【摘要】：目的:鑒于丹參酮化合物有抗腫瘤的作用,本實(shí)驗(yàn)課題主要檢測(cè)丹參酮類化合物(二氫丹參酮I、TanⅡA、CPT及Tan I)作用于T細(xì)胞淋巴瘤細(xì)胞株(Jurkat)的增殖抑制、周期阻滯和誘導(dǎo)凋亡作用,并在此基礎(chǔ)上探討NF-κB、MAPK及PI3K三條信號(hào)通路在介導(dǎo)丹參酮類化合物對(duì)整個(gè)血液系統(tǒng)惡性腫瘤細(xì)胞(多發(fā)性骨髓瘤細(xì)胞株U266、髓系白血病細(xì)胞株NB4及K562、T細(xì)胞淋巴瘤細(xì)胞株Jurkat)的增殖抑制、誘導(dǎo)凋亡及周期阻滯中的作用。方法:(1)體外培養(yǎng)Jurkat細(xì)胞。刺激Jurkat細(xì)胞株的每種丹參酮類化合物設(shè)計(jì)六個(gè)濃度梯度(1.25、2.5、5、10、20、30μmol/L),將實(shí)驗(yàn)組分為24個(gè)組。以等質(zhì)量的六個(gè)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(xì)胞株作為陽性對(duì)照組,同時(shí)設(shè)一個(gè)陰性對(duì)照組。采用CCK8法分別檢測(cè)培養(yǎng)24h、48h、72h的上述各組細(xì)胞的抑制率。PI單染法檢測(cè)20μmol/L的藥物作用于Jurkat細(xì)胞株24h后對(duì)細(xì)胞周期阻滯的影響,Annexin V/PI雙染法檢測(cè)20μmol/L的藥物作用于Jurkat細(xì)胞24h后對(duì)誘導(dǎo)凋亡的作用。(2)培養(yǎng)血液惡性腫瘤細(xì)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(xì)胞,抽提出總蛋白之后用BCA法檢測(cè)其濃度符合要求則置-80℃保存?zhèn)溆?然后用Westernblot方法分別檢測(cè)上述4種細(xì)胞的NF-κB、MAPK及PI3K三條信號(hào)通路蛋白表達(dá)。結(jié)果:1.二氫丹參酮I、TanⅡA、CPT及Tan I對(duì)Jurkat細(xì)胞株均有明顯的增殖抑制作用,且作用效果呈時(shí)間和劑量依賴性。四種丹參酮化合物作用于Jurkat細(xì)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(xì)胞術(shù)檢測(cè)20μmol/L的四種丹參酮藥作用于Jurkat細(xì)胞株24h后周期阻滯作用。與空白對(duì)照細(xì)胞組比較,四種丹參酮類化合物作用于Jurkat細(xì)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(xì)胞比例均明顯增加,P0.01;S期與G2/M期細(xì)胞減少,CPT組與空白對(duì)照細(xì)胞組的S期細(xì)胞比例相當(dāng),P0.05,無顯著差異;二氫丹參酮組對(duì)Jurkat細(xì)胞株的作用最強(qiáng)。Tan IIA組的S期細(xì)胞比例增加,G0/G1期和G2/M期的細(xì)胞比例減少。3.流式細(xì)胞術(shù)檢測(cè)20μmol/L的四種丹參酮藥物作用于Jurkat細(xì)胞株24h后誘導(dǎo)凋亡作用。與空白對(duì)照細(xì)胞組比較,四種丹參酮類化合作用于Jurkat細(xì)胞株后,二氫丹參酮I組、TanⅡA組、CPT組及Tan I組早期凋亡細(xì)胞比率、晚期凋亡和壞死細(xì)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(duì)Jurkat細(xì)胞株作用最為顯著,其次為Tan IIA組Tan I組CPT組,CPT組對(duì)Jurkat細(xì)胞株的作用最弱。4.Westernblot方法分別檢測(cè)NB4、K562、U266、Jurkat經(jīng)20μmol/L的4種丹參酮類化合物刺激24h后對(duì)NF-κB、MAPK及PI3K三條信號(hào)通路蛋白表達(dá)顯示:丹參酮類化合物可明顯抑制血液惡性腫瘤細(xì)胞株NF-κB和PI3K兩條信號(hào)通路,但對(duì)MAPK信號(hào)通路沒有作用。結(jié)論:1、丹參酮類化合物可以抑制Jurkat細(xì)胞株的生長(zhǎng)活力,其效應(yīng)呈劑量及時(shí)間依賴性。2、增殖抑制作用最強(qiáng)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(duì)Jurkat細(xì)胞株的增殖抑制作用明顯弱于柔紅霉素;作用72h后,兩藥對(duì)Jurkat細(xì)胞株增殖抑制作用相當(dāng)。3、丹參酮類化合物作用于Jurkat細(xì)胞株,均有周期阻滯及誘導(dǎo)凋亡作用,且以二氫丹參酮組作用最強(qiáng)。4、丹參酮類化合物可以影響Jurkat細(xì)胞株的周期分布,二氫丹參酮I組、CPT組及Tan I組將Jurkat細(xì)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(xì)胞株(K562、NB4、U266、Jurkat)可以明顯抑制PI3K、NF-κB兩條信號(hào)通路蛋白的表達(dá),介導(dǎo)血液惡性腫瘤細(xì)胞的增殖抑制、周期阻滯及誘導(dǎo)凋亡;對(duì)MAPK信號(hào)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(xué)位授予單位】：川北醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R733

【參考文獻(xiàn)】

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(dǎo)白血病干細(xì)胞凋亡的體外研究[J];華西藥學(xué)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(duì)馬兜鈴酸致人腎小管上皮細(xì)胞損害的保護(hù)機(jī)制研究[J];河北醫(yī)藥;2016年07期

3 劉長(zhǎng)姣;李明春;;阻斷MAPK信號(hào)通路抗癌藥物的研究進(jìn)展[J];解放軍藥學(xué)學(xué)報(bào);2015年06期

4 杜文婷;劉萍;;丹參單體對(duì)動(dòng)脈粥樣硬化作用機(jī)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(duì)髓系白血病的作用機(jī)制研究進(jìn)展[J];實(shí)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(yīng)用研究進(jìn)展[J];神經(jīng)藥理學(xué)報(bào);2014年05期

7 單卿卿;郭勇;龔玉萍;;丹參酮ⅡA對(duì)白血病細(xì)胞株K562增殖抑制及機(jī)制研究[J];四川大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2014年03期

8 朱小鳳;李曉明;;外周T細(xì)胞淋巴瘤的治療進(jìn)展[J];華西醫(yī)學(xué);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(hào)：2249903