本文選題：平滑肌組細(xì)胞 + 內(nèi)皮祖細(xì)胞��； 參考：《第二軍醫(yī)大學(xué)》2012年博士論文

【摘要】：原發(fā)性瓣膜功能不全是臨床上常見病，多發(fā)病，靜脈瓣移植被認(rèn)為是最后的選擇。但自體靜脈瓣移植來源有限，限制了靜脈瓣移植的應(yīng)用，應(yīng)用細(xì)胞生物學(xué)與工程學(xué)原理開發(fā)出具有生物活性、無免疫原性的組織工程化靜脈瓣是修復(fù)與功能重建深靜脈功能不全的理想方案。 目前構(gòu)建組織工程化靜脈瓣研究處尚處于起步階段。Teebken等（2003）應(yīng)用組織工程學(xué)原理，用受體靜脈壁來源的肌纖維母細(xì)胞和內(nèi)皮細(xì)胞構(gòu)建組織工程化靜脈瓣，但是肌纖維母細(xì)胞沒能長入支架內(nèi)部；該課題組在2009年又以人大隱靜脈內(nèi)皮細(xì)胞為種子細(xì)胞，大隱靜脈脫細(xì)胞支架為支架材料，構(gòu)建組織工程化靜脈瓣，三維立體培養(yǎng)8天，發(fā)現(xiàn)細(xì)胞能在瓣膜兩側(cè)和血管壁表面生長并內(nèi)皮化，但該研究血管壁內(nèi)沒種植細(xì)胞，沒有進(jìn)行體內(nèi)移植。 我們實驗室在國家、軍隊和上海市基金的資助下，近年來一直進(jìn)行組織工程化靜脈瓣的研究，我們已[1]利用骨髓來源的多能成體祖細(xì)胞（Multipotent adult progenitor cells，MAPC）和內(nèi)皮祖細(xì)胞（Endothelial prigenitor cell，EPC）作為種子細(xì)胞，以綿羊脫細(xì)胞靜脈瓣支架為支架材料，成功構(gòu)建了組織工程化靜脈，并進(jìn)行了犬、綿羊在體效用性研究，及安全性評價。但是，靜脈瓣在體內(nèi)長期功能欠佳，我們認(rèn)為，其原因可能與瓣膜的內(nèi)皮化程度、內(nèi)皮細(xì)胞體內(nèi)黏附強(qiáng)度、脫細(xì)胞支架材料在制備過程中損傷、變性等因素有關(guān)。擬提高組織工程化靜脈瓣的質(zhì)量，縮短與生理性靜脈瓣的差距，優(yōu)化構(gòu)建程序，有必要對組織工程化靜脈瓣的構(gòu)建中所用材料和構(gòu)建技術(shù)進(jìn)行再探討。在種子細(xì)胞研究方面，我們實驗室利用骨髓源性MAPC和EPC為種子細(xì)胞，成 功構(gòu)建了組織工程化靜脈瓣，但是，細(xì)胞分離培養(yǎng)過程復(fù)雜，需采用全骨髓血培養(yǎng)加免疫磁珠分選，免疫磁珠價格昂貴、不經(jīng)濟(jì)，細(xì)胞分選過程復(fù)雜，易污染。為優(yōu)化組織工程化靜脈瓣構(gòu)建程序、降低成本，有必要對種子細(xì)胞的種類及分離培養(yǎng)方法進(jìn)行再研究。在支架材料制備方法上，先前的研究采用了兩種不同的脫細(xì)胞支架制備方法，但其中哪一種方法較好還未曾研究。近期有研究報道的凍融+生物酶的方法，在其他組 織脫細(xì)胞支架制備中對支架材料結(jié)構(gòu)損傷小，但目前該方法還未見在靜脈瓣膜脫細(xì)胞支架制備中應(yīng)用。在支架材料制備方法上，我們先前的研究采用TritonX-100+NH4OH+DNase+RNase的脫細(xì)胞方法，支架無細(xì)胞殘留，纖維連續(xù)，支架內(nèi)布滿大小不等的孔隙，無免疫原性。但利用該支架材料構(gòu)建的組織工程化靜脈瓣體內(nèi)長期功能欠佳，這是否與該脫細(xì)胞方法有關(guān)尚不得而知。近期研究報道，凍融+生物酶的方法在其他組織脫細(xì)胞中發(fā)現(xiàn)對支架材料結(jié)構(gòu)損傷小，利用該方法制備的瓣膜脫細(xì)胞支架是否能提高組織工程化靜脈瓣體內(nèi)長期功能，也值得研究。 在種子細(xì)胞種植方法上，我們先前的研究采用多點注射和加壓灌注的方法，程序復(fù)雜、需要較高的專門技術(shù)、且發(fā)現(xiàn)利用該方法構(gòu)建的組織工程化靜脈瓣體內(nèi)超過6個月，有內(nèi)皮細(xì)胞脫落、血栓形成的現(xiàn)象，嚴(yán)重影響了組織工程化靜脈瓣的體內(nèi)長期功能，迫切需要尋找新的方法。 本研究針對我們組織工程化靜脈瓣研究中遇到到的問題，擬通過簡化種子細(xì)胞誘導(dǎo)培養(yǎng)方法，獲取活性好，純度高的種子細(xì)胞；獲得一種脫細(xì)胞完全、對支架材料結(jié)構(gòu)損害較小的組織工程化靜脈瓣脫細(xì)胞支架的方法；以及研發(fā)平滑肌細(xì)胞種植器，提高平滑肌細(xì)胞粘附率等三個方面的研究；以提高組織工程化靜脈瓣的構(gòu)建效果，改善組織工程化靜脈瓣遠(yuǎn)期性能。 第一部分同時培養(yǎng)兔骨髓源性EPCs和SPCs 研究目的：同時分離和培養(yǎng)兔骨髓來源的EPCs和平滑肌祖細(xì)胞（Smoothprogenitor cells,SPCs），研究其生物學(xué)特性，評估其作為組織工程化靜脈瓣種子細(xì)胞的可能性。 材料和方法：密度梯度離心法獲取兔骨髓血單個核細(xì)胞沉淀，分別用含5%FBS的EGM-2完全培養(yǎng)基向EPC方向誘導(dǎo)培養(yǎng)；用含5%FBS，20ng/mlPDGF-BB，不含VEGF的EBM-2培養(yǎng)基向SPC方向誘導(dǎo)培養(yǎng)。細(xì)胞培養(yǎng)48h后首次換液，相差顯微鏡下觀察細(xì)胞形態(tài)特征，透射電鏡觀察兩類細(xì)胞超微結(jié)構(gòu)的特點。誘導(dǎo)第7天，14天細(xì)胞免疫熒光、流式細(xì)胞儀檢測EPCs/SPCs表面標(biāo)志陽性率表達(dá)情況；檢測細(xì)胞攝取DiI-ac-LDL和結(jié)合FITC-UEA-1功能，以及在MatriGel上成血管功能情況，將第3代細(xì)胞進(jìn)行凍存和復(fù)蘇，測定細(xì)胞凍存前后的細(xì)胞活性變化。 結(jié)果：EPCs生物學(xué)特性：EPCs培養(yǎng)10天左右，細(xì)胞單層融合呈“鋪路石”狀；EPC表達(dá)CD34，VEGFR-2，弱表達(dá)CD133；透射電鏡可見EPCs胞漿內(nèi)特征性W-P小體；細(xì)胞生物學(xué)功能檢測可見EPCs在Matrigel上呈現(xiàn)血管狀；EPCs具有攝取DiI-ac-LDL和結(jié)合FITC-UEA-1的功能。凍存細(xì)胞復(fù)蘇前后細(xì)胞生長特性方面無明顯變化；SPCs生物學(xué)特性：SPCs培養(yǎng)14天左右出現(xiàn)血管平滑肌特異的生長特征“峰—谷”樣生長特性；表達(dá)CD34，SMA，不表達(dá)Ⅷ和VEGF-2；在透射電鏡下可見細(xì)胞內(nèi)含有與細(xì)胞縱軸平行排列的肌絲；無攝取DiI-ac-LDL和結(jié)合FITC-UEA-1功能；在Matrigel上 無血管狀結(jié)構(gòu)形成。 結(jié)論：骨髓血梯度密度離心得到的單個核細(xì)胞，在不同誘導(dǎo)培養(yǎng)基的誘導(dǎo)下，可同時獲到高純度的EPCs和SPCs，SPCs可自然分化為平滑肌樣細(xì)胞，不需要經(jīng)向平滑肌細(xì)胞誘導(dǎo)分化，省時、經(jīng)濟(jì)、不易污染。 第二部分不同方法制備組織工程化靜脈瓣脫細(xì)胞支架 研究目的：比較三種不同方法制備的帶瓣靜脈脫細(xì)胞支架的組織學(xué)，生物學(xué)特性，以期獲得一種較好的帶瓣靜脈脫細(xì)胞支架材料。 材料和方法:采用以下三種不同方法制備帶瓣靜脈脫細(xì)胞支架。 1.脫氧膽酸鈉組：Beagle犬帶瓣靜脈，浸入4%去氧膽酸鈉溶液，4℃振蕩1h進(jìn)行脫細(xì)胞處理，然后于37℃以50mL生理鹽水反復(fù)沖洗，得到脫細(xì)胞帶瓣靜脈支架，于4℃PBS液中保存?zhèn)溆茫?2.Triton組：Beagle犬帶瓣靜脈，浸入0.5%Triton-100+0.05%NH4OH溶液，4℃振搖3d；超純水4℃振搖3d；DNase+RNase處理（37℃）12h；超純水漂洗，將脫細(xì)胞支架60CO輻照消毒，-80℃保存?zhèn)溆茫?3.凍融+生物酶組：Beagle犬帶瓣靜脈，浸入4℃低滲液浸泡11小時，-80℃3小時，37℃水浴30min，PBS振蕩沖洗。0.05％胰酶+0．02％EDTA處理8h，DNase0.2mg/mL、Nase0.02mg/mL消化8h，PBS漂洗，上述步驟重復(fù)3次，支架材料冷凍干燥，輻照消毒，-80℃保存?zhèn)溆�；隨機(jī)選取各組支架材料檢測，病理切片分別行HE染色觀察組織結(jié)構(gòu)；掃描電鏡觀察支架材料表面及內(nèi)部超微結(jié)構(gòu)；透射電鏡DAPI染色觀察DNA殘留；體外EPCs細(xì)胞種植檢測細(xì)胞相容性。 結(jié)果：三種脫細(xì)胞方法均能徹底去除細(xì)胞，DAPI熒光檢測顯示各組支架材料細(xì) 胞核，均無DNA成分殘留；HE染色及掃描電鏡顯示，凍融+生物酶組膠原纖維排列整齊，未見明顯的膠原纖維結(jié)構(gòu)改變，其他兩組可見膠原纖維斷裂，及結(jié)構(gòu)紊亂現(xiàn)象；與其他兩組脫細(xì)胞支架材料相比凍融+生物酶組支架皮下埋置炎細(xì)胞浸潤較少，，EPCs與凍融生物酶組支架材料粘附性好。 結(jié)論：結(jié)合滲透壓改變的反復(fù)凍融加上低濃度胰酶，核酸酶脫細(xì)胞法，既可以較徹底除去帶瓣膜靜脈細(xì)胞成分，又保留了較完整的細(xì)胞外基質(zhì)結(jié)構(gòu)，具有良好的組織和細(xì)胞相容性，是較理想的帶瓣膜靜脈脫細(xì)胞支架制備方法。 第三部分平滑肌種植器構(gòu)建組織工程化靜脈瓣 研究目的：研制平滑肌細(xì)胞種植器，并利用該裝置種植SPCs，加壓灌注旋轉(zhuǎn)種植EPCs，以提高細(xì)胞的種植率，構(gòu)建性能良好的組織工程化靜脈瓣。 材料和方法： 平滑肌細(xì)胞種植器的研制：改革傳統(tǒng)的從官腔內(nèi)種植平滑肌細(xì)胞的方法，利用真空吸引的作用將平滑肌細(xì)胞從官腔外壁較均勻的種植平滑肌細(xì)于在內(nèi)膜下。 組織工程化靜脈瓣構(gòu)建：選第3代SPCs/EPCs為種子細(xì)胞，實驗組用自制的平滑肌種植器種植SPCs；對照組用加壓灌注多點注射的方法種植SPCs。培養(yǎng)三天后，以加壓灌注種植EPCs，繼續(xù)培養(yǎng)四天。SPCs種植4h，冰凍切片，DAPI染色觀察SPCs種植密度；24h掃描電鏡、甲苯胺藍(lán)染色觀察SPCs在支架材料上的粘附情況；MTT檢測、細(xì)胞粘附實驗檢測種子細(xì)胞在支架材料內(nèi)的增殖性能。一周后HE染色，免疫組織化學(xué)染色觀察構(gòu)建的組織工程化靜脈瓣的組織學(xué)結(jié)構(gòu)。 結(jié)果：平滑肌種植器種植的細(xì)胞密度較均勻、支架材料內(nèi)膜層結(jié)構(gòu)破壞較小、種植平滑肌細(xì)胞的數(shù)量可進(jìn)行量化控制；在無菌的細(xì)胞懸液筒中進(jìn)行，減少了構(gòu)建過程中的污染幾率。種植4h后DAPI染色顯示，實驗組SPCs種植密度均勻，對照組細(xì)胞種植密度不均勻，僅局部有細(xì)胞聚集。掃描電鏡顯示，實驗組血管外表面細(xì)胞粘附較多，已有少量的細(xì)胞外基質(zhì)分泌，支架材料表面平滑；對照組有少量細(xì)胞粘附，支架材料表面有蜂窩狀。甲苯胺藍(lán)染色顯示，實驗組SPCs在支架材料上粘附較多，細(xì)胞密度較大。MTT、細(xì)胞粘附實驗顯示，實驗組細(xì)胞在支架材料上有較好的增殖活性，與對照組相比差別有統(tǒng)計學(xué)意義。組織學(xué)檢查可見實驗組內(nèi)皮細(xì)胞已經(jīng)完全覆蓋支架材料的內(nèi)表面，較對照組內(nèi)皮化完整。結(jié)論：平滑肌細(xì)胞種植裝置是有效的平滑肌種植工具，可高效、均勻種植SPCs， 種植的SPCs產(chǎn)生的細(xì)胞外基質(zhì)，可修復(fù)脫細(xì)胞支架材料表面結(jié)構(gòu)，細(xì)胞外基質(zhì)所含的生長因子，促進(jìn)EPCs的粘附增殖，促進(jìn)了支架材料的內(nèi)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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R329

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