机械拉伸对大鼠骨髓间充质干细胞迁移行为的调节及其相关分子机理研究
发布时间：2018-08-26 12:47
【摘要】：骨髓间充质干细胞（bone marrow derived mesenchymal stem cells, MSCs）是一类具有增殖和多向分化潜能的特殊细胞，在特定环境条件下，MSCs可增殖并定向分化为多种细胞。同时，MSCs具有趋化特性，可以响应炎症或组织损伤（包括肿瘤组织），，进行动员、迁移。MSCs在机体组织损伤修复和再生中具有重要作用，是临床细胞治疗的种子细胞，也是目前应用最多的干细胞类型。 机体中的细胞和组织处于复杂的力学微环境中，MSCs在移出骨髓进入外周血循环的过程中，受到血液流动产生的剪应力和张应变的影响。力学因素可调节活细胞的生理、病理过程，有关机械刺激对MSCs增殖、表型和分化影响的报道比较多，相关的分子机理在深入研究中。但到目前为止，人们对影响MSCs迁移行为的力学因素及其分子机理还知之甚少。本文试图揭示机械拉伸对大鼠MSCs（ratMSCs, rMSCs）迁移行为的影响及其相关分子机理。 本文选用原代分离培养的rMSCs，运用单轴拉伸加载装置对培养在弹性硅胶膜上的rMSCs施加不同条件（频率1Hz，形变量5%～15%，拉伸时间4～12h）的机械拉伸。采用Transwell法和划痕法评价拉伸前后细胞迁移能力的变化，基于明胶酶谱法检测基质金属蛋白酶-2,-（9Matrix metalloproteinase-2,-9）表达的变化。用MMPs抑制剂GM6001抑制拉伸刺激对rMSCs MMP-2和MMP-9表达的激活，考察抑制MMP-2和MMP-9对拉伸促进rMSCs的迁移的影响。实验结果如下： ①rMSCs的原代分离、培养及鉴定 本实验采用Percoll密度梯度离心法，结合rMSCs的贴壁特性，体外分离、培养rMSCs，rMSCs形状一致，呈长梭样、漩涡状分布。流式细胞仪检测结果显示，rMSCs原代细胞表达CD44和CD90，不表达CD34，与rMSCs表面标记抗原一般规律一致，说明分离培养的rMSCs表型均一，符合rMSCs特点。 ②机械拉伸加载对rMSCs迁移行为的影响 将细胞培养在弹性硅胶膜上，采用自制的细胞拉伸加载装置拉伸硅胶膜，对细胞实施加载。运用Transwell法检测细胞迁移，研究发现频率1Hz、形变量10%、拉伸时间8h加载条件促进rMSCs的迁移效果较明显。划痕实验进一步说明，频率1Hz、形变量10%、拉伸时间8h机械刺激对rMSCs迁移能力有促进作用。 ③MMP-2和MMP-9参与机械拉伸诱导的rMSCs迁移 采用明胶酶谱法检测频率1Hz、形变量10%、拉伸时间8h加载条件下MMP-2和MMP-9活性变化，发现拉伸加载组，rMSCs中MMP-2和MMP-9活性分别增加到对照组的235%和248%。 在机械拉伸前加入MMP-2和MMP-9抑制剂GM6001，检测细胞迁移和MMP-2和MMP-9表达变化，结果发现25μM GM6001显著抑制机械刺激诱导的MMP-2和MMP-9分泌增加，同时机械刺激诱导的rMSCs迁移受到抑制。结果提示：频率1Hz、形变量10%、拉伸时间8h的拉伸加载促进rMSCs迁移可能通过刺激rMSCs分泌MMP-2和MMP-9来实现。 本文研究结果表明，适宜的机械拉伸加载可诱导rMSCs分泌MMP-2和MMP-9，促进rMSCs迁移能力。该研究结果为体外调控rMSCs的高效迁移从而更好发挥其组织修复作用提供了实验依据和理论基础，也为干细胞治疗、组织工程及再生医学的相关研究提供了新思路和新方法。
[Abstract]:Bone marrow derived mesenchymal stem cells (MSCs) are a kind of special cells with the potential of proliferation and multi-directional differentiation. Under certain environmental conditions, MSCs can proliferate and differentiate into a variety of cells. Migration. MSCs play an important role in tissue repair and regeneration. MSCs are the seed cells of clinical cell therapy and the most widely used stem cell types.
Cells and tissues in the organism are in a complex mechanical microenvironment. During the process of removing bone marrow and entering the peripheral blood circulation, MSCs are affected by the shear stress and tensile strain produced by the blood flow. However, little is known about the mechanical and molecular mechanisms that affect the migration behavior of MSCs. This paper attempts to reveal the effects of mechanical stretching on the migration behavior of rat MSCs (ratMSCs, rMSCs) and the related molecular mechanisms.
In this paper, the primary isolated and cultured rMSCs were mechanically stretched under different conditions (frequency 1 Hz, deformation 5%-15%, stretching time 4-12 h) by a uniaxial tensile loading device. The migration ability of the cells before and after stretching was evaluated by Transwell method and scratch method, and the matrix was determined by gelatin zymography. The expression of metalloproteinase-2, -(9 Matrix metalloproteinase-2, -9) was altered. MMPs inhibitor GM6001 was used to inhibit the activation of MMP-2 and MMP-9 in rMSCs by stretching stimulation. The effects of inhibiting MMP-2 and MMP-9 on the migration of rMSCs by stretching were investigated.
Isolation, culture and identification of rMSCs
Percoll density gradient centrifugation combined with the adherence characteristics of rMSCs was used to isolate and culture rMSCs in vitro. The results of flow cytometry showed that the primary cells of rMSCs expressed CD44 and CD90, but did not express CD34, which was consistent with the general rule of surface marker antigen of rMSCs. The phenotype is homogeneous and conforms to rMSCs characteristics.
Effect of mechanical tensile loading on migration behavior of rMSCs
Cells were cultured on elastic silica gel membrane and loaded with a self-made stretching loading device. Cell migration was detected by Transwell method. The results showed that the loading conditions of 1 Hz frequency, 10% deformation and 8 h stretching time could promote the migration of rMSCs. Stretching time 8h mechanical stimulation promoted the migration of rMSCs.
MMP-2 and MMP-9 participate in mechanical stretching induced rMSCs migration.
The activities of MMP-2 and MMP-9 were measured by gelatin zymogram at 1 Hz, 10% deformation and 8 h tensile time. The results showed that the activities of MMP-2 and MMP-9 in rMSCs increased to 235% and 248% of the control group, respectively.
MMP-2 and MMP-9 inhibitor GM6001 were added before mechanical stretching to detect cell migration and MMP-2 and MMP-9 expression. The results showed that 25 mu M GM6001 significantly inhibited the increase of MMP-2 and MMP-9 secretion induced by mechanical stimulation, and the migration of rMSCs induced by mechanical stimulation was inhibited. The promotion of rMSCs migration may be achieved by stimulating rMSCs to secrete MMP-2 and MMP-9.
The results of this study indicate that suitable mechanical tensile loading can induce the secretion of MMP-2 and MMP-9 by rMSCs and promote the migration of rMSCs. These results provide experimental and theoretical basis for regulating the efficient migration of rMSCs in vitro and thus play a better role in tissue repair, and also provide relevant research for stem cell therapy, tissue engineering and regenerative medicine. The research provides new ideas and new methods.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
本文编号：2204913
[Abstract]:Bone marrow derived mesenchymal stem cells (MSCs) are a kind of special cells with the potential of proliferation and multi-directional differentiation. Under certain environmental conditions, MSCs can proliferate and differentiate into a variety of cells. Migration. MSCs play an important role in tissue repair and regeneration. MSCs are the seed cells of clinical cell therapy and the most widely used stem cell types.
Cells and tissues in the organism are in a complex mechanical microenvironment. During the process of removing bone marrow and entering the peripheral blood circulation, MSCs are affected by the shear stress and tensile strain produced by the blood flow. However, little is known about the mechanical and molecular mechanisms that affect the migration behavior of MSCs. This paper attempts to reveal the effects of mechanical stretching on the migration behavior of rat MSCs (ratMSCs, rMSCs) and the related molecular mechanisms.
In this paper, the primary isolated and cultured rMSCs were mechanically stretched under different conditions (frequency 1 Hz, deformation 5%-15%, stretching time 4-12 h) by a uniaxial tensile loading device. The migration ability of the cells before and after stretching was evaluated by Transwell method and scratch method, and the matrix was determined by gelatin zymography. The expression of metalloproteinase-2, -(9 Matrix metalloproteinase-2, -9) was altered. MMPs inhibitor GM6001 was used to inhibit the activation of MMP-2 and MMP-9 in rMSCs by stretching stimulation. The effects of inhibiting MMP-2 and MMP-9 on the migration of rMSCs by stretching were investigated.
Isolation, culture and identification of rMSCs
Percoll density gradient centrifugation combined with the adherence characteristics of rMSCs was used to isolate and culture rMSCs in vitro. The results of flow cytometry showed that the primary cells of rMSCs expressed CD44 and CD90, but did not express CD34, which was consistent with the general rule of surface marker antigen of rMSCs. The phenotype is homogeneous and conforms to rMSCs characteristics.
Effect of mechanical tensile loading on migration behavior of rMSCs
Cells were cultured on elastic silica gel membrane and loaded with a self-made stretching loading device. Cell migration was detected by Transwell method. The results showed that the loading conditions of 1 Hz frequency, 10% deformation and 8 h stretching time could promote the migration of rMSCs. Stretching time 8h mechanical stimulation promoted the migration of rMSCs.
MMP-2 and MMP-9 participate in mechanical stretching induced rMSCs migration.
The activities of MMP-2 and MMP-9 were measured by gelatin zymogram at 1 Hz, 10% deformation and 8 h tensile time. The results showed that the activities of MMP-2 and MMP-9 in rMSCs increased to 235% and 248% of the control group, respectively.
MMP-2 and MMP-9 inhibitor GM6001 were added before mechanical stretching to detect cell migration and MMP-2 and MMP-9 expression. The results showed that 25 mu M GM6001 significantly inhibited the increase of MMP-2 and MMP-9 secretion induced by mechanical stimulation, and the migration of rMSCs induced by mechanical stimulation was inhibited. The promotion of rMSCs migration may be achieved by stimulating rMSCs to secrete MMP-2 and MMP-9.
The results of this study indicate that suitable mechanical tensile loading can induce the secretion of MMP-2 and MMP-9 by rMSCs and promote the migration of rMSCs. These results provide experimental and theoretical basis for regulating the efficient migration of rMSCs in vitro and thus play a better role in tissue repair, and also provide relevant research for stem cell therapy, tissue engineering and regenerative medicine. The research provides new ideas and new methods.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
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