本文選題：骨髓基質(zhì)干細胞 + 細胞分化　； 參考：《中國協(xié)和醫(yī)科大學(xué)》2008年博士論文

【摘要】： 目的:探討自體骨髓基質(zhì)干細胞移植在股骨頭缺損壞死部位,移植的骨髓細胞能否成活,能否停留在骨壞死、缺損部位生長增殖,能否分化為成骨細胞,及能否促進成骨、修復(fù)股骨頭缺損壞死。 方法:試驗分三部分。①抽取雙側(cè)狗髂骨骨髓。骨髓細胞行貼壁細胞分離法分離骨髓基質(zhì)干細胞(BMSCs),進行體外培養(yǎng)擴增,試驗用第二代細胞。移植前,用5-溴脫氧尿嘧啶核苷(BrdU)標(biāo)記BMSCs。在狗雙側(cè)股骨頭后外側(cè)軟骨面近頭頸交界處,用直徑8mm的環(huán)鉆向股骨頭中心方向鉆孔10mm深度,造成股骨頭直徑8mm、深度10mm的骨缺損模型。環(huán)鉆取出的骨質(zhì)經(jīng)微波滅活后,與標(biāo)記的BMSCs復(fù)合,植入一側(cè)自體股骨頭缺損處,另一側(cè)單純植入微波滅活骨作為對照,術(shù)后5周取材。②抽取雙側(cè)髂骨骨髓,行密度梯度離心法分離BMSCs,用熒光染料羧基熒光素二醋酸鹽琥珀酰亞胺酯(CFSE)標(biāo)記BMSCs,用明膠海綿吸附分離出的BMSCs,植入一側(cè)自體股骨頭缺損處,另一側(cè)單純植入明膠海綿作為對照,術(shù)后3周取材。③將體外培養(yǎng)擴增的BMSCs,與脫抗原小牛松質(zhì)骨復(fù)合植入一側(cè)自體股骨頭缺損處,另一側(cè)單純植入脫抗原小牛松質(zhì)骨作為對照,術(shù)后12周取材。利用激光共聚焦顯微鏡和免疫組織化學(xué)法分別觀察CFSE和Brdu的標(biāo)記和體內(nèi)定位情況。行X射線片,大體組織觀察,組織化學(xué)染色,圖像分析,及通過免疫組織化學(xué)方法和熒光免疫組織化學(xué)方法檢測成骨細胞特異性分化標(biāo)記核心結(jié)合因子a1(Cbfa1)、骨鈣素、骨橋素和Ⅰ型膠原,以了解BMSCs體內(nèi)分化狀況和成骨效果。 結(jié)果:大體組織學(xué)觀察顯示,BMSCs移植側(cè)骨缺損成骨程度明顯強于對照側(cè)。顯微組織學(xué)研究的結(jié)果進一步顯示,BMSCs移植側(cè)骨缺損區(qū)內(nèi)的成骨量、骨基質(zhì)礦化的程度、骨成熟度明顯高于對照側(cè),含有更加豐富的梭形間充質(zhì)細胞和成骨細胞,成骨細胞分化特異性標(biāo)記Cbfa1、骨鈣素、骨橋素、Ⅰ型膠原表達也明顯強于對照側(cè)。股骨頭缺損內(nèi)發(fā)現(xiàn)大量BrdU陽性細胞,新骨形成越活躍的地方,BrdU陽性細胞數(shù)目越多,很多成骨細胞BrdU陽性,新骨形成區(qū)的很多血管壁也分布著BrdU陽性細胞。熒光顯微鏡下觀察,見BMSCs移植側(cè)帶有CFSE綠色熒光的細胞定位于骨缺損內(nèi),與抗RunX2、Osteocalcin、Osteopontin一抗反應(yīng)的二抗(帶羅丹明標(biāo)記)在綠色熒光集中的地方也集中發(fā)出紅色熒光。 結(jié)論:移植的骨髓細胞在股骨頭內(nèi)能成活,并能停留在骨缺損部位生長增殖。移植的BMSCs能分化成為多種細胞,包括成骨細胞、軟骨細胞及血管壁細胞等。BMSCs能分化成成骨細胞參與成骨,也能參與血管形成。移植的BMSCs能顯著促進骨壞死和骨缺損的修復(fù)。
[Abstract]:Objective: to investigate whether autologous bone marrow stromal cells can survive, remain in osteonecrosis, grow and proliferate, differentiate into osteoblasts and promote osteogenesis after transplantation of bone marrow stromal cells in the necrotic site of femoral head. Repair of femoral head defect and necrosis. Methods: the bone marrow of bilateral iliac bone was extracted in three parts. Bone marrow mesenchymal stem cells (BMSCs) were separated by adherent cells and cultured in vitro. The second generation cells were used in the experiment. BMSCs were labeled with 5-bromodeoxyuridine (BrdU) before transplantation. At the point of bilateral femoral head posterolateral cartilage near the junction of head and neck, the 10mm depth of diameter 8mm was drilled to the center of femoral head, and the bone defect model with diameter of 8mm and depth of 10mm was made. After the bone was inactivated by microwave, the bone was combined with labeled BMSCs and implanted into the defect of one side of the femoral head, and the other side was simply implanted into the microwave inactivated bone as a control. The bone marrow of bilateral iliac bone was extracted from the bone marrow of bilateral ilium 5 weeks after operation. BMSCs were separated by density gradient centrifugation. BMSCs were labeled with fluorescent dye carboxyl fluorescein diacetate succinimide (CFSE). BMSCs were adsorbed by gelatin sponge and implanted into the defect of one side of femoral head. The other side was simply implanted with gelatin sponge as control. Three weeks after operation, the expanded BMSCs were implanted into the defect of one side of the femoral head and the other side was used as the control. 12 weeks after the operation, the samples were taken from the cultured calf cancellous bone and the deantigen calf cancellous bone were implanted into the defect of one side of the femoral head. The labeling and in vivo localization of CFSE and Brdu were observed by laser confocal microscopy and immunohistochemistry respectively. X-ray, gross tissue observation, histochemical staining, image analysis, and detection of osteoblast specific differentiation marker core binding factor A1Cbfa1, osteocalcin, and osteocalcin were performed by immunohistochemistry and fluorescence immunohistochemistry. Osteopontin and type I collagen were used to understand the differentiation and osteogenic effect of BMSCs in vivo. Results: gross histological observation showed that the osteogenic degree of bone defect of BMSCs was significantly stronger than that of control. The results of microhistology further showed that the amount of bone formation, the degree of mineralization of bone matrix and the maturity of bone in the bone defect area of BMSCs were significantly higher than those in the control side, and contained more fusiform mesenchymal cells and osteoblasts. The expression of Cbfa1, osteocalcin, osteopontin and type I collagen in osteoblasts was significantly higher than that in the control group. A large number of BrdU positive cells were found in the defect of femoral head. The more active the new bone formation was, the more BrdU positive cells were, and many osteoblasts were positive for BrdU. BrdU positive cells were also distributed in many vascular walls of the new bone formation area. Under the fluorescence microscope, it was found that the cells with green fluorescence of CFSE in the BMSCs graft were located in the bone defect, and the second antibody (labeled with Rhodamine), which reacted with the first antibody against RunX2Osteocalcinn Osteopontin, also emitted red fluorescence in the place where the green fluorescence was concentrated. Conclusion: the transplanted bone marrow cells can survive in the femoral head and remain at the site of bone defect. BMSCs can differentiate into a variety of cells, including osteoblasts, chondrocytes and vascular parietal cells. BMSCs can differentiate into osteoblasts and participate in angiogenesis. Graft BMSCs can significantly promote bone necrosis and bone defect repair.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R329;R68

【引證文獻】

相關(guān)碩士學(xué)位論文 前1條

1 李富強;寧亞功教授治療股骨頭缺血性壞死學(xué)術(shù)經(jīng)驗總結(jié)及實驗研究[D];中國人民解放軍軍醫(yī)進修學(xué)院;2012年

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本文編號：1906913