剪应力对骨髓间充质干细胞生长状态的影响及其作用机制的研究

发布时间：2018-06-23 22:27

本文选题：组织工程 + 剪应力　；参考：《第四军医大学》2012年博士论文

【摘要】：研究背景相对于机械瓣膜和普通生物瓣膜来说，组织工程心脏瓣膜（TEHV）自身有很多优点，但目前TEHV的最大缺点是植入人体后难以耐受高速高压的血流冲击，功能上难以满足需要，且容易衰坏，使用寿命短。其主要原因是种子细胞如骨髓间充质干细胞（MSCs）在高剪应力下难以存活，不能对TEHV组织结构进行保护和修复，因此提高高剪应力条件下的细胞存活及改善其生长状态研制TEHV的关键方向。人体内血流动力是瓣膜发育的重要起始信号，并在瓣膜发育过程中通过力的大小变化对瓣膜组织结构和功能进行调节[1-2]。从胚胎期至成人，人体内的流体力是逐步增加的，因此研究渐进增加的流体力对TEHV种子细胞的生物学效应具有重要意义，并可能通过渐进增加的流体力训练从而实现TEHV对高速、高压血流的耐受. 研究目的本课题研究渐进性增加的剪应力对MSCs生长状态的影响及其可能的作用机制，并研究渐进性增加剪应力对MSCs进行流体力适应性训练的结果。研究方法和结果第一部分：将大鼠MSCs在0、1、3、8、15dyn/cm~2剪应力水平分别作用24h，观察细胞生长状态变化，并通过MTT、流式细胞技术、Hoechst/PI、透射电子显微镜等检查方法观察MSCs的生长状态和凋亡、坏死情况。结果：1dyn/cm~2剪应力对MSCs无明显影响，3dyn/cm~2的剪应力可以促进细胞生长，而8和15dyn/cm~2的剪应力促使细胞凋亡明显增多。说明合适的剪应力（3dyn/cm~2）对细胞生长有益，而细胞不能耐受高的剪应力作用（8和15dyn/cm~2）。第二部分：以渐进增加的剪应力作用于MSCs，设相应的剪应力高值为对照组，观察细胞生长状态变化，通过MTT测定细胞增殖及活性，流式细胞技术测定细胞死亡百分比。取作用后的细胞培养基，以酶联免疫吸附测定法（Elisa）检测其中细胞生长因子bFGF、TGF-β1、PDGF、VEGF变化，根据生长因子浓度变化制作相应的载药纳米微球，加入培养基。结果：渐进增加的剪应力相较于恒定高剪应力（8和15dyn/cm~2）显著改善了细胞增殖及活力，降低了细胞死亡百分比，并且相较于高剪应力组使细胞生长因子分泌水平升高。载药纳米微球的使用可以改善力作用后的细胞存活。说明渐进性力学训练可以改善MSCs对高剪应力的耐受，改善因高剪应力作用导致的细胞因子分泌减少，通过纳米微球载生长因子缓释系统可以增强力作用后的细胞存活。第三部分：以0、1、3、8、15、1-15dyn/cm~2的剪应力作用于MSCs，通过Western方法测定ERK磷酸化（P-ERK）以及JNK（c-JunN-terminalkinases）底物c-Jun的磷酸化水平，即P-c-Jun的水平。结果：高剪应力（8和15dyn/cm~2）减低了P-ERK，而增加了P-c-Jun的表达。而1-15dyn/cm~2渐进性增加的剪应力相较于高剪应力可以升高P-ERK水平，降低细胞内P-c-Jun的水平。说明剪应力对MSCs的促生长、凋亡作用可能通过ERK和JNK通路进行。第四部分：设计可以模拟人体血流的仿生脉动生物反应器。将1-15dyn/cm~2渐进增加的剪应力预训练过的MSCs种植于脱细胞的猪主动脉壁上（预训练组），对照组接种静态培养的MSCs。将两组均固定于体外脉动反应器，在15dyn/cm~2水平作用72h。取标本进行苏木素-伊红染色（HE），同时以分光光度法和电泳测定组织内DNA含量，以western方法检测组织内I型胶原含量。结果：研制成仿生脉动生物反应器，可以输出近似于心脏血液循环特征的搏动性血流，可持续稳定运转72h。HE结果显示在15dyn/cm~2剪应力作用72h后，训练组的MSCs在动脉壁内留存较多，，而对照组的MSCs从动脉壁基本脱落。分光光度法及电泳显示训练组组织内DNA含量明显高于对照组。Western检测I型胶原含量训练组明显高于对照组。说明力学训练后的MSCs相对于静态培养的MSCs接种于支架材料后可较好耐受高速、高压流体力的作用。结论本研究系统地研究了不同大小剪应力对MSCs的作用，发现适度的剪应力（3dyn/cm~2）促进MSCs生长，过低的剪应力（1dyn/cm~2）对MSCs生长无明显影响,而过高剪应力（8、15dyn/cm~2）抑制MSCs生长并促使细胞死亡。首先提出了对MSCs进行渐进性增加剪应力训练的概念，发现渐进性增加的剪应力相较于恒定高剪应力更容易被细胞耐受，细胞活力增强、存活比例增加、MSCs分泌细胞生长因子增加。探索了剪应力对MSCs作用的机制，发现ERK和JNK通路可能参与剪应力对细胞生长、凋亡的调控机制。将受剪应力预训练的MSCs接种于脱细胞猪主动脉壁在仿生脉动生物反应器内受高水平流体力作用后，MSCs可以较未受力训练的MSCs更好地存活和附着，并能分泌胶原蛋白而发挥组织重塑功能。
[Abstract]:Research background
Tissue engineered heart valve (TEHV) has many advantages over mechanical valves and common biological valvular valves, but the biggest disadvantage of TEHV is that it is difficult to tolerate high velocity and high pressure blood flow shock after implantation. It is difficult to meet the needs, and is easy to decline and use short life. The main reason is the seed cells such as bone marrow mesenchymal stem cells. The cell (MSCs) is difficult to survive under high shear stress and can not protect and repair the tissue structure of TEHV. Therefore, the key direction of TEHV development is to improve the survival of cells under high shear stress and to improve the growth state of the cells.
The flow of blood flow in the human body is an important starting signal of valve development, and changes in the structure and function of the valve during the development of the valve during the development of the valve from the embryonic period to the adult, and the fluid force in the human body is gradually increased. Therefore, the biological effects of the gradual increase of flow strength on the TEHV seed cells are studied. Importantly, it is possible to achieve TEHV tolerance to high speed and high blood pressure through gradual increase in fluid force training.
research objective
This topic studies the effect of the incremental shear stress on the growth state of MSCs and its possible mechanism of action, and studies the results of the gradual increase of shear stress on the adaptive training of fluid force for MSCs.
Research methods and results
The first part: the growth state of the rat MSCs was observed at the 0,1,3,8,15dyn/cm~2 shear stress level respectively, and the growth state of the cells was observed, and the growth state and apoptosis and necrosis of MSCs were observed by MTT, flow cytometry, Hoechst/PI, transmission electron microscope and so on. Fruit: 1dyn/cm~2 shear stress had no obvious effect on MSCs, 3dyn/cm~2, 3dyn/cm~2. 3dyn/cm~2 Shear stress can promote cell growth, and the shear stress of 8 and 15dyn/cm~2 increases cell apoptosis significantly. It shows that appropriate shear stress (3dyn/cm~2) is beneficial to cell growth, but cells can not tolerate high shear stress (8 and 15dyn/cm~2).
The second part: with the incremental shear stress acting on MSCs, the corresponding high shear stress was set as the control group, the cell growth state was observed, the cell proliferation and activity were measured by MTT, the percentage of cell death was measured by flow cytometry. The cell culture medium after action was taken, and the cell growth was detected by enzyme linked immunosorbent assay (Elisa). The change of long factor bFGF, TGF- beta 1, PDGF, VEGF, according to the change of growth factor concentration, make the corresponding drug loaded nanospheres and add the medium. Results: the gradual increase of shear stress significantly improved the cell proliferation and activity compared with the constant high shear stress (8 and 15dyn/cm~2), reduced the percentage of cell death, and compared with the high shear stress group. The use of drug loaded nanospheres can improve the survival of cells after force action. It shows that progressive mechanical training can improve the tolerance of MSCs to high shear stress, improve the decrease of cytokine secretion caused by high shear stress, and enhance the force effect through the sustained release system of nanospheres growth factor. The cells survive.
The third part: with the shear stress of 0,1,3,8,15,1-15dyn/cm~2 acting on MSCs, the phosphorylation level of ERK phosphorylation (P-ERK) and JNK (c-JunN-terminalkinases) substrate c-Jun is measured by Western method, that is, the level of P-c-Jun. Results: high shear stress (8 and 15dyn/cm~2) reduced P-ERK, and increased the expression of P-c-Jun. The increased shear stress can increase the level of P-ERK and decrease the level of P-c-Jun in the cell compared to the high shear stress. It shows that the shear stress can promote the growth of MSCs, and the effect of apoptosis may be carried out through the ERK and JNK pathway.
The fourth part: a bionic bioreactor, which can simulate human blood flow, is designed to grow the pre trained MSCs on the porcine aorta wall of the cell (pre training group), and the control group is inoculated with the static culture of MSCs., and the two groups are fixed to the external pulsation reactor and 72h. at the level of 15dyn/cm~2. The specimens were stained with hematoxylin eosin (HE), and the content of DNA in tissue was measured by spectrophotometry and electrophoresis. The content of I collagen in the tissue was detected by western. Results: a bionic pulsating bioreactor was developed to produce pulsating blood flow similar to the blood circulation of the heart. The result of sustainable and stable operation of 72h.HE showed at 15 After the effect of dyn/cm~2 shear stress for 72h, the MSCs in the training group remained more in the arterial wall, while the MSCs in the control group dropped from the arterial wall. The content of DNA in the training group was significantly higher than that of the control group, the content of the.Western in the training group was significantly higher than that in the control group. The MSCs was relatively quiet after the mechanical training. After inoculation with MSCs scaffolds, the cultured cells can tolerate high speed and high pressure.
conclusion
The effect of shear stress on MSCs is systematically studied in this study. It is found that moderate shear stress (3dyn/cm~2) promotes MSCs growth, and the low shear stress (1dyn/cm~2) has no obvious effect on the growth of MSCs, and the high shear stress (8,15dyn/cm~2) inhibits the growth of MSCs and causes cell death. First, the shear stress is incrementally increased to MSCs. The concept of training shows that progressive shear stress is more easily tolerated by cells than constant high shear stress, cell viability is enhanced, survival ratio increases, and MSCs secretes growth factors. The mechanism of shear stress on MSCs is explored, and the regulatory mechanism of ERK and JNK pathways may be involved in the regulation of cell growth and apoptosis by shear stress. After being inoculated with the shear stress pre trained MSCs in the porcine aorta wall in the bionic pulsating bioreactor, the MSCs can survive and attach better than the untrained MSCs, and can secrete collagen to play the tissue remodeling function.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.11

【参考文献】