Tafazzin基因剔除和修復對粒線體心磷脂的變異分析

Tunghai University Institutional Repository > 理學院 > 化學系所 > 碩博士論文 > Item 310901/29949

Please use this identifier to cite or link to this item: http://140.128.103.80:8080/handle/310901/29949

Title:	Tafazzin基因剔除和修復對粒線體心磷脂的變異分析
Other Titles:	The analysis of tafazzin knockout and restoration on mitochondrial cardiolipin
Authors:	朱怡 CHU,I
Contributors:	許員豪 HSU,YUAN-HAO 化學系
Keywords:	心磷脂 Tafazzin;Cardiolipin;Hap1 cell
Date:	2018
Issue Date:	2018-03-30T01:08:01Z (UTC)
Abstract:	巴氏綜合症（Barth syndrome）為一種心磷脂（Cardiolipin，CL）異常造成的粒線體相關疾病。現今使用多種細胞模型作為研究，然而細胞粒線體機制上的研究尚不明確。本實驗使用Hap1人類類纖維單倍染色體細胞，將細胞中TAZ（Tafazzin）剔除作為ΔTAZ細胞模型。我們利用電子顯微鏡觀察發現細胞粒線體形狀產生泡狀變異。當以海馬能量測定細胞基礎耗氧率發現Hap1ΔTAZ細胞粒線體功能降低。利用質譜鑑定CL與單水解心磷脂monolyso-Cardiolipin （MLCL）組成，發現ΔTAZ細胞CL合成含量降低而MLCL含量增加，且CL組成也產生變異。TAZ剔除後，細胞中CL68:2及CL70:3明顯提高。不同基因調控影響CL組成，定量ΔTAZ細胞mRNA表現量磷脂醯甘油合成酶PGS1（Phosphatidylglycerophosphate synthase 1）降低，導致ΔTAZ細胞無法足夠合成PG以供CL合成。CL水解相關基因PLA2G6 （Phospholipase A2 group VI, transcript variant 2）和PNPLA8（Patatin like phospholipase domain containing 8）表達量增加導致ΔTAZ細胞MLCL累積。接著我們嘗試將TAZ基因轉染到ΔTAZ細胞，以測試其基因剔除後的恢復狀態。使用質譜儀觀察CL70:3與CL70:2和CL72:4與CL72:3含量皆減少。證實TAZ醯基轉移酶恢復重塑CL，將飽和CL連接醯基鏈置換成不飽和的(18:1)醯基鏈，但也使飽和心磷脂總含量降低。定量粒線體相關蛋白mRNA表現量後，發現CL合成基因CRLS1 （Cardiolipin synthase）增加，可見CL的缺乏已刺激細胞合成新CL。因為我們發現TAZ基因剔除導致PG合成酶表達下降，PG合成降低，因此添加PG(18:1)2作為CL合成的原料。鑑定細胞CL與MLCL組成發現由18:1醯基鏈的CL含量皆增加，證實PG(18:1)2參與CL合成。水解CL基因PLA2G6與PNPLA8基因表達量皆降低，為CL含量增加之因素。 Barth syndrome (BTHS) can cause the abnormality of cardiolipin (CL). Even though there are multiple cellular models available, the detailed mechanism is still not clear. Thehuman fibroblast haploid cells (Hap1 cell) were selected as our cellular model. By applying mass spectrometry (MS) to identify the components of CL and monolyso-Cardiolipin (MLCL), we discovered that CL synthesis decreased, while MLCL synthesis increased in the Hap1ΔTAZ cell. CL68:2and CL70:3 have significant increase in total CL. By quantifying the mRNA expression of Hap1ΔTAZ cell, we found the gene expression of PGS1 (phosphatidylglycerophosphate synthase 1) decreased, indicating that the ΔTAZ cell might not have enough PG for CL synthesis. Upon the increasof the expression of CL hydrolysis related genes, PLA2G6 (phospholipase A2 group VI, transcript variant 2) and PNPLA8 (patatin like phospholipase domain containing 8) showed a cumulative increase in MLCL in the ΔTAZ cell. When we attempted to transfect TAZ gene into ΔTAZ cell to recover cell, the quantity of CL70:3, CL70:2, CL72:4 and CL72:3 were all decreased. We discovered that the gene expression of CRLS1 (cardiolipin synthase) increased, which can stimulat CL synthsis. When TAZ was knocked out, the expression of PG synthase decreased. Therefore, adding 50μM of PG (18:1)2 can stimulate CL synthesis. We discovered that the CL content of the 18:1 acyl chains and total CL quantity increased, confirming PG (18:1)2 was involved in CL synthesis. Gene down-regulation of both PLA2G6 and PNPLA8, which decreased the overall hydrolysis of CL and can be the main reason for the increase of CL concentration.
Appears in Collections:	[化學系所] 碩博士論文

Files in This Item:

File	Description	Size	Format
106THU00065007-001.pdf		3016Kb	Adobe PDF	62	View/Open

Loading...