機(jī)械拉伸對(duì)大鼠骨髓間充質(zhì)干細(xì)胞遷移行為的調(diào)節(jié)及其相關(guān)分子機(jī)理研究
發(fā)布時(shí)間：2018-08-26 12:47
【摘要】：骨髓間充質(zhì)干細(xì)胞（bone marrow derived mesenchymal stem cells, MSCs）是一類(lèi)具有增殖和多向分化潛能的特殊細(xì)胞，在特定環(huán)境條件下，MSCs可增殖并定向分化為多種細(xì)胞。同時(shí)，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(jìn)行動(dòng)員、遷移。MSCs在機(jī)體組織損傷修復(fù)和再生中具有重要作用，是臨床細(xì)胞治療的種子細(xì)胞，也是目前應(yīng)用最多的干細(xì)胞類(lèi)型。 機(jī)體中的細(xì)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(jìn)入外周血循環(huán)的過(guò)程中，受到血液流動(dòng)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(xì)胞的生理、病理過(guò)程，有關(guān)機(jī)械刺激對(duì)MSCs增殖、表型和分化影響的報(bào)道比較多，相關(guān)的分子機(jī)理在深入研究中。但到目前為止，人們對(duì)影響MSCs遷移行為的力學(xué)因素及其分子機(jī)理還知之甚少。本文試圖揭示機(jī)械拉伸對(duì)大鼠MSCs（ratMSCs, rMSCs）遷移行為的影響及其相關(guān)分子機(jī)理。 本文選用原代分離培養(yǎng)的rMSCs，運(yùn)用單軸拉伸加載裝置對(duì)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(shí)間4～12h）的機(jī)械拉伸。采用Transwell法和劃痕法評(píng)價(jià)拉伸前后細(xì)胞遷移能力的變化，基于明膠酶譜法檢測(cè)基質(zhì)金屬蛋白酶-2,-（9Matrix metalloproteinase-2,-9）表達(dá)的變化。用MMPs抑制劑GM6001抑制拉伸刺激對(duì)rMSCs MMP-2和MMP-9表達(dá)的激活，考察抑制MMP-2和MMP-9對(duì)拉伸促進(jìn)rMSCs的遷移的影響。實(shí)驗(yàn)結(jié)果如下： ①rMSCs的原代分離、培養(yǎng)及鑒定 本實(shí)驗(yàn)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(zhǎng)梭樣、漩渦狀分布。流式細(xì)胞儀檢測(cè)結(jié)果顯示，rMSCs原代細(xì)胞表達(dá)CD44和CD90，不表達(dá)CD34，與rMSCs表面標(biāo)記抗原一般規(guī)律一致，說(shuō)明分離培養(yǎng)的rMSCs表型均一，符合rMSCs特點(diǎn)。 ②機(jī)械拉伸加載對(duì)rMSCs遷移行為的影響 將細(xì)胞培養(yǎng)在彈性硅膠膜上，采用自制的細(xì)胞拉伸加載裝置拉伸硅膠膜，對(duì)細(xì)胞實(shí)施加載。運(yùn)用Transwell法檢測(cè)細(xì)胞遷移，研究發(fā)現(xiàn)頻率1Hz、形變量10%、拉伸時(shí)間8h加載條件促進(jìn)rMSCs的遷移效果較明顯。劃痕實(shí)驗(yàn)進(jìn)一步說(shuō)明，頻率1Hz、形變量10%、拉伸時(shí)間8h機(jī)械刺激對(duì)rMSCs遷移能力有促進(jìn)作用。 ③MMP-2和MMP-9參與機(jī)械拉伸誘導(dǎo)的rMSCs遷移 采用明膠酶譜法檢測(cè)頻率1Hz、形變量10%、拉伸時(shí)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(duì)照組的235%和248%。 在機(jī)械拉伸前加入MMP-2和MMP-9抑制劑GM6001，檢測(cè)細(xì)胞遷移和MMP-2和MMP-9表達(dá)變化，結(jié)果發(fā)現(xiàn)25μM GM6001顯著抑制機(jī)械刺激誘導(dǎo)的MMP-2和MMP-9分泌增加，同時(shí)機(jī)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(shí)間8h的拉伸加載促進(jìn)rMSCs遷移可能通過(guò)刺激rMSCs分泌MMP-2和MMP-9來(lái)實(shí)現(xiàn)。 本文研究結(jié)果表明，適宜的機(jī)械拉伸加載可誘導(dǎo)rMSCs分泌MMP-2和MMP-9，促進(jìn)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(shí)驗(yàn)依據(jù)和理論基礎(chǔ)，也為干細(xì)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(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R329
本文編號(hào)：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(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R329
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