Establishment of segawa disease specific iPS cells and use to create a drug screening platform

• Main Authors: Yu-Chen Lin, 林毓貞
• Other Authors: 劉詩平
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/zr57j7

碩士 === 中國醫藥大學 === 生物醫學研究所碩士班 === 106 === Segawa disease is a rare disorder caused by GTP cyclohydrolase 1 (GCH1) gene mutation presenting gait disturbance and dystonia. The clinical symptoms like Parkinson''s disease. Segawa disease lacked effective therapeutic methods. The traditional drug is Levodopa but could not rescue Segawa disease. In all type of stem cells, induced pluripotent stem (iPS) cells are the most powerful cells that could differentiate into all three-layer cells, including neural stem cells and with less ethical issues. iPS cell technology is possible to use patient’s own somatic cells to generate patient’s specific iPS cells. In our study, we generated a Seagawa disease specific iPS cells with GCH1 gene mutation and we will use these iPS cells for drug screening to find out a novel pure compound that could increase dopamine secretion. First, we isolated the human fibroblast cells from Segawa disease patient with GCH1 mutation and reprogrammed into iPS cells by using sendai virus infection. The characterization results showed that the morphology of iPS-GCH1 cell colonies are similar to embryonic stem cells. Also, we demonstrated the pluripotent markers , the results shows positive expressed in iPS-GCH1 cells by immunofluorescent staining (IFA), RT-PCR, and flow cytometry. Embryonic bodies (EB) formation was used to detect the three germ layers differentiate ability of iPS-GCH1 cells by TUJ1, SMA, and GATA4 staining . Taken together, we generated a novel iPS-GCH1 cell line with GCH1 mutation and characterized this iPS-GCH1 cell line as a pluripotenty stem cells. In addition, we find out a pure compound that could increase dopamine secretion in SH-SY5Y cells. These study hope to figure out a new therapeutic method for Segawa disease treatment.