急性前骨髓性白血病，是一種造血組織的惡性疾病，其治病原因乃骨髓性前趨細胞不正常的大量增生，並有細胞分化停止的病變。在之前的實驗利用劉氏染色、cDNA microarry、RT-PCR、NBT還原實驗、流式細胞儀、西方墨點法等實驗得知，全反式維生素甲酸 (all-trans retinoic acid)誘導U937細胞分化成巨噬細胞時，BNip3蛋白質的表現量明顯增加，而且把BNip3 antisense送入細胞中以RT-PCR與NBT還原實驗等證實抑制BNip3蛋白質的表現會造成細胞分化的停止，但經由流式細胞儀偵測CD11b的表現量結果不具一致性，所以我們必須確認抑制BNip3蛋白質的表現時是否真的會造成細胞分化能力下降或當BNip3蛋白質的表現上升時是否會促進細胞的分化。本研究利用電穿孔（electroporation）的方式把BNip3 antisense或BNip3表現的載體（BNip3 expression vector）送入細胞內，藉由流式細胞儀偵測細胞表面CD11b的表現量與西方墨點法偵測BNip3蛋白質的表現，結果顯示出BNip3 antisense會抑制全反式維生素甲酸誘導U937細胞分化，與當送入BNip3表現的載體表達BNip3蛋白質時會造成細胞分化。此外我們為了瞭解在全反式維生素甲酸誘導U937細胞分化中有哪些轉錄因子在調控BNip3基因的表現，我們利用電穿孔的方式，將含有luciferase基因（reporter gene）的不同長度BNip3啟動子載體轉染（transfect）到U937細胞中，以觀察在全反式維生素甲酸刺激下BNip3啟動子的活性表現。結果顯示以全反式維生素甲酸誘導U937細胞分化時，BNip3啟動子 (promoter) 被兩個轉錄因子所調控，其中MZF-1轉錄因子，其為BNip3 啟動子的活化子 (activactor)，但我們利用gel retardation的方式，觀察MZF-1轉錄因子與MZ-1探針結合的情形，沒有明確的結果，有待進一步的實驗探討。另一個轉錄因子為AML-1α，其有可能為BNip3 啟動子的抑制子 (repressor)，但也須進一步的實驗確認。由我們的實驗結果得到，當全反式維生素甲酸誘導U937細胞分化時會引起MZF-1與AML-1α轉錄因子產生，這兩個轉錄因子調控BNip3的蛋白表現，以及BNip3蛋白的表現會誘導U937細胞分化。 Acute myelocytic leukemia (AML) is a malignant neoplasm of hematopoietic cells characterized is an abnormal proliferation of myeloid precursor cells. Previous study showed that the BNip3 gene expression of ATRA-induced U937 cell differentiation was detected in cDNA, RT-PCR, and western blot. BNip3 antisense could inhibit the BNip3 gene expression, but could not be confirmed that it inhibited U937 cell differentiation of flow cytometry assay. We investigated whether BNip3 affects differentiation of U937 cell by flow cytometry assay, and found out that the possible BNip3 promoter regions involved in the ATRA-mediated action on this gene. In this study, the result by using flow cytometry assay, we found BNip3 antisense inhibited the ATRA-induced U937 differetiation and overexpression of BNip3 induced U937 differetiation. This indicated that BNip3 plays an important role in cell differentiation. Moreover, we analyzed the activity of progressive deletions of BNip3 promoter by transient trnasfection in U937 cells. The results of the luciferase assay showed that MZF-1 (myeloid zinc finger gene-1) is an ativator of BNip3 promoter and AML-1α (acute myeloid leukemia-1α) is a good candidate of repressor in the ATRA-induced U937 differentiation. To investigate the mechanism involved in ATRA-mediated increase in MZF-1 transcriptional activity, gel retardation assay was performed on nuclear extracts prepared form U937 cells treated with ATRA. The gel retardation assay showed that there were proteins to bind probes, but we could not confirm that these bound proteins were specific. Therefore, further studies are required to understand the transcription regulation mechanism in this region. Take together, these data suggest that MZF-1 and AML-1α induction of BNip3 may play crucial role during ATRA-induced U937 differentiation.
