saRNA介导的TRPV5表达对HK2细胞钙离子转运的作用研究
发布时间：2019-06-05 13:50
【摘要】：泌尿系结石的高复发率凸显了预防其复发在避免患者反复接受手术治疗中的重要意义。而高钙尿是泌尿系统含钙结石发生的重要危险因素。高选择性钙离子通道TRPV5主要分布于肾远端小管上皮细胞顶膜侧，其介导的钙离子由顶膜侧向细胞内的转运是肾脏钙离子主动重吸收的限速步骤。TRPV5基因敲除小鼠表现出明显的高钙尿。我们的前期研究显示，TRPV5在草酸钙结石模型大鼠和遗传性高钙尿结石大鼠肾脏中显著低表达。我们进一步的研究显示泌尿系含钙结石患者肾组织TRPV5表达较对照组显著降低。因而，TRPV5成为预防肾含钙结石的理想标靶基因。上调TRPV5表达水平有助于增加尿钙重吸收，从而降低尿钙水平，为泌尿系含钙结石的预防提供新的手段。近年，人工合成的小双链RNA（dsRNA）及微小RNA（microRNA）均被证明可以有效上调基因表达水平，而非沉默靶基因表达。这种新发现的机制被称为“RNA激活”（RNAa）。在本研究中，靶向基因启动子区域的dsRNA显著激活了TRPV5钙离子通道表达，并上调了细胞钙离子转运水平。 研究目的 本研究利用靶向基因启动子区域的小激活RNA激活TRPV5表达，研究其对细胞钙离子转运的影响。同时探究小激活RNA对二氢睾酮诱导的细胞钙离子转运抑制效应的恢复作用。从而探讨靶向TRPV5基因启动子的小激活RNA在泌尿系含钙结石预防中的应用价值。 研究内容和方法 1.设计合成3个靶向TRPV5启动子区域的dsRNA序列，利用脂质体转染法将之转染入HK2细胞中。使用Western Blot、PCR和细胞免疫荧光的方法检测TRPV5表达变化，从而筛选出可有效上调TRPV5表达的dsRNA。并使用细胞免疫荧光检测筛选出的dsRNA对TRPV5膜丰度的影响。 2.将筛选出的有效的dsRNA以不同时间和浓度作用于HK2细胞。以检测其时间和浓度依从性。 3.将筛选出的dsRNA分别转染入PC3、HEK293和HK2细胞中，以检测其细胞特异性。同时检测上述3种细胞系TRPV5基础表达水平。 4.在不同浓度和不同时间下使用二氢睾酮处理HK2细胞，并使用Western Blot检测其对TRPV5表达的影响。 5.使用Fura-2钙离子荧光探针检测HK2细胞小激活RNA、二氢睾酮以及二者共处理下细胞钙离子转运水平变化。 结果 1.在设计合成的3个靶向TRPV5基因启动子的dsRNA中ds-2939有效上调了HK2细胞TRPV5的mRNA水平和蛋白质表达水平。免疫荧光结果显示ds-2939显著增加了TRPV5在HK2细胞的膜丰度。 2. ds-2939对TRPV5表达水平的激活作用在1天至6天和5nM至80nM具有时间和浓度依从性。 3. ds-2939在TRPV5基础表达量较低的PC3和HK2细胞中激活效果明显。在靶基因基础表达量较高的HEK293细胞中未能观察到激活效果。 4.二氢睾酮在12至48小时和5nM至20nM依从时间和浓度对HK2细胞TRPV5表达水平产生抑制作用。 5.与对照组相比，ds-2939可有效增加HK2细胞钙离子转运水平。二氢睾酮可抑制HK2细胞钙离子转运水平。ds-2939可部分逆转二氢睾酮对HK2细胞钙离子转运水平的抑制效应。 结论 靶向基因启动子区域的小激活RNA可以用于上调TRPV5表达水平，并可激活细胞钙离子转运。同时可部分逆转二氢睾酮对HK2细胞钙离子转运水平的抑制效应。该技术有望成为预防泌尿系含钙结石发生及复发的新方法。
[Abstract]:The high recurrence rate of the urinary system of the urinary system highlights the importance of the prevention of the recurrence of the urinary system in the prevention of repeated surgical treatment of the patient. And high-calcium-urine is an important risk factor for the occurrence of calcium-containing stones in the urinary system. The high-selective calcium ion channel TRPV5 is mainly distributed on the apical membrane side of the renal tubular epithelial cell, and the mediated calcium ion is the speed-limiting step of the active reabsorption of the calcium ions in the kidney by the transport of the calcium ions in the lateral cells of the apical membrane. The TRPV5 knockout mice showed clear high-calcium-urine. Our previous studies have shown that the TRPV5 is significantly lower in the kidney of the calcium oxalate stone model rats and in the hereditary high-calcium urolithiasis rats. Our further study showed that the expression of TRPV5 in the renal tissue of the urinary system with calcium-containing stone was significantly lower than that in the control group. Thus, the TRPV5 is an ideal target gene for the prevention of renal calcium-containing stones. The up-regulation of the expression level of the TRPV5 can help to increase the calcium absorption of the urine, thereby reducing the level of the urine calcium and providing a new means for the prevention of the calcium-containing stone in the urinary system. In recent years, the synthetic small double-stranded RNA (dsRNA) and microRNA (microRNA) have been proved to be able to increase the gene expression level effectively, while the non-silent target gene is expressed. This newly discovered mechanism is referred to as "RNA activation" (RNAa). In this study, the dsRNA of the target gene promoter region significantly activated the expression of the TRPV5 calcium ion channel and up-regulated the cell calcium ion transport level. study Objective The purpose of this study was to study the expression of TRPV5 by using small activated RNA in the promoter region of the target gene, and to study the transport of calcium in the cells. The effects of small activated RNA on the inhibitory effect of dihydrotestosterone on the transport of calcium and calcium ions were also investigated. To study the effect of small activated RNA targeting the TRPV5 gene promoter in the prevention of urinary calcium-containing calculus. use value Contents and methods 1. Design of a dsRNA sequence for 3 targeted TRPV5 promoter regions, transfected with a liposome transfection method, The expression of TRPV5 was detected by Western Blot, PCR and cell immunofluorescence, so that the TRPV5 table could be effectively up-regulated The dsRNA of the invention, and the TRPV is detected by using the cell immunofluorescence to detect the screened dsRNA. 5. Effect of membrane abundance.2. The effective dsRNA screened for different time and concentration to act on the HK2 cells to detect 3. Transfection of the screened dsRNA into PC3, HEK293 and HK2 cells, respectively. so as to detect the cell specificity of the cell line, and simultaneously detect the three cell lines TRPV5 basal expression level.4. HK2 cells were treated with dihydrotestosterone at different concentrations and at different times, and Western Blot test was used 5. Using the Fura-2 calcium ion fluorescence probe to detect the small activated RNA, dihydrotestosterone, and both of the HK2 cells co-existence Results 1. The ds-2939 of the three-target TRPV5 gene promoter which was designed and synthesized effectively up-regulated the HK2 cells T. MRNA level and protein expression level of RPV5. The increase of the membrane abundance of the TRPV5 in the HK2 cells. The activation of the expression level of the TRPV5 on the TRPV5 is from 1 day to Time and concentration compliance for 6 days and 5 nM to 80 nM. The effect of activation in PC3 and HK2 cells with low level of expression of the target gene is obvious. The effect of activation was not observed in HEK293 cells with a high content.4. The dihydrotestosterone was in compliance with 12 to 48 hours and from 5 nM to 20 nM The expression level of TRPV5 in HK2 cells was inhibited by the concentration and concentration of 5.5. The ds-2939 can effectively increase the HK2 cells compared to ds-2939 calcium ion transport level. Dihydrotestosterone can inhibit the calcium ion transport of HK2 cells. ds-2939 Koran The Inhibitory Effect of Dihydrotestosterone on the Transport of Calcium in HK2 Cells. The living RNA can be used to up-regulate the expression level of TRPV5, and can activate the calcium ion of the cell. Transport. At the same time, the calcium ion transport level of the HK2 cells can be partially reversed
【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】：R692.4
本文编号：2493574
[Abstract]:The high recurrence rate of the urinary system of the urinary system highlights the importance of the prevention of the recurrence of the urinary system in the prevention of repeated surgical treatment of the patient. And high-calcium-urine is an important risk factor for the occurrence of calcium-containing stones in the urinary system. The high-selective calcium ion channel TRPV5 is mainly distributed on the apical membrane side of the renal tubular epithelial cell, and the mediated calcium ion is the speed-limiting step of the active reabsorption of the calcium ions in the kidney by the transport of the calcium ions in the lateral cells of the apical membrane. The TRPV5 knockout mice showed clear high-calcium-urine. Our previous studies have shown that the TRPV5 is significantly lower in the kidney of the calcium oxalate stone model rats and in the hereditary high-calcium urolithiasis rats. Our further study showed that the expression of TRPV5 in the renal tissue of the urinary system with calcium-containing stone was significantly lower than that in the control group. Thus, the TRPV5 is an ideal target gene for the prevention of renal calcium-containing stones. The up-regulation of the expression level of the TRPV5 can help to increase the calcium absorption of the urine, thereby reducing the level of the urine calcium and providing a new means for the prevention of the calcium-containing stone in the urinary system. In recent years, the synthetic small double-stranded RNA (dsRNA) and microRNA (microRNA) have been proved to be able to increase the gene expression level effectively, while the non-silent target gene is expressed. This newly discovered mechanism is referred to as "RNA activation" (RNAa). In this study, the dsRNA of the target gene promoter region significantly activated the expression of the TRPV5 calcium ion channel and up-regulated the cell calcium ion transport level. study Objective The purpose of this study was to study the expression of TRPV5 by using small activated RNA in the promoter region of the target gene, and to study the transport of calcium in the cells. The effects of small activated RNA on the inhibitory effect of dihydrotestosterone on the transport of calcium and calcium ions were also investigated. To study the effect of small activated RNA targeting the TRPV5 gene promoter in the prevention of urinary calcium-containing calculus. use value Contents and methods 1. Design of a dsRNA sequence for 3 targeted TRPV5 promoter regions, transfected with a liposome transfection method, The expression of TRPV5 was detected by Western Blot, PCR and cell immunofluorescence, so that the TRPV5 table could be effectively up-regulated The dsRNA of the invention, and the TRPV is detected by using the cell immunofluorescence to detect the screened dsRNA. 5. Effect of membrane abundance.2. The effective dsRNA screened for different time and concentration to act on the HK2 cells to detect 3. Transfection of the screened dsRNA into PC3, HEK293 and HK2 cells, respectively. so as to detect the cell specificity of the cell line, and simultaneously detect the three cell lines TRPV5 basal expression level.4. HK2 cells were treated with dihydrotestosterone at different concentrations and at different times, and Western Blot test was used 5. Using the Fura-2 calcium ion fluorescence probe to detect the small activated RNA, dihydrotestosterone, and both of the HK2 cells co-existence Results 1. The ds-2939 of the three-target TRPV5 gene promoter which was designed and synthesized effectively up-regulated the HK2 cells T. MRNA level and protein expression level of RPV5. The increase of the membrane abundance of the TRPV5 in the HK2 cells. The activation of the expression level of the TRPV5 on the TRPV5 is from 1 day to Time and concentration compliance for 6 days and 5 nM to 80 nM. The effect of activation in PC3 and HK2 cells with low level of expression of the target gene is obvious. The effect of activation was not observed in HEK293 cells with a high content.4. The dihydrotestosterone was in compliance with 12 to 48 hours and from 5 nM to 20 nM The expression level of TRPV5 in HK2 cells was inhibited by the concentration and concentration of 5.5. The ds-2939 can effectively increase the HK2 cells compared to ds-2939 calcium ion transport level. Dihydrotestosterone can inhibit the calcium ion transport of HK2 cells. ds-2939 Koran The Inhibitory Effect of Dihydrotestosterone on the Transport of Calcium in HK2 Cells. The living RNA can be used to up-regulate the expression level of TRPV5, and can activate the calcium ion of the cell. Transport. At the same time, the calcium ion transport level of the HK2 cells can be partially reversed
【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】：R692.4
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