| 摘要: | 本實驗為了瞭解FOXO轉錄蛋白FOXO1、FOXO3、FOCO4及FOXO6在脂肪細胞的生理機制所扮演的角色，我們篩選大量表現FOXO基因的NIH3T3、3T3-L1及C3H10T1/2纖維母細胞之穩定細胞株。利用轉染作用將架構在pMSCV-neo載體的FOXOs基因轉染至GP+E-86反轉錄病毒細胞，再利用G418 (600 μl/ml)抗生素篩選兩週。將此細胞株所分泌的反轉錄病毒培養液置換至NIH3T3、3T3-L1及C3H10T1/2細胞培養液進行感染作用2天，接著利用G418 (600 ~ 800 μl/ml)抗生素篩選2週，然後以反轉錄聚合酶鏈鎖反應及西方墨點法確定FOXOs基因分別在三種細胞內的表現情況。以前者方法已確認在NIH3T3、3T3-L1及C3H10T1/2纖維母細胞篩選出有表達以下野生型及突變型FOXO轉錄因子野生型的human FOXO1 ( hFOXO1-WT)、mouse FoxO3 ( mFoxO3-WT)、mouse FoxO4 ( mFoxO4-WT)及mouse FoxO6 ( mFoxO6-WT)；持續活化突變型human FOXO1-AAA ( hFOXO1-AAA)、human FOXO3-AAA ( hFOXO3-AAA)、human FOXO4-AAA-FLAG ( hFOXO4-AAA-FLAG)及mouse FoxO6-S184A ( mFoxO6-S184A)；以及一個DNA結合位變異突變型的human FOXO1-H215R ( hFOXO1-H215R)。在NIH3T3細胞，表現hFOXO1-WT、hFOXO1-AAA、mFoxO3-WT、hFOXO3-AAA、mFoxO4-WT、mFoxO6-WT或mFoxO6-S184A會抑制細胞生長。在3T3-L1前脂肪細胞，表現hFOXO1-WT、mFoxO3-WT或hFOXO3-AAA會抑制細胞生長，而mFoxO4-WT或mFoxO6-WT則是增加細胞生長。在C3H10T1/2前脂肪細胞，表現hFOXO1-WT、hFOXO1-AAA、hFOXO1-H215R、mFoxO4-WT或mFoxO6-WT亦會抑制細胞生長，但表現mFoxO3-WT, mFoxO4-WT或mFoxO6-S184A則是增加細胞生長速度。這些穩定細胞株的細胞形態分別與其正常細胞的細胞形態以顯微鏡檢視並沒有太大的差異。利用RT-PCR，我們觀察到resistin, adiponectin及aP2基因在大量表現FOXO的NIH3T3, 3T3-L1及C3H10T1/2細胞株與表現在pMSCV-neo空載體的細胞的mRNA表現量有差異。由於NIH3T3、3T3-L1及C3H10T1/2纖維母細胞可以分化成脂肪細胞，故本實驗結果顯示：不同的FOXO轉錄因子對於可分化的纖維母細胞的生長及脂肪激素基因的表現兩方面所扮演的生理及生化方面的角色可能是不同的。 To fully understand the physiological and biochemical roles of the forkhead transcription factors FOXO1, FOXO3, FOXO4, and FOXO6 in fat cells, we stably cloned NIH3T3, 3T3-L1 and C3H10T1/2 fibroblasts with overexpression of the respective FOXO gene. Different FOXO cDNAs inserted into the pMSCV-neo vector (provided by Professor Shen-Liang Chen) were respectively transfected into murine GP+E-86 retrovirus package cells and then selected with G418 (600 μg/ml) for 2 weeks. The harvested retrovirus containing the inserted FOXO gene from the culture medium was directly transferred to NIH3T3, 3T3-L1, or C3H10T1/2 cells and G418 (600 ~ 800 μg/ml) was added to the medium 2 d after the initial infection. After an additional 2-week selection with G418, total RNA isolated from the stable clones was verified with the expression levels of FOXO gene using the methods of RT-PCR and Western blotting. We successfully cloned the NIH3T3, 3T3-L1, and C3H10T1/2 fibroblasts overexpressing with the following wild types and mutants of FOXO transcription factors: the wild types of human FOXO1 (hFOXO1-WT), mouse FoxO3 (mFoxO3-WT), mouse FoxO4 (mFoxO4-WT) , and mouse FoxO6 (mFoxO6-WT); the constitutively active mutants of human FOXO1-AAA (hFOXO1-AAA), human FOXO3-AAA (hFOXO3-AAA), human FOXO4-AAA (hFOXO4-AAA), and mouse FoxO6-S184A (mFoxO6-S184A); and a DNA binding-deficient type of human FOXO1 (hFOXO1-H215R). In NIH3T3 cells, expression with hFOXO1-WT, hFOXO1-AAA, mFoxO3-WT, hFOXO3-AAA, mFoxO6-WT, or mFoxO6-S184A reduced the cell number during a 5-day period of incubation. In 3T3-L1 preadipocytes, expression with either hFOXO1-WT、mFoxO3-WT or hFOXO3-AAA inhibited cell growth, while expression with mFOXO4-WT, or mFoxO6-WT stimulated cell growth. In C3H10T1/2 preadipocytes, expression with hFOXO1-WT, hFOXO1-AAA, hFOXO1-H215R, mFoxO4-WT, or mFoxO6-WT inhibited cell growth, while expression with either mFoxO3-WT, mFoxO4-AAA-FLAG or mFoxO6-S184A stimulated cell growth. The morphology of each clone and parental cell was observed and photographed with no significant change. Using RT-PCR, we further observed that levels of adipogenic resistin, adiponectin, and aP2 mRNAs were altered in the FOXO1-overexpressed NIH3T3, 3T3-L1 and C3H10T1/2 cells when compared to those in the empty pMSCV-neo vector-transfected cells. As NIH3T3, 3T3-L1 and C3H10T1/2 fibroblasts can be differentiated into adipocytes, results of this study suggest the different physiological and biochemical roles of the distinct FOXO transcription factors on preadipocyte growth and adipocytokine gene expression. |
