电解水对试验细胞之影响探讨.doc

蓮花萃取物對大鼠樹突狀細胞免疫調節作用之探討 Evaluation of the immune responses to extracts from Nymphaea rubra Roxb in rat dendritic cells 研究生：李曉芸Lee, Shau-Yu 指導教授：許祥純Sheu, Shyang-Chwen 【摘要】 「醣」對人體生理運作扮演著重要的角色，而負責防禦人體健康的免疫細胞，彼此之間也是靠這些結合在蛋白質表面的醣來傳達免疫訊息。樹突狀細胞(dendritic cells，DCs)為專業的抗原呈現細胞，其可藉由細胞表面之多醣接受器進行訊息傳遞，進而活化T細胞免疫調控路徑。本研究目的為利用誘導單核球分化成樹突狀細胞模式，評估睡蓮多醣(Nymphaea rubra Roxb. Polysaccharides，NR-PS)水解產物對大鼠DCs分化之功效。由6-8週齡大鼠分離骨髓造血幹細胞(Bone marrow hematopoietic cell，BMHC)，以含20 ng/mL rGM-CSF及10 ng/mL rIL-4之完全培養基RPMI-1640共同培養7天後，細胞型態擴大且有典型樹突狀突起之未成熟樹突狀細胞(BMHC-derived immature dendritc cells，BMHC-imDCs )，平均可得細胞總數達3.8±1.50×107 cells/rat。添加不同濃度之NR-PS (0、3.125、6.25、12.5、25、50、100 μg/mL)與1 μg/mL之陽性控制組LPS，處理BMHC-imDCs並於體外培養48小時後，進行細胞型態、細胞表面抗原、吞噬作用與細胞激素含量之分析。經LPS與NR-PS處理後細胞表面型態相較於控制組有更明顯的樹突狀突起。利用流式細胞儀(FACScan flow cytometer)分析其細胞表面抗原與吞噬作用，由結果得知以NR-PS 25 μg/mL刺激後可增加CD80/86、MHC II細胞表面抗原之表現。BMHC-imDCs吞噬FITC–dextran能力方面，NR-PS 25 μg/mL相較於未添加刺激物之控制組有顯著降低趨勢，且與LPS之陽性控制組效果相當。利用酵素免疫分析法(enzyme-linked immunosorbent assay，ELISA)分別測定細胞培養液中IL-12、IFN-γ及IL-10含量，結果IL-12、IFN-γ含量於NR-PS 25 μg/mL處理組相較於控制組有顯著提升；而IL-10含量於NR-PS 12.5-100 μg/mL處理組相較於控制組有顯著降低的趨勢，由上述結果可知，NR-PS 25 μg/mL不僅能刺激大鼠BMHC-imDCs分化趨向BMHC-mDCs之活性，且可增加IL-12、IFN-γ之分泌 關鍵字: 睡蓮、樹狀突細胞、抗原呈獻細胞、細胞激素、免疫評估 【Abstract】 Carbohydrates play a key role in enhancing immune function and facilitating cellular communication. Dendritic cells (DCs) are professional antigen presenting cells (APCs), which are pivotal for initiation of primary immune response. In this study, the regulatory effects of Nymphaea rubra Roxb polysaccharides (NR-PS) on maturation and function of cultured rat bone marrow derived DCs were investigated in vitro. Bone marrow hematopoietic cells (BMHC) were collected from 6-8 week-old rats. BMHC-imDC were grown from BMHC in complete RPMI media 1640 containing 20 ng/mL rGM- CSF and 10 ng/mL rIL-4. The cells enlarged and displayed typical stellate after cultivation for 7 days. An average of 3.8±1.50×107 cells/rat was obtained. BMHC-imDCs were stimulated with NR