【摘要】：泌尿系結(jié)石的高復(fù)發(fā)率凸顯了預(yù)防其復(fù)發(fā)在避免患者反復(fù)接受手術(shù)治療中的重要意義。而高鈣尿是泌尿系統(tǒng)含鈣結(jié)石發(fā)生的重要危險(xiǎn)因素。高選擇性鈣離子通道TRPV5主要分布于腎遠(yuǎn)端小管上皮細(xì)胞頂膜側(cè)，其介導(dǎo)的鈣離子由頂膜側(cè)向細(xì)胞內(nèi)的轉(zhuǎn)運(yùn)是腎臟鈣離子主動(dòng)重吸收的限速步驟。TRPV5基因敲除小鼠表現(xiàn)出明顯的高鈣尿。我們的前期研究顯示，TRPV5在草酸鈣結(jié)石模型大鼠和遺傳性高鈣尿結(jié)石大鼠腎臟中顯著低表達(dá)。我們進(jìn)一步的研究顯示泌尿系含鈣結(jié)石患者腎組織TRPV5表達(dá)較對(duì)照組顯著降低。因而，TRPV5成為預(yù)防腎含鈣結(jié)石的理想標(biāo)靶基因。上調(diào)TRPV5表達(dá)水平有助于增加尿鈣重吸收，從而降低尿鈣水平，為泌尿系含鈣結(jié)石的預(yù)防提供新的手段。近年，人工合成的小雙鏈RNA（dsRNA）及微小RNA（microRNA）均被證明可以有效上調(diào)基因表達(dá)水平，而非沉默靶基因表達(dá)。這種新發(fā)現(xiàn)的機(jī)制被稱為“RNA激活”（RNAa）。在本研究中，靶向基因啟動(dòng)子區(qū)域的dsRNA顯著激活了TRPV5鈣離子通道表達(dá)，并上調(diào)了細(xì)胞鈣離子轉(zhuǎn)運(yùn)水平。 研究目的 本研究利用靶向基因啟動(dòng)子區(qū)域的小激活RNA激活TRPV5表達(dá)，研究其對(duì)細(xì)胞鈣離子轉(zhuǎn)運(yùn)的影響。同時(shí)探究小激活RNA對(duì)二氫睪酮誘導(dǎo)的細(xì)胞鈣離子轉(zhuǎn)運(yùn)抑制效應(yīng)的恢復(fù)作用。從而探討靶向TRPV5基因啟動(dòng)子的小激活RNA在泌尿系含鈣結(jié)石預(yù)防中的應(yīng)用價(jià)值。 研究?jī)?nèi)容和方法 1.設(shè)計(jì)合成3個(gè)靶向TRPV5啟動(dòng)子區(qū)域的dsRNA序列，利用脂質(zhì)體轉(zhuǎn)染法將之轉(zhuǎn)染入HK2細(xì)胞中。使用Western Blot、PCR和細(xì)胞免疫熒光的方法檢測(cè)TRPV5表達(dá)變化，從而篩選出可有效上調(diào)TRPV5表達(dá)的dsRNA。并使用細(xì)胞免疫熒光檢測(cè)篩選出的dsRNA對(duì)TRPV5膜豐度的影響。 2.將篩選出的有效的dsRNA以不同時(shí)間和濃度作用于HK2細(xì)胞。以檢測(cè)其時(shí)間和濃度依從性。 3.將篩選出的dsRNA分別轉(zhuǎn)染入PC3、HEK293和HK2細(xì)胞中，以檢測(cè)其細(xì)胞特異性。同時(shí)檢測(cè)上述3種細(xì)胞系TRPV5基礎(chǔ)表達(dá)水平。 4.在不同濃度和不同時(shí)間下使用二氫睪酮處理HK2細(xì)胞，并使用Western Blot檢測(cè)其對(duì)TRPV5表達(dá)的影響。 5.使用Fura-2鈣離子熒光探針檢測(cè)HK2細(xì)胞小激活RNA、二氫睪酮以及二者共處理下細(xì)胞鈣離子轉(zhuǎn)運(yùn)水平變化。 結(jié)果 1.在設(shè)計(jì)合成的3個(gè)靶向TRPV5基因啟動(dòng)子的dsRNA中ds-2939有效上調(diào)了HK2細(xì)胞TRPV5的mRNA水平和蛋白質(zhì)表達(dá)水平。免疫熒光結(jié)果顯示ds-2939顯著增加了TRPV5在HK2細(xì)胞的膜豐度。 2. ds-2939對(duì)TRPV5表達(dá)水平的激活作用在1天至6天和5nM至80nM具有時(shí)間和濃度依從性。 3. ds-2939在TRPV5基礎(chǔ)表達(dá)量較低的PC3和HK2細(xì)胞中激活效果明顯。在靶基因基礎(chǔ)表達(dá)量較高的HEK293細(xì)胞中未能觀察到激活效果。 4.二氫睪酮在12至48小時(shí)和5nM至20nM依從時(shí)間和濃度對(duì)HK2細(xì)胞TRPV5表達(dá)水平產(chǎn)生抑制作用。 5.與對(duì)照組相比，ds-2939可有效增加HK2細(xì)胞鈣離子轉(zhuǎn)運(yùn)水平。二氫睪酮可抑制HK2細(xì)胞鈣離子轉(zhuǎn)運(yùn)水平。ds-2939可部分逆轉(zhuǎn)二氫睪酮對(duì)HK2細(xì)胞鈣離子轉(zhuǎn)運(yùn)水平的抑制效應(yīng)。 結(jié)論 靶向基因啟動(dòng)子區(qū)域的小激活RNA可以用于上調(diào)TRPV5表達(dá)水平，并可激活細(xì)胞鈣離子轉(zhuǎn)運(yùn)。同時(shí)可部分逆轉(zhuǎn)二氫睪酮對(duì)HK2細(xì)胞鈣離子轉(zhuǎn)運(yùn)水平的抑制效應(yīng)。該技術(shù)有望成為預(yù)防泌尿系含鈣結(jié)石發(fā)生及復(fù)發(fā)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
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R692.4

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