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研究生:李奕慶
研究生(外文):Yi-Ching Lee
論文名稱:黑豆抽出物在人類周邊血單核細胞中促進G-CSF基因表現之探討
論文名稱(外文):G-CSF gene expression in human peripheral blood mononuclear cells stimulated by black soybean extracts
指導教授:楊行義楊行義引用關係郭育綺郭育綺引用關係
指導教授(外文):Hsing-Yi YangYuh-Chi Kuo
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:81
中文關鍵詞:粒細胞集落刺激因子人類周邊血單核細胞
外文關鍵詞:Granulocyte-Colony Stimulating Factor (G-CSF)human peripheral blood mononuclear cells (PBMC)
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黑豆為豆科的植物,在傳統中醫藥方面常被用來治療降脂、抗動脈粥狀硬化、抗腫瘤等。在黑豆抽出物中發現具有抗病毒的能力,而在最近的研究更發現黑豆具有調控免疫藥理的能力。因此本論文的研究目的為:觀察在黑豆抽出物中對於人類周邊血單核細胞增生及G-CSF產生的影響。而在此研究中發現,黑豆抽出物中(EW02-DT-14)具有促進人類周邊血單核細胞產生G-CSF的能力,本論文的研究結果如下:(1) 利用ELISA的方法發現在加入不同濃度的EW02-DT-14作用下,G-CSF蛋白質的產生隨著藥物的濃度增加而增加,並且呈現劑量相關性。(2)在RT-PCR部份,發現在加入不同濃度的EW02-DT-14作用下,G-CSF mRNA的產生也隨著藥物的濃度增加而增加,並且呈現劑量相關性; 而在以real-time PCR來佐證的結果也和RT-PCR的結果是相同的。(3)為了要探討EW02-DT-14是經由何種訊息傳導來促進G-CSF的產生,因此在加入EW02-DT-14之前加入各種的抑制劑觀察,以RT-PCR和real-time PCR來觀察G-CSF在mRNA的表現。結果發現,所加入抑制劑的組別和單獨加入EW02-DT-14的組別相比,此時G-CSF的表現受到了抑制,因此認為EW02-DT-14促進G-CSF的產生的過程中可能和MAPK、 NF-AT 與 NF-□B有關。(4)在NF-AT的部份,使用共軛焦顯微鏡來觀察在EW02-DT-14作用下是否會影響NF-AT translocation。結果發現,在EW02-DT-14作用下的0到2小時並不會影響NF-AT translocation。因為NF-AT會受到上游鈣離子的調控,因此利用了螢光測定法來觀察鈣離子的mobilization。結果發現,EW02-DT-14並不會影響鈣離子的mobilization,因此由實驗的結果可知認為EW02-DT-14並不會參與活化鈣離子到NF-AT的路徑。(5)在NF-□B的部份,使用共軛焦顯微鏡來觀察在EW02-DT-14作用下是否會影響NF-□B translocation。結果發現,在EW02-DT-14作用下的1到2小時會造成NF-□B translocation。 (6)在MAPK部份,利用西方墨點法來觀察在EW02-DT-14作用下是否會影響MAPK的活化。由實驗結果發現,在EW02-DT-14作用下會造成ERK的磷酸化,但是不會影響p38和JNK。而由於ERK會去調控下游的傳訊因子:c-fos。因此以RT-PCR來觀察c-fos的表現,而實驗結果發現,在EW02-DT-14作用下會促進c-fos的表現,因此認為,EW02-DT-14作用下所促進G-CSF的產生的過程中,ERK是參與在其中的因子。(7)在最近的研究指出,G-CSF的產生的過程中可能會受到PI3K的調控,因此以RT-PCR來觀察PI3K是否會影響G-CSF的產生。而實驗的結果發現當加入PI3K的抑制劑時,此時G-CSF的表現明顯的受的抑制,因此推論EW02-DT-14作用下所促進G-CSF的產生的過程中,PI3K是參與在其中的過程。(8)為了要探討EW02-DT-14是否會經由PI3K到PKCθ的路徑而來影響NF-□B translocation, 本論文利用西方墨點法來觀察在EW02-DT-14作用下是否影響這些因子的活化,而在實驗結果中發現,在EW02-DT-14作用下會活化PKCθ,而當使用了PI3K的抑制劑時,此時PKCθ的表現受到了抑制,因此推論EW02-DT-14會透過活化PI3K、PKCθ而造成NF-□B translocation,進而促使G-CSF的產生。(9)為了要探討EW02-DT-14是否會經由PI3K到MAPK的路徑而來影響G-CSF的表現,利用西方墨點法來觀察在EW02-DT-14作用下是否影響這些因子的活化,而在實驗結果中發現,使用了PI3K的抑制劑時,此時ERK的磷酸化受到了抑制,而p38和JNK則不受影響,因此推論,EW02-DT-14會透過活化PI3K、ERK而造促使G-CSF的產生。
Glycine max (L.) merr (Black soybean) belongs to Leguminosae family. It is applied in traditional Chinese medicine for treatment of hyperlipidemia, atheroma, and cancer. Extracts from black soybean has been demonstrated to possess antiviral activity. In the present study, black soybean was selected for immunopharmacologica1 activity test. Objective: The effects of G. max extracted fractions on human peripheral blood mononuclear cells (PBMC) proliferation and granulocyte-colony stimulating factor (G-CSF) production were determined. In this study, it was found that the black soybean extracts (EW02-DT-14) promoted G-CSF production in peripheral blood mononuclear cells (PBMC), and our investigation is shown below:
(1) This study used ELISA to find that after treated different concentration of EW02-DT-14, G-CSF production in protein level was elevated and the effect showed dosage correlation. (2) We used RT-PCR method to find that after treated different concentration of EW02-DT-14, G-CSF expression in mRNA level was promoted and the effect showed dosage correlation. In our real-time PCR results showed the same results as well as the RT-PCR results. (3) In order to investigate that how the EW02-DT-14 promoted G-CSF expression in signal transduction, furthermore, we used added different inhibitors before treated EW02-DT-14 in our experiment. We used RT-PCR and real-time PCR method to investigate the G-CSF mRNA expression. The finding, we demonstrated that when different inhibitors were added before treated EW02-DT-14, G-CSF mRNA production could be reduced compared with EW02-DT-14 alone. Moreover, we showed that the EW02-DT-14 promoted G-CSF production in signal transduction correlated with MAPK、NF-AT、and NF-κB. (4) In NF-AT we used confocal microscopy method to investigate whether the translocation of NF-AT was influenced by EW02-DT-14 or not. We demonstrated that EW02-DT-14 didn’t influence the translocation of NF-AT in 0-2 hours. Because NF-AT might be regulated by upstream Ca2+, We used fluorescence spectrophotometer to investigate the Ca2+ mobilization. We showed that EW02-DT-14 did not influence the Ca2+ mobilization, so we infer that EW02-DT-14 did not activate the Ca2+- NF-AT pathway. (5) In NF-κB, we used confocal microscopy method to investigate whether the translocation of NF-κB was influenced by EW02-DT-14 or not. We showed that EW02-DT-14 activated the translocation of NF-κB within 1-2 hours. (6) In MAPK, we used western blotting to investigate whether EW02-DT-14 activates MAPK or not. We demonstrated that EW02-DT-14 might activate ERK phosphorylation , but p38、JNk did not. The ERk might regulate downstream transcription factor such as c-fos. We used RT-PCR to investigate the c-fos activation. We showed that EW02-DT-14 promoted c-fos production. Therefore, we infer that ERK involved in EW02-DT-14 promoted G-CSF production process. (7) Recent studies demonstrated that G-CSF production might be regulated by PI3K, therefore and we used RT-PCR method to investigate whether PI3K was influenced G-CSF production or not. Our finding showed that when PI3K inhibitors were used, G-CSF production could be decreased. We infer that PI3K involved in EW02-DT-14 promoted G-CSF production signaling cascade. (8) In order to investigate whether the translocation of NF-κB was influenced by PI3K to PKCθ pathway or not. We used western blotting to investigate the effect of EW02-DT-14. The finding, the EW02-DT-14 might activate PKCθ, and the PKCθ expression was inhibited by added PI3K inhibitor. We infer that the G-CSF production might activated by activated PI3K、PKCθ、NF-κB pathway. (9) In order to investigate whether the G-CSF production was influenced by EW02-DT-14 activated the PI3K to MAPK pathway or not. We used western blotting to investigate the effect of EW02-DT-14. In our finding, when we used the PI3K inhibitor could inhibition the ERK phosphrylation, but were not influence the p38 and JNK. So we infer that the EW02-DT-14 might activate the PI3K to ERK pathway and could promote to G-CSF production.
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