發(fā)布時間：2018-06-25 08:52

  本文選題：海藻糖 + 水凝膠��； 參考：《西北大學》2012年碩士論文

【摘要】：組織工程皮膚具有良好的皮膚缺損修復效果,然而產(chǎn)品的生產(chǎn)周期較長,更重要的是由于含有活細胞,其有效保存時間較短,嚴重限制了其臨床應(yīng)用和產(chǎn)業(yè)化。因此迫切需要尋找新的有效保存方法。 深低溫冷凍保存已成功應(yīng)用于活細胞的長期保存,細胞在使用時快速復溫,可以有效維持其存活狀態(tài)。然而對于組織工程皮膚,深低溫冷凍保存并未取得突破性進展,困難在于復溫過程中的重結(jié)晶對細胞產(chǎn)生致命的冰晶損傷,因為生物組織具有較低的熱傳導率和較高的比熱,其內(nèi)部的熱傳導性不均一,復溫速度很難達到避免冰晶損傷所需的速度。海藻糖是一種非還原性二糖,越來越多的研究發(fā)現(xiàn)海藻糖對干燥狀態(tài)下的生物體具有保護作用。 我們設(shè)想先將種子細胞負載海藻糖和DMSO再構(gòu)建組織工程皮膚,海藻糖可以保護細胞在干燥條件下存活,DMSO保護細胞在程序降溫和冷凍過程中存活,組織工程皮膚低溫凍結(jié)后,在真空條件下使其水分升華而成為干燥狀態(tài),為組織工程皮膚的長期保存提供了一種新的方法,這種方法既可以使TES能夠穩(wěn)定儲存,也在復水恢復活性的過程中避免冰晶損傷,目前未見文獻報道。 在本研究中,我們首先優(yōu)化了海藻糖負載種子細胞成纖維細胞的條件并評價了負載海藻糖的成纖維細胞干燥保存的效果。進一步,我們用負載海藻糖的成纖維細胞與鼠尾膠原凝膠構(gòu)建了組織工程皮膚,將其冷凍干燥后,進行了一系列顯微、超微結(jié)構(gòu)觀察,熒光染色,活力測定以及皮膚缺損修復實驗,評價冷凍干燥保存方法對組織工程皮膚的保存效果,初步建立組織工程皮膚保存的新方法。 1.我們從保存液中海藻糖的濃度,孵育時間和孵育溫度三個方面優(yōu)化了成纖維細胞負載海藻糖的條件,獲得最佳導入條件為海藻糖200mM,在37℃條件下孵育8小時。在此條件下負載海藻糖的細胞,經(jīng)過冷凍干燥過程后仍可保存30%的活性,經(jīng)過人工風干過程可保留40%的活性,熒光染色及電鏡觀察顯明細胞膜基本保存完整。 2.我們用負載海藻糖的細胞成功構(gòu)建了組織工程皮膚。經(jīng)過冷凍干燥保存后,組織工程皮膚仍保留有26%的活性；SEM顯示組織工程皮膚的結(jié)構(gòu)基本保持完整；在C57BL/6J小鼠背部全層皮膚缺損修復中,負載海藻糖的組織工程皮膚傷口愈合速率較沒有負載海藻糖的組別提前約30%,而與正常組織工程皮膚無明顯差別。利用海藻糖的干燥保護作用而引入的冷凍干燥保存,能有效保存組織工程皮膚的活性并利于其缺損修復效果的發(fā)揮,為組織工程皮膚的保存提供了新的方法。
[Abstract]:Tissue engineered skin has good skin defect repair effect, but the production cycle of the product is longer, more importantly, because of the existence of living cells, its effective preservation time is shorter, which seriously limits its clinical application and industrialization. Therefore, there is an urgent need to find new effective conservation methods. Cryopreservation has been successfully applied to the long-term preservation of living cells. However, for tissue engineered skin, cryopreservation has not made a breakthrough. The difficulty is that recrystallization in the process of rewarming causes fatal ice crystal damage to cells, because biological tissues have lower thermal conductivity and higher specific heat. The internal heat conductivity is uneven and the rewarming velocity is difficult to avoid ice crystal damage. Trehalose is a kind of non-reducing disaccharide. More and more researches have found that trehalose has protective effect on dry organisms. We imagine that seed cells are first loaded with trehalose and DMSO to construct tissue engineered skin. Trehalose can protect cells from surviving under dry conditions. DMSO protects cells from surviving during programmed cooling and freezing. Tissue engineered skin is frozen at low temperature. In vacuum condition, the moisture is sublimated to a dry state, which provides a new method for the long-term preservation of tissue engineering skin. This method can not only make tes stored stably, but also avoid ice crystal damage in the process of rehydration. At present, there is no literature report. In this study, we first optimized the conditions of trehalose loaded seed cell fibroblasts and evaluated the effect of trehalose loaded fibroblast dry preservation. Furthermore, we constructed tissue engineered skin from fibroblasts loaded with trehalose and collagen gel from rat tail. After freeze-drying, we made a series of microscopic, ultrastructural observation and fluorescent staining. To evaluate the effect of freeze-drying preservation on tissue engineered skin and to establish a new method for tissue engineering skin preservation. We optimized the conditions of the fibroblasts loading trehalose from three aspects: the concentration of trehalose, the incubation time and the incubation temperature. The optimum conditions were obtained as follows: trehalose 200 mm, incubated at 37 鈩，

本文編號：2065367