【作者】 刘楠；

【导师】 王海昌； 李飞； 张荣庆；

【作者基本信息】 第四军医大学 ， 内科学， 2010， 硕士

【摘要】 实验背景和目的近年来磁场的作用逐渐受到人们的重视,它是一种安全有效的物理治疗手段,在治疗骨关节等疾病方面已经取得了良好的疗效,早有报道证明体外培养的人脐静脉内皮细胞以及牛主动脉内皮细胞的血管形成可以被脉冲磁场有效地促进,FGF-2的分泌也同样可以被磁场所促进,因此人们不断寻找一种利用磁场这种无创疗法改善心血管疾病的途径。近年来缺血性心脏病的发病率逐年上升,而治疗缺血性心脏病的主要手段就是促进内皮的再生,进一步使新的血管形成。微血管内皮细胞在功能上有许多与内皮细胞相似的地方,它可以参与血管的再生,是心脏的一个重要组成部分,对改善心血管疾病起到重要的作用。但是现在对于这方面的研究很少,我们只能在探索中前进,寻找磁场发挥生物学效应的最佳波形,强度以及时间,使磁场在治疗心血管疾病上迈出一大步,为其在临床的早日应用打下基础。实验方法实验一不同波形低频脉冲磁场对大鼠心肌微血管内皮细胞的影响1.心肌微血管内皮细胞的原代培养,利用胰酶以及胶原酶消化法培养,用差速贴壁原理纯化细胞。2.实验分为两个大组:矩形波组以及三角波形磁场组,每大组又分为对照组和处理组,每个处理组内各分为0.6mT、1.2mT、1.8mT和2.4mT四个磁场强度照射组。对照组不接受磁场辐照,其他条件辐照组均与磁场辐照组相一致。各磁场辐照组的心肌微血管内皮细胞均于第一次传代在培养箱中培养一段时间后镜下观察见细胞贴壁时开始接受磁场辐照,选用脉冲磁场频率为15Hz,辐照时间为4h/d,连续照射5d。CCK-8法检测细胞增殖情况,transwell小室法检测细胞迁移情况,流式细胞仪测定细胞各周期的变化,Hoechst33258染色鉴定细胞凋亡状况,FITC标记的鬼笔环肽对细胞骨架染色,硝酸还原酶法检测细胞培养液中NO含量的变化。实验二三角波形低频脉冲磁场对大鼠心肌微血管内皮细胞增殖、迁移和细胞周期的影响1.胰酶和胶原酶消化法培养原代的心肌微血管内皮细胞,差速贴壁法纯化细胞。2.取心肌微血管内皮2代细胞,将其分为4个组:即对照组、1.0mT组、1.4mT组和1.8mT组。对照组的细胞不曝磁,其他条件均与1.0mT、1.4mT及1.8mT组一致。1.0mT、1.4mT及1.8mT组的CMECs均于传代后,镜下观察见细胞开始贴壁时,接受曝磁,以频率为15 Hz的脉冲磁场刺激4 h/d,连续照射3 d。MTT法检测细胞增殖活性,transwell小室法检测细胞迁移情况,流式细胞仪测定细胞各周期的变化。实验结果实验一不同波形低频脉冲磁场对大鼠心肌微血管内皮细胞的影响1.成功培养出原代心肌微血管内皮细胞并成功纯化。2.矩形波作用后经CCK-8检测显示4个磁场强度作用后与对照组相比细胞增殖水平均有提高,其中以1.8mT组增殖最为明显(0.9679±0.057 vs 0.7552±0.0334;P<0.01)。三角形波干预后0.6mT组与1.8mT组与对照组相比差异有统计学意义(P<0.05),1.2mT组与2.4mT组与对照组相比差异无统计学意义(P>0.05)。3.矩形波辐照CMECs的迁移加快,各组迁移的细胞数(个/视野)与对照组比较均有显著增多,其中以1.8mT组磁场强度作用最为明显(48.00±4.18 vs 23.80±3.11,P<0.01)。三角波0.6mT组和1.8mT组与对照组相比差异有统计学意义(P<0.05),1.2mT组与2.4mT组与对照组相比没有明显增多(P>0.05)。4.经流式细胞仪检测,细胞经矩形波辐照后,各组与对照组比较细胞各周期(G1期,S期以及G2期)均有明显变化,(G2+S)期的细胞明显增多, (P<0.01),其中以1.8mT组变化最为显著。经三角波辐照后1.8mT组和0.6mT组对周期均有一定的影响(P<0.05),1.2mT组和2.4mT组与对照组相比细胞周期几乎没有变化(P>0.05)。5.在荧光显微镜下观察,矩形波辐照后各组凋亡率均有减低(P<0.05)。三角波辐照后0.6mT组和1.8mT组凋亡率同样减低(P<0.05),1.2mT组和2.4mT组凋亡率与对照组相比无明显差异(2.18±0.19 vs 2.30±0.35;2.24±0.27 vs 2.30±0.35;P>0.05)。6.经矩形波辐照后细胞与对照组比较细胞骨架结构发生了重组,镜下观察见绿色荧光示经FITC标记的鬼笔环肽染色的细胞骨架,红色荧光为经碘化吡啶(PI)染色的细胞核,经矩形波磁场辐照后各组的荧光染色强度均增加,细胞膜下的应力纤维增多,聚集形成了绿色束样结构,肌动蛋白清晰可见,其中0.6mT组以及1.8mT组变化最为显著,三角波辐照后0.6mT和1.8mT亦有相同表现。7.经矩形波辐照5d后,NO分泌能力测定显示,与对照组相比,各组NO分泌能力均有明显提高(P<0.01)。经三角波辐照后,0.6mT组与1.8mT组与对照组相比有统计学意义(P<0.05),1.2mT组与2.4mT组与对照组相比NO分泌能力未见明显提高(P>0.05)。实验二三角波形低频脉冲磁场对大鼠心肌微血管内皮细胞增殖、迁移和细胞周期的影响1.用MTT比色法测定表明,1.4mT组和1.8mT组CMECs的增殖能力与对照组相比有显著提高(P<0.05);而1.0mT组经磁场辐照后,细胞增殖与对照组相比差异无统计学意义(P>0.05)。2. transwell小室法检测细胞迁移能力,细胞经结晶紫染色后,显微镜(×200)下观察计数。经统计学分析可见,磁场能够促进CMECs迁移。1.4mT组和1.8mT组迁移的细胞数(个/视野)与对照组比较差异显著(P<0.05),1.0mT组与对照组相比无明显差异(P>0.05)。3.流式细胞仪检测细胞周期结果显示,细胞经3 d曝磁后,1.4mT组和1.8mT组与对照组比较细胞各周期(G1期、S期和G2期)均有统计学意义,细胞处于增殖分化期的数量明显增多,其中1.8mT的组变化最为显著(P<0.05)。结论在固定频率下,大鼠心肌微血管内皮细胞的增殖、细胞周期、凋亡、迁移、细胞骨架以及NO分泌能力等生物学效应与低频脉冲磁场强度以及磁场的波形都是密切相关的,改变其中任何一个条件都会造成生物学效应的改变。更多还原

【Abstract】 Background and purpose of the experimentIn recent years, the role of the magnetic field has been paid gradually attention, it is a safe and effective means of physical therapy in the treatment of some diseases such as bone and joints and it has achieved good effect.Some researches have already showed that with the application of electromagnetic fields (EMFs) human umbilical vein endothelial cells and bovine aortic endothelial cells culture in vitro in blood vessel formation can be effectively promoted, FGF-2 secretion can also be promoted by the magnetic field, so people are constantly looking for a non-invasive therapy using magnetic field to improve cardiovascular disease. In recent years, the incidence of ischemic heart disease increased year by year, the treatment of ischemic heart disease is to promote endothelial regeneration and angiogenesis. Microvascular endothelial cells(CMECs) have many functions similar to endothelial cells , they can participate in the regeneration of blood vessels, they are an important component of the heart, and played an important part in improve cardiovascular disease . But now few studies were reported in this area, and we can only move forward in the exploration to find biological effects of magnetic field to play the best waveform, intensity and time, so that in the treatment of cardiovascular diseases took another step forward for its in lay in the foundation for the clinical application.MethodsPart 1 Effects of different waveforms of pulsed electromagnetic fields on myocardial microvascular endothelial cells1.Using trypsin and collagenase digestion method isolated cardiac microvascular endothelial cells , with differential adhesion method purified adherent cells.2. The cells were randomly divided into two sections: the rectangular and the triangular waveform group,each large group was divided into control and treatment groups ,and each treatment group were divided into four magnetic fields intensity,they are 0.6mT, 1.2mT, 1.8mT and 2.4mT .Control group did not receive magnetic field exposure, and other conditions of control group were consistent with the treatment group. The treatment groups were passaged and then cultured in the incubator for a period of time, when cell adhesion were observed we start to radiat. The frequency of pulsed magnetic field is 15Hz, irradiation time was 4h / d, continuous exposure 5d. CCK-8 was used to detected cell proliferation,cell migration was detected by transwell, the flow cytometry assayed cell cycle changes, FITC labeled phalloidin staining of the cytoskeleton and NO secretion was measured by Griess assay.Part 2 Effects of triangular waveform pulsed magnetic field on proliferation, migration and cell cycle of myocardial microvascular endothelial cell 1. Using trypsin and collagenase digestion method isolated cardiac microvascular endothelial cells , with differential adhesion method purified adherent cells.2. Cardiac microvascular endothelial cells were divided into four groups: control group, 1.0mT group, 1.4mT group and 1.8mT group. Control group did not receive magnetic field exposure, and other conditions of control group were consistent with the treatment group. When cell adhesion were observed we start to radiat. The frequency of pulsed magnetic field is 15Hz, irradiation time was 4h / d, continuous exposure 3d. MTT was used to detected cell proliferation,cell migration was detected by transwell, the flow cytometry assayed cell cycle changes. ResultsPart 1 Effects of different waveforms of pulsed electromagnetic fields on myocardial microvascular endothelial cells1. Successfully cultured cardiac microvascular endothelial cells and successfully purified.2. CCK-8 detected that after exposuring to the rectangular waveform magnetic field the levels of cell proliferation had raised compared with the control group, the most significant proliferation group is 1.8mT group (0.9679±0.057 vs 0.7552±0.0334; P <0.01 ).Triangle waveform magnetic filed significantly accelerated the proliferation of 0.6mT group and 1.8mT group compared with the control group (P <0.05) ,but 1.2mT group and 2.4mT group compared with the control group showed no significant difference (P> 0.05).3. Rectangular waveform magnetic field accelerated the migration of CMECs in each group (pre/view) .Compared with the control group the migrated number of CMECs significantly increased, in which the 1.8mT group was the most effective (48.00±4.18 vs 23.80±3.11, P <0.01).Compared with the control group, triangle waveform magnetic filed accelerated the migration of 0.6mT group and 1.8mT group significantly (P <0.05), compared with the control group 1.2mT group and 2.4mT group did not significantly increased (P> 0.05).4. By flow cytometry, cells irradiated by rectangular waveform magnetic field compared with the control group cells G1 phase, S phase and G2 phase were significant changes, (G2 + S) phase cells were increased ( P <0.01), the most significant change group is 1.8mT group.Triangle waveform magnetic filed of 1.8mT group and 0.6mT group had some effects on the cell cycle (P <0.05), the cell cycle of 1.2mT group and 2.4mT compared with the control group is almost no change (P> 0.05).5. With the fluorescence microscope, rectangular waveform magnetic field reduce the rate of apoptosis in each group (P <0.05). Triangle waveform magnetic filed of 1.8mT group and 0.6mT group had similar effect in reduce the apoptosis rate of CMECs(P <0.05), The apoptosis rate of 1.2mT group and 2.4mT group show no significant difference compared with the control group (2.18±0.19 vs 2.30±0.35; 2.24±0.27 vs 2.30±0.35; P> 0.05).6. After expose to the rectangular waveform magnetic field cells the cytoskeletal structure of the CMECs reorganized compared with control group , Immunofluorescent staining of each group showed that the stress of fiber membrane increased, the changes of 0.6mT group and 1.8mT group is the most significant, Triangle waveform magnetic filed of 0.6mmT group and 1.8mT group also has the same performance.7. After 5d, NO secretion tests showed that compared with the control group, NO secretion in each group were significantly increased (P <0.01).Triangle waveform magnetic filed of 0.6mT group and 1.8mT group compared with the control group were also accelerated (P <0.05), 1.2mT group and 2.4mT showed no significant increase on NO secretion compared with the control group (P>0.05).Part 2 Effects of triangular waveform pulsed magnetic field on proliferation, migration and cell cycle of myocardial microvascular endothelial cell1. MTT colorimetric assay showed that, the proliferation of 1.4mT group and 1.8mT group of CMECs were significantly increased compared with the control group (P <0.05); No obvious difference were detected in the 1.0mT group of proliferation(P>0.05).2. Transwell assaied the migration of CMECs, cells were treated with crystal violet staining. The statistical analysis shows that the magnetic field can promote the migration of the cells. The migration of 1.4mT group and 1.8mT group was significantly different compared with the control group (P <0.05), 1.0mT group and the control group showed no significant difference (P> 0.05).3. Cell cycle by flow cytometry showed that cells exposed to 3 days, The G1 phase, S phase and G2phase of 1.4mT group and 1.8mT was statistically difference, cell proliferation and differentiation increased significantly. The change of 1.8mT group is the most significant (P <0.05).ConclusionAt a fixed frequency, the biological effects of rat cardiac microvascular endothelial cell are closely related to the intension and the waveforms of pulsed magnetic fields.更多还原