左旋紫草素抗白血病作用機制研究
發(fā)布時間：2019-02-26 13:50
【摘要】：目的體外研究左旋紫草素的抗白血病作用及可能的作用機制,為左旋紫草素用于白血病的臨床治療提供理論依據。方法1、臺盼藍拒染:以左旋紫草素不同濃度(0.5、1、2、4μmol/L)作用于人白血病K562細胞及白血病原代細胞,分不同時段(12h、24h、36h、48h、72h)采用臺盼藍拒染方法計數活細胞數,并繪制細胞生長曲線;2、噻唑藍實驗(MTT)觀察不同濃度左旋紫草素(0.5、1、2、4μmol/L)對人白血病K562細胞及白血病原代細胞的增殖抑制作用,并繪制抑制率曲線;3、采用4',6-二脒基-2苯基吲哚染色法(DAPI)于熒光顯微鏡下觀察不同濃度左旋紫草素(0.5、1、2、4μmol/L)對人白血病K562細胞核的形態(tài)變化;4、流式細胞術(FCM)檢測不同濃度左旋紫草素(0.5、1、2、4μmol/L)對人白血病K562細胞凋亡影響的時間和劑量相關性;以及Fas/FasL表達的變化;5、蛋白印記法(Western blot法)檢測左旋紫草素對人白血病K562細胞及白血病原代細胞的凋亡相關蛋白表達及凋亡相關信號通路分子活性,初步闡明左旋紫草素抗白血病作用及相關機制。結果1、臺盼藍拒染法:不同濃度的左旋紫草素(0.5、1、2、4μmol/l)在12h、24h、36h、48h、72h均能抑制人白血病K562細胞增殖,抑制增殖的作用具有時間-濃度依賴性。2、DAPI染色:隨著左旋紫草素藥物濃度的加大和作用時間逐步延長,人白血病K562細胞出現不同程度的凋亡,表現為細胞核固縮,核邊集,逐步發(fā)展至核碎裂,且其作用具有時間-濃度依賴性。3、MTT實驗:不同濃度左旋紫草素(0.5,1,2,4μmol/L)均能抑制人白血病K562細胞增殖,增殖曲線具有時間-濃度依賴性。4、流式細胞術檢測凋亡率:結果顯示左旋紫草素在一定時間(12h、24h)及濃度范圍內(0.5,1,2,4μmol/L)能誘導人白血病K562細胞凋亡,作用隨藥物濃度及作用時間的增加而有逐漸增強趨勢,且呈現明顯的時間-濃度依賴性。5、流式細胞儀檢測Fas/FasL表達:不同濃度左旋紫草素(0.5,1,2,4μmol/L)作用于人白血病K562細胞24h,流式細胞術檢測Fas/FasL變化,結果表明左旋紫草素能上調Fas/FasL表達,且其作用具有濃度依賴性。6、Western blot法檢測蛋白表達:結果表明左旋紫草素能誘導凋亡蛋白bax表達上調,抗凋亡蛋白bcl-2表達下調,激活Caspase-8、Caspase-3,誘導PARP蛋白發(fā)生活化剪切,激活促有絲分裂原活化蛋白激酶(MAPK),使JNK/SAPK及p38MAPK蛋白發(fā)生磷酸化,其作用具有時間-濃度依賴性。結論1、微摩爾濃度的左旋紫草素于體外能抑制人白血病K562細胞及白血病原代細胞增殖并誘導其凋亡,其抑制增殖與誘導凋亡作用具有時間-濃度依賴性;2、左旋紫草素誘導人白血病K562細胞及白血病原代細胞凋亡的作用可能與上調調亡蛋白bax表達,下調抗調亡蛋白bcl-2表達,并上調Fas/FasL表達,激活Caspase-8、Caspase-3,剪切PARP蛋白等機制相關,并與絲裂原活化蛋白激酶(MAPK信號通路)相關蛋白JNK/SAPK及p38 MAPK蛋白磷酸化相關,作用趨勢具有時間-濃度依賴性。
[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.
【學位授予單位】：延安大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R733.7
本文編號：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.
【學位授予單位】：延安大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R733.7
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