慢病毒介導(dǎo)的人SLC2A9基因過表達(dá)對人近曲小管上皮細(xì)胞尿酸轉(zhuǎn)運(yùn)功能的影響
發(fā)布時(shí)間：2018-09-05 15:30
【摘要】：背景：腎臟尿酸結(jié)石是泌尿系結(jié)石中常見的類型，僅次于含鈣結(jié)石，約占泌尿系結(jié)石的10%-40%。尿酸結(jié)石的成石機(jī)制主要包括3個(gè)方面：尿pH值降低、尿量減少及高尿酸尿，其中高尿酸尿是誘發(fā)尿酸結(jié)石的關(guān)鍵因素之一。在人體，尿酸排泄主要是通過腎臟進(jìn)行代謝的。經(jīng)腎臟濾過的尿酸有90%被腎臟上皮細(xì)胞重吸收，只有10%的尿酸隨尿液排出體外。因而尿液中尿酸水平的高低取決于腎對尿酸的重吸收程度。近年來的研究表明SLC2A9基因編碼的GLUT9蛋白可以將尿酸吸收至腎小管上皮細(xì)胞胞漿內(nèi)，從而調(diào)控尿酸的重吸收，并且已有多項(xiàng)人群基因篩查研究顯示出SLC2A9基因與尿酸轉(zhuǎn)運(yùn)之間存在很強(qiáng)的相關(guān)性，因此，SLC2A9基因被認(rèn)為是尿酸轉(zhuǎn)運(yùn)的一個(gè)重要的調(diào)控元件。本研究利用高效轉(zhuǎn)染的慢病毒作為載體構(gòu)建SLC2A9過表達(dá)質(zhì)粒，將其轉(zhuǎn)染入永生化人腎臟近曲小管上皮細(xì)胞株HK-2中，驗(yàn)證SLC2A9基因過表達(dá)對尿酸轉(zhuǎn)運(yùn)功能的影響，同時(shí)為后期探討SLC2A9基因遺傳變異對尿酸結(jié)石的影響奠定基礎(chǔ)。 目的：將SLC2A9基因載入慢病毒顆粒，探討SLC2A9基因過表達(dá)慢病毒顆粒感染人近曲小管上皮細(xì)胞HK-2后對其尿酸轉(zhuǎn)運(yùn)功能的影響。 方法：PCR反應(yīng)擴(kuò)增目的基因后載入慢病毒表達(dá)載體中構(gòu)建重組載體pLEX-SLC2A9，使用PCR及測序的方法對其進(jìn)行鑒定，并與輔助包裝質(zhì)粒共感染293T細(xì)胞。慢病毒顆粒感染HK-2細(xì)胞后，用PCR和Western blot檢測SLC2A9基因的過表達(dá)效率，并檢測SLC2A9過表達(dá)對其尿酸轉(zhuǎn)運(yùn)功能的影響。 結(jié)果：PCR及測序結(jié)果表明重組慢病毒載體pLEX-SLC2A9的插入序列完全正確，，重組慢病毒載體感染HK-2細(xì)胞后，細(xì)胞內(nèi)的mRNA及蛋白水平增高，并且可以增強(qiáng)HK-2細(xì)胞對尿酸的轉(zhuǎn)運(yùn)。 結(jié)論：成功構(gòu)建了pLEX-SLC2A9慢病毒表達(dá)載體，并證實(shí)了這一慢病毒轉(zhuǎn)染后SLC2A9基因的過表達(dá)可以顯著增強(qiáng)HK-2細(xì)胞的尿酸轉(zhuǎn)運(yùn)能力，為以后進(jìn)一步研究SLC2A9基因的功能、尿酸轉(zhuǎn)運(yùn)機(jī)制及在尿酸結(jié)石形成過程中的作用奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R691.4
本文編號：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.
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R691.4
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1 孫明霞;SLC2A9基因多態(tài)性與男性原發(fā)性痛風(fēng)患者認(rèn)知功能相關(guān)性研究[D];青島大學(xué);2014年
2 吳文正;慢病毒介導(dǎo)的人SLC2A9基因過表達(dá)對人近曲小管上皮細(xì)胞尿酸轉(zhuǎn)運(yùn)功能的影響[D];廣州醫(yī)科大學(xué);2014年
本文編號：2224691
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