本文關(guān)鍵詞： 骨髓 間充質(zhì)干細胞 原代培養(yǎng) 傳代培養(yǎng) 生長曲線 骨髓間充質(zhì)干細胞 氣管粘膜上皮細胞 細胞裂解 射線照射 共培養(yǎng) 家兔氣管 環(huán)氧化合物 戊二醛 理化性能 體外 細胞毒性　出處：《北京協(xié)和醫(yī)學(xué)院》2010年博士論文　論文類型：學(xué)位論文

【摘要】：目的本實驗通過體外細胞培養(yǎng)的方法,從骨髓中分離、純化、鑒定骨髓間充質(zhì)干細胞,研究其體外培養(yǎng)的增殖和生長特性,探討為組織工程化氣管的構(gòu)建提供種子細胞的可能性。 方法收集成人骨髓血,采用密度梯度離心和貼壁生長的方法分離并提取單個核細胞進行體外培養(yǎng)；收集P2-P4代細胞進行流式細胞儀檢測細胞抗原表達；計數(shù)并繪制細胞原代培養(yǎng)和傳代培養(yǎng)的細胞生長曲線。 結(jié)果通過密度梯度離心和貼壁生長的方法可以獲取具有一定形態(tài)特點、貼壁生長、增殖活躍的骨髓間充質(zhì)干細胞；該細胞可表達CD29,CD44,CD105,不表達CD54,CD45;原代培養(yǎng)曲線顯示,第2d-6d為生長的潛伏期,第7-9d進入對數(shù)生長期,10d后進入平臺期；傳代培養(yǎng)的潛伏期為24h-36h,4d-5d后進入對數(shù)增長期,9d左右進入平臺期。在對數(shù)生長期,細胞倍增時間約為48h,傳代培養(yǎng)的細胞在P9-P1o出現(xiàn)衰老現(xiàn)象。 結(jié)論1、利用密度梯度離心和貼壁的方法可以有效地分離、純化骨髓間充質(zhì)干細胞； 2、在適當(dāng)?shù)臈l件下,BMMSCs在體外表現(xiàn)出旺盛的增殖能力,可滿足組織工程學(xué)種子細胞的需要 3、BMMSCs高表達CD29、CD44、CD105,不表達CD34、CD45。 目的本實驗通過體外誘導(dǎo)人骨髓間充質(zhì)干細胞向呼吸道上皮樣細胞的分化并進行形態(tài)學(xué)的初步鑒定,探討為組織工程氣管提供呼吸道上皮種子細胞的可能性。 方法取P2-P4代生長狀態(tài)良好的BMMSCs細胞,用含有家兔氣管粘膜上皮細胞內(nèi)容物的培養(yǎng)基進行誘導(dǎo)培養(yǎng),對誘導(dǎo)、分化的細胞進行形態(tài)學(xué)觀察。 結(jié)果BMMSCs細胞在含有家兔氣管粘膜上皮細胞內(nèi)容物的培養(yǎng)基誘導(dǎo)培養(yǎng)下,經(jīng)過3d時間,細胞形態(tài)由長梭形變成多角形,HE染色可見吞飲小泡。 結(jié)論骨髓間充質(zhì)干細胞經(jīng)體外誘導(dǎo)可以向氣管粘膜上皮樣細胞分化。 目的采用戊二醛和環(huán)氧化合物交聯(lián)制備家兔氣管,評價家兔氣管的生物學(xué)性能,探討環(huán)氧化合物作為新型制備技術(shù)的可行性。 方法新鮮家兔氣管12根,隨機分為戊二醛組處理組(GA組,n=4)、環(huán)氧化合物處理組(PC組,n=4)及新鮮對照組(Fresh組,n=4)。測量各組處理前后的顏色、彈性、管壁厚度的變化。測量并計算各組處理前后的組織含水量,交聯(lián)固定指數(shù)以評價交聯(lián)特點。通過觀察組織斷裂強度和斷裂延伸率評價其機械性能。采用膠原酶降解并測量降解后的斷裂強度以分析組織穩(wěn)定性。通過氣管與BMMSCs共培養(yǎng)、不同濃度交聯(lián)制備劑對BMMSC存活率的影響這兩種方法評價戊二醛和環(huán)氧化合物對交聯(lián)制備家兔氣管產(chǎn)生的細胞毒性。 結(jié)果PC組外觀顏色、管壁彈性、管腔變化更接近天然氣管；PC組和GA組的機械強度相當(dāng),均明顯高于Fresh組,PC組斷裂延伸率大于GA組,且兩組均低于Fresh組；PC組和GA組固定100小時的固定指數(shù)相當(dāng)；體外Ⅱ型膠原酶的降解結(jié)果顯示,PC組和GA組的組織穩(wěn)定性均高于Fresh組,GA組和PC組之間沒有明顯統(tǒng)計學(xué)差異；根據(jù)與處理后氣管共培養(yǎng)的BMMSCs細胞生長情況所得曲線顯示,PC組細胞可繼續(xù)穩(wěn)定增長,GA組細胞3d內(nèi)迅速死亡；原有濃度的PC和GA交聯(lián)制備劑對細胞均有很強的毒性,處理后不超過15%的細胞能夠存活,稀釋8倍以后,可以有超過70%的細胞存活。 結(jié)論1、戊二醛和環(huán)氧化合物均可有效地交聯(lián)制備家兔氣管。 2、環(huán)氧化合物交聯(lián)技術(shù)和戊二醛相比,能夠改善家兔氣管的部分性能(機械性能和細胞毒性效應(yīng))。
[Abstract]:Objective To study the proliferation and growth characteristics of bone marrow mesenchymal stem cells ( MSCs ) from bone marrow by means of in vitro cell culture , and to explore the possibility of providing seed cells for the construction of engineered trachea . Methods Adult bone marrow blood was collected by density gradient centrifugation and adherent growth . Single nuclear cells were isolated and cultured in vitro . The expression of cell antigens was detected by flow cytometry in P2 - P4 cells . Cell growth curves of primary culture and subculture were counted and plotted . Results Bone marrow mesenchymal stem cells with certain morphological characteristics , adherent growth and proliferation were obtained by density gradient centrifugation and adherent growth . The cells expressed CD29 , CD44 , CD105 , did not express CD54 , CD54 ; primary culture curves showed that days 2d - 6d were the latent periods of growth . The incubation period of subculture was 24 h - 36h , 4 days - 5 days . Conclusion 1 . Bone marrow mesenchymal stem cells can be purified and purified by using density gradient centrifugation and adherent method . 2 . Under appropriate conditions , BMMSCs exhibit a strong proliferative capacity in vitro , which can meet the needs of tissue engineering seed cells . 3 . The expression of CD29 , CD44 and CD105 in BMMSCs did not express CD34 , CD45 . Objective To explore the possibility of providing airway epithelial seed cells to tracheal epithelial cells in vitro by inducing differentiation of human bone marrow mesenchymal stem cells into respiratory epithelium - like cells and morphology . Methods BMMSCs with good growth status of P2 - P4 were induced and cultured with medium containing the contents of tracheal mucosa epithelial cells in rabbits . Results The cells of BMMSCs were cultured under the culture medium containing the contents of tracheal mucosa epithelial cells in rabbits . After 3d time , the cell morphology changed from long shuttle shape to polygonal shape , HE staining showed that the cells were swallowed . Conclusion In vitro induction of bone marrow mesenchymal stem cells can differentiate into tracheal mucosa epithelioid cells . Objective To evaluate the biological properties of rabbit tracheal and evaluate the feasibility of using epoxy compound as a new preparation technique by cross - linking glutaraldehyde and epoxy compound . Methods 12 rabbits were randomly divided into glutaraldehyde group treated group ( GA group , n = 4 ) , epoxy compound treatment group ( PC group , n = 4 ) and fresh control group ( Fresh group , n = 4 ) . The changes of color , elasticity and wall thickness before and after treatment were measured and calculated . The water content and cross - linking fixation index before and after treatment were measured and calculated to evaluate the cross - linking characteristics . By observing the fracture strength and elongation at break , the mechanical properties were evaluated . The effects of glutaraldehyde and epoxy compound on the survival of BMMSC were evaluated by the co - culture of tracheal and BMMSCs , and the cytotoxicity of glutaraldehyde and epoxy compounds on the tracheal production of rabbits was evaluated . Results The mechanical strength of PC group and GA group was significantly higher than that in fresh group , PC group and GA group were significantly higher than that in fresh group , and that in PC group and GA group were higher than that in fresh group . Conclusion 1 銆，

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