电刺激诱导骨髓间充质干细胞分化为神经细胞作用的研究
发布时间：2019-06-20 01:51
【摘要】：周围神经损伤和脊髓损伤是骨科最常见的神经损伤性疾病，具有发生率高、损伤程度重、修复效果差、致残率高、治疗费用高等特点。“重建结构连续性和加快神经生长速度”是国际公认的神经损伤修复的关键，但这些问题至今尚未得到有效解决。组织工程细胞移植技术为神经再生及修复带来了希望，但由于取材来源有限和引发免疫排斥反应等问题，其应用受到限制。 骨髓间充质干细胞具有高度自我更新能力和多向分化潜能，可被诱导分化为神经细胞，被认为是理想的组织工程种子细胞。目前常用的化学诱导剂诱导法，易对细胞造成毒性损伤，导致诱导后细胞存活时间缩短、功能下降，无法满足移植需求。因此，亟需寻找一种安全、有效的诱导分化方法。 电刺激在神经损伤修复中的应用日益增加。有研究表明，电刺激可诱导脂肪干细胞分化成成骨细胞，并可促进骨髓间充质干细胞向心肌细胞分化，提示电刺激可干预干细胞的分化行为。但至今为止，有关电刺激对骨髓间充质干细胞诱导分化为神经细胞的研究未见报道。因此，研究其有效性和可行的实施方法，对于构建组织工程种子细胞、促进神经损伤修复具有重要意义。 本课题拟采用电刺激这一物理方法诱导骨髓间充质干细胞分化，在筛选并确定适宜电刺激参数的基础上，从细胞形态、细胞增殖能力、细胞凋亡情况、细胞分化等多个方面进行系统研究，为构建组织工程种子细胞、促进神经损伤修复提供新的思路。具体内容如下： 第一部分大鼠骨髓间充质干细胞的分离、培养及鉴定 目的：分离培养并纯化大鼠骨髓间充质干细胞，采用形态学观察、流式细胞技术和诱导分化成骨实验进行骨髓间充质干细胞的鉴定。 方法：应用全骨髓贴壁分离培养法培养纯化SD大鼠骨髓间充质干细胞，倒置显微镜观察细胞形态学变化；CCK-8法检测不同代数细胞的增殖能力；流式细胞技术检测CD29、CD90、CD45表达情况；使用成骨诱导培养基诱导P3代BMSCs成骨分化，明确其分化能力。 结果：应用全骨髓贴壁分离培养法，成功培养出生长状态较好的BMSCs，，CCK-8法检测显示细胞增殖能力良好。培养的细胞经流式细胞检测，大部分细胞呈现CD29、CD90阳性，CD45阴性，证明本实验所培养的细胞为骨髓间充质干细胞。进一步计数统计发现，CD29和CD90双阳性、CD45阴性的细胞数占总培养细胞数的96.89%，说明本实验获得的细胞纯度较高。采用常规化学法诱导培养细胞成骨分化实验结果显示，诱导后的细胞呈茜素红、ALP染色阳性，证明所培养的细胞可被诱导分化为成骨细胞，具有良好的分化潜能。 结论：通过细胞增殖能力检测、流式细胞鉴定以及成骨诱导分化实验证实，本实验所采用的全骨髓贴壁分离培养法，可稳定获得活性良好、具有自我更新能力和分化潜能的骨髓间充质干细胞，可为后续研究提供充足的细胞来源。 第二部分电刺激对于骨髓间充质干细胞诱导分化过程的作用研究 目的：采用电刺激诱导骨髓间充质干细胞向神经细胞分化，探索诱导分化的最适电刺激参数，初步阐明电刺激对骨髓间充质干细胞诱导分化为神经细胞的作用，为应用种子细胞移植修复神经损伤提供新方法。 方法：（1）应用直流电刺激，选取不同刺激时间（A：15min/d、B：30min/d、C：1h/d、D：2h/d、E：空白对照）和不同电压（A：1V、B：3V、C：5V、D：10V、E：空白对照），对骨髓间充质干细胞进行干预，CCK-8法检测细胞增殖情况变化，流式细胞技术检测电刺激对细胞凋亡的影响，筛选最适宜的电刺激参数。（2）采用筛选出的适宜电刺激参数进行诱导分化研究。分别设立电刺激诱导组、化学诱导组、复合诱导组和空白对照组，MAP-2免疫荧光染色鉴定细胞诱导分化情况，并计算诱导分化比率。 结果：(1)刺激时间筛选实验结果表明，当电压设定为3V时，采用刺激时间为30min/d（B组）的参数刺激后，细胞形态及数量均明显优于其余各组；细胞增殖能力较其余各组明显提高，且差异具有统计学意义；细胞凋亡检测发现该参数的电刺激未明显增加细胞凋亡。刺激电压筛选实验结果表明，当刺激时间为30min/d，采用刺激电压为3V（B组）的参数刺激后，细胞形态及数量优于其他各组，细胞增殖能力明显提高，且差异具有统计学意义；细胞凋亡检测表明该参数的电刺激未明显增加细胞凋亡。从而确定了3V、30min/d为适宜电刺激参数。(2)应用常规化学诱导法作为阳性对照，对BMSCs进行化学诱导，通过诱导率和免疫荧光染色结果的比较，发现电刺激诱导组与化学诱导组，均可诱导BMSCs分化为神经元，统计学无显著差异（P＞0.05）；而复合诱导组的诱导率，优于电刺激诱导组和化学诱导组，且具有统计学差异（P＜0.05）。但复合诱导组和化学诱导组，在细胞诱导成功后，出现细胞破裂、崩解及细胞残片，而电刺激组未见此现象。 结论：通过对不同刺激时间和不同电压参数进行筛选，确定3V，30min/d，连续刺激2d为适宜电刺激参数，应用于骨髓间充质干细胞诱导分化为神经细胞实验，研究结果表明，电刺激不仅可达到与化学诱导剂相同的诱导分化效果，而且毒性及损伤作用小，可更加安全有效的对骨髓间充质干细胞发挥诱导分化作用。本实验为应用电刺激干预干细胞提供实验依据，为组织工程种子细胞的构建提供了新的思路。
[Abstract]:Peripheral nerve injury and spinal cord injury are the most common nerve injury diseases in the department of orthopedics. It has the characteristics of high incidence, heavy injury degree, poor repair effect, high residual rate, high treatment cost and the like. "Reconstruction of structural continuity and speed of nerve growth" is the key to the internationally recognized repair of nerve injury, but these problems have not yet been effectively resolved. Tissue engineering cell transplantation technology has brought hope for nerve regeneration and repair, but its application is limited due to the limited source of material and immune rejection. The bone marrow mesenchymal stem cells have high self-renewal ability and multi-directional differentiation potential, can be induced to differentiate into nerve cells, and are considered to be ideal tissue engineering seeds The present general chemical inducers are easy to cause toxic damage to the cells, and the survival time of the cells after induction is shortened, the function is reduced, and the transplantation needs can not be met. Please, therefore, there is an urgent need to find a safe and effective method of inducing differentiation The application of electrical stimulation in the repair of nerve injury The results show that the electrical stimulation can induce the differentiation of the adipose-derived stem cells into the osteoblast and promote the differentiation of the bone marrow-derived mesenchymal stem cells into the cardiac muscle cells, suggesting that the electrical stimulation can interfere with the differentiation of the stem cells. The effect of electrical stimulation on the induction and differentiation of bone marrow-derived mesenchymal stem cells into nerve cells has not been studied. See the report. Therefore, to study its effectiveness and feasible implementation methods, for the construction of tissue engineering seed cells, it is of great importance to promote the repair of nerve injury The purpose of this study is to induce the differentiation of bone marrow-derived mesenchymal stem cells by the physical method of electrical stimulation. On the basis of screening and determining appropriate electric stimulation parameters, the cell morphology, cell proliferation ability, cell apoptosis, cell differentiation and so on are introduced. The study of the line system in order to construct the seed cells of tissue engineering and promote the repair of nerve injury To supply new ideas. The contents of the body are as follows: the first part of the rat bone marrow mesenchymal stem cells The purpose of isolation, culture and identification was to separate and culture and purify the rat bone marrow-derived mesenchymal stem cells. Identification of mesenchymal stem cells. Methods: The bone marrow mesenchymal stem cells of SD rats were cultured by full-bone marrow adherent culture method. The morphological changes of cells were observed by inverted microscope. The proliferation ability of different algebraic cells was detected by CCK-8 method. CD29 and CD were detected by flow cytometry. 90, CD45 expression; using osteogenic induction medium to induce P3 generation BMSCs to become The results showed that BMSCs and CCK-8 with better growth status were successfully cultured by the method of full-bone marrow adherent separation and culture. The cultured cells were detected by flow cytometry. Most of the cells exhibited CD29, CD90 positive and CD45 negative. The cultured cells were bone marrow mesenchymal stem cells. The results showed that the number of CD29 and CD90 positive and CD45-negative cells accounted for 96.89% of the total number of cultured cells. The results of induction of osteogenic differentiation of cultured cells by conventional chemical method showed that the induced cells showed a positive staining and the ALP staining was positive, and that the cultured cells could be induced to differentiate into the cells. Osteoclasts, with good differentiation potential. Conclusion: Through cell proliferation, flow cytometry and osteogenic differentiation, the whole bone marrow adherent culture method is used in this experiment, which can be stably obtained with good activity and self-renewal. the mesenchymal stem cells of the force and the differentiation potential, Adequate source of cells can be provided for subsequent studies. The second part of the electrical stimulation is for bone The Role of Mesenchymal Stem Cells in the Differentiation of Mesenchymal Stem Cells: Induction of Mesenchymal Stem Cells from Mesenchymal Stem Cells to the Nerves by Electrostimulation The effects of electrical stimulation on the induction and differentiation of bone marrow-derived mesenchymal stem cells into nerve cells were discussed, and the effects of electrical stimulation on the induction and differentiation of bone marrow-derived mesenchymal stem cells into nerve cells were discussed. Methods: (1) DC stimulation was applied to select different stimulation time (A:15 min/ d, B:30 min/ d, C:1 h/ d, D: 2h/ d, E: blank control) and different voltage (A:1 V, B: 3 V, C:5 V, D:10 V, E: blank control), the bone marrow mesenchymal stem cells were intervened, the proliferation of the cells was detected by the CCK-8 method, and the electropuncture was detected by flow cytometry. The most appropriate parameters of the electrical stimulation were selected by the effect of the stimulation on the apoptosis of the cells. (2) The induced differentiation was induced by the appropriate electrical stimulation parameters. The electrical stimulation induction group, the chemical induction group, the compound induction group, the blank control group and the MAP-2 immunofluorescence staining were respectively established. Results: (1) The experimental results of the stimulation time showed that, when the voltage was set to 3 V, the cell morphology and number of the cells were significantly superior to the other groups after the stimulation time was 30 min/ d (group B). The colonizing ability was significantly higher than the other groups, and the difference was of statistical significance. It was found that the electrical stimulation of the parameters did not significantly increase the apoptosis of the cells. The experimental results of the stimulation of the voltage showed that the cell morphology and number were superior to that of the other cells after the stimulation time was 30 min/ day and the stimulation voltage was 3 V (group B). The cell proliferation ability of the group was significantly improved, and the difference was of statistical significance; fine The cell apoptosis test showed that the electrical stimulation of the parameter did not significantly increase the apoptosis of the cells. 3 V and 30 min/ d were determined to be suitable for electrical stimulation. (2) BMSCs were induced by chemical induction, induction rate and immunofluorescence staining, and BMSCs were induced by the comparison of induction rate and immunofluorescence staining. The induction rate of the compound induction group was superior to that of the electric stimulation induction group. And the combination induction group and the chemical induction group, after the cell induction was successful, appeared. The results showed that 3 V,30 min/ d and 2 d of continuous stimulation were suitable for stimulation of bone by screening different stimulation time and different voltage parameters. The induction and differentiation of the mesenchymal stem cells into the nerve cell experiments show that the electrical stimulation can not only achieve the same induction and differentiation effect as the chemical inducers, but also the toxicity and the damage effect are small, The invention can more safely and effectively induce differentiation of the bone marrow mesenchymal stem cells.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
[Abstract]:Peripheral nerve injury and spinal cord injury are the most common nerve injury diseases in the department of orthopedics. It has the characteristics of high incidence, heavy injury degree, poor repair effect, high residual rate, high treatment cost and the like. "Reconstruction of structural continuity and speed of nerve growth" is the key to the internationally recognized repair of nerve injury, but these problems have not yet been effectively resolved. Tissue engineering cell transplantation technology has brought hope for nerve regeneration and repair, but its application is limited due to the limited source of material and immune rejection. The bone marrow mesenchymal stem cells have high self-renewal ability and multi-directional differentiation potential, can be induced to differentiate into nerve cells, and are considered to be ideal tissue engineering seeds The present general chemical inducers are easy to cause toxic damage to the cells, and the survival time of the cells after induction is shortened, the function is reduced, and the transplantation needs can not be met. Please, therefore, there is an urgent need to find a safe and effective method of inducing differentiation The application of electrical stimulation in the repair of nerve injury The results show that the electrical stimulation can induce the differentiation of the adipose-derived stem cells into the osteoblast and promote the differentiation of the bone marrow-derived mesenchymal stem cells into the cardiac muscle cells, suggesting that the electrical stimulation can interfere with the differentiation of the stem cells. The effect of electrical stimulation on the induction and differentiation of bone marrow-derived mesenchymal stem cells into nerve cells has not been studied. See the report. Therefore, to study its effectiveness and feasible implementation methods, for the construction of tissue engineering seed cells, it is of great importance to promote the repair of nerve injury The purpose of this study is to induce the differentiation of bone marrow-derived mesenchymal stem cells by the physical method of electrical stimulation. On the basis of screening and determining appropriate electric stimulation parameters, the cell morphology, cell proliferation ability, cell apoptosis, cell differentiation and so on are introduced. The study of the line system in order to construct the seed cells of tissue engineering and promote the repair of nerve injury To supply new ideas. The contents of the body are as follows: the first part of the rat bone marrow mesenchymal stem cells The purpose of isolation, culture and identification was to separate and culture and purify the rat bone marrow-derived mesenchymal stem cells. Identification of mesenchymal stem cells. Methods: The bone marrow mesenchymal stem cells of SD rats were cultured by full-bone marrow adherent culture method. The morphological changes of cells were observed by inverted microscope. The proliferation ability of different algebraic cells was detected by CCK-8 method. CD29 and CD were detected by flow cytometry. 90, CD45 expression; using osteogenic induction medium to induce P3 generation BMSCs to become The results showed that BMSCs and CCK-8 with better growth status were successfully cultured by the method of full-bone marrow adherent separation and culture. The cultured cells were detected by flow cytometry. Most of the cells exhibited CD29, CD90 positive and CD45 negative. The cultured cells were bone marrow mesenchymal stem cells. The results showed that the number of CD29 and CD90 positive and CD45-negative cells accounted for 96.89% of the total number of cultured cells. The results of induction of osteogenic differentiation of cultured cells by conventional chemical method showed that the induced cells showed a positive staining and the ALP staining was positive, and that the cultured cells could be induced to differentiate into the cells. Osteoclasts, with good differentiation potential. Conclusion: Through cell proliferation, flow cytometry and osteogenic differentiation, the whole bone marrow adherent culture method is used in this experiment, which can be stably obtained with good activity and self-renewal. the mesenchymal stem cells of the force and the differentiation potential, Adequate source of cells can be provided for subsequent studies. The second part of the electrical stimulation is for bone The Role of Mesenchymal Stem Cells in the Differentiation of Mesenchymal Stem Cells: Induction of Mesenchymal Stem Cells from Mesenchymal Stem Cells to the Nerves by Electrostimulation The effects of electrical stimulation on the induction and differentiation of bone marrow-derived mesenchymal stem cells into nerve cells were discussed, and the effects of electrical stimulation on the induction and differentiation of bone marrow-derived mesenchymal stem cells into nerve cells were discussed. Methods: (1) DC stimulation was applied to select different stimulation time (A:15 min/ d, B:30 min/ d, C:1 h/ d, D: 2h/ d, E: blank control) and different voltage (A:1 V, B: 3 V, C:5 V, D:10 V, E: blank control), the bone marrow mesenchymal stem cells were intervened, the proliferation of the cells was detected by the CCK-8 method, and the electropuncture was detected by flow cytometry. The most appropriate parameters of the electrical stimulation were selected by the effect of the stimulation on the apoptosis of the cells. (2) The induced differentiation was induced by the appropriate electrical stimulation parameters. The electrical stimulation induction group, the chemical induction group, the compound induction group, the blank control group and the MAP-2 immunofluorescence staining were respectively established. Results: (1) The experimental results of the stimulation time showed that, when the voltage was set to 3 V, the cell morphology and number of the cells were significantly superior to the other groups after the stimulation time was 30 min/ d (group B). The colonizing ability was significantly higher than the other groups, and the difference was of statistical significance. It was found that the electrical stimulation of the parameters did not significantly increase the apoptosis of the cells. The experimental results of the stimulation of the voltage showed that the cell morphology and number were superior to that of the other cells after the stimulation time was 30 min/ day and the stimulation voltage was 3 V (group B). The cell proliferation ability of the group was significantly improved, and the difference was of statistical significance; fine The cell apoptosis test showed that the electrical stimulation of the parameter did not significantly increase the apoptosis of the cells. 3 V and 30 min/ d were determined to be suitable for electrical stimulation. (2) BMSCs were induced by chemical induction, induction rate and immunofluorescence staining, and BMSCs were induced by the comparison of induction rate and immunofluorescence staining. The induction rate of the compound induction group was superior to that of the electric stimulation induction group. And the combination induction group and the chemical induction group, after the cell induction was successful, appeared. The results showed that 3 V,30 min/ d and 2 d of continuous stimulation were suitable for stimulation of bone by screening different stimulation time and different voltage parameters. The induction and differentiation of the mesenchymal stem cells into the nerve cell experiments show that the electrical stimulation can not only achieve the same induction and differentiation effect as the chemical inducers, but also the toxicity and the damage effect are small, The invention can more safely and effectively induce differentiation of the bone marrow mesenchymal stem cells.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
【参考文献】
相关期刊论文 前10条
1 龙乾发;刘卫平;玉石;刘阳;韩蕊;王孝安;李娟;;体外诱导大鼠骨髓间充质干细胞向GABA能神经元分化[J];中华神经外科疾病研究杂志;2009年06期
2 伍长学;杨思远;马建e
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