INF2在家族性局灶节段肾小球硬化中发病机制研究

发布时间：2018-05-01 20:36

本文选题：家族性 + 局灶节段性肾小球硬化　；参考：《上海交通大学》2014年博士论文

【摘要】：局灶节段性肾小球硬化（focal segmental glomerulosclerosis, FSGS）是一种独立的肾脏病理诊断。光镜下主要表现为部分肾小球中毛细血管袢小叶的非炎症性硬化，电镜下主要表现为足突的广泛融合。家族遗传性FSGS（familial FSGS， FFSGS）是FSGS的一种主要类型，其主要表现为家族中一人肾穿证实为FSGS，同时伴有家族其他成员大量蛋白尿或不明原因的肾功能不全。关于其临床预后的研究，仅有少量文章报道了其临床表现重及预后不良。而对于与散发性FSGS（sporadic FSGS，SFSGS）相比，FFSGS临床表现及预后有何特征，目前尚没有大组数据的相关报道。FFSGS的发病机制目前广泛认为与足细胞相关蛋白的基因突变相关。目前已经发现了数种可以导致FFSGS的致病基因，但这些已经发现的致病基因仅能解释部分FFSGS的发病，尤其是在中国汉族人群乃至整个亚洲人群中，已知致病基因的突变率非常低。提示可能存在新的致病基因需要我们进一步去探索发现。基因的连锁分析及定位克隆在新致病基因探索中发挥了重要的作用，是目前寻找新致病基因的主要方法。INF2（inverted formin2）是2010年新近发现的可以导致常染色体显性遗传性FSGS的主要致病基因之一，在欧美人群中其基因突变率可高达12%-17%，而在中国汉族人群中其基因突变率如何，目前为止还没有相关的报道。此外INF2作为一种重要的成蛋白家族成员，其在维持细胞骨架方面存在重要的作用，但其作用机制目前仍不清楚。本研究第一部分收集了我科收治的FFSGS家系83个，患者合计共124名，以及同期所收治的性别年龄相匹配的SFSGS患者124名，通过对其临床资料的回顾性整理分析，观察FFSGS与SFSGS相比其临床特征如何。结果显示与SFSGS相比，FFSGS患者发病较早，少数患者表现为肾病综合征，多数患者可合并血尿，基线水平上患者肾功能及肾小球滤过率与SFSGS患者无显著性差异。病理表现上，FFSGS患者不论小球病变还是小管病变，与SFSGS患者相比其损伤均较严重。此外，FFSGS患者对治疗反应不良，与SFSGS患者相比预后较差。肾移植可能是FFSGS的有效治疗方法。本研究第二部分，通过对一个大FFSGS家系进行连锁分析，寻找其致病基因，连锁分析结果定位于14q32，为目前已知的AD FFSGS致病基因INF2所在位点，通过基因测序确定p.S85W为其致病基因突变。此外我们对所收集的70个FFSGS家系进行INF2基因突变的筛查，在另一个家系中发现另一新的致病基因突变p.S129_Q130ins VRQLS。由此在中国汉族人群首次筛查INF2基因突变率为2.8%，远低于国外报道的相关突变率，提示我们仍需要进一步探索针对中国人乃至亚洲人群的常见致病基因。本研究第三部分利用质粒转染的方法，研究p.S85W和p.S129_Q130ins VRQLS两个新发现的基因突变在足细胞损伤方面的作用机制。结果显示p.S85W质粒转染的足细胞INF2的蛋白表达水平较野生型质粒显著下调，免疫荧光结果显示野生型INF2呈颗粒状均质分布于细胞浆，而p.S85W突变型足细胞INF2呈团块状聚集于胞浆或核周。此外p.S85W突变型足细胞α-actinin4蛋白表达水平较野生型显著下降，其mRNA水平较野生型质粒同样显著性下降。血清反应因子（serum reactive factor，SRF）表达水平在野生型及突变型质粒没有显著性变化，而p-SRF水平较野生型质粒显著下降。Cdc42表达水平在突变型及野生型足细胞没有显著性差异。此外p.S85W突变型质粒还可改变足细胞骨架F-actin、α-actinin4的细胞内纤维状结构为弥散性颗粒状分布；野生型足细胞SRF主要分布于细胞核，少量分布于细胞浆，而p.S85W突变型足细胞则主要分布于胞浆，提示SRF转录活性的下降。免疫共沉淀结果显示正常足细胞及野生型足细胞INF2与Cdc42可相互结合，而p.S85W突变型足细胞两者的相互结合显著下降，与此相对应在激光共聚焦显微镜观察下，野生型及正常足细胞INF2与Cdc42分布于细胞浆，且两者呈现较好的共定位，而p.S85W突变型足细胞两者的共定位消失。以上结果在p.S129_Q130ins VRQLS突变型质粒中，与正常对照组及野生型足细胞均没有显著性的差别。而细胞黏附实验结果显示两种突变型质粒细胞黏附能力较野生型足细胞显著下降，而划痕实验结果显示两种基因突变型细胞迁移能力较野生型显著上升，提示生理状态下，基因突变型足细胞可能比较容易从基底膜脱落。此外Hoechst方法检测该两种新发现的基因突变可以加重足细胞的凋亡。综上所述，与SFSGS相比，，FFSGS发病较早，主要表现为中等量蛋白尿，其病理表现较重，对治疗反应较差，预后不好，而肾移植是其有效的治疗方法。对所收集的FFSGS患者进行INF2基因突变筛查，发现p.S85W和p.S129_Q130ins VRQLS两种新的基因突变，中国汉族人群中INF2基因突变率仅2.8%，突变率显著低于欧美人群。在作用机制方面p.S85W可通过影响INF2与Cdc42之间的相互作用，降低SRF的活化，进而影响足细胞骨架蛋白的正常结构与功能，而p.S129_Q130ins VRQLS对以上改变无影响。此外两种基因突变均可降低足细胞的黏附能力，并促进足细胞凋亡。
[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.

【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692.6

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