【摘要】：目的:該研究為了驗(yàn)證白芍總苷脂質(zhì)體(TGP)對(duì)白血病的治療效果,并從細(xì)胞形態(tài)以及分子水平上明曉其藥用機(jī)理,并為后階段TGP在白血病治療中應(yīng)用規(guī)程的制定提供理論依據(jù)�？疾霻GP對(duì)于K562、HL-60以及人骨髓白血病細(xì)胞的抑制效果,觀察K562、HL-60以及人骨髓白血病細(xì)胞的凋亡過程中形態(tài)學(xué)改變,考察HL60細(xì)胞在TGP誘導(dǎo)下相關(guān)凋亡基因與蛋白表達(dá)的變化規(guī)律,探究其細(xì)胞凋亡過程中的信息分子傳遞過程。方法:該研究以K562、HL60細(xì)胞系以及人骨髓白血病細(xì)胞為材料,并采用同時(shí)設(shè)置培養(yǎng)時(shí)間、TGP濃度兩個(gè)自變量進(jìn)行白血病抑制性細(xì)胞培養(yǎng)實(shí)驗(yàn),并以CCK8法檢測(cè)其細(xì)胞增殖抑制率表現(xiàn)。對(duì)TGP濃度、培養(yǎng)時(shí)間以及白血病細(xì)胞種類3個(gè)因素的抑制效應(yīng)進(jìn)行方差分析。使用200ug/ml白芍總脂質(zhì)體培養(yǎng)HL60,并對(duì)空白對(duì)照組與6h、12h處理組鏡檢觀察其形態(tài)學(xué)變化。分別使用RT-PCR與Western Blot方法,鑒定TGP對(duì)于內(nèi)源性細(xì)胞凋亡因子Bcl-2、Bax、Caspase3和Caspase9的基因與蛋白質(zhì)表達(dá)水平的變化。結(jié)果:(1)TGP對(duì)K562、HL60和人骨髓白血病細(xì)胞表現(xiàn)出隨著培養(yǎng)時(shí)間的增加,抑制率不斷的上升(R=0.797,P=0.0000.05);隨著TGP濃度的上升,抑制率亦不斷上升(R=0.196,P=0.0130.05)。通過三因子方差分析表明,TGP濃度、白血病細(xì)胞種類、培養(yǎng)時(shí)間均對(duì)抑制率表現(xiàn)出極顯著效應(yīng),其中TGP濃度的F值表現(xiàn)最大,達(dá)到了1629.132。(2)在細(xì)胞形態(tài)學(xué)上觀察12h之后的細(xì)胞可以發(fā)現(xiàn),HL60細(xì)胞已經(jīng)明顯的表現(xiàn)出固縮、邊集、碎裂等典型的凋亡細(xì)胞核形態(tài)特征,細(xì)胞核的形態(tài)上不規(guī)則程度也在不斷的加大。使用流式細(xì)胞儀觀察其凋亡率,結(jié)果表明:K562細(xì)胞凋亡率由3.0%顯著上升至42.49%(X2=74.526,P=0.0000.05);HL60由3.87%顯著上升至46.73%(X2=71.331,P=0.0000.05);人骨髓白血病細(xì)胞由4.15%顯著上升至52.46%(X2=81.800,P=0.0000.01),均具有統(tǒng)計(jì)學(xué)意義。(3)在m RNA的表達(dá)水平上,隨著培養(yǎng)時(shí)間的增加,Bcl-2的表達(dá)水平顯著下降(R=-0.880,P=0.0000.05)、Bax則顯著上升(R=0.825,P=0.0000.05)、Bcl-2/Bax比值顯著下降(R=-0.957,P=0.0000.05);Caspase3(R=0.676,P=0.0080.05)和Caspase9(R=0.606,P=0.0180.05)表現(xiàn)出明顯的上升趨勢(shì)。在蛋白質(zhì)的表達(dá)水平上,同樣表現(xiàn)出Bcl-2的表達(dá)水平不斷下降(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)則不斷上升;在Bcl-2/Bax比值之上,表現(xiàn)出不斷下降趨勢(shì)(R=-0.757,P=0.0020.05)。這一結(jié)果與一般的內(nèi)源性細(xì)胞凋亡結(jié)論一致。結(jié)論:TGP對(duì)K562、HL60和人骨髓白血病細(xì)胞具有良好抑制效果,并且伴隨著良好的時(shí)間依賴性和劑量依賴性。隨著培養(yǎng)時(shí)間延長(zhǎng),白血病細(xì)胞凋亡形態(tài)變化而越明顯。分子機(jī)理研究表明TGP可能通過激活內(nèi)源性細(xì)胞凋亡機(jī)制,從而達(dá)到誘導(dǎo)白血病細(xì)胞凋亡的。該研究認(rèn)為TGP能成為臨床中一種優(yōu)良的白血病治療藥物。
[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.
【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R733.7

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