左旋紫草素抗白血病作用机制研究
发布时间：2019-02-26 13:50
【摘要】：目的体外研究左旋紫草素的抗白血病作用及可能的作用机制,为左旋紫草素用于白血病的临床治疗提供理论依据。方法1、台盼蓝拒染:以左旋紫草素不同浓度(0.5、1、2、4μmol/L)作用于人白血病K562细胞及白血病原代细胞,分不同时段(12h、24h、36h、48h、72h)采用台盼蓝拒染方法计数活细胞数,并绘制细胞生长曲线;2、噻唑蓝实验(MTT)观察不同浓度左旋紫草素(0.5、1、2、4μmol/L)对人白血病K562细胞及白血病原代细胞的增殖抑制作用,并绘制抑制率曲线;3、采用4',6-二脒基-2苯基吲哚染色法(DAPI)于荧光显微镜下观察不同浓度左旋紫草素(0.5、1、2、4μmol/L)对人白血病K562细胞核的形态变化;4、流式细胞术(FCM)检测不同浓度左旋紫草素(0.5、1、2、4μmol/L)对人白血病K562细胞凋亡影响的时间和剂量相关性;以及Fas/FasL表达的变化;5、蛋白印记法(Western blot法)检测左旋紫草素对人白血病K562细胞及白血病原代细胞的凋亡相关蛋白表达及凋亡相关信号通路分子活性,初步阐明左旋紫草素抗白血病作用及相关机制。结果1、台盼蓝拒染法:不同浓度的左旋紫草素(0.5、1、2、4μmol/l)在12h、24h、36h、48h、72h均能抑制人白血病K562细胞增殖,抑制增殖的作用具有时间-浓度依赖性。2、DAPI染色:随着左旋紫草素药物浓度的加大和作用时间逐步延长,人白血病K562细胞出现不同程度的凋亡,表现为细胞核固缩,核边集,逐步发展至核碎裂,且其作用具有时间-浓度依赖性。3、MTT实验:不同浓度左旋紫草素(0.5,1,2,4μmol/L)均能抑制人白血病K562细胞增殖,增殖曲线具有时间-浓度依赖性。4、流式细胞术检测凋亡率:结果显示左旋紫草素在一定时间(12h、24h)及浓度范围内(0.5,1,2,4μmol/L)能诱导人白血病K562细胞凋亡,作用随药物浓度及作用时间的增加而有逐渐增强趋势,且呈现明显的时间-浓度依赖性。5、流式细胞仪检测Fas/FasL表达:不同浓度左旋紫草素(0.5,1,2,4μmol/L)作用于人白血病K562细胞24h,流式细胞术检测Fas/FasL变化,结果表明左旋紫草素能上调Fas/FasL表达,且其作用具有浓度依赖性。6、Western blot法检测蛋白表达:结果表明左旋紫草素能诱导凋亡蛋白bax表达上调,抗凋亡蛋白bcl-2表达下调,激活Caspase-8、Caspase-3,诱导PARP蛋白发生活化剪切,激活促有丝分裂原活化蛋白激酶(MAPK),使JNK/SAPK及p38MAPK蛋白发生磷酸化,其作用具有时间-浓度依赖性。结论1、微摩尔浓度的左旋紫草素于体外能抑制人白血病K562细胞及白血病原代细胞增殖并诱导其凋亡,其抑制增殖与诱导凋亡作用具有时间-浓度依赖性;2、左旋紫草素诱导人白血病K562细胞及白血病原代细胞凋亡的作用可能与上调调亡蛋白bax表达,下调抗调亡蛋白bcl-2表达,并上调Fas/FasL表达,激活Caspase-8、Caspase-3,剪切PARP蛋白等机制相关,并与丝裂原活化蛋白激酶(MAPK信号通路)相关蛋白JNK/SAPK及p38 MAPK蛋白磷酸化相关,作用趋势具有时间-浓度依赖性。
[Abstract]:Aim to study the anti-leukemia effect of L-shikonin in vitro and its possible mechanism in order to provide a theoretical basis for the clinical therapy of L-shikonin in leukemia. Methods 1, trypan blue exclusion: human leukemia K562 cells and primary leukemic cells were treated with different concentrations of L-violet (0.5, 1,2,4 渭 mol / L) at different time points (12 h, 24 h, 36 h, 48 h), and the cells were treated with L-violet at different concentrations (0.5, 1,2,4 渭 mol / L) at different time points (12 h, 24 h, 36 h, 48 h). The number of living cells was counted by trypan blue exclusion method and the cell growth curve was drawn. 2. The inhibitory effects of L-violet (0.5, 1,2,4 渭 mol / L) on the proliferation of human leukemia K562 cells and primary leukemia cells were observed by (MTT) assay, and the inhibitory rate curve was drawn. 3. The morphological changes of K562 cell nuclei were observed by 4-diamino-2-phenylindole staining (DAPI) at different concentrations (0.5, 1, 2, 4 渭 mol / L) under fluorescence microscope. 4, flow cytometry (FCM) was used to detect the time-and dose-dependent effect of L-violet (0.5, 1, 2, 4 渭 mol / L) on apoptosis of K562 cells and the changes of Fas/FasL expression. (5) the expression of apoptosis-related protein and the activity of apoptosis-related signaling pathway in human leukemia K562 cells and primary leukemic cells were detected by (Western blot method. To elucidate the anti-leukemic effect of L-shikonin and its related mechanism. Results 1, trypan blue exclusion assay: different concentrations of L-shikonin (0.5, 1,2,4 渭 mol / l) at 12 h, 24 h, 36 h, 48 h, 72 h inhibited the proliferation of human leukemia K562 cells in a time-concentration dependent manner. 2, 2, 4 渭 mol / l L-shikonin (0.5 渭 mol / l, 1 渭 mol / l, 2,4 渭 mol / l) for 12 h, 24 h, 36 h, 48 h, 72 h. DAPI staining: with the increase of the concentration of L-shikonin and the gradual prolongation of the action time, human leukemic K562 cells showed apoptosis in varying degrees, showing karyon condensation, nuclear side aggregation, and gradually developing to nuclear fragmentation. 3. MTT assay showed that different concentrations of L-violet (0.5, 1,2,4 渭 mol / L) could inhibit the proliferation of human leukemia K562 cells in a time-concentration dependent manner. 4, 4, 2, 4 渭 mol / L L-cypermethrin could inhibit the proliferation of human leukemia K562 cells in a time-concentration dependent manner. 4, 2, 4 渭 mol / L. The apoptosis rate of K562 cells was detected by flow cytometry. The results showed that L-shikonin could induce the apoptosis of K562 cells within a certain time (12 h, 24 h) and concentration range (0.5, 1, 2, 4 渭 mol / L). The effect increased gradually with the increase of drug concentration and time, and showed a significant time-concentration dependence. 5. Flow cytometry was used to detect the expression of Fas/FasL: different concentrations of L-violet (0.5, 1, 1, 5) were detected by flow cytometry. Human leukemia K562 cells were treated with 2,4 渭 mol / L for 24 h, and the changes of Fas/FasL were detected by flow cytometry. The results showed that L-shikonin could up-regulate the expression of Fas/FasL in a concentration-dependent manner. The results showed that L-shikonin could up-regulate the expression of apoptotic protein bax, down-regulate the expression of anti-apoptotic protein bcl-2 and activate Caspase-8,Caspase-3, to induce the activation of PARP protein. Activated mitogen-activated protein kinase (MAPK), phosphorylated JNK/SAPK and p38MAPK proteins in a time-concentration dependent manner. Conclusion 1. The micromolar concentration of L-shikonin can inhibit the proliferation and induce apoptosis of human leukemia K562 cells and primary leukemia cells in vitro. The inhibitory effect of L-shikonin is time-concentration dependent on the inhibition of proliferation and inducing apoptosis of K562 cells and primary leukemic cells in a time-concentration dependent manner. 2. The apoptosis of K562 cells and primary leukemic cells induced by L-shikonin may be related to up-regulating the expression of apoptosis protein bax, down-regulating the expression of anti-apoptosis protein bcl-2 and up-regulating the expression of Fas/FasL and activating Caspase-8,Caspase-3,. The mechanism of cleavage of PARP protein was related to the phosphorylation of JNK/SAPK and p38 MAPK proteins associated with mitogen-activated protein kinase (MAPK signaling pathway) in a time-concentration dependent manner.
【学位授予单位】：延安大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R733.7
本文编号：2430832
[Abstract]:Aim to study the anti-leukemia effect of L-shikonin in vitro and its possible mechanism in order to provide a theoretical basis for the clinical therapy of L-shikonin in leukemia. Methods 1, trypan blue exclusion: human leukemia K562 cells and primary leukemic cells were treated with different concentrations of L-violet (0.5, 1,2,4 渭 mol / L) at different time points (12 h, 24 h, 36 h, 48 h), and the cells were treated with L-violet at different concentrations (0.5, 1,2,4 渭 mol / L) at different time points (12 h, 24 h, 36 h, 48 h). The number of living cells was counted by trypan blue exclusion method and the cell growth curve was drawn. 2. The inhibitory effects of L-violet (0.5, 1,2,4 渭 mol / L) on the proliferation of human leukemia K562 cells and primary leukemia cells were observed by (MTT) assay, and the inhibitory rate curve was drawn. 3. The morphological changes of K562 cell nuclei were observed by 4-diamino-2-phenylindole staining (DAPI) at different concentrations (0.5, 1, 2, 4 渭 mol / L) under fluorescence microscope. 4, flow cytometry (FCM) was used to detect the time-and dose-dependent effect of L-violet (0.5, 1, 2, 4 渭 mol / L) on apoptosis of K562 cells and the changes of Fas/FasL expression. (5) the expression of apoptosis-related protein and the activity of apoptosis-related signaling pathway in human leukemia K562 cells and primary leukemic cells were detected by (Western blot method. To elucidate the anti-leukemic effect of L-shikonin and its related mechanism. Results 1, trypan blue exclusion assay: different concentrations of L-shikonin (0.5, 1,2,4 渭 mol / l) at 12 h, 24 h, 36 h, 48 h, 72 h inhibited the proliferation of human leukemia K562 cells in a time-concentration dependent manner. 2, 2, 4 渭 mol / l L-shikonin (0.5 渭 mol / l, 1 渭 mol / l, 2,4 渭 mol / l) for 12 h, 24 h, 36 h, 48 h, 72 h. DAPI staining: with the increase of the concentration of L-shikonin and the gradual prolongation of the action time, human leukemic K562 cells showed apoptosis in varying degrees, showing karyon condensation, nuclear side aggregation, and gradually developing to nuclear fragmentation. 3. MTT assay showed that different concentrations of L-violet (0.5, 1,2,4 渭 mol / L) could inhibit the proliferation of human leukemia K562 cells in a time-concentration dependent manner. 4, 4, 2, 4 渭 mol / L L-cypermethrin could inhibit the proliferation of human leukemia K562 cells in a time-concentration dependent manner. 4, 2, 4 渭 mol / L. The apoptosis rate of K562 cells was detected by flow cytometry. The results showed that L-shikonin could induce the apoptosis of K562 cells within a certain time (12 h, 24 h) and concentration range (0.5, 1, 2, 4 渭 mol / L). The effect increased gradually with the increase of drug concentration and time, and showed a significant time-concentration dependence. 5. Flow cytometry was used to detect the expression of Fas/FasL: different concentrations of L-violet (0.5, 1, 1, 5) were detected by flow cytometry. Human leukemia K562 cells were treated with 2,4 渭 mol / L for 24 h, and the changes of Fas/FasL were detected by flow cytometry. The results showed that L-shikonin could up-regulate the expression of Fas/FasL in a concentration-dependent manner. The results showed that L-shikonin could up-regulate the expression of apoptotic protein bax, down-regulate the expression of anti-apoptotic protein bcl-2 and activate Caspase-8,Caspase-3, to induce the activation of PARP protein. Activated mitogen-activated protein kinase (MAPK), phosphorylated JNK/SAPK and p38MAPK proteins in a time-concentration dependent manner. Conclusion 1. The micromolar concentration of L-shikonin can inhibit the proliferation and induce apoptosis of human leukemia K562 cells and primary leukemia cells in vitro. The inhibitory effect of L-shikonin is time-concentration dependent on the inhibition of proliferation and inducing apoptosis of K562 cells and primary leukemic cells in a time-concentration dependent manner. 2. The apoptosis of K562 cells and primary leukemic cells induced by L-shikonin may be related to up-regulating the expression of apoptosis protein bax, down-regulating the expression of anti-apoptosis protein bcl-2 and up-regulating the expression of Fas/FasL and activating Caspase-8,Caspase-3,. The mechanism of cleavage of PARP protein was related to the phosphorylation of JNK/SAPK and p38 MAPK proteins associated with mitogen-activated protein kinase (MAPK signaling pathway) in a time-concentration dependent manner.
【学位授予单位】：延安大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R733.7
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