论文标题:苦参碱对增殖心肌成纤维细胞的促凋亡作用及其机制
Apoptotic Effect of Matrine on Proliferative Cardiac Fibroblasts and Its Mechanism
论文作者 周成慧
论文导师 欧阳静萍,论文学位 硕士,论文专业 病理与病理生理
论文单位 武汉大学,点击次数 771,论文页数 51页File Size4094k
2004-04-01论文网 http://www.lw23.com/lunwen_1226132/ AngII,苦参碱,凋亡,BcI-2,Bax,Caspase-3
angiotensin II, matrine, apoptosis, Bcl-2, Bax, Caspase-3
目的 本研究以血管紧张素Ⅱ(Angiotensin Ⅱ，AngⅡ)作用的小鼠心肌成纤维细胞(cardiac fibrolasts，CFs)为对象，探讨中药苦参碱(matrine，Mat)的促凋亡作用及其机制。 方法 1．体外分离、培养小鼠心肌成纤维细胞。 2．不同浓度的(1.0～4.0mmol／L)Mat分别作用于正常CFs和增殖CFs24h，采用流式细胞术检测CFs的凋亡率，筛选有效浓度。 3．根据结果2，选用10~(-7)mmol／LAngⅡ，2.0mmol／LMat+AngⅡ，3.0mmol／LMat+AngⅡ，4.0mmol／LMat+AngⅡ处理体外培养的CFs 24h，以含1％新生牛血清(NCS)的DMEM培养的CFs为对照组；利用倒置相差显微镜、PI／Hoechst33342双染结合荧光显微镜技术、细胞记数观察细胞形态和生长状况；使用流式细胞术检测CFs的凋亡率、细胞周期及凋亡相关基因Bcl-2、Bax蛋白表达的改变；采用Western blotting检测Caspase-3蛋白的表达及活化Caspase-3片段的产生。 4．根据以上结果选用3.0mmol／LMat+AngⅡ分别处理CFs 12h、24h、48h、72h；利用倒置相差显微镜、PI／Hoechst33342双染结合荧光显微镜技术、细胞记数观察不同时间CFs形态和生长状况；使用流式细胞术检测各时间段内CFs的凋亡率、细胞周期及凋亡相关基因Bcl-2、Bax蛋白表达的改变；采用Western blotting检测Caspase-3蛋白的表达及活化Caspase-3片段的产生。 结果 1．不同浓度的(1.0～4.0mmol／L)Mat分别作用于正常CFs和增殖CFs24h后，流式细胞术检测显示Mat浓度为1.0 mmol／L时，正常CFs和增殖CFs未出现凋亡；Mat浓度为2.0 mmol／L时，正常CFs未出现凋亡，而增殖CFs出现凋亡；Mat浓度为3.0 mmol／L时，正常CFs和增殖CFs均出现凋亡，但增殖CFs凋亡率明显高于正常CFs(P＜0.05)；Mat浓度为4.0 mmol／L时，正常CFs和增殖CFs均出现凋亡，但两者无显著差别。 2．根据结果1．选用浓度范围为2.0～4.0mmol／LMat处理增殖CFs24h后，细胞数减少；PI／Hoechst33342双染显示在Mat浓度为3.0mmol／L时细胞染色质浓缩、边集、碎裂；流式细胞术检测示，凋亡率随浓度的升高而升高;细胞周期分析显示不同浓度Mat处理组与对照组和Angl工组比较，Gl期细胞数量增加，但各Mat处理组之间无明显变化，S期细胞数量逐渐减少，而GZ/M期无明显变化;流式细胞术检测显示不同浓度Mat处理组与对照组和Angll组比较，Bcl一2的表达量减少，但各Mat处理组之间无明显变化;与对照组和Angll比较，不同浓度Mat处理组Bax的表达量增加且随浓度的升高而逐渐增加呈剂量效应;Westem blotting检测显示在Mat浓度为3 .ommol/L和4.ommol/L时出现活化Caspase一3片段。 3.3.0们nrnol/LMat作用增殖CFs不同时间段内，Pl/Hoeehst33342双染显示在24h、48h、72h均细胞出现染色质浓缩、边集、碎裂;流式细胞术检测示凋亡率随时间的延长而升高:细胞周期分析显示不同时间段Mat处理组与对照组和Angll组比较，Gl期细胞数量增加，但各Mat处理组之间无明显变化，S期细胞数量减少，而GZ/M期无明显变化;流式细胞术检测显示不同时间段Mat处理组与对照组和Angn组比较，Bcl一2的表达量减少，但各Mat处理组之间无明显变化;与对照组和Angll组比较，不同时间段Mat处理组Bax的表达量增加且随时间的延长而逐渐增加呈时间效应;W已stem blot检测显示在24h、48h、72h时均出现活化Caspase一3片段。 结论: 1.2.0一3.Ommol/L Mat范围内促进增殖CFS凋亡，并且其凋亡作用强于对正常CFs促凋亡的作用。 2.2.0 mmol/L Mat具有促进增殖CFs凋亡的作用，在2.0一30 mmol几Mat范围内呈浓度依赖效益; 3.在3，0 mmol/L Mat浓度作用24h后，苦参碱对增殖CFs的促凋亡作用在24一48h内呈时间依赖效应;CasPas一3苦参碱促进增殖的CFs凋亡的机制可能与Bcl一2低水平表达，Bax高表达，活化有关。
AIM:The aim of this study is to investigate apoptotic effect of matrine on proliferative mouse cardiac fibroblasts and it"s mechanism.METHODS:Cardiac fibroblasts were isolated from 3-day-old Kunming mice by collagenase disruption and cultured. Firstly, normal cardiac fibroblasts and angiotensin II -intreated cardiac fibroblasts were respectively treated for 24 hours by 1.0~4.0mmol/L matrine and their apoptosis ratios were measured by flow cytometry.Secondly, according to the results of above experiments, the conscentrations frome 2.0mmol/L to 4.0mmol/L were selected. Then the cardiac fibroblasts were respectively treated for 24 hours with 10-7mmol/LangiotensinII, 2.0mmol/Lmatrine+angiotensin II, 3.0mmol/L matrine+angiotensin II and 4.0mmol/L matrine+angiotensin II . The cardie fibroblasts maintained in Dulbecco"s modified Eagle"s medium(DEME) containing 1% new born calf serum were as control. Morphology of cell were observed by inverted phase contrast microscope and cell numbers were measured by use of a Coulter Counter. Morphology of apoptotic cardiac fibroblasts were observed by fluorescence microscope with PI/Hoechst33342 double staining. Cell cycle phases, apoptotic ratio of cardiac fibroblasts and expression of the apoptosis-related gene Bcl-2, Bax were measured by flow cytometry. Activity of Caspase-3 and expression of activated fragment of Caspase-3 from the total cell extracts were detected by Western blotting analysis.Finally, the cardiac fibroblasts were respectively treated for 12 hours, 24hours, 48hours and 72hours by 3.0mmol/L matrine+angiotensin II. The cardie fibroblasts maintained in Dulbecco"s modified Eagle"s medium(DEME) containing 1% new born calf serum were as control. All the detections and their methods were as the same as above.RESULTS:1.Treatment on normal cardiac fibrolblasts and proliferative cardiac fibroblasts with 1~4 mmol/Lmatrine for 24 hours revealed that when the concentratin is 1.0mmol/L, neither normal cardiac fibrolblasts nor proliferative cardiac fibroblasts did appear apoptosis; when 2.0mmol/L, proliferative cardiac fibroblasts started apoptosis; when3.0mmol/L, both normal cardiac fibrolblasts and proliferative cardiac fibroblasts appeared apoptosis and the apoptotic ratio of proliferative cardiac fibroblasts was markedly higher than that of normal cardiac fibrolblasts; when 4.0mmol/L, both normal cardiac fibrolblasts and proliferative cardiac fibroblasts also appeared apoptosis, but the apoptotic ratios of these two groups were no significant difference.2. Treatment on proliferative cardiac fibroblasts with 2~4 mmol/Lmatrine for 24 hours revealed that cell numbers were decreased and the apoptotic ratios were increased gradually with the concentration increased(P<0.05); PI/Hoechst33342 double staining could distinguish apoptotic cells and normal cells, typical apoptosis features of nucleolus were observed under fluorescence microscope in 3.0 mmol/Lmatrine+ angiotensin II and the features were more prominent with the concentration increased. Matrine also affected cell cycle obviously, compared with control and angiotensin II. Matrine mainly causing the number of cell in Gl phase was increased (P<0.05) and that of in S phase was decreased (P<0.05). Expression of the apoptosis-suppression gene Bcl-2 was reduced (P<0.05), while expression of the apoptosis-induction gene Bax was enhanced and the effect was dose dependented (P<0.05). At the same time activity of Caspase 3 was induced and expression of activated fragments of Caspase-3 were detected in 3.0mmol/L matrine+angiotensin II and 4.0mmol/L matrine+angiotensin II.3.Treatment on proliferative cardiac fibroblasts with 3.0 mmol/Lmatrine for 12h,24h,48h and 72h revealed that the apoptosis ratio was increased gradually from 24 hours to 48 hours (P0.05); PI/Hoechst33342 double staining could distinguish apoptosis cells and normal cells, typical apoptosis features of nucleolus were observed under fluorescence microscope when proliferative cardiac fibroblasts were treated by 3.0 mmol/Lmatrine for 24hours and the feat
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