脐带血间充质干细胞的分离培养和鉴定.docVIP

脐带血间充质干细胞的分离培养和鉴定 目录 TOC \o 1-9 \h \z \u 目录 1 正文 1 文1：脐带血间充质干细胞的分离培养和鉴定 1 1 材料与方法 2 2 结 果 4 3 讨 论 5 文2：简述梨黑星病菌的分离培养和基质研究 6 1 材料与方法 6 2 结果与分析 8 3 讨论 8 参考文摘引言： 9 原创性声明（模板） 10 文章致谢（模板） 11 正文 脐带血间充质干细胞的分离培养和鉴定 文1：脐带血间充质干细胞的分离培养和鉴定 Abstract: Objective Isolation and cultivation of mesenchymal stem cells (MSCs) in human umbilical cord in vitro， and determine their biological properties. Methods The mononuclear cells were isolated by deity gradient centrifugation from human umbilical cord blood in sterile condition， and cultured in DMEM medium containing 10% fetal bovine serum. After the adherent mononuclear cells were obtained， the shape of cells were observed by microscope， then the cell growth curve， the cell cycle and the cell surface antige were obtained by immunocytochemistry and flow cytometry methods. Results MSCs obtained by Percoll ( g/mL) were similar in size， spindle-shaped or star-shaped fibroblasts-liked cells. Cell growth curve analysis indicated that MSCs were in the exponential stage after 5d and in the stationary stages after 9d. Flow cytometry analysis showed that the CD29 and CD44 positive cells were about 50%～70%. Conclusio The human umbilical cord derived mesenchymal stem cells were grown stably in vitro and can be used as the seed-cells in tissue engineering。 Key words:human umbilical cord blood; mesenchymal stem cells; cell cycle; immunocytochemistry 间充质干细胞(mesenchymal stem cells，MSCs)在一定条件下具有多向分化的潜能，是组织工程研究中重要的种子细胞来源。寻找来源丰富并不受伦理学制约的间充质干细胞成为近年来的研究热点[1]。脐血(umbilical cord blood， UCB)在胚胎娩出后，与胎盘一起存在的医疗废物。与骨髓相比，UCB来源更丰富，取材方便，具有肿瘤和微生物污染机会少等优点。有人认为脐血中也存在间充质干细胞(Umbilical cord blood-derived mesenchymal stem cells，UCB-MSCs)。如果从脐血中培养出MSCs，与胚胎干细胞相比，应用和研究则不受伦理的制约，蕴藏着巨大的临床应用价值[2，3]。本研究将探讨人UCB-MSCs体外培养的方法、细胞的生长曲线、增殖周期和细胞表面标志等方面，分析UCB-MSCs 作为间充质干细胞来源的可行性。 1 材料与方法 UCB-MSCs的分离与培养 9 份脐血标本均来源于辽宁医学院附属第一医院，均获得产妇同意。以等量的PBS缓冲液混匀，缓慢滴入到等量的淋巴细胞分离液(Fercoll， g/mL，美国Sigma公司)，650 g离心15 min。抽取白膜层细胞，以等量的PBS缓冲液洗涤离心，基础培养液(DMEM/F12，10%胎牛血清、100 U/mL