白芍总苷脂质体诱导白血病细胞凋亡与分子机理的研究
发布时间：2018-11-05 13:18
【摘要】：目的:该研究为了验证白芍总苷脂质体(TGP)对白血病的治疗效果,并从细胞形态以及分子水平上明晓其药用机理,并为后阶段TGP在白血病治疗中应用规程的制定提供理论依据。考察TGP对于K562、HL-60以及人骨髓白血病细胞的抑制效果,观察K562、HL-60以及人骨髓白血病细胞的凋亡过程中形态学改变,考察HL60细胞在TGP诱导下相关凋亡基因与蛋白表达的变化规律,探究其细胞凋亡过程中的信息分子传递过程。方法:该研究以K562、HL60细胞系以及人骨髓白血病细胞为材料,并采用同时设置培养时间、TGP浓度两个自变量进行白血病抑制性细胞培养实验,并以CCK8法检测其细胞增殖抑制率表现。对TGP浓度、培养时间以及白血病细胞种类3个因素的抑制效应进行方差分析。使用200ug/ml白芍总脂质体培养HL60,并对空白对照组与6h、12h处理组镜检观察其形态学变化。分别使用RT-PCR与Western Blot方法,鉴定TGP对于内源性细胞凋亡因子Bcl-2、Bax、Caspase3和Caspase9的基因与蛋白质表达水平的变化。结果:(1)TGP对K562、HL60和人骨髓白血病细胞表现出随着培养时间的增加,抑制率不断的上升(R=0.797,P=0.0000.05);随着TGP浓度的上升,抑制率亦不断上升(R=0.196,P=0.0130.05)。通过三因子方差分析表明,TGP浓度、白血病细胞种类、培养时间均对抑制率表现出极显著效应,其中TGP浓度的F值表现最大,达到了1629.132。(2)在细胞形态学上观察12h之后的细胞可以发现,HL60细胞已经明显的表现出固缩、边集、碎裂等典型的凋亡细胞核形态特征,细胞核的形态上不规则程度也在不断的加大。使用流式细胞仪观察其凋亡率,结果表明:K562细胞凋亡率由3.0%显著上升至42.49%(X2=74.526,P=0.0000.05);HL60由3.87%显著上升至46.73%(X2=71.331,P=0.0000.05);人骨髓白血病细胞由4.15%显著上升至52.46%(X2=81.800,P=0.0000.01),均具有统计学意义。(3)在m RNA的表达水平上,随着培养时间的增加,Bcl-2的表达水平显著下降(R=-0.880,P=0.0000.05)、Bax则显著上升(R=0.825,P=0.0000.05)、Bcl-2/Bax比值显著下降(R=-0.957,P=0.0000.05);Caspase3(R=0.676,P=0.0080.05)和Caspase9(R=0.606,P=0.0180.05)表现出明显的上升趋势。在蛋白质的表达水平上,同样表现出Bcl-2的表达水平不断下降(R=-0.899,P=0.0000.05)、Bax(R=0.695,P=0.0050.05)、Caspase3(R=0.807,P=0.0010.05)和Caspase9(R=0.597,P=0.0200.05)则不断上升;在Bcl-2/Bax比值之上,表现出不断下降趋势(R=-0.757,P=0.0020.05)。这一结果与一般的内源性细胞凋亡结论一致。结论:TGP对K562、HL60和人骨髓白血病细胞具有良好抑制效果,并且伴随着良好的时间依赖性和剂量依赖性。随着培养时间延长,白血病细胞凋亡形态变化而越明显。分子机理研究表明TGP可能通过激活内源性细胞凋亡机制,从而达到诱导白血病细胞凋亡的。该研究认为TGP能成为临床中一种优良的白血病治疗药物。
[Abstract]:Objective: to verify the therapeutic effect of total glucoside of paeony liposome (TGP) on leukemia and to understand its medicinal mechanism from cell morphology and molecular level. It also provides the theoretical basis for the formulation of the application protocols of TGP in the treatment of leukemia in the later stage. To investigate the inhibitory effect of TGP on K562 HL-60 and human myeloid leukemia cells, and to observe the morphological changes of K562 HL-60 and human myeloid leukemia cells during apoptosis. To investigate the expression of apoptotic genes and proteins in HL60 cells induced by TGP, and to explore the process of signaling in the process of apoptosis. Methods: K562HL-60 cell line and human bone marrow leukemia cell line were used as materials, and two independent variables of TGP concentration and simultaneous culture time were used to carry out the leukemia inhibitory cell culture experiment. The inhibition rate of cell proliferation was detected by CCK8 assay. The inhibitory effects of TGP concentration, culture time and leukemic cell types were analyzed by ANOVA. HL60, was cultured with total liposome of Paeonia lanceolata (200ug/ml) and the morphological changes were observed under microscope in the blank control group and the 6 h ~ (12 h) treatment group. RT-PCR and Western Blot methods were used to identify the changes of gene and protein expression of endogenous apoptosis factor Bcl-2,Bax,Caspase3 and Caspase9 by TGP. Results: (1) the inhibitory rate of TGP on K562HL-60 cells and human myeloid leukemia cells increased with the increase of culture time (RP0. 797). With the increase of TGP concentration, the inhibition rate was also increased (R _ (0.196) P _ (0.013) 0.05). The results of three factor variance analysis showed that the concentration of TGP, the type of leukemic cells and the culture time all showed significant effects on the inhibition rate, and the F value of TGP concentration was the largest. It reached 1629.132. (2) after 12 h observation of cell morphology, HL60 cells showed typical morphological characteristics of apoptotic nuclei, such as pyknosis, edge aggregation, fragmentation and so on. The degree of morphological irregularity of the nucleus is also increasing. The apoptosis rate of K562 cells increased significantly from 3.0% to 42.49% by flow cytometry. HL60 increased significantly from 3.87% to 46.73% (X2O71.331P0. 0000. 05). Human bone marrow leukemic cells increased significantly from 4.15% to 52.46% (X2O81.800). (3) at the level of m RNA, the expression of m RNA increased with the increase of culture time. The expression level of Bcl-2 was significantly decreased (RP0. 00000. 05), Bax) and the ratio of Bcl-2/Bax was significantly decreased (RP0. 957 P0. 0000. 05). Caspase3 (RD676 P0. 0080.05) and Caspase9 (RH0. 606 P0. 018. 05) showed an obvious upward trend. In the protein expression level, the expression level of Bcl-2 was also decreased (RP0. 899P0. 0000.05), Bax (), Caspase3 (RH0. 807P0. 0010.05) and Caspase9 (RH0. 597), and the protein expression level was also decreased (RP0. 899), Bax (), Caspase3 (RH0. 807P0. 0010. 05) and Caspase9 (RD0. 597). (P = 0.0200.05). Above the Bcl-2/Bax ratio, it showed a decreasing trend (RV-0. 757 P0. 0020. 05). This result is consistent with the general conclusion of endogenous apoptosis. Conclusion: TGP has a good inhibitory effect on K562 HL-60 and human myeloid leukemia cells, and has a good time and dose dependence. With the prolongation of culture time, the morphological changes of leukemia cell apoptosis became more obvious. Molecular mechanism studies indicate that TGP may induce apoptosis of leukemia cells by activating endogenous apoptosis mechanism. This study suggests that TGP can be a good clinical treatment for leukemia.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.7
本文编号：2312209
[Abstract]:Objective: to verify the therapeutic effect of total glucoside of paeony liposome (TGP) on leukemia and to understand its medicinal mechanism from cell morphology and molecular level. It also provides the theoretical basis for the formulation of the application protocols of TGP in the treatment of leukemia in the later stage. To investigate the inhibitory effect of TGP on K562 HL-60 and human myeloid leukemia cells, and to observe the morphological changes of K562 HL-60 and human myeloid leukemia cells during apoptosis. To investigate the expression of apoptotic genes and proteins in HL60 cells induced by TGP, and to explore the process of signaling in the process of apoptosis. Methods: K562HL-60 cell line and human bone marrow leukemia cell line were used as materials, and two independent variables of TGP concentration and simultaneous culture time were used to carry out the leukemia inhibitory cell culture experiment. The inhibition rate of cell proliferation was detected by CCK8 assay. The inhibitory effects of TGP concentration, culture time and leukemic cell types were analyzed by ANOVA. HL60, was cultured with total liposome of Paeonia lanceolata (200ug/ml) and the morphological changes were observed under microscope in the blank control group and the 6 h ~ (12 h) treatment group. RT-PCR and Western Blot methods were used to identify the changes of gene and protein expression of endogenous apoptosis factor Bcl-2,Bax,Caspase3 and Caspase9 by TGP. Results: (1) the inhibitory rate of TGP on K562HL-60 cells and human myeloid leukemia cells increased with the increase of culture time (RP0. 797). With the increase of TGP concentration, the inhibition rate was also increased (R _ (0.196) P _ (0.013) 0.05). The results of three factor variance analysis showed that the concentration of TGP, the type of leukemic cells and the culture time all showed significant effects on the inhibition rate, and the F value of TGP concentration was the largest. It reached 1629.132. (2) after 12 h observation of cell morphology, HL60 cells showed typical morphological characteristics of apoptotic nuclei, such as pyknosis, edge aggregation, fragmentation and so on. The degree of morphological irregularity of the nucleus is also increasing. The apoptosis rate of K562 cells increased significantly from 3.0% to 42.49% by flow cytometry. HL60 increased significantly from 3.87% to 46.73% (X2O71.331P0. 0000. 05). Human bone marrow leukemic cells increased significantly from 4.15% to 52.46% (X2O81.800). (3) at the level of m RNA, the expression of m RNA increased with the increase of culture time. The expression level of Bcl-2 was significantly decreased (RP0. 00000. 05), Bax) and the ratio of Bcl-2/Bax was significantly decreased (RP0. 957 P0. 0000. 05). Caspase3 (RD676 P0. 0080.05) and Caspase9 (RH0. 606 P0. 018. 05) showed an obvious upward trend. In the protein expression level, the expression level of Bcl-2 was also decreased (RP0. 899P0. 0000.05), Bax (), Caspase3 (RH0. 807P0. 0010.05) and Caspase9 (RH0. 597), and the protein expression level was also decreased (RP0. 899), Bax (), Caspase3 (RH0. 807P0. 0010. 05) and Caspase9 (RD0. 597). (P = 0.0200.05). Above the Bcl-2/Bax ratio, it showed a decreasing trend (RV-0. 757 P0. 0020. 05). This result is consistent with the general conclusion of endogenous apoptosis. Conclusion: TGP has a good inhibitory effect on K562 HL-60 and human myeloid leukemia cells, and has a good time and dose dependence. With the prolongation of culture time, the morphological changes of leukemia cell apoptosis became more obvious. Molecular mechanism studies indicate that TGP may induce apoptosis of leukemia cells by activating endogenous apoptosis mechanism. This study suggests that TGP can be a good clinical treatment for leukemia.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.7
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