组织工程化静脉瓣构建的关键技术

发布时间：2018-05-09 19:23

本文选题：平滑肌组细胞 + 内皮祖细胞　；参考：《第二军医大学》2012年博士论文

【摘要】：原发性瓣膜功能不全是临床上常见病，多发病，静脉瓣移植被认为是最后的选择。但自体静脉瓣移植来源有限，限制了静脉瓣移植的应用，应用细胞生物学与工程学原理开发出具有生物活性、无免疫原性的组织工程化静脉瓣是修复与功能重建深静脉功能不全的理想方案。目前构建组织工程化静脉瓣研究处尚处于起步阶段。Teebken等（2003）应用组织工程学原理，用受体静脉壁来源的肌纤维母细胞和内皮细胞构建组织工程化静脉瓣，但是肌纤维母细胞没能长入支架内部；该课题组在2009年又以人大隐静脉内皮细胞为种子细胞，大隐静脉脱细胞支架为支架材料，构建组织工程化静脉瓣，三维立体培养8天，发现细胞能在瓣膜两侧和血管壁表面生长并内皮化，但该研究血管壁内没种植细胞，没有进行体内移植。我们实验室在国家、军队和上海市基金的资助下，近年来一直进行组织工程化静脉瓣的研究，我们已[1]利用骨髓来源的多能成体祖细胞（Multipotent adult progenitor cells，MAPC）和内皮祖细胞（Endothelial prigenitor cell，EPC）作为种子细胞，以绵羊脱细胞静脉瓣支架为支架材料，成功构建了组织工程化静脉，并进行了犬、绵羊在体效用性研究，及安全性评价。但是，静脉瓣在体内长期功能欠佳，我们认为，其原因可能与瓣膜的内皮化程度、内皮细胞体内黏附强度、脱细胞支架材料在制备过程中损伤、变性等因素有关。拟提高组织工程化静脉瓣的质量，缩短与生理性静脉瓣的差距，优化构建程序，有必要对组织工程化静脉瓣的构建中所用材料和构建技术进行再探讨。在种子细胞研究方面，我们实验室利用骨髓源性MAPC和EPC为种子细胞，成功构建了组织工程化静脉瓣，但是，细胞分离培养过程复杂，需采用全骨髓血培养加免疫磁珠分选，免疫磁珠价格昂贵、不经济，细胞分选过程复杂，易污染。为优化组织工程化静脉瓣构建程序、降低成本，有必要对种子细胞的种类及分离培养方法进行再研究。在支架材料制备方法上，先前的研究采用了两种不同的脱细胞支架制备方法，但其中哪一种方法较好还未曾研究。近期有研究报道的冻融+生物酶的方法，在其他组织脱细胞支架制备中对支架材料结构损伤小，但目前该方法还未见在静脉瓣膜脱细胞支架制备中应用。在支架材料制备方法上，我们先前的研究采用TritonX-100+NH4OH+DNase+RNase的脱细胞方法，支架无细胞残留，纤维连续，支架内布满大小不等的孔隙，无免疫原性。但利用该支架材料构建的组织工程化静脉瓣体内长期功能欠佳，这是否与该脱细胞方法有关尚不得而知。近期研究报道，冻融+生物酶的方法在其他组织脱细胞中发现对支架材料结构损伤小，利用该方法制备的瓣膜脱细胞支架是否能提高组织工程化静脉瓣体内长期功能，也值得研究。在种子细胞种植方法上，我们先前的研究采用多点注射和加压灌注的方法，程序复杂、需要较高的专门技术、且发现利用该方法构建的组织工程化静脉瓣体内超过6个月，有内皮细胞脱落、血栓形成的现象，严重影响了组织工程化静脉瓣的体内长期功能，迫切需要寻找新的方法。本研究针对我们组织工程化静脉瓣研究中遇到到的问题，拟通过简化种子细胞诱导培养方法，获取活性好，纯度高的种子细胞；获得一种脱细胞完全、对支架材料结构损害较小的组织工程化静脉瓣脱细胞支架的方法；以及研发平滑肌细胞种植器，提高平滑肌细胞粘附率等三个方面的研究；以提高组织工程化静脉瓣的构建效果，改善组织工程化静脉瓣远期性能。第一部分同时培养兔骨髓源性EPCs和SPCs 研究目的：同时分离和培养兔骨髓来源的EPCs和平滑肌祖细胞（Smoothprogenitor cells,SPCs），研究其生物学特性，评估其作为组织工程化静脉瓣种子细胞的可能性。材料和方法：密度梯度离心法获取兔骨髓血单个核细胞沉淀，分别用含5%FBS的EGM-2完全培养基向EPC方向诱导培养；用含5%FBS，20ng/mlPDGF-BB，不含VEGF的EBM-2培养基向SPC方向诱导培养。细胞培养48h后首次换液，相差显微镜下观察细胞形态特征，透射电镜观察两类细胞超微结构的特点。诱导第7天，14天细胞免疫荧光、流式细胞仪检测EPCs/SPCs表面标志阳性率表达情况；检测细胞摄取DiI-ac-LDL和结合FITC-UEA-1功能，以及在MatriGel上成血管功能情况，将第3代细胞进行冻存和复苏，测定细胞冻存前后的细胞活性变化。结果：EPCs生物学特性：EPCs培养10天左右，细胞单层融合呈“铺路石”状；EPC表达CD34，VEGFR-2，弱表达CD133；透射电镜可见EPCs胞浆内特征性W-P小体；细胞生物学功能检测可见EPCs在Matrigel上呈现血管状；EPCs具有摄取DiI-ac-LDL和结合FITC-UEA-1的功能。冻存细胞复苏前后细胞生长特性方面无明显变化；SPCs生物学特性：SPCs培养14天左右出现血管平滑肌特异的生长特征“峰—谷”样生长特性；表达CD34，SMA，不表达Ⅷ和VEGF-2；在透射电镜下可见细胞内含有与细胞纵轴平行排列的肌丝；无摄取DiI-ac-LDL和结合FITC-UEA-1功能；在Matrigel上无血管状结构形成。结论：骨髓血梯度密度离心得到的单个核细胞，在不同诱导培养基的诱导下，可同时获到高纯度的EPCs和SPCs，SPCs可自然分化为平滑肌样细胞，不需要经向平滑肌细胞诱导分化，省时、经济、不易污染。第二部分不同方法制备组织工程化静脉瓣脱细胞支架研究目的：比较三种不同方法制备的带瓣静脉脱细胞支架的组织学，生物学特性，以期获得一种较好的带瓣静脉脱细胞支架材料。材料和方法:采用以下三种不同方法制备带瓣静脉脱细胞支架。 1.脱氧胆酸钠组：Beagle犬带瓣静脉，浸入4%去氧胆酸钠溶液，4℃振荡1h进行脱细胞处理，然后于37℃以50mL生理盐水反复冲洗，得到脱细胞带瓣静脉支架，于4℃PBS液中保存备用； 2.Triton组：Beagle犬带瓣静脉，浸入0.5%Triton-100+0.05%NH4OH溶液，4℃振摇3d；超纯水4℃振摇3d；DNase+RNase处理（37℃）12h；超纯水漂洗，将脱细胞支架60CO辐照消毒，-80℃保存备用； 3.冻融+生物酶组：Beagle犬带瓣静脉，浸入4℃低渗液浸泡11小时，-80℃3小时，37℃水浴30min，PBS振荡冲洗。0.05％胰酶+0．02％EDTA处理8h，DNase0.2mg/mL、Nase0.02mg/mL消化8h，PBS漂洗，上述步骤重复3次，支架材料冷冻干燥，辐照消毒，-80℃保存备用；随机选取各组支架材料检测，病理切片分别行HE染色观察组织结构；扫描电镜观察支架材料表面及内部超微结构；透射电镜DAPI染色观察DNA残留；体外EPCs细胞种植检测细胞相容性。结果：三种脱细胞方法均能彻底去除细胞，DAPI荧光检测显示各组支架材料细胞核，均无DNA成分残留；HE染色及扫描电镜显示，冻融+生物酶组胶原纤维排列整齐，未见明显的胶原纤维结构改变，其他两组可见胶原纤维断裂，及结构紊乱现象；与其他两组脱细胞支架材料相比冻融+生物酶组支架皮下埋置炎细胞浸润较少，，EPCs与冻融生物酶组支架材料粘附性好。结论：结合渗透压改变的反复冻融加上低浓度胰酶，核酸酶脱细胞法，既可以较彻底除去带瓣膜静脉细胞成分，又保留了较完整的细胞外基质结构，具有良好的组织和细胞相容性，是较理想的带瓣膜静脉脱细胞支架制备方法。第三部分平滑肌种植器构建组织工程化静脉瓣研究目的：研制平滑肌细胞种植器，并利用该装置种植SPCs，加压灌注旋转种植EPCs，以提高细胞的种植率，构建性能良好的组织工程化静脉瓣。材料和方法：平滑肌细胞种植器的研制：改革传统的从官腔内种植平滑肌细胞的方法，利用真空吸引的作用将平滑肌细胞从官腔外壁较均匀的种植平滑肌细于在内膜下。组织工程化静脉瓣构建：选第3代SPCs/EPCs为种子细胞，实验组用自制的平滑肌种植器种植SPCs；对照组用加压灌注多点注射的方法种植SPCs。培养三天后，以加压灌注种植EPCs，继续培养四天。SPCs种植4h，冰冻切片，DAPI染色观察SPCs种植密度；24h扫描电镜、甲苯胺蓝染色观察SPCs在支架材料上的粘附情况；MTT检测、细胞粘附实验检测种子细胞在支架材料内的增殖性能。一周后HE染色，免疫组织化学染色观察构建的组织工程化静脉瓣的组织学结构。结果：平滑肌种植器种植的细胞密度较均匀、支架材料内膜层结构破坏较小、种植平滑肌细胞的数量可进行量化控制；在无菌的细胞悬液筒中进行，减少了构建过程中的污染几率。种植4h后DAPI染色显示，实验组SPCs种植密度均匀，对照组细胞种植密度不均匀，仅局部有细胞聚集。扫描电镜显示，实验组血管外表面细胞粘附较多，已有少量的细胞外基质分泌，支架材料表面平滑；对照组有少量细胞粘附，支架材料表面有蜂窝状。甲苯胺蓝染色显示，实验组SPCs在支架材料上粘附较多，细胞密度较大。MTT、细胞粘附实验显示，实验组细胞在支架材料上有较好的增殖活性，与对照组相比差别有统计学意义。组织学检查可见实验组内皮细胞已经完全覆盖支架材料的内表面，较对照组内皮化完整。结论：平滑肌细胞种植装置是有效的平滑肌种植工具，可高效、均匀种植SPCs，种植的SPCs产生的细胞外基质，可修复脱细胞支架材料表面结构，细胞外基质所含的生长因子，促进EPCs的粘附增殖，促进了支架材料的内皮化。
[Abstract]:Primary valvular insufficiency is a common clinical disease, which is often a common disease. Venous valve transplantation is considered to be the final choice. However, autologous venous flap transplantation is limited and limited the application of venous valve transplantation. Biological activity is developed by the principle of cell biology and engineering. Tissue engineered vein petals without immunogenicity are repair and work. An ideal solution for the reconstruction of deep venous insufficiency.
At present, the construction of tissue engineering vein valve research department is still in the initial stage of.Teebken, etc. (2003) the application of tissue engineering principle to construct tissue engineered vein petals with muscle fibroblasts and endothelial cells derived from the recipient vein wall, but myofibroblast failed to grow into the inner part of the stent. In 2009, the group was also in the human great saphenous vein. The skin cell is the seed cell and the large saphenous vein decellular scaffold is the scaffold material. The tissue engineered venous valve is constructed for 8 days. The cells can grow and endothelialization on the surface of the valvular and vascular walls, but there is no cell in the vessel wall and no body transplantation is carried out.
Under the support of the national, military and Shanghai municipal funds, our laboratory has been studying tissue engineered venous valves in recent years. We have [1] using Multipotent adult progenitor cells (MAPC) and endothelial progenitor cells (Endothelial prigenitor cell, EPC) as seed cells. The tissue engineered vein was successfully constructed with the cellular vein stent as the scaffold material, and the body utility and safety evaluation of the dog and sheep were carried out. However, the long-term function of the venous valve was not good in the body. We think that the reason may be the degree of endothelialization of the valve, the adhesion strength of the inner skin cells, and the preparation of the scaffold material. We should improve the quality of the tissue engineering venous valve, shorten the gap with the physiological venous valve, and optimize the construction procedure. It is necessary to discuss the materials and construction techniques used in the construction of tissue engineered venous valve. In the field of seed cell research, our laboratory uses bone marrow derived MAPC and EPC as a method. Seed cells
The tissue engineering venous valve is constructed, but the process of cell separation and culture is complicated. The whole bone marrow blood culture and immunomagnetic beads are needed. The immune magnetic beads are expensive, uneconomical, and the process of cell sorting is complex and easy to pollute. It is necessary to optimize the construction of the engineering vein petal construction program and reduce the cost. It is necessary to make the seed cell types and separate the culture. In the preparation of scaffold materials, two different scaffold preparation methods have been used in the preparation of scaffold materials, but one of them has not been studied. Recently, the methods of freezing thawing + biologic enzyme have been reported in the other groups.
The structure of the scaffold has little damage to the scaffold material in the preparation of the acellular scaffold. But the method has not yet been used in the preparation of the venular valvular acellular scaffold. In the preparation of scaffold material, our previous study adopted the TritonX-100+NH4OH+DNase+RNase decellular method, the stents have no fine cell residue, the fiber is continuous, and the size of the stent is different. But recent studies have reported that the method of freezing thawing + biologic enzyme has been found in other tissue decellular deactivation of the scaffold material to be less damaged and the valve prepared by this method. Whether decellularized scaffolds can improve the long-term function of tissue-engineered venous valves is also worth studying.
In the method of seed cell planting, our previous study using multi point injection and pressure perfusion method is complex and requires high specific techniques. It is found that the tissue engineered venous valve constructed by this method has been in the body for more than 6 months, with endothelial cells falling off and thrombosis, which seriously affects the tissue engineered venous valve. The long-term function of the body is urgently needed to find new methods.
In this study, we aim at the problems encountered in the study of the engineering venous valve study. The method of simplifying the induction and culture method of seed cells to obtain the seed cells with good activity and high purity is obtained, and a method of tissue engineering veno de cell scaffold with little damage to the structure of scaffold material and the development of smooth muscle are obtained. To improve the construction effect of tissue engineered venous valve and to improve the long-term performance of tissue engineered venous valve, three aspects such as cell implant, increase of adhesion rate of smooth muscle cells and so on.
In the first part, rabbit bone marrow derived EPCs and SPCs were cultured at the same time
The purpose of this study was to isolate and cultivate EPCs and Smoothprogenitor cells (SPCs) from rabbit bone marrow, and to study its biological characteristics and evaluate its possibility as a tissue engineered vein petal seed cell.
Materials and methods: density gradient centrifugation was used to obtain the rabbit bone marrow blood mononuclear cells and induce culture in the direction of EPC, respectively, with the EGM-2 complete medium containing 5%FBS. The culture medium containing 5%FBS, 20ng/mlPDGF-BB, EBM-2 without VEGF was induced in the direction of SPC, and the cells were cultured for the first time after 48h, and the morphological characteristics of the cells were observed under the phase contrast microscope. The ultrastructural characteristics of the two types of cells were observed by transmission electron microscopy. Induced seventh days, 14 days of cell immunofluorescence, flow cytometry was used to detect the expression of the positive rate of the EPCs/SPCs surface markers, and the cell uptake of DiI-ac-LDL and FITC-UEA-1, as well as the vascular function on MatriGel, were frozen and resuscitation of the third generation cells. Cell activity changes before and after cryopreservation.
Results: EPCs biological characteristics: EPCs culture for about 10 days, cell monolayer fusion was "pave" shape, EPC expressed CD34, VEGFR-2, weak expression of CD133; transmission electron microscope showed the characteristic W-P corpuscle in the cytoplasm of EPCs; cell biological function test showed that EPCs on Matrigel showed vascular; EPCs has DiI-ac-LDL and binding DiI-ac-LDL 1 function. There was no obvious change in the cell growth characteristics of the frozen cells before and after the resuscitation. SPCs biological characteristics: the growth characteristics of the vascular smooth muscle specific growth characteristic "peak valley" like growth characteristics were appeared in the SPCs culture about 14 days, and the expression of CD34, SMA, not expressed VIII and VEGF-2; under transmission electron microscope, the cells contained parallel rows with the longitudinal axis of the cells. The myocutaneous fibers of the column; no uptake of DiI-ac-LDL and binding of FITC-UEA-1; on Matrigel.
No vascular structure was formed.
Conclusion: the mononuclear cells derived from bone marrow blood gradient density centrifugation can obtain high purity EPCs and SPCs at the same time under the induction of different inducible medium. SPCs can naturally differentiate into smooth muscle like cells. It does not need to induce differentiation to smooth muscle cells, time saving, economic and not easy to pollute.
The second part is to prepare tissue-engineered venous valve acellular scaffolds by different methods.
Objective: To compare the histological and biological characteristics of the three different methods of venovalal venovenous decellular scaffold, in order to obtain a better material for the venal venovenous decellular scaffold.
Materials and methods: the decellularized scaffolds were prepared by following three different methods.
1. sodium deoxycholate group: Beagle canine with valved vein, immersed in 4% deoxycholate sodium solution, 4 degrees centigrade oscillating 1h to remove cell treatment, and then at 37 centigrade 50mL saline rinse repeatedly, get the decellular vena valvale stent, stored in 4 centigrade PBS solution;
Group 2.Triton: Beagle canine with petal vein, immerse in 0.5%Triton-100+0.05%NH4OH solution, shake 3D at 4 C, shake 3D at 4 C; DNase+RNase treatment (37) 12h, ultra pure water rinsing, irradiate the acellular scaffold 60CO, and save at -80;
3. freeze-thaw + biologic enzyme group: Beagle canine belt vein, immerse in 4 centigrade hypotonic liquid for 11 hours, -80 C for 3 hours, 37 centigrade water bath 30min, PBS oscillation rinse.0.05% pancreatin +0.02%EDTA to treat 8h, DNase0.2mg/mL, Nase0.02mg/mL digesting 8h, PBS rinsing, the above steps are repeated 3 times, the scaffold materials freeze drying, irradiation disinfection, -80 C preservation reserve; random The scaffold materials were detected in each group. The histological structure was observed by HE staining, and the surface and internal ultrastructure of the scaffold materials were observed by scanning electron microscopy. The residual DNA was observed by transmission electron microscopy (TEM) DAPI staining, and the cell compatibility was detected by EPCs cells in vitro.
Results: the three kinds of acellular methods could completely remove the cells. DAPI fluorescence detection showed that the scaffold materials of each group were fine.
There was no residual DNA in the nucleus. HE staining and scanning electron microscopy showed that the collagen fibers in the freeze thawing + biological enzyme group were arranged neatly, and there was no obvious structural change of collagen fibers. The other two groups showed collagen fibrils breaking and structural disorder; and the other two groups of scaffolds were buried under the freeze-thaw + biologic enzyme group and buried inflammatory cells subcutaneously. Less wetting, EPCs and freeze-thaw enzyme group scaffold materials good adhesion.
Conclusion: combined with repeated osmotic pressure, combined with low concentration of trypsin and low concentration of pancreatin, the nuclease decellular method can not only remove the components of the valvular venous cells, but also retain a complete extracellular matrix structure. It has good tissue and cell compatibility. It is an ideal method for the preparation of the valvular vein decellular scaffold.
Construction of tissue-engineered venous valve by third parts of smooth muscle implants
Objective: to develop a smooth muscle cell implant, and to use the device to plant SPCs and pressure perfusion to grow EPCs in order to improve the planting rate of cells and to construct a tissue engineered venous valve with good performance.
Materials and methods:
The development of smooth muscle cell implant: to reform the traditional method of planting smooth muscle cells from the functional cavity, and use the effect of vacuum to attract smooth muscle cells from the outer wall to the intima.
Tissue engineered venous valve construction: third generation of SPCs/EPCs were selected as seed cells, and SPCs was planted by self-made smooth muscle implants in the experimental group. The control group was cultivated for three days after SPCs. cultivation with pressure perfusion, and EPCs was planted by pressure perfusion..SPCs planting 4H was continued for four days, frozen section, and DAPI staining was used to observe the density of SPCs cultivation; 2 4h scanning electron microscopy, toluidine blue staining was used to observe the adhesion of SPCs on the scaffold material; MTT detection, cell adhesion test was used to detect the proliferation of seed cells in the scaffold material. After a week, HE staining was used to observe the histological structure of tissue engineered venous valve constructed by immunohistochemical staining.
Results: the density of the cells planted by the smooth muscle implant is more uniform, the destruction of the intima structure of the scaffold material is smaller, the number of the cultured smooth muscle cells can be quantified, and the pollution probability in the construction process is reduced in the sterile cell suspension tube. After 4h, the DAPI staining shows that the density of SPCs in the experimental group is uniform and the control group is in the control group. The cell density was uneven and the cell aggregation was only localized. The scanning electron microscope showed that the outer surface cells of the experimental group had more adhesion, a small amount of extracellular matrix was secreted and the surface of the scaffold was smooth; a small amount of cells adhered to the control group, and the surface of the scaffold material was honeycomb. Methylene blue staining showed that the experimental group SPCs was on the scaffold material. The cell adhesion experiment showed that the cells in the experimental group had better proliferation activity on the scaffold material, and there was a significant difference between the experimental group and the control group. The histological examination showed that the endothelial cells in the experimental group had completely covered the inner surface of the scaffold material, and the endothelialization of the control group was complete. Conclusion: smooth muscle cells were more complete than the control group. Planting device is an effective tool for smooth muscle cultivation, which can efficiently and evenly plant SPCs.
The extracellular matrix produced by SPCs can repair the surface structure of the scaffold material and the growth factor contained in the extracellular matrix, which promotes the adhesion and proliferation of EPCs, and promotes the endothelialization of the scaffold materials.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

【参考文献】