本文關(guān)鍵詞： 組織工程 原位轉(zhuǎn)染 基因活性材料 骨髓間充質(zhì)干細(xì)胞 骨軟骨修復(fù) 軟骨下骨　出處：《南京大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：由于炎癥、外傷、腫瘤等各種疾病導(dǎo)致的關(guān)節(jié)軟骨及其下方骨組織破壞甚至大范圍的組織缺損,經(jīng)常會(huì)導(dǎo)致關(guān)節(jié)功能的部分或者全部喪失；而且病損無論原發(fā)于關(guān)節(jié)軟骨或骨,到后期均表現(xiàn)為骨軟骨同時(shí)破壞。前期的組織工程更多的關(guān)注了軟骨組織的構(gòu)建,而對(duì)具有重要生理作用的軟骨下骨關(guān)注較少。 基因活化材料的原位傳輸方式,改變了以往基因輸送系統(tǒng)中基因成分尋找靶細(xì)胞的轉(zhuǎn)染方式,取而代之的是種子細(xì)胞或修復(fù)細(xì)胞在材料內(nèi)部“捕獲”或“遭遇”到DNA成分,因此這種轉(zhuǎn)染方式適用于局部區(qū)域修復(fù)。 本研究利用骨髓干細(xì)胞的多向分化性能,原位轉(zhuǎn)染誘導(dǎo)其在同一支架的不同區(qū)域分別向軟骨細(xì)胞和成骨細(xì)胞分化,構(gòu)建骨軟骨復(fù)合體。在前期工作的基礎(chǔ)上,我們選用殼聚糖/明膠制作模擬軟骨組織的多孔支架,TGF-β1質(zhì)�；罨笳T導(dǎo)骨髓干細(xì)胞(MSC)向軟骨細(xì)胞分化；殼聚糖/明膠/羥基磷灰石支架模擬成骨組織,BMP-2質(zhì)�；罨笳T導(dǎo)MSC向成骨細(xì)胞分化；分開培養(yǎng)一周后利用纖維蛋白膠黏合構(gòu)成骨軟骨復(fù)合體。通過電鏡觀察,ELISA檢測(cè)因子表達(dá),Q-PCR, HE染色,免疫組化染色觀察MSC在支架上的生長(zhǎng)和分化；體內(nèi)構(gòu)建兔膝關(guān)節(jié)骨軟骨全層缺損,植入復(fù)合體觀察修復(fù)效果。 掃描電鏡顯示,我們構(gòu)建的支架具有很好的成孔性,并且兩層之間具有很好的黏合；基因活化材料能夠很好的支持MSC的貼附和增殖；ELISA測(cè)定TGF-β1和BMP-2因子的表達(dá)量顯示該系統(tǒng)是一個(gè)很好的原位轉(zhuǎn)染系統(tǒng),基因活化組兩種因子都有較高的表達(dá)；相比未基因活化的支架,Q-PCR結(jié)果顯示在軟骨層生長(zhǎng)的MSC表達(dá)的蛋白聚糖和Ⅱ型膠原mRNA量有所上調(diào),成骨層生長(zhǎng)的MSC表達(dá)的骨橋蛋白,骨連接蛋白和Ⅰ型膠原nRNA量有所上調(diào)；免疫組化顯示軟骨層高表達(dá)Ⅱ型膠原而成骨層高表達(dá)Ⅰ型膠原,說明在骨軟骨復(fù)合體上同時(shí)誘導(dǎo)了MSC向軟骨和成骨細(xì)胞分化；利用此復(fù)合體進(jìn)行的兔膝關(guān)節(jié)全層缺損修復(fù),HE,免疫組化都顯示出比單層支架更好的修復(fù)效果,促進(jìn)了軟骨和軟骨下骨的再生以及與原生組織的整合。 本研究說明,雙基因活化的雙層支架能夠在不同層次上同時(shí)誘導(dǎo)MSC分別向軟骨細(xì)胞和成骨細(xì)胞分化；此雙層復(fù)合體能夠促進(jìn)骨軟骨缺損的修復(fù)。
[Abstract]:Because of inflammation, trauma, tumor and other diseases caused by articular cartilage and underlying bone destruction even large tissue defects, often leads to joint function of all or part of the loss; and whether the primary lesion of articular cartilage or bone, to the late show at the same time. The early destruction of bone and cartilage tissue engineering more attention the cartilage tissue, and plays an important physiological role of the subchondral bone less attention.
In situ gene activation transmission materials, changed the genetic components for gene delivery system targeting cells, instead of the seed cells or repair cells inside the material "capture" or "experience" to the composition of DNA, so this was suitable for local repair.
This research using stem cell differentiation properties of bone marrow, in situ transfection induced in the different areas of the same scaffold to chondrogenic and osteogenic differentiation, construction of bone cartilage complex. On the basis of previous work, we use chitosan / gelatin production simulation of cartilage tissue scaffolds, bone marrow TGF- beta 1 after the activation of stem cells (MSC) differentiation into chondrocytes; chitosan / gelatin / hydroxyapatite scaffold simulating bone tissue, BMP-2 was activated after MSC was induced to differentiate into osteoblasts; separate culture after a week of using fibrin adhesive composition of bone cartilage complex. By electron microscopy, the expression of ELISA, detection of factor Q-PCR, HE staining, growth and differentiation of MSC was observed by immunohistochemistry on the stent; full-thickness osteochondral defects in the rabbit knee joint in vivo implantation of complex construction, to observe the repair effect.
Scanning electron microscopy showed that we constructed with hole bracket as well, and has good adhesion between the two layers; gene activation and proliferation on materials can be well supported by MSC; expression of ELISA was determined by TGF- beta 1 and BMP-2 factor shows that the system is a good primary transfection system expression, gene activation group of two kinds of factors are higher; compared with the stent was not gene activation, Q-PCR results showed that the proteoglycan and collagen type mRNA expression in cartilage growth layer MSC increased expression of osteogenic layer growth MSC osteopontin, osteonectin and collagen type nRNA content increased; immunohistochemistry showed that the expression of type II collagen and cartilage layer of bone high expression of collagen I, in osteochondral and MSC was induced to chondrocytes and osteoblasts of rabbit knee joint; the complex whole layer Defect repair, HE and immunohistochemistry showed better repair effect than monolayer scaffold, promoting the regeneration of cartilage and subchondral bone and integrating with primary tissue.
This study shows that double gene activated bilayer scaffolds can induce MSC to differentiate into chondrocytes and osteoblasts at different levels. The bilayer complex can promote the repair of osteochondral defects.

【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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本文編號(hào)：1522802