【摘要】：脂肪源性干細(xì)胞在溫度敏感性支架材料上培養(yǎng)特性的實(shí)驗(yàn)研究 [目的]在溫度敏感性支架材料(Temperature-responsive scaffolds, TRSs)上培養(yǎng)兔脂肪源性干細(xì)胞(Adipose-derived stem cells, ADSCs)、兔口腔黏膜上皮細(xì)胞(Oral mucosal epithelial cells, OMECs),通過比較兩種細(xì)胞體外的生長情況和生長特性,探求脂肪源干細(xì)胞作為重建眼表的種子細(xì)胞的可能性。 [方法]異丙基丙烯酰胺溶于異丙醇加入35mm直徑的培養(yǎng)皿,利用電子束照射制備溫度敏感性培養(yǎng)皿。取兔脂肪組織及口腔黏膜上皮組織,分別消化、離心獲取原代細(xì)胞,ADSCs體外培養(yǎng)至二代后接種于TRSs,原代OMECs直接接種于有絲裂霉素C處理過的有鼠3T3生長的TRSs中,比較兩種細(xì)胞的形態(tài)、生長速度、細(xì)胞層片脫附時間、層片細(xì)胞計(jì)數(shù)、層片HE染色、細(xì)胞免疫組化染色(角蛋白CK12及干細(xì)胞標(biāo)志物p63、ABCG2)及其掃描電鏡檢查。 [結(jié)果]二代ADSCs細(xì)胞呈長梭形編織狀,局部呈漩渦狀排列,細(xì)胞形態(tài)單一,活性強(qiáng)。原代OMECs呈不規(guī)則圓形、折光性強(qiáng),完全伸展的細(xì)胞呈扁平的卵圓形,胞核清晰。二代ADSCs覆蓋率達(dá)100%所需的生長周期為12-14天,層片脫附時間為(46±9.6)min,細(xì)胞總計(jì)數(shù)為(7.9±1.1)×105個/片；而原代OMECs所需生長周期為14-16天,層片脫附時間為(91.9±10.9)min,計(jì)數(shù)為(45.8±26.5)×105個/片,兩者脫附時間及細(xì)胞總計(jì)數(shù)具有統(tǒng)計(jì)學(xué)差別。HE染色示ADSCs細(xì)胞核呈1-3層排列,而OMECs細(xì)胞核呈4-5層覆層結(jié)構(gòu)。兩者角蛋白CK12及干細(xì)胞標(biāo)志物P63、ABCG2均呈陽性。掃描電鏡可見兩者細(xì)胞均具有上皮典型微絨毛、細(xì)胞間連接緊密。 [結(jié)論]通過在TRSs上培養(yǎng)獲取的ADSCs層片可作為眼表重建的新種子來源。
[Abstract]:Experimental study on the characteristics of Adipose-derived Stem cells cultured on Temperature-Sensitive scaffolds [objective] Rabbit adipose stem cells (Adipose-derived stem cells, ADSCs),) were cultured on the thermosensitive scaffold (Temperature-responsive scaffolds, TRSs). By comparing the growth and growth characteristics of two kinds of rabbit oral mucosal epithelial cells (Oral mucosal epithelial cells, OMECs),) in vitro, the possibility of using adipose derived stem cells as seed cells for ocular surface reconstruction was explored. [methods] Isopropyl acrylamide was dissolved in isopropanol and added into 35mm diameter petri dish. Temperature sensitive petri dish was prepared by electron beam irradiation. Rabbit adipose tissue and oral mucosal epithelium were digested, primary cells were obtained by centrifugation. ADSCs was cultured in vitro and then inoculated with TRSs, primary OMECs directly into TRSs grown by rat 3T3 treated with mitomycin C. The morphology, growth rate, desorption time, cell count, lamellar HE staining, cell immunohistochemical staining (keratin CK12 and stem cell marker p63 ABCG2) and scanning electron microscopy (SEM) were compared. [results] the second generation of ADSCs cells showed long fusiform braided shape, local whirlpool arrangement, single morphology and strong activity. The primary OMECs was irregular and round with strong refraction. The fully stretched cells were flat oval and the nucleus was clear. The growth cycle of the second generation with 100% ADSCs coverage was 12-14 days, the desorption time of the lamellar was (46 鹵9.6) min, cells, the total number of min, cells was (7.9 鹵1.1) 脳 105 / tablet. The growth cycle of primary OMECs was 14-16 days, and the desorption time of lamellar was (91.9 鹵10.9) min, count was (45.8 鹵26.5) 脳 105 / tablet. The desorption time and the total number of cells were significantly different between the two groups. HE staining showed that the nucleus of ADSCs was arranged in 1-3 layers, while the nucleus of OMECs had a structure of 4-5 layers. Both keratin CK12 and stem cell marker P63 + ABCG2 were positive. Scanning electron microscopy showed that both cells had typical microvilli of epithelium and close intercellular junctions. [conclusion] the ADSCs layer obtained from TRSs can be used as a new seed source for ocular surface reconstruction.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R318.0

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本文編號：2380349