發(fā)布時(shí)間：2018-06-23 22:27

  本文選題：組織工程 + 剪應(yīng)力�。� 參考：《第四軍醫(yī)大學(xué)》2012年博士論文

【摘要】：研究背景 相對(duì)于機(jī)械瓣膜和普通生物瓣膜來(lái)說(shuō)，組織工程心臟瓣膜（TEHV）自身有很多優(yōu)點(diǎn)，但目前TEHV的最大缺點(diǎn)是植入人體后難以耐受高速高壓的血流沖擊，功能上難以滿足需要，且容易衰壞，使用壽命短。其主要原因是種子細(xì)胞如骨髓間充質(zhì)干細(xì)胞（MSCs）在高剪應(yīng)力下難以存活，不能對(duì)TEHV組織結(jié)構(gòu)進(jìn)行保護(hù)和修復(fù)，因此提高高剪應(yīng)力條件下的細(xì)胞存活及改善其生長(zhǎng)狀態(tài)研制TEHV的關(guān)鍵方向。 人體內(nèi)血流動(dòng)力是瓣膜發(fā)育的重要起始信號(hào)，并在瓣膜發(fā)育過(guò)程中通過(guò)力的大小變化對(duì)瓣膜組織結(jié)構(gòu)和功能進(jìn)行調(diào)節(jié)[1-2]。從胚胎期至成人，人體內(nèi)的流體力是逐步增加的，因此研究漸進(jìn)增加的流體力對(duì)TEHV種子細(xì)胞的生物學(xué)效應(yīng)具有重要意義，并可能通過(guò)漸進(jìn)增加的流體力訓(xùn)練從而實(shí)現(xiàn)TEHV對(duì)高速、高壓血流的耐受. 研究目的 本課題研究漸進(jìn)性增加的剪應(yīng)力對(duì)MSCs生長(zhǎng)狀態(tài)的影響及其可能的作用機(jī)制，并研究漸進(jìn)性增加剪應(yīng)力對(duì)MSCs進(jìn)行流體力適應(yīng)性訓(xùn)練的結(jié)果。 研究方法和結(jié)果 第一部分：將大鼠MSCs在0、1、3、8、15dyn/cm~2剪應(yīng)力水平分別作用24h，觀察細(xì)胞生長(zhǎng)狀態(tài)變化，并通過(guò)MTT、流式細(xì)胞技術(shù)、Hoechst/PI、透射電子顯微鏡等檢查方法觀察MSCs的生長(zhǎng)狀態(tài)和凋亡、壞死情況。結(jié)果：1dyn/cm~2剪應(yīng)力對(duì)MSCs無(wú)明顯影響，3dyn/cm~2的剪應(yīng)力可以促進(jìn)細(xì)胞生長(zhǎng)，而8和15dyn/cm~2的剪應(yīng)力促使細(xì)胞凋亡明顯增多。說(shuō)明合適的剪應(yīng)力（3dyn/cm~2）對(duì)細(xì)胞生長(zhǎng)有益，而細(xì)胞不能耐受高的剪應(yīng)力作用（8和15dyn/cm~2）。 第二部分：以漸進(jìn)增加的剪應(yīng)力作用于MSCs，設(shè)相應(yīng)的剪應(yīng)力高值為對(duì)照組，觀察細(xì)胞生長(zhǎng)狀態(tài)變化，通過(guò)MTT測(cè)定細(xì)胞增殖及活性，流式細(xì)胞技術(shù)測(cè)定細(xì)胞死亡百分比。取作用后的細(xì)胞培養(yǎng)基，以酶聯(lián)免疫吸附測(cè)定法（Elisa）檢測(cè)其中細(xì)胞生長(zhǎng)因子bFGF、TGF-β1、PDGF、VEGF變化，根據(jù)生長(zhǎng)因子濃度變化制作相應(yīng)的載藥納米微球，加入培養(yǎng)基。結(jié)果：漸進(jìn)增加的剪應(yīng)力相較于恒定高剪應(yīng)力（8和15dyn/cm~2）顯著改善了細(xì)胞增殖及活力，降低了細(xì)胞死亡百分比，并且相較于高剪應(yīng)力組使細(xì)胞生長(zhǎng)因子分泌水平升高。載藥納米微球的使用可以改善力作用后的細(xì)胞存活。說(shuō)明漸進(jìn)性力學(xué)訓(xùn)練可以改善MSCs對(duì)高剪應(yīng)力的耐受，改善因高剪應(yīng)力作用導(dǎo)致的細(xì)胞因子分泌減少，通過(guò)納米微球載生長(zhǎng)因子緩釋系統(tǒng)可以增強(qiáng)力作用后的細(xì)胞存活。 第三部分：以0、1、3、8、15、1-15dyn/cm~2的剪應(yīng)力作用于MSCs，通過(guò)Western方法測(cè)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。結(jié)果：高剪應(yīng)力（8和15dyn/cm~2）減低了P-ERK，而增加了P-c-Jun的表達(dá)。而1-15dyn/cm~2漸進(jìn)性增加的剪應(yīng)力相較于高剪應(yīng)力可以升高P-ERK水平，降低細(xì)胞內(nèi)P-c-Jun的水平。說(shuō)明剪應(yīng)力對(duì)MSCs的促生長(zhǎng)、凋亡作用可能通過(guò)ERK和JNK通路進(jìn)行。 第四部分：設(shè)計(jì)可以模擬人體血流的仿生脈動(dòng)生物反應(yīng)器。將1-15dyn/cm~2漸進(jìn)增加的剪應(yīng)力預(yù)訓(xùn)練過(guò)的MSCs種植于脫細(xì)胞的豬主動(dòng)脈壁上（預(yù)訓(xùn)練組），對(duì)照組接種靜態(tài)培養(yǎng)的MSCs。將兩組均固定于體外脈動(dòng)反應(yīng)器，在15dyn/cm~2水平作用72h。取標(biāo)本進(jìn)行蘇木素-伊紅染色（HE），同時(shí)以分光光度法和電泳測(cè)定組織內(nèi)DNA含量，以western方法檢測(cè)組織內(nèi)I型膠原含量。結(jié)果：研制成仿生脈動(dòng)生物反應(yīng)器，可以輸出近似于心臟血液循環(huán)特征的搏動(dòng)性血流，可持續(xù)穩(wěn)定運(yùn)轉(zhuǎn)72h。HE結(jié)果顯示在15dyn/cm~2剪應(yīng)力作用72h后，訓(xùn)練組的MSCs在動(dòng)脈壁內(nèi)留存較多，，而對(duì)照組的MSCs從動(dòng)脈壁基本脫落。分光光度法及電泳顯示訓(xùn)練組組織內(nèi)DNA含量明顯高于對(duì)照組。Western檢測(cè)I型膠原含量訓(xùn)練組明顯高于對(duì)照組。說(shuō)明力學(xué)訓(xùn)練后的MSCs相對(duì)于靜態(tài)培養(yǎng)的MSCs接種于支架材料后可較好耐受高速、高壓流體力的作用。 結(jié)論 本研究系統(tǒng)地研究了不同大小剪應(yīng)力對(duì)MSCs的作用，發(fā)現(xiàn)適度的剪應(yīng)力（3dyn/cm~2）促進(jìn)MSCs生長(zhǎng)，過(guò)低的剪應(yīng)力（1dyn/cm~2）對(duì)MSCs生長(zhǎng)無(wú)明顯影響,而過(guò)高剪應(yīng)力（8、15dyn/cm~2）抑制MSCs生長(zhǎng)并促使細(xì)胞死亡。首先提出了對(duì)MSCs進(jìn)行漸進(jìn)性增加剪應(yīng)力訓(xùn)練的概念，發(fā)現(xiàn)漸進(jìn)性增加的剪應(yīng)力相較于恒定高剪應(yīng)力更容易被細(xì)胞耐受，細(xì)胞活力增強(qiáng)、存活比例增加、MSCs分泌細(xì)胞生長(zhǎng)因子增加。探索了剪應(yīng)力對(duì)MSCs作用的機(jī)制，發(fā)現(xiàn)ERK和JNK通路可能參與剪應(yīng)力對(duì)細(xì)胞生長(zhǎng)、凋亡的調(diào)控機(jī)制。將受剪應(yīng)力預(yù)訓(xùn)練的MSCs接種于脫細(xì)胞豬主動(dòng)脈壁在仿生脈動(dòng)生物反應(yīng)器內(nèi)受高水平流體力作用后，MSCs可以較未受力訓(xùn)練的MSCs更好地存活和附著，并能分泌膠原蛋白而發(fā)揮組織重塑功能。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R318.11

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