抗多囊蛋白1氨基端单克隆抗体的制备和鉴定

目的 :用杂交瘤技术制备抗多囊蛋白 1胞外区氨基端的单克隆抗体 (mAb) ,并对其特异性进行鉴定。方法 :以肾组织总RNA为模板 ,用RT PCR扩增多囊蛋白 1胞外区氨基端的编码基因PKD1cDNA。将该基因克隆到融合蛋白表达载体pQE3 0中 ,转染大肠杆菌M15。以异丙基硫代半乳糖苷 (IPTG)诱导表达多囊蛋白 1胞外区氨基端的组氨酸融合蛋白 (PC1 e2 ) ,用亲和层析法纯化。以纯化的融合蛋白作为抗原免疫BALB/c小鼠 ,取小鼠脾细胞与小鼠骨髓瘤细胞株Sp2 / 0进行细胞融合 ,间接ELISA筛选阳性克隆 ,有限稀释法进行单克隆化。mAb的特异性用间接ELISA和Westernblot鉴定。结果 :克隆到两个编码多囊蛋白 1氨基端的cDNA片段 (50 2bp和 471bp)。构建的表达质粒经酶切和DNA测序证实 ,为所需要的质粒。表达出相对分子质量 (Mr)分别为 1980 0和 1890 0的融合蛋白 ,经Westernblot鉴定 ,均为多囊蛋白 1的融合蛋白。用Mr 为 1890 0的融合蛋白免疫小鼠 ,得到杂交瘤细胞株 7B1。Westernblot分析表明 ,该细胞株分泌的mAb能特异地与多囊蛋白 1氨基端结合。结论 :本实验表达了PKD1多拷贝区所编码的PC1 e2 ,成功地制备了抗多囊蛋白 1胞外区N端的mAb。     

Polycystin-1相关的信号转导通路

已知85%～90%的常染色体显性遗传性多囊肾病是由pkd1突变引起的,因此pkd1编码的多囊蛋白-1的功能受到研究者的关注。多囊蛋白-1是一个具有长的细胞外氨基末端,11个跨膜区,短的细胞内羧基末端的跨膜蛋白。近年来发现多囊蛋白-1与多条信号转导通路有关。文章主要介绍多囊蛋白-1相关的PI3-K/Akt/mTOR、Wnt/β-catenin和JAK-STAT等信号转导通路、各信号通路之间的联系及其在常染色体显性遗传性多囊肾病致病中的作用。     

多囊肾病的临床实践指南

多囊肾病(polycystic kidney disease,PKD)是由基因突变所导致的一类遗传性肾病,按其遗传方式又分为常染色体显性多囊肾病(autosomal dominant polycystic kidney disease,ADPKD)和常染色体隐性多囊肾病(autosom al recessive polycystic kidney disease,ARPKD)。该病的主要病理特点是肾脏囊肿进行性增大、增多,破坏正常的肾脏结构,最终导致终末期肾病(end stage renal disease,ESRD),患者只能依靠透析或肾移植维持生命。我们在参考国内外本领域的基础研究、临床研究和相关指南共识的基础上,结合中国人群的实际情况编写了该项指南,旨在总结多囊肾病的医学遗传学知识和临床处置要点,以提高临床医师的认识水平,为该病的诊治提供规范化建议。     

小鼠多囊蛋白1氨基端多克隆抗体的制备和其生物特性分析

目的 制备针对多囊蛋白1(PC1)胞外区的抗体,为分析PC1胞外区生物特性和功能提供工具.方法 根据生物信息学分析结果,PCR扩增编码小鼠Pkd1的氨基端474E-640L的eDNA片段.将该片段克隆到GST融合蛋白表达载体上,在IPTG诱导下产生小鼠Pkdl抗原(mPkd1-N).纯化浓缩目的蛋白并制备兔抗PC1氨基端多克隆抗体(mPkd1-Np),并以Western blot法、免疫组化以及免疫荧光方法分析该兔抗PC1氨基端抗体的特异性.结果 成功地构建了mPkd1-N片段真核及原核表达载体,在大肠杆菌中实现表达,并制备了抗小鼠PC1氨基端的多克隆抗体mPkd1-Np.通过生化和细胞学方法证实了该抗体针对PC1的特异性.结论 成功制备了高效价、特异性的兔抗mPkdl-Np多克隆抗体.     

两个中国常染色体显性遗传性多囊肾病家系的表型与基因型分析

目的研究中国人多囊肾病基因1（polycystic kidney disease 1 gene，PKD1）突变的特点，检测基因突变位点。方法25例多囊肾患者，正常对照16名，扩增PKD1基因的第44、45外显子的基因片段，变性梯度凝胶电泳突变检测系统进行初筛，然后测序。结果发现1个移码突变（12431delCT）、1个无义突变（C12217T）、1个多态性（A50747C），突变检测率为8％（2／25）。结论检测到2个新的可能的致病突变：1个移码突变（12431delCT）、1个无义突变（C12217T）。     

常染色体显性多囊肾病家系的 PDK1基因变异检测与产前诊断

目的探讨8个多囊肾病家系的致病变异位点, 为常染色体显性多囊肾病(autosomal dominant polycystic kidney disease, ADPKD)的遗传咨询和产前诊断提供理论依据。方法应用全外显子组测序和高通量测序技术检测8个独立家系中先证者的PKD1、PKD2基因, 通过Sanger测序进行位点验证和家系分析, 结合多囊肾疾病数据库和蛋白变异预测软件进行生物信息学分析。结果检测出8个PDK1变异, 包括5个无义变异和3个错义变异。其中4个无义变异PDK1:c.7555C>T, c.7288C>T, c.4957C>T和c.11423G>A已报道为ADPKD的致病变异, 1个错义变异PDK1:c.2180T>G(p.Leu727Arg)报道为可能致病的变异;3个变异位点未见报道, c.6781G>T(p.Glu2261*), c.109T>G(p.Cys37Gly), c.8495A>G(p.Asn2832Ser), 其中无义变异PDK1 c.6781G>T(p.Glu2261*)为致病变异, 错义变异PDK...     

常染色体显性遗传性多囊肾病临床及基因突变分析

目的 分析常染色体显性遗传性多囊肾病（ADPKD）患者临床特征及基因突变特点。方法 入选ADPKD患者23例,收集临床数据,并进行家系调查;抽取外周血经高通量测序方法进行多囊肾基因检测。结果 23例ADPKD患者主要临床表现为腰腹痛、血尿、感染,肾功能不全;与女性患者相比,男性ADPKD患者血尿酸水平明显增高;基因检测PKD1基因突变19例;PKD2基因突变4例。同处于慢性肾脏病（CKD）5期的ADPKD患者,PKD1基因突变患者血红蛋白明显低于PKD2基因突变患者（65.89±13.59 vs 97.5±17.02,P<0.01）。结论 ADPKD可进展至肾功能衰竭,基因检测有助于早期诊断和预后评估,终末期ADPKD患者,PKD1基因突变患者预后更差。     

常染色体显性成人多囊肾病两家系PKD1、PKD2基因的突变鉴定

目的 鉴定两个常染色体显性成人多囊肾病家系的致病突变.方法 采用酚氯仿法提取家系成员及无亲缘关系的100名健康对照个体的外周血白细胞DNA,PCR扩增先证者致病基因PKD1、PKD2的所有外显子序列及其侧翼内含子剪切区域,直接测序确定DNA序列的变异.通过家系和正常对照的比较分析,对检测到的变异是否与疾病相关进行了初步探讨.结果 在两个家系中共检测到5个序列变异:PKD1:c.2469G＞A,PKD1:c.5014_5015 delAG,PKD1:c.10529C＞T,PKD2:c.568G＞A和PKD2:c.2020-1_2020 delAG.其中PKD1:c.2469G＞A和PKD2:c.2020-1_2020 delAG为新发现的变异.此外,检测到的移码突变和剪切突变未见于家系中健康成员及无亲缘关系的正常对照.结论 PKD1:c.5014_5015 delAG和PKD2:c.2020-1_2020 delAG分别为家系A和B的致病突变,且PKD2:c.2020-1_2020 delAG为先证者新发生的突变.     

常染色体显性遗传性多囊肾病的基因诊断及其临床应用

常染色体显性遗传性多囊肾 (autosomaldominantpoly cystickidneydisease ,ADPKD)是人类最常见的单基因遗传病之一 ,发病率约为 1‰[1] 。本病通过常染色体显性方式遗传 ,男女发病率相等 ,外显率几乎 10 0 %。其主要临床特征是双侧肾脏形成多个液性囊泡 ,囊肿进行性长大 ,造成肾脏结构和功能的损害 ,最终可导致终末期肾衰。除肾脏外 ,还常伴有肝、胰、脑、心等多个器官受累。ADPKD属延迟显性遗传 ,一般在 4 0岁左右才出现临床症状 ,得以诊断。而此时患者多已将致病基因传给下一代。ADPKD目前尚无有效治疗方法。因此 ,产前和症状前基因…     

常染色体显性多囊肾组织差异表达基因的初步研究

目的应用基因芯片技术及最新公共数据库，筛选常染色体显性多囊肾组织中差异表达的基因，对其进行功能分类，并对其中1条基因利用原位杂交技术进行验证。方法将代表8398条人类基因的PCR产物制成基因芯片。将等量的多囊肾组织和正常肾组织mRNA分别用Cy5、Cy3荧光标记，逆转录合成cDNA探针，混合后与上述基因芯片杂交。扫描杂交信号荧光强度，找出差异表达基因，对获得的基因进行分子生物信息学分析。并对其中的上调表达基因IGF1 mRNA进行原位杂交，验证基因芯片结果的准确性。结果（1）在进入研究的8398条基因中，共发现357条差异表达基因。94条基因在多囊肾组织中低表达，263条基因高表达；（2）上调表达基因主要属于原癌基因，细胞骨架蛋白和运动相关蛋白，凋亡相关蛋白，细胞信号和传递蛋白，细胞因子；下调表达基因主要属于抑癌基因，DNA结合、转录和转录因子，细胞信号和传递蛋白，参与代谢的基因；（3）IGF1 mRNA原位杂交结果与芯片结果一致。结论基因表达谱芯片可快速、高效地筛选差异表达基因；多囊肾病的发生、发展中存在着多种不同功能基因表达调控的改变。     

Cellular localization and tissue distribution of polycystin-1

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of fluid-filled cysts in both kidneys, in addition to a variety of extra-renal manifestations. The PKD1 gene product, polycystin-1, encodes a novel protein with a putative role in cell–cell/cell–matrix interactions. The present study we focused on the (sub)cellular localization of polycystin-1 in cultured cells, and on its tissue distribution in various organs. In Madin Darby canine kidney (MDCK) cells, several polyclonal antibodies showed intense staining at the sites of interaction between adjacent cells, which remained after Triton extraction. Weak cytoplasmic staining was observed. No signal was detected at the free borders of cell aggregates, supporting a role for polycystin-1 in cell–cell interactions. At the tissue level, polycystin-1 expression was observed in specific cell types in tissues with known manifestations of the disease, but also in tissues of organs which have not been reported to be affected in ADPKD. Expression was frequently seen in epithelia, but also in endocrine cells (pancreatic islets, parathyroid-producing cells, clusters in the adenohypophysis, clusters in the adrenal gland, and Leydig cells in the testis). In addition, expression was observed in myocardium and more weakly in myocytes of cardiac valves, of the cerebral arteries, and of skeletal muscles. Copyright © 1999 John Wiley & Sons, Ltd.     

两例常染色体显性多囊肾患者PKD1基因的突变检测

目的研究两例常染色体显性多囊肾患者的致病原因。方法对常染色体显性多囊肾患者的多囊肾病1基因(PKD1)3′端单拷贝区进行了聚合酶链反应-变性高效液相色谱(PCR-denaturing high-per-formance liquid chromatography,DHPLC)分析,并对有异常峰形的PCR产物进行测序。结果在1例患者中发现第42外显子的C11901A有一个无义突变,导致原丝氨酸3897变为终止密码子;而另一例患者第35外显子的C10737T有一个错义突变,导致原苏氨酸3509变为甲硫氨酸。在正常对照中发现两种同义突变分别为第42外显子的G11824A及C11860T。结论用DHPLC和DNA测序方法对两名患者进行PKD1的突变检测中,发现一个新的无义突变、一个错义突变以及两种同义突变。     

Autosomal dominant polycystic kidney disease: comprehensive mutation analysis of PKD1 and PKD2 in 700 unrelated patients

Autosomal dominant polycystic kidney disease (ADPKD), the most common inherited kidney disorder, is caused by mutations in PKD1 or PKD2. The molecular diagnosis of ADPKD is complicated by extensive allelic heterogeneity and particularly by the presence of six highly homologous sequences of PKD1 exons 1-33. Here, we screened PKD1 and PKD2 for both conventional mutations and gross genomic rearrangements in up to 700 unrelated ADPKD patients--the largest patient cohort to date--by means of direct sequencing, followed by quantitative fluorescent multiplex polymerase chain reaction or array-comparative genomic hybridization. This resulted in the identification of the largest number of new pathogenic mutations (n = 351) in a single publication, expanded the spectrum of known ADPKD pathogenic mutations by 41.8% for PKD1 and by 23.8% for PKD2, and provided new insights into several issues, such as the population-dependent distribution of recurrent mutations compared with founder mutations and the relative paucity of pathogenic missense mutations in the PKD2 gene. Our study, together with others, highlights the importance of developing novel approaches for both mutation detection and functional validation of nondefinite pathogenic mutations to increase the diagnostic value of molecular testing for ADPKD.     

Primary carcinoid tumor in a polycystic kidney

A case of a primary carcinoid tumor within a polycystic kidney is reported. A 51-year-old woman with autosomal dominant polycystic kidney disease (ADPKD) underwent a bilateral nephrectomy because of an infection in her polycystic kidneys. A tumor (1.5 cm in diameter) was incidentally found close to the hilum of the left kidney. Histologically, the tumor exhibited a predominantly trabecular pattern of cuboidal or columnar cells. Grimelius staining showed numerous silver-stained neurosecretory granules in most of the tumor cells; the tumor cells were also positive for chromogranin, synaptophysin, prostatic acid phosphatase (PAP) and neuron-specific enolase (NSE). The tumor was diagnosed as a primary carcinoid tumor of the kidney. Primary renal carcinoid is a very rare neoplasm, and this is the first report of such a lesion arising in a polycystic kidney.     

PGD for autosomal dominant polycystic kidney disease type 1

Autosomal dominant polycystic kidney disease (ADPKD) is primarily characterized by renal cysts and progression to renal failure. It is a genetically heterogeneous disease, with mutations in the PKD1 gene accounting for the majority of cases. Direct mutation detection for PKD1-linked ADPKD or type 1 is complicated by the large size and complex genomic structure of PKD1. This paper describes a microsatellite marker-based assay for PGD in couples at risk of transmitting ADPKD type 1. During PGD, genetic analysis is carried out on single blastomeres biopsied from preimplantation embryos obtained after IVF, and only embryos unaffected by the disease under investigation are selected for transfer. Single-cell genetic analysis relied on a fluorescent duplex-PCR of linked polymorphic markers followed by fragment length determination on an automated sequencer. The co-amplification of the intragenic KG8 and the extragenic D16S291 marker at the single-cell level was evaluated in pre-clinical tests on lymphoblasts and research blastomeres. The developed assay proved to be efficient (96.1% amplification) and accurate (1.4% allele drop-out and 4.3% contamination), and can be applied in all informative ADPKD type 1 couples. From five clinical cycles carried out for three couples, two pregnancies ensued, resulting in the birth of two healthy children.