本文關(guān)鍵詞： 骨髓 間充質(zhì)干細(xì)胞 原代培養(yǎng) 傳代培養(yǎng) 生長(zhǎng)曲線 骨髓間充質(zhì)干細(xì)胞 氣管粘膜上皮細(xì)胞 細(xì)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(xì)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(shí)驗(yàn)通過體外細(xì)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(xì)胞,研究其體外培養(yǎng)的增殖和生長(zhǎng)特性,探討為組織工程化氣管的構(gòu)建提供種子細(xì)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(zhǎng)的方法分離并提取單個(gè)核細(xì)胞進(jìn)行體外培養(yǎng)；收集P2-P4代細(xì)胞進(jìn)行流式細(xì)胞儀檢測(cè)細(xì)胞抗原表達(dá)；計(jì)數(shù)并繪制細(xì)胞原代培養(yǎng)和傳代培養(yǎng)的細(xì)胞生長(zhǎng)曲線。 結(jié)果通過密度梯度離心和貼壁生長(zhǎng)的方法可以獲取具有一定形態(tài)特點(diǎn)、貼壁生長(zhǎng)、增殖活躍的骨髓間充質(zhì)干細(xì)胞；該細(xì)胞可表達(dá)CD29,CD44,CD105,不表達(dá)CD54,CD45;原代培養(yǎng)曲線顯示,第2d-6d為生長(zhǎng)的潛伏期,第7-9d進(jìn)入對(duì)數(shù)生長(zhǎng)期,10d后進(jìn)入平臺(tái)期；傳代培養(yǎng)的潛伏期為24h-36h,4d-5d后進(jìn)入對(duì)數(shù)增長(zhǎng)期,9d左右進(jìn)入平臺(tái)期。在對(duì)數(shù)生長(zhǎng)期,細(xì)胞倍增時(shí)間約為48h,傳代培養(yǎng)的細(xì)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(xì)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(xì)胞的需要 3、BMMSCs高表達(dá)CD29、CD44、CD105,不表達(dá)CD34、CD45。 目的本實(shí)驗(yàn)通過體外誘導(dǎo)人骨髓間充質(zhì)干細(xì)胞向呼吸道上皮樣細(xì)胞的分化并進(jìn)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(xì)胞的可能性。 方法取P2-P4代生長(zhǎng)狀態(tài)良好的BMMSCs細(xì)胞,用含有家兔氣管粘膜上皮細(xì)胞內(nèi)容物的培養(yǎng)基進(jìn)行誘導(dǎo)培養(yǎng),對(duì)誘導(dǎo)、分化的細(xì)胞進(jìn)行形態(tài)學(xué)觀察。 結(jié)果BMMSCs細(xì)胞在含有家兔氣管粘膜上皮細(xì)胞內(nèi)容物的培養(yǎng)基誘導(dǎo)培養(yǎng)下,經(jīng)過3d時(shí)間,細(xì)胞形態(tài)由長(zhǎng)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(xì)胞經(jīng)體外誘導(dǎo)可以向氣管粘膜上皮樣細(xì)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(píng)價(jià)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(jī)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(duì)照組(Fresh組,n=4)。測(cè)量各組處理前后的顏色、彈性、管壁厚度的變化。測(cè)量并計(jì)算各組處理前后的組織含水量,交聯(lián)固定指數(shù)以評(píng)價(jià)交聯(lián)特點(diǎn)。通過觀察組織斷裂強(qiáng)度和斷裂延伸率評(píng)價(jià)其機(jī)械性能。采用膠原酶降解并測(cè)量降解后的斷裂強(qiáng)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(duì)BMMSC存活率的影響這兩種方法評(píng)價(jià)戊二醛和環(huán)氧化合物對(duì)交聯(lián)制備家兔氣管產(chǎn)生的細(xì)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(jī)械強(qiáng)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(shí)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(jì)學(xué)差異；根據(jù)與處理后氣管共培養(yǎng)的BMMSCs細(xì)胞生長(zhǎng)情況所得曲線顯示,PC組細(xì)胞可繼續(xù)穩(wěn)定增長(zhǎng),GA組細(xì)胞3d內(nèi)迅速死亡；原有濃度的PC和GA交聯(lián)制備劑對(duì)細(xì)胞均有很強(qiáng)的毒性,處理后不超過15%的細(xì)胞能夠存活,稀釋8倍以后,可以有超過70%的細(xì)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(jī)械性能和細(xì)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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