系統識別號 U0026-0309201818595900
論文名稱(中文) RASIP1在心血管細胞分化的調節機制
論文名稱(英文) Regulatory role of RASIP1 in cardiovascular cell differentiation
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Institute of Clinical Medicine
學年度 106
學期 2
出版年 107
研究生(中文) 孫惟政
研究生(英文) Wei-Cheng Sun
學號 S96051079
學位類別 碩士
語文別 英文
論文頁數 55頁
口試委員 指導教授-陳芃潔
中文關鍵字 努南氏症  RASIP1  CRISPR  誘導性多功能幹細胞 
英文關鍵字 Noonan syndrome  RASIP1  CRISPR  iPSCs 
中文摘要 努南氏症(Noonan syndrome, NS)是一種體染色體顯性和遺傳異質性疾病,其特徵有身材矮小,發育遲緩和先天性心臟異常。我們已經透過外顯子體定序在努南氏症的病人裡找到 RASIP1 的突變。努南氏症病人帶有 RASIP1 的突變會有較少見的心臟缺陷表現型,像是主動脈根部擴張症、主動脈縮窄、大動脈瘤。這些表現型有可能只發生在帶有 RASIP1 突變的努南氏症病人身上。為了研究 RASIP1 突變而導致努南氏症的分子機制,我們的目標在透過基因組編輯產生帶有努南氏症 RASIP1 突變誘導性多功能幹細胞(iPS細胞)。因為iPS細胞有能力分化成多種心血管細胞,因此我們可以透過iPS細胞的分化來研究突變在功能上的影響。先前的研究已經成功的運用群聚且有規律間隔的短迴文重複序列(CRISPR)系統在iPS細胞上。然而 CRISPR 系統在iPS細胞所造成的基因敲入之效率只有2~3%。在這個研究裡,我目標在改善iPS細胞同源性定向修復(Homology directed repair, HDR)的頻率並產生帶有病人突變。我用RNA定序的方式在iPS細胞建立雙股DNA斷裂相關基因的表現量檔案。此外也做出了雙股DNA模板並送到人胚胎腎細胞293(Human Embryonic Kidney Cells 293)和iPS細胞,也比較了單股模板和雙股模板HDR的效率。此外,我用了RASIP1敲除的iPS細胞分化到不同心血管細胞,發現RASIP1敲除的iPS細胞分化能力和功能下降,磷酸化的GSK3β表現量也下降。
英文摘要 Noonan syndrome (NS), an autosomal dominant and genetically heterogeneous disorder, is characterized by short stature, developmental delay and congenital heart diseases. Previously, we identified candidate NS-associated variants in RASIP1 through exome sequencing. Patients carrying RASIP1 variants showed atypical cardiac phenotypes for NS, such as dilation of aortic root, coarctation of aorta and aortic aneurysms, which raise the possibility that these cardiac phenotypes occurred specifically to NS patients carrying RASIP1. To investigate the molecular mechanisms through which RASIP1 mutations lead to NS, we aim to generate cell models that carry patient-specific RASIP1 mutations through genome editing in the induced pluripotent stem cells (iPSCs). Because iPSCs have the ability to differentiate into many cell lineages of cardiovascular cells, we can study the impact of mutations on the function of cardiovascular cells. Previous studies have successfully utilized CRISPR system in iPSCs, however, the efficiency of CRISPR/Cas9 mediated knock-in was only 2-3% in iPSCs. In this study, I aim to improve the homology-direct repair (HDR) rates in iPSCs and generate iPSCs carrying patient-specific mutations. I used RNA-sequencing (RNA-seq) to establish double strand break-related gene profile in iPSCs. I also generated a reporter system in iPSCs. In addition, I compared the HDR rate in cells supplied with long dsDNA donor and that with single strand oligodeoxy nucleotides. Moreover, I differentiated RASIP1 knock-out (RKO) iPSCs into cardiac cell lineages and found that the differentiation ability of RKO iPSCs was decreased. The ability of forming plexus like vascular structures was also decrease. Furthermore, results of RNA-seq suggested that several signaling pathways were altered in RKO cells. Lastly, I found that phosphorylated-GSK3β level was altered in RKO cells. Taken together, these results revealed the possible mechanisms through which RASIP1 mutation affect the cardiovascular cell differentiation and function.
論文目次 abstract 1~2
摘要 3
致謝 4
Contents 5~6
Introduction 7
Noonan syndrome 8
Congenital heart diseases in NS 9
To investigate cardiac defects in cell models 10
Genome editing via CRISPR system 11
The challenge of CRIPSR-mediated genome editing 12
Materials and Methods 14
Culture of cell 15
CRIPSR plasmid 15
dsDNA donor construction 16
Transfection and electroporation 16
Genomic DNA preparation, PCR and NGS sequencing 17
The differentiation of iPS cells 17
RNA library preparation 18
Flow cytometry 18
Real-time PCR 18
Western blot 19
Results 20
Establish the DSB repair related-gene profile in iPSCs 21
Establish the traffic light reporter iPSCs 21
Investigate HDR efficiency in cells with knock down of NHEJ-related gene 22
Construction of long dsDNA donor 22
Comparing the HDR efficiency between the ssODN and dsDNA donor 23
The differentiation of CRISPR engineered iPSCs to mesodermal cells 24
The differentiation of CRISPR engineered iPSCs to endothelial cells 25
The differentiation of CRISPR engineered iPSCs to ectodermal cells 25
RASIP1 mutation alters signaling pathways in the cell 26
Discussion 27~31
Figures 32~46
Tables 47~51
References 52~56
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