局灶節(jié)段腎小球硬化癥的基因復查及蛋白質(zhì)組學研究
發(fā)布時間：2018-09-03 09:09
【摘要】：局灶節(jié)段腎小球硬化（focal segmental glomerulosclerosis，F(xiàn)SGS)是一組病理學上以僅累及部分腎小球及腎小球毛細血管袢部分小葉的硬化性病變?yōu)樘卣鞯呐R床病理綜合征1，是成人腎病綜合征（nephrotic syndrome，NS）和慢性腎衰竭最常見的原因之一，在經(jīng)腎活檢診斷的原發(fā)性腎小球腎炎中約占5.6~37.8%2-5。盡管存在一定地區(qū)差異，F(xiàn)SGS在各地區(qū)的發(fā)病率總體呈持續(xù)上升趨勢6-8。FSGS常以大量蛋白尿為主要臨床表現(xiàn)，可伴有鏡下血尿、高血壓和腎功能不全，治療效果相對較差�；颊叽_診后5年和10年腎存活率分別約為83.5%和43.8%9。尤其是激素抵抗、激素依賴、反復復發(fā)的所謂“難治性”FSGS，如不能得到有效治療，超過50%的患者將在3-8年內(nèi)進展至終末期腎功能衰竭5。正因如此，F(xiàn)SGS已成為目前國際國內(nèi)研究的熱點之一。 按不同病因，F(xiàn)SGS可分為原發(fā)性和繼發(fā)性10，繼發(fā)性FSGS的病因中包括家族性11。足細胞相關基因變異是家族性FSGS發(fā)病的重要原因，目前已知導致成人發(fā)病型家族性FSGS的基因主要有ACTN4、TRPC6和INF2基因12，但ACTN4、TRPC6基因在中國成人發(fā)病型家族性FSGS中的確切突變概率仍未可知。 原發(fā)性FSGS約占80%13，而其發(fā)病機制尚有諸多未明。蛋白質(zhì)組學(proteomics)技術的發(fā)展為尋找FSGS發(fā)病過程中的關鍵性物質(zhì)，進一步闡明其發(fā)病機制和尋找新的治療靶點提供了可能。近年來，采用相對和絕對定量同位素標記(isobaric tags for relative and absolute quantitation，iTRAQ)技術聯(lián)合串聯(lián)質(zhì)譜的方法，可以對蛋白質(zhì)的肽段進行比較精確的鑒別和定量14，與二維電泳相比，它具有重復性好，靈敏度高，標記效率高，可以進行定量檢測等優(yōu)點15-17。目前蛋白質(zhì)組學已被廣泛應用于腎臟疾病的診斷，發(fā)病機制的探索，疾病活動度以及預后的預判等方面18-23。其中尿液因取材簡單，性質(zhì)穩(wěn)定，產(chǎn)生和排泄的途徑與腎臟和泌尿道密切相關，是腎臟疾病蛋白質(zhì)組學研究的理想標本。 在本研究的第一部分，我們進行了國際單中心最大組的成年發(fā)病型家族性FSGS的TRPC6和ACTN6基因突變篩查（共80個家系），在80個先證者中共篩查出TRPC6基因Q889K突變2例，發(fā)現(xiàn)TRPC6基因SNP14種，未檢出ACTN4突變，，篩出ACTN4基因SNP22種。報道了TRPC6基因在本組中國成年發(fā)病型家族性FSGS中的突變率為2.5%，ACTN4基因的突變率為0%。完善了成年發(fā)病型家族性FSGS中TRPC6和ACTN6基因突變率的數(shù)據(jù)。 在本研究的第二部分，采用四聯(lián)iTRAQ聯(lián)合2D-nano-HPLC-MS/MS的方法在原發(fā)性FSGS、MCD患者、正常對照組尿液中檢出并鑒定出蛋白質(zhì)200種。其中包括細胞漿、細胞核、細胞膜、細胞外的多種酶類、生長因子、轉錄調(diào)節(jié)因子、受體蛋白、轉運蛋白和離子通道蛋白等。應用Ingenuity Pathway Analysis（IPA）軟件的Top canonical pathway對差異蛋白進行分析后發(fā)現(xiàn)FSGS組paxillin信號轉導、肌動細胞細胞骨架信號轉導通路受影響，提示這兩條通路可能與FSGS發(fā)病機制有關。差異蛋白之一的vinculin經(jīng)ELISA驗證發(fā)現(xiàn)在原發(fā)性FSGS患者尿液中較對照組升高，結果有統(tǒng)計學差異，并與質(zhì)譜結果一致。 在本研究的第三部分，通過體外實驗發(fā)現(xiàn)，阿霉素刺激24小時后足細胞中vinculin蛋白的表達水平升高；同時，免疫熒光結果顯示FSGS患者腎組織vinculin熒光強度較輕微病變對照組高，提示vinculin蛋白可能參與FSGS的發(fā)病過程，或是FSGS腎損傷后表達量改變的蛋白之一。此外，我們對原發(fā)性FSGS患者尿vinculin水平與尿蛋白、血白蛋白、血肌酐值進行了相關分析，但未發(fā)現(xiàn)尿vinculin與上述指標存在相關性。 總之，ACTN4和TRPC6基因突變的發(fā)病率較低，并不是成人發(fā)病型家族性FSGS的常見原因。蛋白質(zhì)組學的發(fā)展為探索FSGS的發(fā)病機制帶來新的啟發(fā)，而vinculin蛋白可能參與FSGS的發(fā)病過程。
[Abstract]:Focal segmental glomerulosclerosis (FSGS) is a group of clinicopathological syndromes characterized by sclerotic lesions involving only part of the glomerular and part of the glomerular capillary loops. FSGS is one of the most common causes of adult nephrotic syndrome (NS) and chronic renal failure. Although there are some regional differences, the incidence of FSGS in various regions is generally on the rise 6-8. FSGS is often characterized by a large number of proteinuria as the main clinical manifestations, may be accompanied by microscopic hematuria, hypertension and renal insufficiency, the treatment effect is relatively poor. The annual and 10-year renal survival rates are about 83.5% and 43.8% respectively. Especially the so-called "refractory" FSGS with hormone resistance, hormone dependence and recurrence, if not effectively treated, more than 50% of the patients will progress to end-stage renal failure within 3-8 years. Therefore, FSGS has become one of the hot spots of international and domestic research.
According to different etiologies, FSGS can be divided into primary and secondary 10. Familial 11 is included in secondary FSGS. Podocyte-related gene mutation is an important cause of familial FSGS. It is known that ACTN4, TRPC6 and INF2 gene 12 are the main genes causing familial FSGS in adults, but ACTN4 and TRPC6 genes are found in Chinese adult pathogenic countries. The exact mutation probability in the family FSGS is still unknown.
The development of proteomics technology has made it possible to find the key substances in the pathogenesis of FSGS, further elucidate its pathogenesis and find new therapeutic targets. In recent years, relative and absolute quantitative isobaric tags for rela have been used. Compared with two-dimensional electrophoresis, it has the advantages of good reproducibility, high sensitivity, high labeling efficiency, and can be used for quantitative detection. At present, proteomics has been widely used in kidney. Diagnosis, pathogenesis, activity and prognosis of the disease are 18-23. Urine is an ideal specimen for proteomics of kidney diseases because of its simple material, stable nature, and the way of production and excretion is closely related to the kidney and urinary tract.
In the first part of this study, we screened for TRPC6 and ACTN6 gene mutations in adult onset familial FSGS in the largest international single center group (80 families). Two cases of Q889K mutations in TRPC6 gene were screened out among 80 probands. Fourteen kinds of SNP14 mutations were found in TRPC6 gene, no ACTN4 mutations were detected, and 22 kinds of SNP22 of ACTN4 gene were screened out. The mutation rates of TRPC6 and ACTN4 genes in this group of Chinese adult onset familial FSGS were 2.5% and 0%, respectively.
In the second part of this study, we detected and identified 200 proteins in urine of patients with primary FSGS, MCD and normal controls by quadruple iTRAQ and two-dimensional-nano-HPLC-MS/MS. These proteins include cytoplasm, nucleus, cell membrane, extracellular enzymes, growth factors, transcription regulators, receptor proteins, transporters and ion channels. Top canonical pathway of Ingenuity Pathway Analysis (IPA) software was used to analyze the differential proteins. It was found that paxillin signal transduction and actinocyte cytoskeleton signal transduction pathway were affected in FSGS group, suggesting that the two pathways may be related to the pathogenesis of FSGS. In patients with primary FSGS, the urine levels were higher than those in the control group, and the results were statistically significant.
In the third part of this study, we found that the expression of vinculin protein in podocytes increased after 24 hours of adriamycin stimulation. At the same time, the immunofluorescence results showed that vinculin fluorescence intensity in renal tissue of FSGS patients was higher than that of the control group with mild lesions, suggesting that vinculin protein may participate in the pathogenesis of FSGS, or FSGS renal injury. In addition, we analyzed the correlation between urinary vinculin and urinary protein, serum albumin and serum creatinine in patients with primary FSGS, but found no correlation between urinary vinculin and the above indexes.
In conclusion, the low incidence of ACTN4 and TRPC6 mutations is not a common cause of familial FSGS in adults. The development of proteomics provides new insights into the pathogenesis of FSGS, and vinculin may be involved in the pathogenesis of FSGS.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R692.6
本文編號：2219528
[Abstract]:Focal segmental glomerulosclerosis (FSGS) is a group of clinicopathological syndromes characterized by sclerotic lesions involving only part of the glomerular and part of the glomerular capillary loops. FSGS is one of the most common causes of adult nephrotic syndrome (NS) and chronic renal failure. Although there are some regional differences, the incidence of FSGS in various regions is generally on the rise 6-8. FSGS is often characterized by a large number of proteinuria as the main clinical manifestations, may be accompanied by microscopic hematuria, hypertension and renal insufficiency, the treatment effect is relatively poor. The annual and 10-year renal survival rates are about 83.5% and 43.8% respectively. Especially the so-called "refractory" FSGS with hormone resistance, hormone dependence and recurrence, if not effectively treated, more than 50% of the patients will progress to end-stage renal failure within 3-8 years. Therefore, FSGS has become one of the hot spots of international and domestic research.
According to different etiologies, FSGS can be divided into primary and secondary 10. Familial 11 is included in secondary FSGS. Podocyte-related gene mutation is an important cause of familial FSGS. It is known that ACTN4, TRPC6 and INF2 gene 12 are the main genes causing familial FSGS in adults, but ACTN4 and TRPC6 genes are found in Chinese adult pathogenic countries. The exact mutation probability in the family FSGS is still unknown.
The development of proteomics technology has made it possible to find the key substances in the pathogenesis of FSGS, further elucidate its pathogenesis and find new therapeutic targets. In recent years, relative and absolute quantitative isobaric tags for rela have been used. Compared with two-dimensional electrophoresis, it has the advantages of good reproducibility, high sensitivity, high labeling efficiency, and can be used for quantitative detection. At present, proteomics has been widely used in kidney. Diagnosis, pathogenesis, activity and prognosis of the disease are 18-23. Urine is an ideal specimen for proteomics of kidney diseases because of its simple material, stable nature, and the way of production and excretion is closely related to the kidney and urinary tract.
In the first part of this study, we screened for TRPC6 and ACTN6 gene mutations in adult onset familial FSGS in the largest international single center group (80 families). Two cases of Q889K mutations in TRPC6 gene were screened out among 80 probands. Fourteen kinds of SNP14 mutations were found in TRPC6 gene, no ACTN4 mutations were detected, and 22 kinds of SNP22 of ACTN4 gene were screened out. The mutation rates of TRPC6 and ACTN4 genes in this group of Chinese adult onset familial FSGS were 2.5% and 0%, respectively.
In the second part of this study, we detected and identified 200 proteins in urine of patients with primary FSGS, MCD and normal controls by quadruple iTRAQ and two-dimensional-nano-HPLC-MS/MS. These proteins include cytoplasm, nucleus, cell membrane, extracellular enzymes, growth factors, transcription regulators, receptor proteins, transporters and ion channels. Top canonical pathway of Ingenuity Pathway Analysis (IPA) software was used to analyze the differential proteins. It was found that paxillin signal transduction and actinocyte cytoskeleton signal transduction pathway were affected in FSGS group, suggesting that the two pathways may be related to the pathogenesis of FSGS. In patients with primary FSGS, the urine levels were higher than those in the control group, and the results were statistically significant.
In the third part of this study, we found that the expression of vinculin protein in podocytes increased after 24 hours of adriamycin stimulation. At the same time, the immunofluorescence results showed that vinculin fluorescence intensity in renal tissue of FSGS patients was higher than that of the control group with mild lesions, suggesting that vinculin protein may participate in the pathogenesis of FSGS, or FSGS renal injury. In addition, we analyzed the correlation between urinary vinculin and urinary protein, serum albumin and serum creatinine in patients with primary FSGS, but found no correlation between urinary vinculin and the above indexes.
In conclusion, the low incidence of ACTN4 and TRPC6 mutations is not a common cause of familial FSGS in adults. The development of proteomics provides new insights into the pathogenesis of FSGS, and vinculin may be involved in the pathogenesis of FSGS.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R692.6
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