【作者】 周娅蕾；

【导师】 罗二平； 申广浩；

【作者基本信息】 第四军医大学 ， 生物物理学， 2007， 硕士

【摘要】 20世纪70年代以来,脉冲电磁场(pulsed electromagnetic fields,PEMFs)作为一种非侵入性治疗方法被用于骨折延迟愈合、骨不连、骨质疏松症等骨科疾病。电磁场能促进活细胞增殖及分化,刺激骨局部因子的产生,并能改善股骨的骨密度和生物力学特性。但是电磁场对骨的作用机理还不是很清楚,电磁场的频率、场强、作用时间以及脉宽波形等对治疗效果也有不同程度的影响。间充质干细胞(mesenchymal stem cells, MSCs)是存在于骨髓基质系统中的一种组织干细胞,具有自我更新能力和多向分化潜能,一定的诱导条件下可分化为成骨细胞。而且成体干细胞具有注入体内后的趋化性特征,较明显的集中到受损部位,到达受损伤部位后,在局部微环境的诱导下,成体干细胞发生明显的诱导分化,使成体干细胞向损伤组织修复所急需的组织细胞分化,从而促进损伤组织的修复[1]。这对骨科疾病的自体细胞治疗甚至异体细胞的治疗提供了可能性。本课题主要研究低强度PEMFs对体外培养大鼠骨髓间充质干细胞(rat mesenchymal stem cells, rMSCs)增殖及向成骨细胞分化的影响。应用MTT法、ALP测定、四环素荧光标记、钙结节染色法测定MSCs增殖及向成骨细胞分化后的体外骨形成情况。初步探讨了频率和强度对rMSCs增殖和成骨分化的影响。为临床应用PEMFs治疗骨折、骨不连、骨质疏松症等骨科疾病提供了新的思路与方法。主要工作概括如下:1、rMSCs的原代培养目的:探讨rMSCs简单有效的体外原代培养方法。方法:用全骨髓贴壁法分离rMSCs,差速黏附法进行纯化,绘制原代及生长良好的第3代细胞的生长曲线,进行PAS-过碘酸雪夫氏染色、油红-O染色和碱性磷酸酶(ALP)染色。结果:细胞形态良好,具有MSCs外观,生长曲线呈典型的S型,染色结果均为阴性,提示细胞未分泌肝糖元,未向脂肪细胞及成骨细胞方向分化。结论:实验采用的方法可以获得一定量的rMSCs,方法简单可行,为进一步实验提供了物质基础。2、PEMFs对rMSCs增殖的影响目的:探讨不同频率,不同磁场强度的PEMFs对rMSCs的影响。方法:用全骨髓贴壁法分离rMSCs,对生长良好的第3代细胞进行15Hz ,0.2mT; 15Hz ,0.4mT;15Hz ,0.8mT;1Hz,0.2mT; 1Hz,0.4mT; 1Hz,0.8mT的PEMFs刺激,MTT法测定细胞增殖水平,流式细胞仪法测定细胞周期变化。结果:rMSCs经PEMFs刺激5d(1次/1d,每次3h)后,各实验组细胞增殖水平和(S+G2/M)期细胞数量均有不同程度提高,差异有统计学意义(P<0.05)。其中MTT水平以1Hz,0.4mT组变化最为明显(3d,0.323±0.051;5d,0.714±0.058),和对照组相比(3d,0.246±0.044;5d,0.487±0.064),差异具统计学意义(P<0.01);细胞周期变化也以1Hz,0.4mT组变化最为明显(G0/G1:75.3±0.17,S:8.5±0.12,G2/M:16.4±0.20,PI:24.9),相对于对照组(G0/G1:83.3±0.33,S:7.0±0.29,G2/M:9.8±0.23,PI:16.8),差异具有统计学意义(P<0.05)。结论:rMSCs经PEMFs场刺激后,能促进体外培养该细胞的增殖水平,磁场参数对实验结果有影响,以1Hz,0.4mT的PEMFs效果最佳。3、PEMFs对rMSCs成骨分化的影响目的:探讨PEMFs刺激对rMSCs向成骨细胞分化的影响。方法:用全骨髓贴壁法分离rMSCs,经差速黏附法筛选,对生长良好的第3代细胞进行频率1Hz、磁感应强度0.4mT的PEMFs刺激,检测碱性磷酸酶活性,进行四环素荧光染色和钙结节染色。结果:rMSCs经PEMFs刺激(1次/1d,每次3h)后,实验组碱性磷酸酶水平升高,差异有统计学意义(P<0.05),四环素荧光染色和钙结节染色结果均呈阳性。结论:推测PEMFs能影响rMSCs向成骨细胞的分化。更多还原

【Abstract】 From the 1960s, pulsed electromagnetic fields(PEMFs) is used to treat many kinds of bone diseases such as delayed union, nonunion and osteoporosis without any incursive damage. PEMFs can improve the proliferation and differentiation of live cells, stimulate the appearance of the local factors, and meliorate the bone density and biomechanics trait of thighbone. But the mechanism of electromagnetic fields on bone is unclear. The frequency, stimulation time, intensity and pulse shape of PEMFs can influence the therapeutic in different degree. Mesenchymal stem cells (MSCs) is one kind of tissue stem cells, they indwelled in marrow stroma system. MSCs have the ability to update themselves and the potential to transform into many kinds of other cells. And they can transform into osteoblast under specifical conditions. Furthermore, adult stem cells put up their chemotaxis when they were injected into one’s body. In other words they concentrate in where is injured. Under the local microenvironment, adult stem cells turn into certain tissue cells which is urgently need to repair the injured tissue. And then, the injured tissue repaired. This offer the possibility that one will be cured with his own cells even with xenogenous cells.This experiment is to observe the effects of PEMFs on MSCs of SD rat in vitro. This experiment is to observe the effects of PEMFs on osteoblast of SD rat in vitro. MTT assay, Alkaline phosphatatase- specific activity assay, tetracycline fluorescence labeling assay,calcium nodal stain were used to observe the effects of PEMFs on MSCs’proliferation, differentiation and bone tissue-like formation. We design some experiments to observe the effect of PEMFs’frequency and intensity on MSCs’proliferation, differentiation. The experiment results offer us some evidence to treat bone diseases such as delayed union, nonunion and osteoporosis.The work can be summarized as follows:1. MSCs isolated and culturedOBJECTIVE: To study a simple and effective method to isolate MSCs form rat. METHTHODS: The mesenchymal stem cells were obtained by using whole marrow attachment method and differential speed attachment method was used to pure them. Draw the growth carve of MSCs of primary generation and third generation. Periodic acid-Schiff’s stain, oil red-O stain and alkaline phosphatase stain were practiced. RESULTS: The configuration of MSCs is favorable, the growth carve show typical“S”.The results of stains are all negative, it means the cells can’s excrete hepatin, include no lipocyte and osteoblast. CONCLUSION: The method can obtained MSCs and it’s simple and effective. It’s a preparation for the next experiment.2. The effects of pulsed electromagnetic fields rat mesenchymal stem cellOBJECTIVE: To study the effects of pulsed electromagnetic fields (PEMFs) with different frequency and intensity on mesenchymal stem cells of rat. METHTHODS: The mesenchymal stem cells were obtained by using whole marrow attachment method. The third generation cells were irradiated by use of pulsed electromagnetic fields with 15Hz,0.2mT; 15Hz,0.4mT; 15Hz,0.8mT; 1Hz,0.2mT; 1Hz,0.4mT; 1Hz,0.8mT. The method of MTT was employed to evaluate the levels of proliferation. The parameters of the cells cycle were detected with the flow cytometer (FCM). RESULTS: After 5 days of irradiation by pulsed electromagnetic fields (once a day, 3h each time), the proliferation of the mesenchymal stem cells in rat was accelerated. The result of MTT is chaned significantly(P<0.01). The group of 1Hz ,0.4mT are most are most effective(3d,0.323±0.051;5d,0.714±0.058), they are increased significantly compare with the sham group(3d,0.246±0.044;5d,0.487±0.064)(P<0.01). Effective and the percentage of cells at the (S+G2/M) phase was increased significantly (P<0.05). And the group of 1Hz ,0.4mT are most effective(G0/G1:75.3±0.17, S : 8.5±0.12, G2/M : 16.4±0.20, PI : 24.9), they are increased significantly compare with the sham group (G0/G1:83.3±0.33,S:7.0±0.29, G2/M:9.8±0.23, PI : 16.8) (P<0.01). CONCLUSION: Irradiation of pulsed electromagnetic fields could increase the proliferation of mesenchymal stem cells of rat in vitro, and the results could be affected by the parameters of PEMFs.3. The effects of pulsed electromagnetic fields on inducing rat mesenchymal stem cells into osteoblastOBJECTIVE: To study the effects of pulsed electromagnetic fields (PEMFs) on inducing the mesenchymal stem cells (MSCs) into osteoblast. METHTHODS: The rat mesenchymal stem cells were obtained by using whole marrow attachment method, and then selected by the adhesive method. The third generation cells were irradiated by PEMFs of 1Hz, 0.4mT, then the activity of Alkaline phosphatase (ALP) was measured, tetracycline fluorescence labeling assay and bone nodule formation were used. RESULTS: After being exposed in PEMFs (once a day,3h each time), activity of ALP was increased significantly (P<0.05), and tetracycline fluorescence labeling assay and bone nodule formation were positive. CONCLUSION: Exposing in PEMFs could induce rat mesenchymal stem cells into osteoblast.更多还原