慢病毒介导的人SLC2A9基因过表达对人近曲小管上皮细胞尿酸转运功能的影响
发布时间：2018-09-05 15:30
【摘要】：背景：肾脏尿酸结石是泌尿系结石中常见的类型，仅次于含钙结石，约占泌尿系结石的10%-40%。尿酸结石的成石机制主要包括3个方面：尿pH值降低、尿量减少及高尿酸尿，其中高尿酸尿是诱发尿酸结石的关键因素之一。在人体，尿酸排泄主要是通过肾脏进行代谢的。经肾脏滤过的尿酸有90%被肾脏上皮细胞重吸收，只有10%的尿酸随尿液排出体外。因而尿液中尿酸水平的高低取决于肾对尿酸的重吸收程度。近年来的研究表明SLC2A9基因编码的GLUT9蛋白可以将尿酸吸收至肾小管上皮细胞胞浆内，从而调控尿酸的重吸收，并且已有多项人群基因筛查研究显示出SLC2A9基因与尿酸转运之间存在很强的相关性，因此，SLC2A9基因被认为是尿酸转运的一个重要的调控元件。本研究利用高效转染的慢病毒作为载体构建SLC2A9过表达质粒，将其转染入永生化人肾脏近曲小管上皮细胞株HK-2中，验证SLC2A9基因过表达对尿酸转运功能的影响，同时为后期探讨SLC2A9基因遗传变异对尿酸结石的影响奠定基础。 目的：将SLC2A9基因载入慢病毒颗粒，探讨SLC2A9基因过表达慢病毒颗粒感染人近曲小管上皮细胞HK-2后对其尿酸转运功能的影响。 方法：PCR反应扩增目的基因后载入慢病毒表达载体中构建重组载体pLEX-SLC2A9，使用PCR及测序的方法对其进行鉴定，并与辅助包装质粒共感染293T细胞。慢病毒颗粒感染HK-2细胞后，用PCR和Western blot检测SLC2A9基因的过表达效率，并检测SLC2A9过表达对其尿酸转运功能的影响。 结果：PCR及测序结果表明重组慢病毒载体pLEX-SLC2A9的插入序列完全正确，，重组慢病毒载体感染HK-2细胞后，细胞内的mRNA及蛋白水平增高，并且可以增强HK-2细胞对尿酸的转运。 结论：成功构建了pLEX-SLC2A9慢病毒表达载体，并证实了这一慢病毒转染后SLC2A9基因的过表达可以显著增强HK-2细胞的尿酸转运能力，为以后进一步研究SLC2A9基因的功能、尿酸转运机制及在尿酸结石形成过程中的作用奠定了基础。
[Abstract]:Background: renal uric acid calculus is a common type of urinary calculi, second only to calcium stones, accounting for about 10-40 of urinary calculi. The lithogenesis mechanism of uric acid stone mainly includes three aspects: the decrease of urinary pH, the decrease of urine volume and the high uric acid urine, among which high uric acid urine is one of the key factors to induce uric acid stone. In humans, uric acid excretion is primarily metabolized through the kidneys. 90% of the uric acid filtered through the kidney was reabsorbed by the renal epithelial cells, and only 10% of the uric acid was excreted from the urine. The level of uric acid in urine therefore depends on the degree of reabsorption of uric acid by the kidney. Recent studies have shown that the GLUT9 protein encoded by SLC2A9 gene can absorb uric acid into the cytoplasm of renal tubular epithelial cells, thereby regulating the reabsorption of uric acid. Many studies on gene screening have shown that there is a strong correlation between SLC2A9 gene and uric acid transport. Therefore, SLC2A9 gene is considered to be an important regulatory element of uric acid transport. In this study, SLC2A9 overexpression plasmid was constructed by using highly transfected lentivirus as vector, and transfected into HK-2 cell line of immortalized human kidney proximal convoluted tubule to verify the effect of SLC2A9 gene overexpression on uric acid transport function. At the same time, it will lay a foundation for studying the effect of genetic variation of SLC2A9 gene on uric acid stone. Aim: to investigate the effect of lentivirus particles overexpression of SLC2A9 gene on uric acid transport in human proximal tubule epithelial cells (HK-2). Methods the target gene was amplified by 1: PCR and inserted into the lentivirus expression vector to construct the recombinant vector pLEX-SLC2A9,. The recombinant vector was identified by PCR and sequenced and co-infected with the auxiliary packaging plasmid 293T cells. After lentivirus particles were infected with HK-2 cells, PCR and Western blot were used to detect the overexpression efficiency of SLC2A9 gene and the effect of SLC2A9 overexpression on the uric acid transport function. Results the insertion sequence of the recombinant lentivirus vector pLEX-SLC2A9 was completely correct. After the recombinant lentivirus vector was infected with HK-2 cells, the mRNA and protein levels in the cells increased, and the transport of uric acid in HK-2 cells was enhanced. Conclusion: pLEX-SLC2A9 lentivirus expression vector was successfully constructed, and it was proved that overexpression of SLC2A9 gene after lentivirus transfection could significantly enhance the uric acid transport ability of HK-2 cells, and further study the function of SLC2A9 gene in the future. The mechanism of uric acid transport and its role in the formation of uric acid stones lay the foundation.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R691.4
本文编号：2224691
[Abstract]:Background: renal uric acid calculus is a common type of urinary calculi, second only to calcium stones, accounting for about 10-40 of urinary calculi. The lithogenesis mechanism of uric acid stone mainly includes three aspects: the decrease of urinary pH, the decrease of urine volume and the high uric acid urine, among which high uric acid urine is one of the key factors to induce uric acid stone. In humans, uric acid excretion is primarily metabolized through the kidneys. 90% of the uric acid filtered through the kidney was reabsorbed by the renal epithelial cells, and only 10% of the uric acid was excreted from the urine. The level of uric acid in urine therefore depends on the degree of reabsorption of uric acid by the kidney. Recent studies have shown that the GLUT9 protein encoded by SLC2A9 gene can absorb uric acid into the cytoplasm of renal tubular epithelial cells, thereby regulating the reabsorption of uric acid. Many studies on gene screening have shown that there is a strong correlation between SLC2A9 gene and uric acid transport. Therefore, SLC2A9 gene is considered to be an important regulatory element of uric acid transport. In this study, SLC2A9 overexpression plasmid was constructed by using highly transfected lentivirus as vector, and transfected into HK-2 cell line of immortalized human kidney proximal convoluted tubule to verify the effect of SLC2A9 gene overexpression on uric acid transport function. At the same time, it will lay a foundation for studying the effect of genetic variation of SLC2A9 gene on uric acid stone. Aim: to investigate the effect of lentivirus particles overexpression of SLC2A9 gene on uric acid transport in human proximal tubule epithelial cells (HK-2). Methods the target gene was amplified by 1: PCR and inserted into the lentivirus expression vector to construct the recombinant vector pLEX-SLC2A9,. The recombinant vector was identified by PCR and sequenced and co-infected with the auxiliary packaging plasmid 293T cells. After lentivirus particles were infected with HK-2 cells, PCR and Western blot were used to detect the overexpression efficiency of SLC2A9 gene and the effect of SLC2A9 overexpression on the uric acid transport function. Results the insertion sequence of the recombinant lentivirus vector pLEX-SLC2A9 was completely correct. After the recombinant lentivirus vector was infected with HK-2 cells, the mRNA and protein levels in the cells increased, and the transport of uric acid in HK-2 cells was enhanced. Conclusion: pLEX-SLC2A9 lentivirus expression vector was successfully constructed, and it was proved that overexpression of SLC2A9 gene after lentivirus transfection could significantly enhance the uric acid transport ability of HK-2 cells, and further study the function of SLC2A9 gene in the future. The mechanism of uric acid transport and its role in the formation of uric acid stones lay the foundation.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R691.4
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1 孙明霞;SLC2A9基因多态性与男性原发性痛风患者认知功能相关性研究[D];青岛大学;2014年
2 吴文正;慢病毒介导的人SLC2A9基因过表达对人近曲小管上皮细胞尿酸转运功能的影响[D];广州医科大学;2014年
本文编号：2224691
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