APOL1基因變異對(duì)中國(guó)FSGS患者影響的研究
發(fā)布時(shí)間：2018-08-14 11:33
【摘要】：局灶節(jié)段腎小球硬化(FSGS)是難治性腎病綜合征和終末期腎病最常見的原因之一,遺傳因素在其發(fā)病和進(jìn)展中起重要作用。前期研究顯示APOL1的G1和G2變異與FSGS的發(fā)病與進(jìn)展密切相關(guān)。但該結(jié)果并沒有在中國(guó)人群中得到重復(fù)。提示我國(guó)FSGS可能存在其他APOL1變異。本研究的目的是:(1)對(duì)APOL1基因單核苷酸多態(tài)性和單體型進(jìn)行分析,探索APOL1基因單體型對(duì)不同人群中疾病的易感性。(2)檢測(cè)FSGS患者和正常對(duì)照組中APOL1基因拷貝數(shù)的情況,通過(guò)比較拷貝數(shù)變異在兩組的分布情況,探索APOL1基因拷貝數(shù)變異對(duì)中國(guó)FSGS患者臨床治療與預(yù)后之間的可能意義。(3)利用本課題組前期發(fā)現(xiàn)的APOL1新突變,構(gòu)建突變質(zhì)粒和野生型質(zhì)粒轉(zhuǎn)染到永生化的足細(xì)胞,研究該APOL1突變對(duì)細(xì)胞標(biāo)志蛋白、細(xì)胞骨架和離子通道的影響,并探索其參與FSGS足細(xì)胞損傷的可能機(jī)制。我們的研究方法包括(1)從千人基因組數(shù)據(jù)中篩選APOL1基因SNP,統(tǒng)計(jì)不同區(qū)域APOL1單體型,并比較每個(gè)單體型在不同人群中的分布情況。(2)篩選符合條件的FSGS患者DNA樣本;通過(guò)設(shè)計(jì)APOL1引物和探針進(jìn)行Taqman拷貝數(shù)變異實(shí)驗(yàn);使用Copy Caller軟件分析結(jié)果,SPSS分析FSGS患者與正常對(duì)照組中APOL1基因拷貝數(shù)分布差異及不同拷貝數(shù)的FSGS患者臨床預(yù)后的差異。(3)將突變質(zhì)粒轉(zhuǎn)染至細(xì)胞后動(dòng)態(tài)追蹤細(xì)胞形態(tài)的改變,western blot檢測(cè)足細(xì)胞標(biāo)志蛋白,自噬標(biāo)志物,信號(hào)轉(zhuǎn)導(dǎo)蛋白分子的表達(dá)水平;激光共聚焦檢測(cè)足細(xì)胞骨架的改變及線粒體損傷情況,并用膜片鉗技術(shù)檢測(cè)足細(xì)胞膜離子通道的變化。研究結(jié)果顯示(1)APOL1基因各個(gè)區(qū)域的單體型在不同人群的分布都具有明顯差異。大部分單體型在混合人群和歐洲人群的頻率相對(duì)較高,有2個(gè)單體型在亞洲人群中的分布頻率高于其他種群。(2)FSGS患者和正常組之間APOL1基因拷貝數(shù)分布情況沒有統(tǒng)計(jì)學(xué)意義。多拷貝數(shù)和低拷貝數(shù)的FSGS患者之間治療后的緩解率無(wú)顯著差異。(3)轉(zhuǎn)染突變質(zhì)粒的足細(xì)胞細(xì)胞骨架蛋白排列紊亂,細(xì)胞發(fā)生溶解,細(xì)胞間隙增大,間接反映了足細(xì)胞足突的收縮;足細(xì)胞標(biāo)志蛋白表達(dá)下降,P38MAPK、SAPKs和JNK等信號(hào)通路被激活;此外,APOL1新突變可能破壞了細(xì)胞膜氯離子通道蛋白。在本研究中,我們從三個(gè)層面分析了APOL1基因變異與FSGS的關(guān)系。結(jié)果表明,APOL1基因的單核苷酸多態(tài)性在不同人群中分布有明顯差異。其拷貝數(shù)變異與我國(guó)FSGS易感性無(wú)密切相關(guān)性。我們前期發(fā)現(xiàn)的APOL1位點(diǎn)突變可能從多方面影響足細(xì)胞的形態(tài)和功能,表明APOL1基因突變可能參與我國(guó)FSGS的發(fā)病,其機(jī)制還需要深入研究。
[Abstract]:Focal segmental glomerulosclerosis (FSGS) is one of the most common causes of refractory nephrotic syndrome and end-stage nephropathy. Genetic factors play an important role in its pathogenesis and progression. Previous studies have shown that G 1 and G 2 mutations of APOL1 are closely related to the pathogenesis and progression of FSGS. But the results were not repeated in Chinese. These results suggest that there may be other APOL1 variations in FSGS in China. The purpose of this study was: (1) to analyze the mononucleotide polymorphism and haplotype of APOL1 gene, and to explore the susceptibility of APOL1 haplotype to disease in different population. (2) to detect the copy number of APOL1 gene in FSGS patients and normal controls. By comparing the distribution of copy number variation in the two groups, the possible significance of copy number variation of APOL1 gene to the clinical treatment and prognosis of Chinese patients with FSGS was explored. (3) A new mutation of APOL1 was found in the early stage of our research group. The mutant plasmids and wild-type plasmids were constructed and transfected into immortalized podocytes. The effects of the APOL1 mutation on cell marker proteins, cytoskeleton and ion channels were studied, and the possible mechanisms involved in the damage of FSGS podocytes were explored. Our research methods include: (1) screening APOL1 gene from thousands of human genome data, counting APOL1 haplotypes in different regions, and comparing the distribution of APOL1 haplotypes in different populations. (2) screening DNA samples from eligible FSGS patients; Taqman copy number variation experiments were carried out by designing APOL1 primers and probes. The results of Copy Caller software were used to analyze the difference of copy number distribution of APOL1 gene between FSGS patients and normal controls and the difference of clinical prognosis between FSGS patients with different copy numbers. (3) the mutant plasmid was transfected into the cells and the morphology of the cells was dynamically tracked. Western blot was used to detect podocyte marker protein. The changes of cytoskeleton and mitochondrial damage of podocytes were detected by laser confocal focusing and ion channels of podocyte membrane were detected by patch clamp technique. The results showed that (1) the distribution of haplotypes in different regions of APOL1 gene was significantly different in different populations. The frequencies of most haplotypes were relatively high in mixed population and European population, and the frequencies of two haplotypes in Asian population were higher than those in other populations. (2) there was no significant difference in the distribution of APOL1 gene copy number between FSGS patients and normal population. There was no significant difference in remission rate between FSGS patients with multiple copy number and low copy number. (3) the cytoskeleton protein of podocyte transfected with mutated plasmids was disordered, the cells dissolved and the cell gap increased, which indirectly reflected the contraction of podocyte foot process. The expression of P38 MAPK- SAPKs and JNK were activated, and the new mutation of APOL1 may destroy the membrane chloride channel protein. In this study, we analyzed the relationship between APOL1 gene variation and FSGS on three levels. The results showed that the single nucleotide polymorphisms of APOL1 gene were significantly different in different populations. There was no close correlation between copy number variation and susceptibility to FSGS in China. The mutation of APOL1 locus discovered in our earlier period may affect the morphology and function of podocyte in many ways, which indicates that the mutation of APOL1 gene may be involved in the pathogenesis of FSGS in China, and its mechanism needs further study.
【學(xué)位授予單位】：電子科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R692
本文編號(hào)：2182724
[Abstract]:Focal segmental glomerulosclerosis (FSGS) is one of the most common causes of refractory nephrotic syndrome and end-stage nephropathy. Genetic factors play an important role in its pathogenesis and progression. Previous studies have shown that G 1 and G 2 mutations of APOL1 are closely related to the pathogenesis and progression of FSGS. But the results were not repeated in Chinese. These results suggest that there may be other APOL1 variations in FSGS in China. The purpose of this study was: (1) to analyze the mononucleotide polymorphism and haplotype of APOL1 gene, and to explore the susceptibility of APOL1 haplotype to disease in different population. (2) to detect the copy number of APOL1 gene in FSGS patients and normal controls. By comparing the distribution of copy number variation in the two groups, the possible significance of copy number variation of APOL1 gene to the clinical treatment and prognosis of Chinese patients with FSGS was explored. (3) A new mutation of APOL1 was found in the early stage of our research group. The mutant plasmids and wild-type plasmids were constructed and transfected into immortalized podocytes. The effects of the APOL1 mutation on cell marker proteins, cytoskeleton and ion channels were studied, and the possible mechanisms involved in the damage of FSGS podocytes were explored. Our research methods include: (1) screening APOL1 gene from thousands of human genome data, counting APOL1 haplotypes in different regions, and comparing the distribution of APOL1 haplotypes in different populations. (2) screening DNA samples from eligible FSGS patients; Taqman copy number variation experiments were carried out by designing APOL1 primers and probes. The results of Copy Caller software were used to analyze the difference of copy number distribution of APOL1 gene between FSGS patients and normal controls and the difference of clinical prognosis between FSGS patients with different copy numbers. (3) the mutant plasmid was transfected into the cells and the morphology of the cells was dynamically tracked. Western blot was used to detect podocyte marker protein. The changes of cytoskeleton and mitochondrial damage of podocytes were detected by laser confocal focusing and ion channels of podocyte membrane were detected by patch clamp technique. The results showed that (1) the distribution of haplotypes in different regions of APOL1 gene was significantly different in different populations. The frequencies of most haplotypes were relatively high in mixed population and European population, and the frequencies of two haplotypes in Asian population were higher than those in other populations. (2) there was no significant difference in the distribution of APOL1 gene copy number between FSGS patients and normal population. There was no significant difference in remission rate between FSGS patients with multiple copy number and low copy number. (3) the cytoskeleton protein of podocyte transfected with mutated plasmids was disordered, the cells dissolved and the cell gap increased, which indirectly reflected the contraction of podocyte foot process. The expression of P38 MAPK- SAPKs and JNK were activated, and the new mutation of APOL1 may destroy the membrane chloride channel protein. In this study, we analyzed the relationship between APOL1 gene variation and FSGS on three levels. The results showed that the single nucleotide polymorphisms of APOL1 gene were significantly different in different populations. There was no close correlation between copy number variation and susceptibility to FSGS in China. The mutation of APOL1 locus discovered in our earlier period may affect the morphology and function of podocyte in many ways, which indicates that the mutation of APOL1 gene may be involved in the pathogenesis of FSGS in China, and its mechanism needs further study.
【學(xué)位授予單位】：電子科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R692
【參考文獻(xiàn)】
相關(guān)期刊論文 前6條
1 Alex N Kasembeli;Raquel Duarte;Michèle Ramsay;Saraladevi Naicker;;African origins and chronic kidney disease susceptibility in the human immunodeficiency virus era[J];World Journal of Nephrology;2015年02期
2 陳亭宇;李貴森;;局灶節(jié)段性腎小球硬化患者致病基因突變檢測(cè)[J];臨床腎臟病雜志;2014年06期
3 杜仁騫;金力;張鋒;;基因組拷貝數(shù)變異及其突變機(jī)理與人類疾病[J];遺傳;2011年08期
4 李貴森;王慶文;;局灶節(jié)段性腎小球硬化的遺傳背景[J];腎臟病與透析腎移植雜志;2010年04期
5 梁燕,程麗靜,黃海長(zhǎng);足細(xì)胞損傷與腎小球疾病[J];中國(guó)病理生理雜志;2005年09期
6 陳惠萍,曾彩虹,胡偉新,王慶文,俞雨生,姚小丹,唐政,王建平,朱茂艷,周虹,劉紅,劉志紅,黎磊石;10594例腎活檢病理資料分析[J];腎臟病與透析腎移植雜志;2000年06期
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