探討Laminin-γ3蛋白質於遺傳性體染色體顯性多囊性腎臟疾病老鼠之異常表現

成人多囊性腎臟疾病（Autosomal Dominant Polycystic Kidney Disease；ADPKD）是一種盛行率約1/1000遺傳性體染色體顯性腎病變，且於所有慢性腎衰竭病患中約占7-10％。由文獻知於美國約有50萬個患者，而於全世界則約有4百萬至6百萬個患者。ADPKD的致病基因目前熟知的有兩型︰第一類型PKD1 gene，85-90％的病患是屬於第一類型；第二類型則為PKD2 gene。PKD1 gene可表現出polycystin-1(pkd-1) 蛋白質，此蛋白在腎臟上皮細胞分佈於三個不同位置上︰細胞基底層、細胞與細胞連接處及細胞頂端絨毛等處，其主要作用是與細胞內液體分泌和吸收有關進而調控細胞的生理功能及活動。由組織切片中觀察到的資料也顯示出腎囊腫上皮細胞相當容易與下方相連的間質組織形成分離。且由吾人之前的互補核酸陣列的研究結果也知laminin γ3蛋白質於4周齡純合子基因結合ADPKD疾病實驗老鼠的腎臟組織會呈現增強的現象。層黏連蛋白（Laminin）是屬於細胞外基質之一，並由三種型態的異構物所組成（α、β、γ）；laminin γ3蛋白質是其中一個異構物，此蛋白質的分佈主要是於皮膚、眼睛、心臟、肺臟與生殖系統，並且Champliaud等學人於1999年曾提及laminin γ3 可表現於具有纖毛組織的上皮細胞的基頂端以及成熟老鼠的腎臟上皮細胞之基底部。為此吾人的實驗計畫將利用已建立好的ADPKD疾病模式實驗老鼠來進行細胞外間質組織的相關研究，且我們的研究標地將置於laminin中的laminin γ3蛋白質於ADPKD疾病的表現分析。因此我們的目的將是利用組織免疫染色與西方墨點實驗來探討：1）laminin γ3於野生型、異合子基因型與純合子基因的表現量差異；2）laminin γ3於不同週齡ADPKD疾病實驗老鼠的發育表現；3）探討laminin γ3與polycystin-1的相關性。在實驗結果中吾人證實了laminin γ3蛋白質於不同週齡（1天、7天、14天、30天）的純合子基因型(2 allele－homozygous；－/－)ADPKD疾病實驗老鼠之腎小管上皮細胞的表現量的確有增強表現，而其表現位置會由原本主要的基底層與細胞質內表現轉為管腔側的細胞膜表現為主；另於雙重組織免疫螢光染色的結果中，吾人也證實了laminin γ3蛋白質會表現於pkd-1 蛋白質表現的位置。所以吾人證實了laminin γ3於實驗老鼠的腎臟組織的表現量的確有所增強，且此表現於異合子基因型初期只見位置的改變而至後期才有明顯量的改變同時伴隨表現位置之差異。然而其表現雖可與pkd-1位於同一位置，但似乎是發生於pkd-1蛋白質的變化之前。因此吾人也可推測laminin γ3蛋白質於ADPKD疾病的致病機轉應屬有關，且未來其相關的生理病理意義也將是吾人繼續探討的一個重要方向。

關鍵字

多囊腎；細胞外基質

並列摘要

Autosomal dominant polycystic kidney disease (ADPKD) has an incidence of 1 in 1000 live births. It is the most common life-threatening inherited cystic kidney diseases characterized by the development of gradually enlarging renal cysts and a progressive loss of normal renal tissue that can lead to chronic renal failure and hemodialysis. Finding of PKD1 and PKD2 genes were major milestones in studying of this specific kidney disease. ADPKD is recognized as a monogenic disorder caused by mutation in two genes, PKD1, accounting for approximately 85% - 90％ of cases, and PKD2, accounting for approximately 10% of cases. Polycystin-1（pkd-1） and polycystin-2（pkd-2） are the proteins of these two genes. Polycystin-1 is a large (>460 KD) membrane protein of about 4300 amino acids with 11 transmembrane domains. Its extensive extracellular N terminus contains a number of adhesive domains that implicate polycystin-1 in cell-cell and cell-matrix interactions. Their functions include cell proliferation, regulation of cell survival, altered tubular cell polarity, fluid secretion, and reaction with extracellular matrix. In our preliminary data, renal tubular cystic epithelium was noted to have a tendency to detach from extracellular matrix. Meanwhile, in renal tissue of cDNA microarray analysis, laminin γ3 upregulated also was noted. About this reason, our purposes will focus on the role of laminin γ3 , element of ECM, in the developmental process of renal cystic formation and the interaction between extracellular matrix abnormalities and PKD disease. That utilizes critical information gained from this genetically manipulated mouse models for the purpose of aberrant expression of laminin γ3 . Overall, in homozygous mutant, the distribution aberrant of laminin γ3 translocated from basement to apical side at day 1 and the expression of laminin γ3 was increased at day 30. Meanwhile, the expression of laminin γ3 and polycystin-1 protein is colocalized in either cytoplasm or apical side of congenital ADPKD mice.

並列關鍵字

ADPKD ； Laminin-γ3 ； ECM

參考文獻

1. Brasier, J.L., Henske, E.P. (1997). Loss of the polycystic kidney disease (PKD1) region of chromosome 16p13 in renal cyst cells supports a loss-of-function model for cyst pathogenesis. J. Clin. Invest. 99, 194-199

2. Delmas, P., Nomura, H., Li, X., Lakkis, M., Luo, Y., Segal, Y., Fernández-Fernández, J.M., Harris, P., Frischauf, A., Brown, D.A., and Zhou, J. (2002). Constitutive Activation of G-proteins by Polycystin-1 Is Antagonized by Polycystin-2. J. Biol. Chem. 277, 11276-11283

3. Dorien J. M. Peters, et al. （1999）. Cellular localization and tissue distribution of polycystin-1. J. Pathol. 118: 439-446

5. Jiang, S.T., Chiou, Y.Y., Wang, E., Lin, H.K., Lin, S.G., Lin, Y.T., Wang, C.K.L., Tang, M.J., Li, H. (2006). Defining a Link with Autosomal Dominant Polycystic Kidney Disease in Mice Congenitally Low Expression of Pkd1 Am J Pathol

6. Koch M, Olson PF, Albus A, Jin W, Hunter DD, Brunken WJ, Burgeson RE, Champliaud MF (1999). Characterization and expression of the laminin gamma3 chain: a novel, non-basement membrane-associated, laminin chain. Journal of Cell Biology 145:605-618

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探討Laminin-γ3蛋白質於遺傳性體染色體顯性多囊性腎臟疾病老鼠之異常表現

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