2的HaCaT细胞株(HaCaT-A), Western blot方法鉴定稳定株的构建； (4)免疫沉淀(Immunoprecipitation, IP)检测CD147与ABCG2在HaCaT-A中是否形成复合物； (5)利用HEK293FT细胞共转染高表达ABCG2质粒及不同CD147片段质粒,IP分析ABCG2与CD147相互结合位点； (6) Western blot检测MX诱导的ABCG2在HaCaT-C及对照组HaCaT细胞株(HaCaT-N)的表达差异； (7)采用Alarm blue染色方法分析HaCaT-C及HaCaT-N对MX的敏感性(IC50)； (8) Western blot分析TNF-α及对照组PBS诱导HaCaT细胞株中CD147与ABCG2表达水平的差异。结果 (1)银屑病皮损与正常皮肤相比,CD147及ABCG2在银屑病角质形成细胞中的表达全层上调,并且主要表达在细胞膜； (2)激光共聚焦发现银屑病皮损角质形成细胞中CD147与ABCG2存在共定位现象； (3)成功构建高表达CD147的HaCaT-C及高表达ABCG2的HaCaT-A细胞株； (4)IP显示CD147与ABCG2在HaCaT-A细胞中以复合物形式存在； (5)成功建立共同转染ABCG2质粒及不同CD147片段质粒的8组293FT细胞,IP揭示CD147与ABCG2结合位点位于CD147跨膜区； (6)MX诱导HaCaT-C细胞株中ABCG2的表达为对照组HaCaT-N细胞株的1.56±0.16倍。(P0.05)； (7) HaCaT-C细胞株对MX的敏感性显著低于对照组HaCaT-N细胞株,IC50分别为6.83+0.14nM,3.20+0.17nM(P0.05),提示CD147对MX诱导的ABCG2(?)勺蛋白水平及药物转运功能均有调控作用； (8)TNF-α诱导HaCaT细胞中ABCG2及CD147表达水平升高。结论 (1)银屑病皮损角质形成细胞中CD147与ABCG2的表达增高并存在共定位现象； (2)CD147和ABCG2相结合,结合域位于CD147跨膜区； (3)CD147对ABCG2蛋白表达及功能具有调控作用。
[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- 伪 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 卤0.16 times higher than that of the control HaCaT-N cell line, and the expression of ABCG2 was 1.56 卤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- 伪 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.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R758.63

【相似文献】