脂肪源性干细胞在温度敏感性支架材料上培养特性的实验研究
发布时间：2018-12-15 08:37
【摘要】：脂肪源性干细胞在温度敏感性支架材料上培养特性的实验研究 [目的]在温度敏感性支架材料(Temperature-responsive scaffolds, TRSs)上培养兔脂肪源性干细胞(Adipose-derived stem cells, ADSCs)、兔口腔黏膜上皮细胞(Oral mucosal epithelial cells, OMECs),通过比较两种细胞体外的生长情况和生长特性,探求脂肪源干细胞作为重建眼表的种子细胞的可能性。 [方法]异丙基丙烯酰胺溶于异丙醇加入35mm直径的培养皿,利用电子束照射制备温度敏感性培养皿。取兔脂肪组织及口腔黏膜上皮组织,分别消化、离心获取原代细胞,ADSCs体外培养至二代后接种于TRSs,原代OMECs直接接种于有丝裂霉素C处理过的有鼠3T3生长的TRSs中,比较两种细胞的形态、生长速度、细胞层片脱附时间、层片细胞计数、层片HE染色、细胞免疫组化染色(角蛋白CK12及干细胞标志物p63、ABCG2)及其扫描电镜检查。 [结果]二代ADSCs细胞呈长梭形编织状,局部呈漩涡状排列,细胞形态单一,活性强。原代OMECs呈不规则圆形、折光性强,完全伸展的细胞呈扁平的卵圆形,胞核清晰。二代ADSCs覆盖率达100%所需的生长周期为12-14天,层片脱附时间为(46±9.6)min,细胞总计数为(7.9±1.1)×105个/片；而原代OMECs所需生长周期为14-16天,层片脱附时间为(91.9±10.9)min,计数为(45.8±26.5)×105个/片,两者脱附时间及细胞总计数具有统计学差别。HE染色示ADSCs细胞核呈1-3层排列,而OMECs细胞核呈4-5层覆层结构。两者角蛋白CK12及干细胞标志物P63、ABCG2均呈阳性。扫描电镜可见两者细胞均具有上皮典型微绒毛、细胞间连接紧密。 [结论]通过在TRSs上培养获取的ADSCs层片可作为眼表重建的新种子来源。
[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 卤9.6) min, cells, the total number of min, cells was (7.9 卤1.1) 脳 105 / tablet. The growth cycle of primary OMECs was 14-16 days, and the desorption time of lamellar was (91.9 卤10.9) min, count was (45.8 卤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.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.0
本文编号：2380349
[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 卤9.6) min, cells, the total number of min, cells was (7.9 卤1.1) 脳 105 / tablet. The growth cycle of primary OMECs was 14-16 days, and the desorption time of lamellar was (91.9 卤10.9) min, count was (45.8 卤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.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.0
【共引文献】
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