【摘要】：目的:鑒于丹參酮化合物有抗腫瘤的作用,本實驗課題主要檢測丹參酮類化合物(二氫丹參酮I、TanⅡA、CPT及Tan I)作用于T細胞淋巴瘤細胞株(Jurkat)的增殖抑制、周期阻滯和誘導凋亡作用,并在此基礎上探討NF-κB、MAPK及PI3K三條信號通路在介導丹參酮類化合物對整個血液系統(tǒng)惡性腫瘤細胞(多發(fā)性骨髓瘤細胞株U266、髓系白血病細胞株NB4及K562、T細胞淋巴瘤細胞株Jurkat)的增殖抑制、誘導凋亡及周期阻滯中的作用。方法:(1)體外培養(yǎng)Jurkat細胞。刺激Jurkat細胞株的每種丹參酮類化合物設計六個濃度梯度(1.25、2.5、5、10、20、30μmol/L),將實驗組分為24個組。以等質(zhì)量的六個濃度梯度的柔紅霉素(0.7、1.4、2.8、5.6、11.2、16.8μmol/L)刺激Jurkat細胞株作為陽性對照組,同時設一個陰性對照組。采用CCK8法分別檢測培養(yǎng)24h、48h、72h的上述各組細胞的抑制率。PI單染法檢測20μmol/L的藥物作用于Jurkat細胞株24h后對細胞周期阻滯的影響,Annexin V/PI雙染法檢測20μmol/L的藥物作用于Jurkat細胞24h后對誘導凋亡的作用。(2)培養(yǎng)血液惡性腫瘤細胞株(NB4、K562、U266、Jurkat),收集經(jīng)20μmol/L的4種丹參酮類化合物刺激24h后的NB4、K562、U266、Jurkat細胞,抽提出總蛋白之后用BCA法檢測其濃度符合要求則置-80℃保存?zhèn)溆?然后用Westernblot方法分別檢測上述4種細胞的NF-κB、MAPK及PI3K三條信號通路蛋白表達。結(jié)果:1.二氫丹參酮I、TanⅡA、CPT及Tan I對Jurkat細胞株均有明顯的增殖抑制作用,且作用效果呈時間和劑量依賴性。四種丹參酮化合物作用于Jurkat細胞株:24h,半數(shù)有效抑制濃度(IC50)分別為16.40、12.53、49.47、54.19μmol/L;48h,IC50分別為8.73、7.28、38.89、15.66μmol/L;72h,IC50分別為2.95、2.65、17.43、10.28μmol/L。2.流式細胞術(shù)檢測20μmol/L的四種丹參酮藥作用于Jurkat細胞株24h后周期阻滯作用。與空白對照細胞組比較,四種丹參酮類化合物作用于Jurkat細胞株后,除Tan IIA組外,二氫丹參酮I組、CPT組、Tan I組的G0/G1期細胞比例均明顯增加,P0.01;S期與G2/M期細胞減少,CPT組與空白對照細胞組的S期細胞比例相當,P0.05,無顯著差異;二氫丹參酮組對Jurkat細胞株的作用最強。Tan IIA組的S期細胞比例增加,G0/G1期和G2/M期的細胞比例減少。3.流式細胞術(shù)檢測20μmol/L的四種丹參酮藥物作用于Jurkat細胞株24h后誘導凋亡作用。與空白對照細胞組比較,四種丹參酮類化合作用于Jurkat細胞株后,二氫丹參酮I組、TanⅡA組、CPT組及Tan I組早期凋亡細胞比率、晚期凋亡和壞死細胞比率均明顯增高,P0.01。四種丹參酮類化合物中,二氫丹參酮對Jurkat細胞株作用最為顯著,其次為Tan IIA組Tan I組CPT組,CPT組對Jurkat細胞株的作用最弱。4.Westernblot方法分別檢測NB4、K562、U266、Jurkat經(jīng)20μmol/L的4種丹參酮類化合物刺激24h后對NF-κB、MAPK及PI3K三條信號通路蛋白表達顯示:丹參酮類化合物可明顯抑制血液惡性腫瘤細胞株NF-κB和PI3K兩條信號通路,但對MAPK信號通路沒有作用。結(jié)論:1、丹參酮類化合物可以抑制Jurkat細胞株的生長活力,其效應呈劑量及時間依賴性。2、增殖抑制作用最強的二氫丹參酮I與等質(zhì)量的柔紅霉素比較,分別作用24h和48h后二氫丹參酮I對Jurkat細胞株的增殖抑制作用明顯弱于柔紅霉素;作用72h后,兩藥對Jurkat細胞株增殖抑制作用相當。3、丹參酮類化合物作用于Jurkat細胞株,均有周期阻滯及誘導凋亡作用,且以二氫丹參酮組作用最強。4、丹參酮類化合物可以影響Jurkat細胞株的周期分布,二氫丹參酮I組、CPT組及Tan I組將Jurkat細胞株阻滯于G0/G1期,Tan IIA組將其阻滯于S期。5、丹參酮類化合物作用于血液惡性腫瘤細胞株(K562、NB4、U266、Jurkat)可以明顯抑制PI3K、NF-κB兩條信號通路蛋白的表達,介導血液惡性腫瘤細胞的增殖抑制、周期阻滯及誘導凋亡;對MAPK信號通路無明顯作用。
[Abstract]:OBJECTIVE: In view of the anti-tumor effect of tanshinone compounds, this study mainly examined the effects of tanshinone compounds (dihydrotanshinone I, Tan I I A, CPT and Tan I) on T-cell lymphoma cell line (Jurkat) proliferation inhibition, cycle arrest and apoptosis induction, and on this basis to explore the role of NF-kappa B, MAPK and PI3K signaling pathways in the mediation. METHODS: (1) Jurkat cells were cultured in vitro to stimulate tanshinone analogy of each Jurkat cell line. Six concentration gradients (1.25,2.5,5,10,20,30 micromol/L) were designed to stimulate Jurkat cell lines with the same concentration gradients of daunorubicin (0.7,1.4,2.8,5.6,11.2,16.8 micromol/L) as the positive control group and a negative control group. The inhibition rate of Jurkat cells was detected by PI monoclonal staining. The effect of 20 micromol/L drugs on cell cycle arrest was detected by PI monoclonal staining. The effect of 20 micromol/L drugs on apoptosis of Jurkat cells was detected by Annexin V/PI double staining. (2) Blood malignant tumor cell lines (NB4, K562, U266, Jurkat) were cultured and four kinds of Salvia 20 micromol/L were collected. NB4, K562, U266 and Jurkat cells were stimulated by ginsenone compounds for 24 hours. Total proteins were extracted and then stored at - 80 C when the concentration of total proteins met the requirements by BCA. The expression of NF-kappa B, MAPK and PI3K signal pathway proteins in these four cells was detected by Western blot. Results: 1. Dihydrotanshinone I, Tan I I A, CPT and Tan I pairs. Jurkat cell lines were inhibited by four kinds of tanshinone compounds in a time-and dose-dependent manner. The half effective inhibitory concentrations (IC50) were 16.40, 12.53, 49.47 and 54.19 micromol/L at 24 hours, 8.73, 7.28, 38.89, 15.66 micromol/L at 48 hours, 2.95, 2.65, 17.43, 10.28 micromol/L at 72 hours, respectively. Flow cytometry was used to detect the cell cycle arrest of Jurkat cell line after 24 hours. Compared with the control group, the percentage of G0/G1 phase cells in Tan IIA group, CPT group and Tan I group increased significantly, P 0.01. Compared with G2/M phase, the proportion of S phase cells in CPT group and blank control group was similar, P 0.05, no significant difference was found; the effect of DHT group on Jurkat cell line was the strongest. Compared with the control group, the ratio of early apoptotic cells, late apoptotic cells and necrotic cells in DHT group I, Tan I I A, CPT group and Tan I group were significantly higher than those in the control group (P 0.01). Tan IIA group Tan I group CPT group, CPT group on Jurkat cell line effect is the weakest. 4. Western blot method was used to detect NB4, K562, U266, Jurkat after 20 micromol/L of four tanshinone compounds stimulated 24 hours to NF-kappa B, MAPK and PI3K three signal pathway protein expression showed that tanshinone compounds can significantly inhibit Conclusion: 1. Tanshinone compounds can inhibit the growth of Jurkat cells in a dose-and time-dependent manner. 2. Dihydrotanshinone I has the strongest inhibitory effect on proliferation compared with daunorubicin of the same quality, and it can inhibit the growth of Jurkat cells in a dose-and time-dependent manner. The inhibitory effect of DHT-I on the proliferation of Jurkat cell line was weaker than that of daunorubicin at 4 h and 48 h, and the inhibitory effect of DHT-I on the proliferation of Jurkat cell line was similar to that of daunorubicin at 72 h. Jurkat cell lines were blocked at G0/G1 phase by dihydrotanshinone I, CPT and Tan I, and at S phase by Tan I I I. Tanshinone compounds could inhibit the expression of PI3K and NF-kappa B signal pathway proteins in blood malignant tumor cell lines (K562, NB4, U266, Jurkat). Cell proliferation was inhibited, cell cycle arrest and apoptosis was induced in hematological malignant tumor cells.
【學位授予單位】：川北醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R733

【參考文獻】

相關(guān)期刊論文 前10條

1 李慧;向洪;王銀;卿紅;王春森;;丹參酮類化合物誘導白血病干細胞凋亡的體外研究[J];華西藥學雜志;2016年04期

2 胡波;范紅偉;王燕午;馬廣駿;;丹參單體IH764-3對馬兜鈴酸致人腎小管上皮細胞損害的保護機制研究[J];河北醫(yī)藥;2016年07期

3 劉長姣;李明春;;阻斷MAPK信號通路抗癌藥物的研究進展[J];解放軍藥學學報;2015年06期

4 杜文婷;劉萍;;丹參單體對動脈粥樣硬化作用機制的研究概述[J];中西醫(yī)結(jié)合心腦血管病雜志;2015年07期

5 向洪;王春森;李慧;;丹參酮類化合物對髓系白血病的作用機制研究進展[J];實用醫(yī)院臨床雜志;2015年01期

6 梅艷飛;張丹參;;丹參酮Ⅱ_A的藥理作用及治療應用研究進展[J];神經(jīng)藥理學報;2014年05期

7 單卿卿;郭勇;龔玉萍;;丹參酮ⅡA對白血病細胞株K562增殖抑制及機制研究[J];四川大學學報(醫(yī)學版);2014年03期

8 朱小鳳;李曉明;;外周T細胞淋巴瘤的治療進展[J];華西醫(yī)學;2014年04期

9 李彈彈;馬杰;閆春艷;付雪;李鳳月;侯新s，

本文編號：2249903