本文選題：骨髓間充質(zhì)干細胞 + 內(nèi)皮祖細胞��； 參考：《第四軍醫(yī)大學》2012年博士論文

【摘要】：實驗一骨髓間充質(zhì)干細胞的分離培養(yǎng)及多向誘導分化 [摘要]目的：體外獲取兔骨髓間充質(zhì)干細胞（mesenchymal stem cells，MSCs）、純化、擴增，同時探討其生物學特性及多向分化潛能。方法：全骨髓貼壁法體外分離培養(yǎng)MSCs，觀察其增殖、生長特性和組織學形態(tài)；并向成骨細胞、脂肪細胞和軟骨細胞多向誘導分化，分別采用鈣結(jié)節(jié)茜素紅染色、油紅O染色和甲苯胺藍染色鑒定。結(jié)果：貼壁的骨髓間充質(zhì)干細胞為紡錘形，呈克隆樣生長，性狀穩(wěn)定，堿性磷酸酶（alkaline phosphatase，ALP）染色弱陽性。經(jīng)成骨、成脂和成軟骨誘導培養(yǎng)后，表現(xiàn)出相應(yīng)細胞的形態(tài)學和生物學特征。結(jié)論：全骨髓貼壁篩選法取材方便，骨髓間充質(zhì)干細胞增殖能力強，在不同的條件培養(yǎng)基誘導下能夠多向分化。 實驗二內(nèi)皮祖細胞的培養(yǎng)和鑒定 [摘要]目的：探索分離純化及體外培養(yǎng)內(nèi)皮祖細胞(endothelialprogenitor cells, EPCs)的方法，并進行鑒定。為采用EPCs促進血管化組織工程骨的構(gòu)建策略提供實驗基礎(chǔ)。方法：將骨髓單核細胞(mononuclear cells,MNCs)接種至明膠涂層的培養(yǎng)皿內(nèi)，使用含10%胎牛血清（fetal bovineserum，F(xiàn)BS）、添加50μg/mL ECGS (endothelial cell growth supplements)的M199培養(yǎng)液，置37℃、體積分數(shù)為5%的CO2飽和濕度恒溫培養(yǎng)箱培養(yǎng)。3天后棄去未貼壁細胞，此后每3天換液1次。形成內(nèi)皮祖細胞克隆后，0.05%胰酶-EDTA消化傳代，2-4代細胞用于實驗。通過CD31免疫熒光染色、荊豆凝集素免疫細胞化學染色、毛細血管腔形成能力及透射電子顯微鏡(transmission electron microscope, TEM)檢測進行鑒定。結(jié)果：培養(yǎng)5-7天，內(nèi)皮祖細胞的早期克隆形成，中央為大量圓形細胞。2周后，細胞表現(xiàn)出典型的“鵝卵石”狀。培養(yǎng)過程中可形成管腔狀結(jié)構(gòu)。可與荊豆凝集素特異性相結(jié)合；內(nèi)皮細胞特異性表面標志CD31熒光染色呈陽性表達；透射電鏡觀察細胞質(zhì)內(nèi)見內(nèi)皮細胞特有的細胞器-(W-P)小體。結(jié)論：特定的培養(yǎng)條件下可以獲取足量的EPCs，細胞可迅速體外擴增，并具有內(nèi)皮細胞特異性標志物。 實驗三骨髓間充質(zhì)干細胞膜片的體外構(gòu)建及成骨分化研究 [摘要]目的：探索體外構(gòu)建骨髓間充質(zhì)干細胞膜片的簡便方法，分析膜片組織學結(jié)構(gòu)，并評估其成骨分化潛能。方法：將兔MSCs高密度接種于直徑10cm培養(yǎng)皿，成骨誘導條件下連續(xù)培養(yǎng)2周形成細胞膜片。收獲時，使用細胞刮沿皿底小心刮起，或用鑷子輕輕提起。通過組織學、組織化學、堿性磷酸酶活性定量檢測、透射電鏡、掃描電鏡(scanning electronmicroscope，SEM)等一系列方法，檢測細胞膜片的物理及生物學特性。.結(jié)果：高密度連續(xù)培養(yǎng)后形成的MSCs膜片，呈半透明薄膜狀，有彈性，其內(nèi)多個白色結(jié)節(jié)。HE染色證實骨髓間充質(zhì)細胞膜片是一種由多層細胞和細胞外基質(zhì)（extracellular matrix，ECM）組成的聚集體。堿性磷酸酶、茜素紅及Von kossa等組織化學染色呈陽性；堿性磷酸酶定量分析含量較高；掃描電鏡及透射電鏡檢查均見基質(zhì)中大量的礦化結(jié)節(jié)聚集。結(jié)論：通過體外簡單的連續(xù)培養(yǎng)方法和成骨誘導后，可構(gòu)建具有良好成骨能力MSCs膜片。 實驗四骨髓間充質(zhì)干細胞膜片復合內(nèi)皮祖細胞構(gòu)建血管化組織工程骨 [摘要]目的：探討利用MSCs膜片復合EPCs構(gòu)建血管化的無外支架組織工程骨的可行性,以解決傳統(tǒng)接種方法存在的諸多問題。方法：將骨髓細胞行雙向誘導：骨髓單核細胞中MSCs連續(xù)培養(yǎng)成骨誘導后，細胞增殖、分泌細胞外基質(zhì)，形成成骨性膜片；另一部分骨髓單核細胞在內(nèi)皮生長培養(yǎng)基內(nèi)分化為EPCs。然后將MSCs膜片復合EPCs，折疊、卷曲成圓柱狀的復合細胞聚集體。最后，將復合體移植至免疫缺陷的裸鼠背部皮下。單純MSCs膜片構(gòu)建組織塊作為對照。術(shù)后4周，8周分別進行大體觀察、顯微CT（micro-computed tomography，Micro-CT）、掃描電鏡及組織學檢查。結(jié)果：MSCs膜片復合EPCs構(gòu)建物植入體內(nèi)后，形成血管化骨樣組織，外觀色紅、質(zhì)硬。CT、掃描電鏡及組織學檢查均證實新骨形成，而且所形成的骨密度和血管密度均高于對照組。結(jié)論：首次利用MSCs膜片復合EPCs構(gòu)建較大體積的血管化組織工程骨，無需外支架。實驗證實EPCs的引入不僅能產(chǎn)生功能性血管網(wǎng)，而且能夠促進骨形成。 實驗五骨髓間充質(zhì)干細胞膜片復合內(nèi)皮祖細胞構(gòu)建可注射血管化組織工程骨 [摘要]目的：探討可注射的血管化組織工程新的構(gòu)建策略。方法：利用MSCs膜片復合EPCs，形成具有雙重細胞成分的聚集體。復合種子細胞附著于自身分泌的細胞外基質(zhì)支架上，形成具有骨組織三維結(jié)構(gòu)和生理活性的復合體。再注射至無免疫動物背部皮下，術(shù)后4周，8周分別進行大體觀察、顯微CT、掃描電鏡及組織學檢查，觀察其在體內(nèi)發(fā)育成骨樣組織的能力。結(jié)果：MSCs膜片/EPCs復合體，可以順利通過注射針頭至皮下。8周后形成鮮紅骨樣組織，呈扁圓形，外周密布一層血管網(wǎng)，質(zhì)地硬，最大長度近2.0cm。Micro-CT影像顯示高密度礦化組織形成。橫斷面掃描電鏡觀察呈現(xiàn)松質(zhì)骨樣結(jié)構(gòu)，其內(nèi)大量片狀骨小梁排列，交織為多孔的網(wǎng)格樣結(jié)構(gòu)。組織學檢查證實新生的骨組織和豐富的血管形成。此外，新骨的形成存在兩種方式：組織塊外周以膜內(nèi)骨化為主，骨基質(zhì)內(nèi)可見骨細胞，骨小梁邊緣成行排列矮柱狀的成骨細胞；組織塊內(nèi)部還可見肥大的軟骨細胞和鈣化軟骨，提示軟骨內(nèi)骨化方式。結(jié)論：本研究，首次驗證了將EPCs復合MSCs膜片，構(gòu)建可注射的血管化組織工程骨的可行性，無需外源性載體。為構(gòu)建可注射的血管化組織工程骨提供了全新的思路。 實驗六骨髓間充質(zhì)干細胞膜片/內(nèi)皮祖細胞/珊瑚構(gòu)建大塊管狀骨 [摘要]目的：利用細胞膜片技術(shù)，輔以管狀形態(tài)的內(nèi)支撐體，首次探索移植自體體內(nèi)構(gòu)建大塊的具備特定形態(tài)的組織工程骨。方法：構(gòu)建兔來源MSCs膜片-EPCs，與珊瑚內(nèi)支撐體卷層樣復合，形成管樣結(jié)構(gòu)。體外實驗，置灌注式生物反應(yīng)器動態(tài)培養(yǎng)8周，觀察細胞生長和組織的形成；體內(nèi)實驗，移植自體背部皮下，利用自體的微環(huán)境和自身分泌的各種生長因子調(diào)控來完成骨組織的構(gòu)建。體內(nèi)、外標本取材，進行大體觀察、顯微CT、掃描電鏡及組織學檢查，分析新骨形成。結(jié)果：體外，，隨著灌注式反應(yīng)器動態(tài)培養(yǎng)時間的增長，細胞基質(zhì)分泌旺盛，細胞與細胞產(chǎn)生連接，各層聚集體之間逐漸融合、增厚，質(zhì)地變硬，并與珊瑚材料緊密結(jié)合，呈現(xiàn)出白色的骨樣組織外觀。體內(nèi)培養(yǎng)8周后，構(gòu)建了長度近5.0cm大塊管狀骨。顯微CT、掃描電鏡及組織學檢查均證實，珊瑚表面、材料間隙及深部均有新骨形成。結(jié)論：MSCs膜片-EPCs與珊瑚自體體內(nèi)可構(gòu)建大塊的管狀骨。
[Abstract]:Isolation and multidirectional differentiation of bone marrow mesenchymal stem cells in Experiment 1
[Abstract] Objective: to obtain mesenchymal stem cells (MSCs) in vitro, to purify and amplify, and to explore its biological characteristics and pluripotent differentiation potential. Methods: the whole bone marrow adherent method was used to isolate and culture MSCs in vitro, to observe its proliferation, growth and histology, and to osteoblasts, adipocytes and chondrocytes. Multidirectional differentiation, using alizarin red staining with calcium nodules, oil red O staining and toluidine blue staining. Results: the adherent bone marrow mesenchymal stem cells were spindle shaped, cloned and stable, and alkaline phosphatase (alkaline phosphatase, ALP) dyed weak Yang. After induction of osteogenesis, fat formation and cartilage formation, the phase was displayed. The morphological and biological characteristics of the cells. Conclusion: all bone marrow adherent screening method is convenient, bone marrow mesenchymal stem cells have strong proliferation ability and can be multidifferentiated under the induction of different conditions.
The culture and identification of experiment two inner skin progenitor cells
[Abstract] Objective: To explore the method of isolation and purification and in vitro culture of endothelialprogenitor cells (EPCs), and to identify it. It provides an experimental basis for the use of EPCs to promote the construction of vascularized tissue engineering bone. Methods: bone marrow mononuclear cells (mononuclear cells, MNCs) are inoculated into the culture dish of gelatin coating, so that the bone marrow mononuclear cells (MNCs) are inoculated into the culture dish of gelatin coating. A M199 culture containing 10% fetal bovine serum (fetal BovineSerum, FBS) and 50 g/mL ECGS (endothelial cell growth supplements) was added to the incubator at 37 degrees C and 5% in the volume fraction of CO2 saturated humidity and constant temperature incubator. After.3 days, the non adherent cells were abandoned, and then 1 times every 3 days. After the formation of endothelial progenitor cells, 0.05% pancreatin was digested and passed to 2. 2 -4 cells were used for experiments. By CD31 immunofluorescence staining, immunocytochemical staining of agglutinin, capillary cavity formation and transmission electron microscopy (transmission electron microscope, TEM) detection. Results: 5-7 days of culture, the early cloning of endothelial progenitor cells was formed in the center of a large number of round cells after.2 weeks, The cells show a typical "cobblestone" shape. In the process of culture, a lumen structure can be formed. It can be combined with the specificity of the bean agglutinin; the specific surface of the endothelial cell mark CD31 fluorescent staining is positive; transmission electron microscopy is used to observe the specific organelles - (W-P) corpuscles in the cytoplasm of the cytoplasm. Conclusion: specific culture conditions A sufficient amount of EPCs can be obtained under this condition. The cells can be amplified in vitro and have specific markers of endothelial cells.
Experiment three the construction and osteogenic differentiation of bone marrow mesenchymal stem cell membrane in vitro
[Abstract] Objective: To explore a simple method of constructing bone marrow mesenchymal stem cell membrane in vitro, analyze the histological structure of the diaphragm and evaluate its osteogenic differentiation potential. Methods: the rabbit MSCs was inoculated at high density in the diameter 10cm culture dish, and the cell membrane was formed continuously for 2 weeks under the induction of osteogenesis, and the cell was scraped along the dish bottom carefully. Or gently lift it with tweezers. Through a series of methods such as histology, histochemistry, quantitative detection of alkaline phosphatase activity, transmission electron microscopy, scanning electron microscopy (scanning electronmicroscope, SEM), and so on, the physical and biological properties of the cell diaphragms are detected. Results: the MSCs diaphragm formed after high density continuous culture, is translucent thin film and elastic, The.HE staining of multiple white nodules confirmed that the bone marrow mesenchymal cell membrane was a group of multi-layer cells and extracellular matrix (extracellular matrix, ECM). The histochemical staining of alkaline phosphatase, alizarin red and Von Kossa was positive; the quantitative analysis of alkaline phosphatase was higher; scanning electron microscope and transmission electron microscope examination A large number of mineralized nodules were found in the matrix. Conclusion: MSCs film with good osteogenesis ability can be constructed by a simple continuous culture method and osteogenic induction in vitro.
Experiment four bone marrow mesenchymal stem cell membrane combined with endothelial progenitor cells to construct vascularized tissue-engineered bone
[Abstract] Objective: To explore the feasibility of constructing vascularized non stenting tissue engineered bone with MSCs film combined with EPCs in order to solve the problems existing in traditional inoculation methods. Methods: bone marrow cells were induced by bidirectional induction: bone marrow mononuclear cells were continuously cultured and cultured for bone induction, cell proliferation, extracellular matrix, and formation of osteogenesis. The other bone marrow mononuclear cells were differentiated into EPCs. in the endothelial growth culture base and then the MSCs diaphragm was combined with EPCs, folded and curled into a cylindrical composite cell aggregate. Finally, the complex was transplanted subcutaneously into the back of the immune deficient nude mice. The simple MSCs diaphragm was used as the control. 4 weeks after the operation, the mass was carried out, respectively. Body observation, microscopic CT (micro-computed tomography, Micro-CT), scanning electron microscope and histological examination. Results: after the MSCs diaphragm composite EPCs construction was implanted in the body, the vascularized osteoid tissue was formed. The appearance of the tissue was red and hard, the scanning electron microscope and the histological examination confirmed the formation of the new bone, and the bone density and the density of the blood vessel were higher than that of the pair. Conclusion: a large volume of vascularized tissue engineering bone was constructed with MSCs film combined with EPCs for the first time without external scaffold. It was proved that the introduction of EPCs could not only produce functional vascular network, but also promote bone formation.
Experiment five bone marrow mesenchymal stem cells and endothelial progenitor cells were used to construct injectable tissue-engineered bone.
[Abstract] Objective: To explore a new construction strategy for injectable vascularized tissue engineering. Methods: MSCs membrane composite EPCs was used to form a polymer with double cell components. Compound seed cells attached to the self secreted extracellular matrix scaffold to form a complex of three-dimensional structure and physiological activity of bone tissue. The immune animals were subcutaneously on the back, 4 weeks and 8 weeks after the operation. Microscopical CT, scanning electron microscopy and histological examination were used to observe the ability to develop osteoid tissue in the body. Results: the MSCs diaphragm /EPCs complex could successfully form a red bone like tissue through the injection of the needle to the subcutaneous.8 week, a flat circle, and a layer of blood vessels in the outer circumference. Web, texture hard, the maximum length near 2.0cm.Micro-CT images showed high density mineralized tissue. Cross section scanning electron microscopy showed a loose bone like structure, a large number of trabecular bone trabeculae arranged and interwoven into a porous mesh like structure. Histological examination confirmed the formation of new bone tissue and rich vascularization. In addition, the formation of new bone was two The tissue mass was mainly in the outer membrane of the tissue, bone cells were visible in the bone matrix, the bone trabecular edge was arranged in a row of short columnar osteoblasts, and the hypertrophic chondrocytes and calcified cartilage were also visible within the tissue. Conclusion: the EPCs composite MSCs diaphragm was first verified and the injectable blood was constructed for the first time. The feasibility of tubular tissue-engineered bone does not require exogenous vectors. It provides a new idea for the construction of injectable vascularized tissue-engineered bone.
Experiment six bone marrow mesenchymal stem cells / endothelial progenitor cells / corals construct large tubular bone.
[Abstract] Objective: To explore the construction of a large block of tissue engineered bone with a tubular form of internal support with cell membrane technique and tubular form for the first time. Method: to construct a rabbit source MSCs film -EPCs, combined with the reel sample of the corals, to form a tube like structure. In vitro experiment, the perfusion bioreactor was moved. The growth of cells and the formation of tissue were observed for 8 weeks. In vivo experiments, transplantation of autologous back subcutaneous, autologous microenvironment and the regulation of various growth factors which were secreted by themselves to complete the construction of bone tissue. The body and external specimens were taken for general observation, microscopical CT, scanning electron microscopy and histological examination, and analysis of new bone formation. Results: body results: body results: body In addition, with the growth of the dynamic culture time of the perfusion reactor, the cell matrix was exuberant and the cells were connected with the cells. The aggregates of each layer were gradually fused, thickened and hardened, and the appearance of the white bone like tissue was presented closely with the coral material. After 8 weeks of culture, the length of the tubular bone with a length of nearly 5.0cm was constructed. Microscopical CT, Scanning electron microscopy and histological examination confirmed that there were new bone formation in the coral surface, the material gap and the deep part. Conclusion: the MSCs diaphragm -EPCs and the coral autologous body can build large tubular bones.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R318.08

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1 張文鵬;葉發(fā)剛;y囇澡

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