【作者】 陈兵；

【导师】 欧阳建；

【作者基本信息】 南京中医药大学 ， 中西医结合临床， 2009， 博士

【摘要】 【目的】探讨通关藤提取物（商品名:消癌平）抗恶性血液系统疾病作用及机制,以及通关藤提取物对正常免疫细胞及造血干细胞功能的影响。【方法】以0μL/mL～100μL/mL的通关藤（Marsdenia tenacissima）提取物处理Jurkat、Raji、RPMI8226、HL-60、K562细胞株1～5天,MTT法检测其对5种细胞的抑制作用;流式细胞仪检测分析细胞周期改变。检测Jurkat、Raji、RPMI8226细胞株的凋亡机制,采用Annexin V/PI双染法观察细胞形态并检测其凋亡率,JC-1染色法检测细胞线粒体跨膜电位（ΔΨm）水平;Western blot检测细胞半胱氨酸蛋白酶（Caspase）-3蛋白表达。采用MTT法检测通关藤提取物对正常、ConA及LPS诱导的人淋巴细胞增殖活性的影响,中性红法检测巨噬细胞的吞噬功能,CFU-GM半固体集落培养法检测造血干细胞的集落形成能力。【结果】1.通关藤提取物体外对HL-60、K562、Jurkat、Raji、KPMI8226等5种细胞株具有不同程度的增殖抑制作用。在相同作用条件下,通关藤提取物对Jurkat的抑制率较高,且呈现出剂量依赖性。通关藤提取物可诱导RPMI8226和Raji细胞阻滞在G₂期,对其他细胞无明显周期阻滞。2.通关藤提取物体外对Jurkat、Raji、RPMI8226细胞株具有不同程度的诱导凋亡作用。Jurkat的凋亡率最高,且呈现出剂量、时间依赖性。25、50μL/mL的通关藤提取物能降低Jurkat、Raji细胞内ΔΨm,并促进其凋亡。100μL/mL的通关藤提取物也可降低RPMI8226细胞ΔΨm而诱导其凋亡。50μL/mL的通关藤提取物处理组细胞Caspase-3蛋白的表达水平较对照组无明显改变。3.通关藤提取物体外对ConA诱导的正常淋巴细胞和LPS诱导的正常淋巴细胞有促增殖作用且呈剂量依赖性。通关藤提取物对正常巨噬细胞吞噬中性红染液的能力无明显影响。对正常骨髓/外周血造血干细胞CFU-GM形成能力没有显著影响。【结论】1.通关藤提取物体外对多种恶性血液病细胞株具有不同程度的增殖抑制作用。2.通关藤提取物体外对部分淋巴细胞白血病细胞株和骨髓瘤细胞株有不同程度的诱导凋亡作用,可能是通过线粒体途径诱导这些细胞凋亡。3.通关藤提取物体外对正常免疫细胞和造血干细胞无明显的细胞毒作用,并且有促进T、B细胞增殖作用。更多还原

【Abstract】 【Objective】To investigate the effect of anti-hematological malignances and its mechanisms of extracts from Marsdenia tenacissima（trade name:Xiao aiping）,and the effect of extracts from Marsdenia tenacissima on normal human immunocytes and hemopoietic stem cells（HSCs）.【Methods】MTT method was adopted to observe the inhibiting effect of extracts from Marsdenia tenacissima with 0μL/mL～100μL/mL on Jurkat、Raji、RPMI8226、HL-60、K562 cell lines for 1～5 days.Cell cycle was detected by flow cytometry（FCM）.Cell morphology and apoptosis were measured by Annexin V/PI staining and mitochondrial membrane potential（ΔΨm） was measured by JC-1 assay.Western blot method was used to detect the protein expression of caspase-3.Cell proliferation was measured by MTT assay in normal cells and cells treated with ConA or LPS.Phagocytosis of normal macrophage was measured by the dose of Neutral Red Dye.CFU-GM was assessed by semi-solid culture assay for determination of colony formation capacity of HSCs.【Results】1.Extracts from Marsdenia tenacissima had the most significant inhibition on Jurkat cells with dose-dependent among Jurkat,Raji,RPMI8226,HL-60,K562 cell lines in vitro. Extracts from Marsdenia tenacissima could induce G₂ phase arrest of RPMI8226 and Raji cell lines.2.Extracts from Marsdenia tenacissima had varying degrees of apoptosis induction on Jurkat、Raji、RPMI8226 cell lines in vitro.The apoptosis rate of Jurkat cells was the highest with dose-,time- dependent.Extracts from Marsdenia tenacissima with 25 and 50μL/mL could decrease cellΔΨm and induce apoptosis in Jurkat and Raji cells,while extracts from Marsdenia tenacissima with 100μL/mL could also decrease cellΔΨm and induce apoptosis in RPMI8226 cells.No significant changes in protein expressions of caspase-3 in groups treated by extracts from Marsdenia tenacissima.3.Extracts from Marsdenia tenacissima could enhance proliferation of normal lymphocytes treated with ConA or LPS with dose-dependent in vitro.There was no effect of extracts from Marsdenia tenacissima on macrophage to ingest Neutral Red Dye and CFU-GM colony formation capacity of normal bone marrow/ peripheral HSCs.【Conclusion】1.Extracts from Marsdenia tenacissima have varying degrees of inhibition on various hematological malignance cell lines in vitro.2.Extracts from Marsdenia tenacissima induce apoptosis on some lymphocytic leukemic cell strains and myeloma cell strain in vitro by mitochondrial pathway.3.There is no significant cell toxicity of extracts from Marsdenia tenacissima on normal immunocytes and HSCs in vitro.And extracts from Marsdenia tenacissima can enhance proliferation of normal lymphocytes treated with ConA or LPS in vitro.更多还原