本論文主要的研究在於人類的自體顯性遺傳之多囊腎疾病， 當
polycystin 1 蛋白具有正常功能時可作為調控腎小管新生作用的重
要中心，當異常時會造成自體多囊腎的形成。在許多的腎臟疾病
中囊泡的形成是普遍的特徵，而自體多囊腎疾病最主要是分自體
顯性遺傳之多囊腎疾病Autosomal Dominant Polycystic Kidney
Disease (ADPKD)， 這是一種屬於自體體染色體顯性遺傳之多囊
腎疾病，在自體染色體顯性多囊性腎臟疾病中，因為pkd1 基因突變所造
成的疾病約有85～90%的發生機率，pkd1 基因位於人類染色體16p13.3 的
位置(1, 2)，polycystin 1 蛋白是由pkd1 基因轉譯而成的膜蛋白，在細胞和細
胞間與細胞和基質間進行相互影響。本論文的研究是以miRNA 轉殖至集尿
管上皮細胞（M1）內，藉而調控polycystin 1 蛋白的表現，以建立低表現多
囊腎蛋白之細胞株，並觀察其細胞型態與功能性之表現。
首先，利用RNAi 的實驗技術將含有不同pkd1 基因位子的miRNA 載體
送入M1 細胞後，利用MTT 分析polycystin 1 knock-down 的轉殖 M1 細胞
後之增生作用。結果顯示將M1 細胞培養24 小時至120 小時polycystin 1
knock-down 細胞株中以6820 細胞株的增生作用具有顯著的意義。因此將
6820 細胞株利用螢光強弱以flow cytometry 進行細胞分選實驗，進而分選出
具較高螢光強度和較低螢光強度的兩株polycystin 1 knock-down 細胞株，
II
並分別命名為6820-High GFP 細胞株與6820-Low GFP 細胞株。利用RT-PCR
和real time PCR 的實驗證實polycystin 1 knock-down 的M1細胞株於mRNA
層次確實可被pkd 1 之miRNA 所抑制，若與wild type 相比較無論6820-High
GFP 或6820-Low GFP 細胞株的pkd 1 皆呈現有意義的抑制作用。
最後於功能性的實驗，利用所建立之polycystin 1 knock-down 細胞株進
行3D 立體基質培養基（3D Matrigel）之培養來進行養殖與觀察，將M1、
M1-GFP、6820-High GFP、6820-Low GFP 細胞株培養在Matrigel 中，發現
M1 細胞株與M1-GFP 具有腎小管新生作用的功能，而6820-High GFP 和
6820-Low GFP 細胞株皆有明顯的囊狀結構的形成。最後經由以上的結果吾
人可得知，我們已經成功建立polycystin 1 knock-down 的細胞株以及腎小管
與囊狀結構的形成，並能表現類似於組織中的增生異常與囊狀結構的形
成，未來也將可利用此細胞株進行更進一步的機制探討。本論文主要的研究在於人類的自體顯性遺傳... Our studies on human ADPKD have highlighted the central importance of polycystin 1 in normal developmen of tubulogenesis, and low expression of polycystin 1 would lead to cyst formation. Cyst formation is a common feature of many renal diseases and may be the results of primary genetic diseases, or secondary to hereditary syndromes that were related to the results of developmental abnormality. The most common and best understood autosomal dominant polycystic kidney disease (ADPKD). In a majority of cases (85–90%), the gene involved is pkd1 and encodes polycystin 1, a large receptor like integral membrane protein that contains several extracellular motifs indicative of cell–cell and cell–matrix interaction. In this study, we have established two polycystin 1 knock-down cell lines by miRNA method and they also showed some biological function as we saw in human ADPKD. Our results found that polycystin 1 knock-down cell miRNA 6820 exhibited significant effects on cell proliferation at 24h later to 120h cell cultures. According to the GFP fluorescence intensity, miRNA 6820 cells could be sorted to high fluorescence and low fluorescence of 6820 cell. Down regulation of polycystin 1 protein in transcriptional level were confirmed by RT-PCR and real time PCR investigation and these were parallel to the intensity of GFP fluorescence. Cysts formation were also presented in three-dimensional Matrigel culture system and low expression of polycystin 1 cell line would produce larger cysts than relative high expression of polycystin 1 cell line. These results provided the evidence that polycystin 1 did exhibit significant effect tubulogenesis and its’ knock-down cell would promote cystic formation. Finally, according to our results in vitro polycystin 1 knock-down cell lines were set up successful and their cellular biologic behaviors would present as human ADPKD and our previous polycystic kidney mouse model. Meanwhile, these cell lines will provide as a good in vitro cellular model to investigate further cellular mechanical signal pathway of cyst formation in future.
