柳松菇及鴻喜菇萃取物對人類白血病細胞株 (U937) 生長抑制於G0/G1期之機制

Abstract

菇類中包含了相當多元化的生理活性物質，具抗腫瘤、抑制癌細胞生長、提升免疫力、促進白血病細胞成熟分化等功能。本實驗室之前的研究，已證實柳松菇及鴻喜菇蛋白質萃取物具有誘導人類白血病細胞株U937分化為巨噬細胞及抑制其生長的能力。而在本實驗中，分別採用柳松菇及鴻喜菇硫酸銨沉澱物及其陰離子交換樹脂DEAE Sepharose CL-6B所得之區分物AA-0.1和HM-0.2對人類白血病細胞株U937進行生長抑制測試，及探討對其細胞週期造成的變化和細胞週期調控因子之影響機制。 實驗結果顯示，經DEAE Sepharose CL-6B分離出之AA-0.1及HM-0.2以濃度25μg/mL對U937直接刺激時即具有79.9% 及78.6% 之生長抑制率，而經流式細胞儀檢測得知，其細胞週期於刺激24 hr後即開始停滯於G0/G1期，72 hr後停滯於此期的細胞數比例更分別提高至87.2% 及89.6% 而無細胞凋亡之情形發生。同時也將柳松菇和鴻喜菇之4℃冷萃物、硫酸銨沉澱物分別以100-400μg/mL及25-50μg/mL之不同濃度進行細胞週期檢測，也發現在不同劑量、時間的刺激下，均抑制細胞生長於G0/G1期。 以酵素免疫染色法分析細胞週期調控因子之結果顯示，細胞週期蛋白CDK4在AA-0.1及HM-0.2作用下，均隨時間的增加而有減少之趨勢；對細胞增生有正向調控作用之PCNA則因HM-0.2作用而在24 hr後即迅速降低其表現量。 此外G1期調控相關因子尚包含D type cyclins、CDK2、CDK6，CDKI中之INK4 family、CIP/KIP family及Rb、E2F；尚待更進一步之測試，以更明瞭菇類中活性物質對人類白血病細胞株之生長抑制途徑。

Numberous bioactive components in mushrooms have been demonstarted to be revelant to the antitumor, anticancer, immunoenhancement, and differentiation-inducing effect of leukemia. Researches in the present laboratory have proved that protein extracts from AA（Agrocybe aegerita） and HM（Hypsizigus marmoreus） were responsible for the growth inhibition and differentiation of U937 cells into macrophages/monocytes. As a subsequent experiment to study the cell cycle arrest mechanisms of U937 cells by protein isolates from AA and HM, protein in mushrooms were fractionated by 100 % saurated ammonium sulfate, followed by a DEAE Sepharose CL-6B chromatography, eluted by 0.1 M NaCl for AA protein (AA-0.1) and 0.2 M NaCl for HM protein (HM-0.2). Results showed that AA-0.1 and HM-0.2 at a dosage of 25