發(fā)布時間：2018-05-01 20:36

  本文選題：家族性 + 局灶節(jié)段性腎小球硬化　； 參考：《上海交通大學》2014年博士論文

【摘要】：局灶節(jié)段性腎小球硬化（focal segmental glomerulosclerosis, FSGS）是一種獨立的腎臟病理診斷。光鏡下主要表現(xiàn)為部分腎小球中毛細血管袢小葉的非炎癥性硬化，電鏡下主要表現(xiàn)為足突的廣泛融合。家族遺傳性FSGS（familial FSGS， FFSGS）是FSGS的一種主要類型，其主要表現(xiàn)為家族中一人腎穿證實為FSGS，同時伴有家族其他成員大量蛋白尿或不明原因的腎功能不全。關于其臨床預后的研究，僅有少量文章報道了其臨床表現(xiàn)重及預后不良。而對于與散發(fā)性FSGS（sporadic FSGS，SFSGS）相比，F(xiàn)FSGS臨床表現(xiàn)及預后有何特征，目前尚沒有大組數(shù)據(jù)的相關報道。FFSGS的發(fā)病機制目前廣泛認為與足細胞相關蛋白的基因突變相關。目前已經(jīng)發(fā)現(xiàn)了數(shù)種可以導致FFSGS的致病基因，但這些已經(jīng)發(fā)現(xiàn)的致病基因僅能解釋部分FFSGS的發(fā)病，尤其是在中國漢族人群乃至整個亞洲人群中，已知致病基因的突變率非常低。提示可能存在新的致病基因需要我們進一步去探索發(fā)現(xiàn)�；虻倪B鎖分析及定位克隆在新致病基因探索中發(fā)揮了重要的作用，是目前尋找新致病基因的主要方法。INF2（inverted formin2）是2010年新近發(fā)現(xiàn)的可以導致常染色體顯性遺傳性FSGS的主要致病基因之一，在歐美人群中其基因突變率可高達12%-17%，而在中國漢族人群中其基因突變率如何，目前為止還沒有相關的報道。此外INF2作為一種重要的成蛋白家族成員，其在維持細胞骨架方面存在重要的作用，但其作用機制目前仍不清楚。 本研究第一部分收集了我科收治的FFSGS家系83個，患者合計共124名，以及同期所收治的性別年齡相匹配的SFSGS患者124名，通過對其臨床資料的回顧性整理分析，觀察FFSGS與SFSGS相比其臨床特征如何。結果顯示與SFSGS相比，F(xiàn)FSGS患者發(fā)病較早，少數(shù)患者表現(xiàn)為腎病綜合征，多數(shù)患者可合并血尿，基線水平上患者腎功能及腎小球濾過率與SFSGS患者無顯著性差異。病理表現(xiàn)上，F(xiàn)FSGS患者不論小球病變還是小管病變，與SFSGS患者相比其損傷均較嚴重。此外，F(xiàn)FSGS患者對治療反應不良，與SFSGS患者相比預后較差。腎移植可能是FFSGS的有效治療方法。 本研究第二部分，通過對一個大FFSGS家系進行連鎖分析，尋找其致病基因，連鎖分析結果定位于14q32，為目前已知的AD FFSGS致病基因INF2所在位點，通過基因測序確定p.S85W為其致病基因突變。此外我們對所收集的70個FFSGS家系進行INF2基因突變的篩查，在另一個家系中發(fā)現(xiàn)另一新的致病基因突變p.S129_Q130ins VRQLS。由此在中國漢族人群首次篩查INF2基因突變率為2.8%，遠低于國外報道的相關突變率，提示我們?nèi)孕枰M一步探索針對中國人乃至亞洲人群的常見致病基因。 本研究第三部分利用質(zhì)粒轉(zhuǎn)染的方法，研究p.S85W和p.S129_Q130ins VRQLS兩個新發(fā)現(xiàn)的基因突變在足細胞損傷方面的作用機制。結果顯示p.S85W質(zhì)粒轉(zhuǎn)染的足細胞INF2的蛋白表達水平較野生型質(zhì)粒顯著下調(diào)，免疫熒光結果顯示野生型INF2呈顆粒狀均質(zhì)分布于細胞漿，而p.S85W突變型足細胞INF2呈團塊狀聚集于胞漿或核周。此外p.S85W突變型足細胞α-actinin4蛋白表達水平較野生型顯著下降，其mRNA水平較野生型質(zhì)粒同樣顯著性下降。血清反應因子（serum reactive factor，SRF）表達水平在野生型及突變型質(zhì)粒沒有顯著性變化，而p-SRF水平較野生型質(zhì)粒顯著下降。Cdc42表達水平在突變型及野生型足細胞沒有顯著性差異。此外p.S85W突變型質(zhì)粒還可改變足細胞骨架F-actin、α-actinin4的細胞內(nèi)纖維狀結構為彌散性顆粒狀分布；野生型足細胞SRF主要分布于細胞核，少量分布于細胞漿，而p.S85W突變型足細胞則主要分布于胞漿，提示SRF轉(zhuǎn)錄活性的下降。免疫共沉淀結果顯示正常足細胞及野生型足細胞INF2與Cdc42可相互結合，而p.S85W突變型足細胞兩者的相互結合顯著下降，與此相對應在激光共聚焦顯微鏡觀察下，野生型及正常足細胞INF2與Cdc42分布于細胞漿，且兩者呈現(xiàn)較好的共定位，而p.S85W突變型足細胞兩者的共定位消失。以上結果在p.S129_Q130ins VRQLS突變型質(zhì)粒中，與正常對照組及野生型足細胞均沒有顯著性的差別。而細胞黏附實驗結果顯示兩種突變型質(zhì)粒細胞黏附能力較野生型足細胞顯著下降，而劃痕實驗結果顯示兩種基因突變型細胞遷移能力較野生型顯著上升，提示生理狀態(tài)下，基因突變型足細胞可能比較容易從基底膜脫落。此外Hoechst方法檢測該兩種新發(fā)現(xiàn)的基因突變可以加重足細胞的凋亡。 綜上所述，與SFSGS相比，，F(xiàn)FSGS發(fā)病較早，主要表現(xiàn)為中等量蛋白尿，其病理表現(xiàn)較重，對治療反應較差，預后不好，而腎移植是其有效的治療方法。對所收集的FFSGS患者進行INF2基因突變篩查，發(fā)現(xiàn)p.S85W和p.S129_Q130ins VRQLS兩種新的基因突變，中國漢族人群中INF2基因突變率僅2.8%，突變率顯著低于歐美人群。在作用機制方面p.S85W可通過影響INF2與Cdc42之間的相互作用，降低SRF的活化，進而影響足細胞骨架蛋白的正常結構與功能，而p.S129_Q130ins VRQLS對以上改變無影響。此外兩種基因突變均可降低足細胞的黏附能力，并促進足細胞凋亡。
[Abstract]:Focal segmental glomerulosclerosis (focal segmental glomerulosclerosis, FSGS) is an independent pathological diagnosis of renal pathology. The main manifestation is the non inflammatory sclerosis of the capillary loops of the glomeruli in the partial glomeruli. Under the electron microscope, the main manifestation is the extensive fusion of the foot process. The familial hereditary FSGS (familial FSGS, FFSGS) is a kind of FSGS. The main manifestation is that one of the family members of the family is confirmed to be FSGS, accompanied by a large number of proteinuria or unexplained renal insufficiency in other family members. Only a few articles reported on the clinical prognosis and poor prognosis on the clinical prognosis of the family. Compared with the sporadic FSGS (sporadic FSGS, SFSGS), FFSGS is a clinical case. What is the characteristics of bed performance and prognosis, there is not a large group of data related to the report that the pathogenesis of.FFSGS is currently widely believed to be associated with mutations in the gene for podocyte related proteins. Several pathogenic genes that can lead to FFSGS have been found, but these pathogenetic bases have been found only to explain the incidence of partial FFSGS, especially in the pathogenesis. The mutation rate of the known pathogenic genes is very low in the Chinese Han population and even the whole Asian population. It is suggested that the possible existence of new pathogenic genes needs to be further explored. Linkage analysis and location cloning of genes play an important role in the exploration of new pathogenic genes, which are the main methods to find new pathogenic genes,.INF2 (I Nverted formin2) is one of the major pathogenic genes that can lead to the autosomal dominant hereditary FSGS in 2010. The mutation rate of the gene in the European and American population can be as high as 12%-17%, and the gene mutation rate in the Han population in China has not been reported so far. In addition, INF2 is an important protein family. Members of the family play an important role in maintaining cytoskeleton, but the mechanism is unclear.
The first part of this study collected 83 FFSGS families in our department, a total of 124 patients, and 124 SFSGS patients with matched sex and age in the same period. Through retrospective analysis of their clinical data, the clinical features of FFSGS compared with SFSGS were observed. The results showed that compared with SFSGS, the onset of FFSGS was earlier than that of SFSGS. In a few patients with nephrotic syndrome, most patients can be combined with hematuria. There is no significant difference in renal function and glomerular filtration rate from SFSGS patients at baseline level. In pathological manifestations, FFSGS patients, regardless of small ball lesions or tubules, have more serious injuries compared with SFSGS patients. In addition, FFSGS patients have poor response to treatment and S FSGS patients have poor prognosis. Renal transplantation may be an effective treatment for FFSGS.
In the second part of this study, a linkage analysis of a large FFSGS family was carried out to find its pathogenic gene. The result of linkage analysis was located at 14q32, the locus of the known AD FFSGS pathogenic gene INF2, and the gene sequencing was used to determine the mutation of p.S85W as its pathogenic gene. In addition, we carried out the INF2 gene process of the 70 FFSGS families in which we were collected. In another family, another new genetic mutation p.S129_Q130ins VRQLS. was found in another family. The first screening of INF2 gene mutation rate in Chinese Han population was 2.8%, which was much lower than the related mutation rate reported abroad, suggesting that we still need to further explore the common pathogenic genes for Chinese and Asian people.
The third part of this study, using plasmid transfection method, studied the mechanism of p.S85W and p.S129_Q130ins VRQLS's two newly discovered gene mutations in foot cell injury. The results showed that the protein expression level of INF2 in p.S85W plasmid transfected is significantly lower than that of wild type plasmids, and the immunofluorescence results show that the wild type INF2 is granular. The homoplasm was distributed in the cytoplasm, while the INF2 of p.S85W mutant Poddar was clustered in the cytoplasm or peri. In addition, the expression level of alpha -actinin4 protein in the p.S85W mutant Poda decreased significantly than that of the wild type, and its mRNA level was also significantly lower than that of the wild type. The level of the expression of serum reactive factor (serum reactive factor, SRF) was in the wild. There was no significant change in the type and mutant plasmids, but the level of p-SRF was significantly lower than that of the wild type plasmids. There was no significant difference in the.Cdc42 expression level between the mutant and the wild type podocytes. In addition, the p.S85W mutant plasmids could also change the F-actin of the Poddar cytoskeleton, and the fibrous structure in the cells of the alpha -actinin4 was diffuse granular distribution, and the wild type was found in the wild type. The SRF of the podocyte was mainly distributed in the nucleus and distributed in the cytoplasm, while the p.S85W mutated Poddar was mainly distributed in the cytoplasm, suggesting the decrease of SRF transcriptional activity. The results of immunoprecipitation showed that the normal Poddar and wild type foot cell INF2 and Cdc42 could be combined with each other, and the combination of p.S85W mutant podocytes decreased significantly. Compared with the laser confocal microscope, the INF2 and Cdc42 of the wild and normal podocytes were distributed in the cytoplasm, and both showed good co location, while the co localization of the p.S85W mutable podthe cells disappeared. The above results were not in the normal control group and the wild type foot cells in the p.S129_Q130ins VRQLS mutant plasmids. The results of cell adhesion test showed that the adhesion ability of two mutant plasmids was significantly lower than that of wild type foot cells. The results of scratch test showed that the migration ability of two mutant cells was significantly higher than that of the wild type, suggesting that the gene mutated Poddar may be easier to fall off the basement membrane in physiological state. In addition, the detection of these two new mutations by Hoechst can aggravate podocyte apoptosis.
In summary, compared with SFSGS, FFSGS has an early onset, mainly characterized by moderate proteinuria, its pathological manifestations are heavy, the response to treatment is poor, and the prognosis is poor, and renal transplantation is an effective treatment. The INF2 mutation screening for the collected FFSGS patients and the discovery of two new genetic mutations in p.S85W and p.S129_Q130ins VRQLS, China, China The mutation rate of INF2 gene in the Han population is only 2.8%, and the mutation rate is significantly lower than that in the European and American population. In the mechanism of action, p.S85W can affect the interaction between INF2 and Cdc42, reduce the activation of SRF, and then affect the normal structure and function of the cytoskeleton protein, and the p.S129_Q130ins VRQLS has no effect on the above changes. In addition, the two genes process. All changes can reduce the adhesion ability of podocytes and promote podocyte apoptosis.

【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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