丹参酮化合物对血液系统恶性肿瘤作用及其机制探讨

发布时间：2018-09-19 10:41

【摘要】：目的:鉴于丹参酮化合物有抗肿瘤的作用,本实验课题主要检测丹参酮类化合物(二氢丹参酮I、TanⅡA、CPT及Tan I)作用于T细胞淋巴瘤细胞株(Jurkat)的增殖抑制、周期阻滞和诱导凋亡作用,并在此基础上探讨NF-κB、MAPK及PI3K三条信号通路在介导丹参酮类化合物对整个血液系统恶性肿瘤细胞(多发性骨髓瘤细胞株U266、髓系白血病细胞株NB4及K562、T细胞淋巴瘤细胞株Jurkat)的增殖抑制、诱导凋亡及周期阻滞中的作用。方法:(1)体外培养Jurkat细胞。刺激Jurkat细胞株的每种丹参酮类化合物设计六个浓度梯度(1.25、2.5、5、10、20、30μmol/L),将实验组分为24个组。以等质量的六个浓度梯度的柔红霉素(0.7、1.4、2.8、5.6、11.2、16.8μmol/L)刺激Jurkat细胞株作为阳性对照组,同时设一个阴性对照组。采用CCK8法分别检测培养24h、48h、72h的上述各组细胞的抑制率。PI单染法检测20μmol/L的药物作用于Jurkat细胞株24h后对细胞周期阻滞的影响,Annexin V/PI双染法检测20μmol/L的药物作用于Jurkat细胞24h后对诱导凋亡的作用。(2)培养血液恶性肿瘤细胞株(NB4、K562、U266、Jurkat),收集经20μmol/L的4种丹参酮类化合物刺激24h后的NB4、K562、U266、Jurkat细胞,抽提出总蛋白之后用BCA法检测其浓度符合要求则置-80℃保存备用,然后用Westernblot方法分别检测上述4种细胞的NF-κB、MAPK及PI3K三条信号通路蛋白表达。结果:1.二氢丹参酮I、TanⅡA、CPT及Tan I对Jurkat细胞株均有明显的增殖抑制作用,且作用效果呈时间和剂量依赖性。四种丹参酮化合物作用于Jurkat细胞株:24h,半数有效抑制浓度(IC50)分别为16.40、12.53、49.47、54.19μmol/L;48h,IC50分别为8.73、7.28、38.89、15.66μmol/L;72h,IC50分别为2.95、2.65、17.43、10.28μmol/L。2.流式细胞术检测20μmol/L的四种丹参酮药作用于Jurkat细胞株24h后周期阻滞作用。与空白对照细胞组比较,四种丹参酮类化合物作用于Jurkat细胞株后,除Tan IIA组外,二氢丹参酮I组、CPT组、Tan I组的G0/G1期细胞比例均明显增加,P0.01;S期与G2/M期细胞减少,CPT组与空白对照细胞组的S期细胞比例相当,P0.05,无显著差异;二氢丹参酮组对Jurkat细胞株的作用最强。Tan IIA组的S期细胞比例增加,G0/G1期和G2/M期的细胞比例减少。3.流式细胞术检测20μmol/L的四种丹参酮药物作用于Jurkat细胞株24h后诱导凋亡作用。与空白对照细胞组比较,四种丹参酮类化合作用于Jurkat细胞株后,二氢丹参酮I组、TanⅡA组、CPT组及Tan I组早期凋亡细胞比率、晚期凋亡和坏死细胞比率均明显增高,P0.01。四种丹参酮类化合物中,二氢丹参酮对Jurkat细胞株作用最为显著,其次为Tan IIA组Tan I组CPT组,CPT组对Jurkat细胞株的作用最弱。4.Westernblot方法分别检测NB4、K562、U266、Jurkat经20μmol/L的4种丹参酮类化合物刺激24h后对NF-κB、MAPK及PI3K三条信号通路蛋白表达显示:丹参酮类化合物可明显抑制血液恶性肿瘤细胞株NF-κB和PI3K两条信号通路,但对MAPK信号通路没有作用。结论:1、丹参酮类化合物可以抑制Jurkat细胞株的生长活力,其效应呈剂量及时间依赖性。2、增殖抑制作用最强的二氢丹参酮I与等质量的柔红霉素比较,分别作用24h和48h后二氢丹参酮I对Jurkat细胞株的增殖抑制作用明显弱于柔红霉素;作用72h后,两药对Jurkat细胞株增殖抑制作用相当。3、丹参酮类化合物作用于Jurkat细胞株,均有周期阻滞及诱导凋亡作用,且以二氢丹参酮组作用最强。4、丹参酮类化合物可以影响Jurkat细胞株的周期分布,二氢丹参酮I组、CPT组及Tan I组将Jurkat细胞株阻滞于G0/G1期,Tan IIA组将其阻滞于S期。5、丹参酮类化合物作用于血液恶性肿瘤细胞株(K562、NB4、U266、Jurkat)可以明显抑制PI3K、NF-κB两条信号通路蛋白的表达,介导血液恶性肿瘤细胞的增殖抑制、周期阻滞及诱导凋亡;对MAPK信号通路无明显作用。
[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.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733

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