| 张 磊,刘利敏,刘 婷,张玉明,孙世仁.HE4荧光素酶报告基因载体构建与鉴定及其在肾脏纤维化中的作用[J].,2017,17(31):6006-6009 |
| HE4荧光素酶报告基因载体构建与鉴定及其在肾脏纤维化中的作用 |
| Construction and Identification of Luciferase Reporter Vector for HE4 Promoter and the Role of HE4 in the Renal Fibrosis |
| 投稿时间：2017-05-17 修订日期：2017-06-13 |
| DOI：10.13241/j.cnki.pmb.2017.31.002 |
| 中文关键词: 人附睾蛋白4 荧光素酶 重组质粒 肾脏纤维化 |
| 英文关键词: HE4 Luciferase Recombinant plasmid Renal fibrosis |
| 基金项目:国家自然科学基金项目(81400699) |
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| 中文摘要: |
| 摘要 目的：通过构建HE4荧光素酶报告基因载体以及质粒转染以分析HIF-1α对HE4靶向调控，明确HE4调控肾脏纤维化的分子机制。方法：采用巢式PCR扩增HE4启动子片段，纯化酶切后定向克隆进pGL-3basic报告基因载体，进而与PRL-TK质粒共转染HK2细胞，检测荧光素酶的表达HIF-1α过表达质粒转染HK2细胞，western blot检测HE4分子的表达。HK2细胞转染HE4过表达质粒后检测COL4A1、COL1A1分子的表达。结果：获得HE4启动子荧光素酶报告载体，酶切鉴定结果与初期预测一致，荧光素酶检测结果证实HIF-1α可结合于HE4启动子上，从而启动HE4的表达。HK2细胞转染HIF-1α正义过表达质粒，HE4的mRNA和蛋白水平均显著增加。HK2细胞转染HE4过表达质粒，COL4A1、COL1A1表达水平上调。结论：本研究成功构建HE4启动子荧光素酶报告基因载体，证实HIF-1α为HE4上游靶基因，过表达的HE4可能通过上调COL4A1、COL1A1促进细胞外基质沉积促进肾脏纤维化的发生发展。 |
| 英文摘要: |
| ABSTRACT Objective: To determine the targeted regulating role of HIF-1α on the expression of HE4 based on a constructed lu- ciferase reporter HE4 gene vector and the transfection of pcDNA-HIF-1α, and investigate the mechanisms of HE4 mediated renal fibro- sis. Methods: The sequence of was chemically synthesized and directionally cloned into pGL3-BASIC. The recombinant pGL3-HE4 lu- ciferase reporter vector was identified by PCR, enzyme digestion. Then both pGL3-HE4 and pcDNA3.1-HIF-1α transfected into HK2 cells to detect the activity of luciferase. Western blot qRT-PCR was used to analyzed the relationship between hif-1α and HE4 after HK2 cells transfected with pcDNA3.1-HIF-1α. The mechanisms of HE4 mediated renal fibrosis was analyzed by western blot, qRT-PCR after HK2 cells was transfected with pcDNA3.1-HE4 or added the recombinant protein of HE4. Results: The pGL3-HE4 luciferase reorter vec- tor had been identified by enzyme digestion as expectably. The hif-1α could bind to the region of HE4 promoter to promote the tran- scription of HE4. The expression of HE4 was significantly increased after transfected with pcDNA3.1-HIF-1α in HK2 cells (P＜0.05). After transfected with pcDNA3.1-HE4 in HK2 cells, the expression of COL4A1, COL1A1 was also significantly increased (P＜0.05). Conclusion: The luciferase reporter vector of HE4 was successfully constructed and provided a tool for further studying. HE4 was targeted by HIF-1α and overexpression of HE4 could promote the accumulation of ECM involved in the regulation of renal fibrosis by upregulat- ing the expression of COL4A1 and COL1A1. |
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