【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(jī)制,為左旋紫草素用于白血病的臨床治療提供理論依據(jù)。方法1、臺(tái)盼藍(lán)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(xì)胞及白血病原代細(xì)胞,分不同時(shí)段(12h、24h、36h、48h、72h)采用臺(tái)盼藍(lán)拒染方法計(jì)數(shù)活細(xì)胞數(shù),并繪制細(xì)胞生長曲線;2、噻唑藍(lán)實(shí)驗(yàn)(MTT)觀察不同濃度左旋紫草素(0.5、1、2、4μmol/L)對(duì)人白血病K562細(xì)胞及白血病原代細(xì)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(duì)人白血病K562細(xì)胞核的形態(tài)變化;4、流式細(xì)胞術(shù)(FCM)檢測(cè)不同濃度左旋紫草素(0.5、1、2、4μmol/L)對(duì)人白血病K562細(xì)胞凋亡影響的時(shí)間和劑量相關(guān)性;以及Fas/FasL表達(dá)的變化;5、蛋白印記法(Western blot法)檢測(cè)左旋紫草素對(duì)人白血病K562細(xì)胞及白血病原代細(xì)胞的凋亡相關(guān)蛋白表達(dá)及凋亡相關(guān)信號(hào)通路分子活性,初步闡明左旋紫草素抗白血病作用及相關(guān)機(jī)制。結(jié)果1、臺(tái)盼藍(lán)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(xì)胞增殖,抑制增殖的作用具有時(shí)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(shí)間逐步延長,人白血病K562細(xì)胞出現(xiàn)不同程度的凋亡,表現(xiàn)為細(xì)胞核固縮,核邊集,逐步發(fā)展至核碎裂,且其作用具有時(shí)間-濃度依賴性。3、MTT實(shí)驗(yàn):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(xì)胞增殖,增殖曲線具有時(shí)間-濃度依賴性。4、流式細(xì)胞術(shù)檢測(cè)凋亡率:結(jié)果顯示左旋紫草素在一定時(shí)間(12h、24h)及濃度范圍內(nèi)(0.5,1,2,4μmol/L)能誘導(dǎo)人白血病K562細(xì)胞凋亡,作用隨藥物濃度及作用時(shí)間的增加而有逐漸增強(qiáng)趨勢(shì),且呈現(xiàn)明顯的時(shí)間-濃度依賴性。5、流式細(xì)胞儀檢測(cè)Fas/FasL表達(dá):不同濃度左旋紫草素(0.5,1,2,4μmol/L)作用于人白血病K562細(xì)胞24h,流式細(xì)胞術(shù)檢測(cè)Fas/FasL變化,結(jié)果表明左旋紫草素能上調(diào)Fas/FasL表達(dá),且其作用具有濃度依賴性。6、Western blot法檢測(cè)蛋白表達(dá):結(jié)果表明左旋紫草素能誘導(dǎo)凋亡蛋白bax表達(dá)上調(diào),抗凋亡蛋白bcl-2表達(dá)下調(diào),激活Caspase-8、Caspase-3,誘導(dǎo)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(shí)間-濃度依賴性。結(jié)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(xì)胞及白血病原代細(xì)胞增殖并誘導(dǎo)其凋亡,其抑制增殖與誘導(dǎo)凋亡作用具有時(shí)間-濃度依賴性;2、左旋紫草素誘導(dǎo)人白血病K562細(xì)胞及白血病原代細(xì)胞凋亡的作用可能與上調(diào)調(diào)亡蛋白bax表達(dá),下調(diào)抗調(diào)亡蛋白bcl-2表達(dá),并上調(diào)Fas/FasL表達(dá),激活Caspase-8、Caspase-3,剪切PARP蛋白等機(jī)制相關(guān),并與絲裂原活化蛋白激酶(MAPK信號(hào)通路)相關(guān)蛋白JNK/SAPK及p38 MAPK蛋白磷酸化相關(guān),作用趨勢(shì)具有時(shí)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(xué)位授予單位】：延安大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R733.7

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