機械拉伸對大鼠骨髓間充質(zhì)干細胞遷移行為的調(diào)節(jié)及其相關(guān)分子機理研究
發(fā)布時間：2018-08-26 12:47
【摘要】：骨髓間充質(zhì)干細胞（bone marrow derived mesenchymal stem cells, MSCs）是一類具有增殖和多向分化潛能的特殊細胞，在特定環(huán)境條件下，MSCs可增殖并定向分化為多種細胞。同時，MSCs具有趨化特性，可以響應(yīng)炎癥或組織損傷（包括腫瘤組織），，進行動員、遷移。MSCs在機體組織損傷修復(fù)和再生中具有重要作用，是臨床細胞治療的種子細胞，也是目前應(yīng)用最多的干細胞類型。 機體中的細胞和組織處于復(fù)雜的力學(xué)微環(huán)境中，MSCs在移出骨髓進入外周血循環(huán)的過程中，受到血液流動產(chǎn)生的剪應(yīng)力和張應(yīng)變的影響。力學(xué)因素可調(diào)節(jié)活細胞的生理、病理過程，有關(guān)機械刺激對MSCs增殖、表型和分化影響的報道比較多，相關(guān)的分子機理在深入研究中。但到目前為止，人們對影響MSCs遷移行為的力學(xué)因素及其分子機理還知之甚少。本文試圖揭示機械拉伸對大鼠MSCs（ratMSCs, rMSCs）遷移行為的影響及其相關(guān)分子機理。 本文選用原代分離培養(yǎng)的rMSCs，運用單軸拉伸加載裝置對培養(yǎng)在彈性硅膠膜上的rMSCs施加不同條件（頻率1Hz，形變量5%～15%，拉伸時間4～12h）的機械拉伸。采用Transwell法和劃痕法評價拉伸前后細胞遷移能力的變化，基于明膠酶譜法檢測基質(zhì)金屬蛋白酶-2,-（9Matrix metalloproteinase-2,-9）表達的變化。用MMPs抑制劑GM6001抑制拉伸刺激對rMSCs MMP-2和MMP-9表達的激活，考察抑制MMP-2和MMP-9對拉伸促進rMSCs的遷移的影響。實驗結(jié)果如下： ①rMSCs的原代分離、培養(yǎng)及鑒定 本實驗采用Percoll密度梯度離心法，結(jié)合rMSCs的貼壁特性，體外分離、培養(yǎng)rMSCs，rMSCs形狀一致，呈長梭樣、漩渦狀分布。流式細胞儀檢測結(jié)果顯示，rMSCs原代細胞表達CD44和CD90，不表達CD34，與rMSCs表面標(biāo)記抗原一般規(guī)律一致，說明分離培養(yǎng)的rMSCs表型均一，符合rMSCs特點。 ②機械拉伸加載對rMSCs遷移行為的影響 將細胞培養(yǎng)在彈性硅膠膜上，采用自制的細胞拉伸加載裝置拉伸硅膠膜，對細胞實施加載。運用Transwell法檢測細胞遷移，研究發(fā)現(xiàn)頻率1Hz、形變量10%、拉伸時間8h加載條件促進rMSCs的遷移效果較明顯。劃痕實驗進一步說明，頻率1Hz、形變量10%、拉伸時間8h機械刺激對rMSCs遷移能力有促進作用。 ③MMP-2和MMP-9參與機械拉伸誘導(dǎo)的rMSCs遷移 采用明膠酶譜法檢測頻率1Hz、形變量10%、拉伸時間8h加載條件下MMP-2和MMP-9活性變化，發(fā)現(xiàn)拉伸加載組，rMSCs中MMP-2和MMP-9活性分別增加到對照組的235%和248%。 在機械拉伸前加入MMP-2和MMP-9抑制劑GM6001，檢測細胞遷移和MMP-2和MMP-9表達變化，結(jié)果發(fā)現(xiàn)25μM GM6001顯著抑制機械刺激誘導(dǎo)的MMP-2和MMP-9分泌增加，同時機械刺激誘導(dǎo)的rMSCs遷移受到抑制。結(jié)果提示：頻率1Hz、形變量10%、拉伸時間8h的拉伸加載促進rMSCs遷移可能通過刺激rMSCs分泌MMP-2和MMP-9來實現(xiàn)。 本文研究結(jié)果表明，適宜的機械拉伸加載可誘導(dǎo)rMSCs分泌MMP-2和MMP-9，促進rMSCs遷移能力。該研究結(jié)果為體外調(diào)控rMSCs的高效遷移從而更好發(fā)揮其組織修復(fù)作用提供了實驗依據(jù)和理論基礎(chǔ)，也為干細胞治療、組織工程及再生醫(yī)學(xué)的相關(guān)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.
【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329
本文編號：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.
【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329
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