本文選題：流動切應力 + 間充質(zhì)干細胞　； 參考：《四川大學》2007年博士論文

【摘要】： 目的：通過研究流動切應力因素作用于大鼠骨髓間充質(zhì)干細胞(mesenchymal stem cells, MSCs)后，對其內(nèi)皮誘導所得細胞抗血栓形成、血管活性物質(zhì)生成及粘附因子表達等生物學性質(zhì)的變化，探討流動切應力加載與MSCs內(nèi)皮分化過程相結合后，能否在體外構建出滿足缺血性心臟病(ischemic heart disease, IHD)治療及心血管組織工程需要的種子細胞。 方法：1．采用Percoll分離液密度梯度分離法，體外分離及純化SD大鼠MSCs，經(jīng)過形態(tài)學特征、表面標志物表達及多向分化潛能等多方面鑒定所得細胞。將純化后細胞置入含VEGF及bFGF的內(nèi)皮誘導液中定向誘導2周后，倒置相差顯微鏡觀察細胞形態(tài)變化，免疫熒光染色觀察細胞CD31表達情況，免疫組織化學方法檢測Ⅷ因子表達情況，，透射電子顯微鏡觀察細胞內(nèi)超微結構。2．將分離純化所得的大鼠MSCs按照加載切應力大小分為靜態(tài)對照組、5 dyn／cm~2切應力加載組和10dyn／cm~2切應力加載組，切應力加載組完成3小時切應力加載后，三組均于靜態(tài)下定向內(nèi)皮誘導14天，分別以熒光實時定量逆轉(zhuǎn)錄-聚合酶鏈反應(real-time fluorescent quantitation reverse transcription-polymerase chain reaction,real-time RT-PCR)及流式細胞儀(flow cytometry)觀察所得細胞的t-PA、eNOS、ET-1、ICAM-1、VCAM-1等因子mRNA及蛋白表達水平的變化情況。流動切應力加載組所得細胞培養(yǎng)3天后傳代，再次檢測傳代后細胞上述因子的表達情況。3．同時也觀察所得細胞的NO合成量、總抗氧化能力(T-AOC)、及NADPH氧化酶gp91phox亞單位mRNA的變化情況。 結果：1．內(nèi)皮誘導2周后，培養(yǎng)細胞獲得了內(nèi)皮特異性細胞形態(tài)、排列方式及超微結構，并表達內(nèi)皮特異性抗原CD31及Ⅷ因子相關抗原。2．對MSCs施加生理范圍內(nèi)大小的層流切應力3h后，其內(nèi)皮誘導所得P1代細胞的t-PA、eNOS、ET-1、ICAM-1、VCAM-1等因子mRNA表達水平均明顯升高，且與切應力強度呈依賴關系。傳代后P2代細胞上述因子中，除ICAM-1表達繼續(xù)升高外，其余因子表達下降，其中eNOS和ET-1表達下降明顯，而t-PA和VCAM-1表達下降不明顯。3．所得P1代細胞的eNOS、ET-1、ICAM-1、VCAM-1等因子蛋白表達水平變化趨勢不同。5 dyn／cm~2加載后，eNOS表達下降，而10 dyn／cm~2加載后eNOS表達增加。其余因子無論切應力強度大小，均表達增加。傳代后P2代細胞中，5 dyn／cm~2組eNOS表達仍低于對照水平，而10dyn／cm~2組水平降低至對照水平。同時其余因子蛋白表達下降至對照水平。4．對MSCs施加生理范圍內(nèi)大小的層流切應力3h后，其內(nèi)皮誘導所得細胞的NO產(chǎn)量及T-AOC增加，gp91phox亞單位mRNA表達下降，均與切應力強度呈依賴關系。 結論：1．成年SD大鼠骨髓MSCs能在體外定向誘導為內(nèi)皮細胞，有望成為心血管疾病的細胞治療及相關心血管組織工程理想的種子細胞來源。2．生理范圍內(nèi)流動切應力能對由MSCs分化而來的內(nèi)皮細胞抗血栓能力、抗氧化能力、血管活性物質(zhì)生成及粘附分子表達等多方面進行精細的調(diào)控。其調(diào)控效果及強度因細胞因子的不同而不同。這可能是由于不同的細胞因子是通過各自特異的分子機制來對同一類型切應力作出反應的結果。3．向MSCs階段施加生理范圍大小的層流切應力，可作為一種有效的種子細胞預處理方法及手段，能使種子細胞具有更強的增殖分化能力，更優(yōu)良的抗血栓形成、抗炎癥及抗動脈粥樣硬化性質(zhì)，有助于IHD細胞治療及心血管組織工程的發(fā)展。
[Abstract]:Objective: To investigate the changes in the biological properties of antithrombotic, vasoactive substances and adhesion factor expression induced by endothelial induced cells after the effect of flow shear stress factors on rat bone marrow mesenchymal stem cells (mesenchymal stem cells, MSCs), and explore the combination of flow shear stress loading and MSCs endothelium differentiation. Can seed cells be constructed in vitro to meet the needs of ischemic heart disease (IHD) treatment and cardiovascular tissue engineering.
Methods: 1. the density gradient separation method of Percoll separation liquid was used to separate and purify the SD rat MSCs in vitro. The cells were identified by morphological characteristics, expression of surface markers and multidirectional differentiation potential. After the purified cells were placed in the endothelial induction solution containing VEGF and bFGF for 2 weeks, the cells were inverted with phase contrast microscope to observe the cells. Morphological changes, immunofluorescence staining were used to observe the expression of CD31 in cells. Immunohistochemical method was used to detect the expression of factor VIII factor. The ultrastructure of.2. was observed by transmission electron microscope. The isolated rat MSCs was divided into static control group according to the loading stress size, 5 dyn / cm~2 shear stress loading group and 10dyn / cm~2 shear stress group. In the force loading group, after the shear stress loading group completed the 3 hour shear stress loading, the three groups were induced by the static direction endothelium for 14 days. The real-time quantitative reverse transcription polymerase chain reaction (real-time fluorescent quantitation reverse transcription-polymerase chain reaction, real-time RT-PCR) and the flow cytometry (flow cytometry) were used respectively. The changes of t-PA, eNOS, ET-1, ICAM-1, VCAM-1 and other factors mRNA and protein expression levels were observed. The cell culture of the flow shear stress loading group was subcultured 3 days later, and the expression of the above factors after the passage was detected again..3. also observed the NO synthesis of the cells, the total antioxidant capacity (T-AOC), and NADPH oxidase. Changes in the gp91phox subunit mRNA.
Results: 1. after 2 weeks of endothelium induction, the cultured cells obtained the endothelial specific cell morphology, arrangement and ultrastructure, and expressed the endothelial specific antigen CD31 and factor VIII associated antigen.2. to apply the physiological range of laminar shear stress 3H to MSCs, and the endothelial cells induced the t-PA, eNOS, ET-1, ICAM-1, VCAM-1, etc. of the P1 generation cells. The expression level of factor mRNA increased obviously, and was dependent on the shear stress intensity. Among the above factors, the expression of eNOS and ET-1 decreased obviously, except that the expression of eNOS and ET-1 decreased, but the decrease of t-PA and VCAM-1 expression was not obvious in the eNOS, ET-1, ICAM-1, and other factors of the t-PA and VCAM-1 expressions. After the loading of.5 dyn / cm~2, the expression of eNOS decreased, and the expression of eNOS increased after 10 dyn / cm~2 loading. The expression of the remaining factors increased in both the shear stress intensity and the magnitude of the shear stress intensity. The eNOS expression of the 5 dyn / cm~2 group was still lower than the control level in P2 generation cells after the passage, and the level of 10dyn per cm~2 group decreased to the control level. After the expression of other factor proteins decreased to the control level.4., the NO production and T-AOC increased and the mRNA expression of gp91phox subunit decreased, and all of them were dependent on the shear stress intensity after applying the laminar shear stress 3H in the physiological range of MSCs.
Conclusion: 1. the bone marrow MSCs of adult SD rats can be induced to be endothelial cells in vitro, and it is expected to become a cell therapy for cardiovascular diseases and the ideal seed cell source of cardiovascular tissue engineering. The flow shear stress in the.2. physiological range of MSCs can be used for the anti blood thrombus ability, antioxidant capacity and vasoactive activity of endothelial cells derived from MSCs The effect and intensity of the regulatory effect and intensity vary with the cell factor. This may be because different cytokines are the result of the response to the same type of shear stress by the specific molecular mechanism,.3. exerts a physiological range of laminar cutting to the MSCs stage. Stress, as an effective seed cell preconditioning method and means, can make the seed cells more capable of proliferation and differentiation, better antithrombotic formation, anti-inflammatory and anti atherosclerotic properties, and can help IHD cell therapy and the development of cardiovascular tissue engineering.
【學位授予單位】：四川大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R329

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