臺灣博碩士論文加值系統

為了瞭解在脂肪細胞分化過程中基因表現的改變，本研究室先前利用抑制性扣除雜合法（suppression subtractive hybridization, SSH）得到了6個在脂肪細胞的表現遠高於在脂肪前身細胞的基因。這6個基因中，有2個是身分未知的基因。本試驗利用快速增殖cDNA尾端法（rapid amplified cDNA ends, RACE）發現，這兩個基因都為血管收縮素轉換酶2（angiotensin converting enzyme 2, ACE2），由於豬ACE2基因序列全長並未被發表，本試驗利用已發表物種之序列設計引子，最後得到豬全長ACE2 cDNA序列。
多元不飽和脂肪酸（polyunsaturated fatty acid, PUFA）可被代謝成產物二十碳酸，它們具有調節血壓的功能，此外許多二十碳酸也能影響脂肪細胞的分化，所以我們假設PUFA也能調節同樣能影響血壓的ACE2基因表現。因此我們將取自藍瑞斯豬的脂肪前身細胞分化四天後，以50 μM 的共軛亞麻油酸（conjugted linoleic acid, CLA）、花生四烯酸（arachidonic acid, AA）、二十碳五烯酸（eicosapentaenoic acid, EPA）、二十二碳六烯酸（docosahexaenoic acid, DHA）或硬脂酸（stearic acid, 18:0）處理48小時，發現EPA與18:0不能改變ACE2 mRNA的表現，然而AA、CLA、DHA則會顯著減少ACE2 mRNA的表現。為進一步瞭解何種二十碳酸產物對ACE2有調控作用，我們利用n-6脂肪酸代謝酵素抑制劑clotrimazole、ETYA與indomethacin分別抑制 monooxygenase、 lipooxygenase 與 cyclooxygenase，結果發現添加三種抑制劑都可以恢復部份因AA而降低的ACE2表現量，可見這三類二十碳酸都可能參與了調控ACE2的表現。
為探討ACE2的功能，我們利用慢病毒載體過量表現ACE2於豬脂肪前身細胞，接著誘導分化三天，再分析脂肪細胞分化相關基因（CCATT/enhancer binding protein β、sterol regulatory element binding protein-1C與peroxisome proliferator-activated receptor γ）與脂肪分解相關基因（adipose triglyceride lipase and
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hormone sensitive lipase），結果發現過量表現ACE2並不能影響脂肪細胞分化早期相關基因與脂肪分解相關基因。
綜上所述，我們發現PUFA會透過它們的代謝產物來降低ACE2的基因表現，而ACE2對於分化早期基因與脂肪分解基因並無調控作用，所以ACE2在脂肪細胞中有無其他功能有待證實。

Using suppression subtractive hybridization technique, we found that two unkown genes (AEUG1 and AEUG3) were highly expressed in the adipocytes compared with preadipocytes. We then identified that these two genes were indicated to be angiotensin converting enzyme 2 (ACE2). We applied 3’RACE (rapid amplified of cDNA end), 5’RACE, and PCR based on primers from full length cDNA sequences of other species published on NCBI, to obtain the full length porcine ACE2 cDNA sequence.
Polyunsaturated fatty acids (PUFA) are precursors of 3 series of eicosanoids which are antiaggregators and vasoconstrictors. Because the functions of eicosanoids link PUFA to regulating blood pressure, we hypothesize that PUFA can regulate the expression of the vasodilator, ACE2. Preadipocytes from Landrace pigs were induced to differentiate for 4 days, then treated with 50 μM of different PUFA, conjugted linoleic acid (CLA), arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or stearic acid (18:0). Addition of either EPA or 18:0 for 48 h did not change the ACE2 mRNA level, whereas the treaments of AA、CLA and DHA significantly decreased ACE2 mRNA level after 48 h. To further understand how PUFA metabolites affect ACE2 mRNA expression, we inhibited individual enzymes that involved in the eicosanoid production. The inhibitors for monooxygenase, lipooxygenase, and cyclooxygenase are clotrimazole, ETYA, and indomethacin, respectively. We found all three inhibitors could recover ACE2 expression inhibition by AA treatment, suggesting that three eicosanoids are involved in regulating the expression of ACE2. To further investigate the function of ACE2, we enhanced ACE2 expression in porcine adipocyte by using lentivirus as a vector. After overexpression of ACE2 in preadipocytes, we induced preadipocytes to differentiate into adipocytes for 3 days, then analysed
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differentiation marker genes (CCATT/enhancer binding protein β、sterol regulatory element binding protein-1C and peroxisome proliferator-activated receptor γ) and lipolytic genes (adipose triglyceride lipase and hormone sensitive lipase). The results showed the expression of both differentiation marker genes and lipolytic genes were not modified by overxpression ACE2. Therefore ACE2 may not have a role in regulating early stage adipocyte differentiation and lipolysis.
In conclusion, we found PUFA could down-regulate the expression of ACE2 through its metabolites in porcine adipocytes. ACE2 may not have a function on modifying the expression of early stage adipocyte differentiation gene and lipolytic genes. Taken together, PUFA can down-regulate ACE2 gene expression but the physiological modification by which ACE2 mediating requires further demonstration.

頁次
目錄............................................................... i
中文摘要.......................................................... ii
英文摘要.......................................................... iv
圖次.............................................................. vi
表次............................................................ viii
壹、文獻檢討........................................................ 1
一、前言...................................................... 1
二、脂肪細胞的分化............................................ 2
三、脂肪細胞的分泌功能........................................ 3
四、n-3與n-6多元不飽和脂肪酸................................ 7
貳、材料與方法..................................................... 14
試驗一選殖豬之血管收縮素轉換酶2 cDNA序列全長................ 14
試驗二 多不飽和脂肪酸對脂肪細胞血管收縮素系統相關基因之調.... 20
試驗三 過度表達ACE2基因於脂肪前身細胞對分化之影響.......... 31
參、結果與討論....................................................... 43
試驗一選殖豬之血管收縮素轉換酶2 cDNA序列全長................. 43
試驗二 多不飽和脂肪酸對脂肪細胞血管收縮素系統相關基因之調控.. 50
試驗三 過度表達ACE2基因於脂肪前身細胞對分化之影響.......... 57
肆、結論........................................................... 63
伍、參考文獻....................................................... 64

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