三维及二维培养大鼠胰腺导管来源干细胞的体外对比实验研究.docVIP

  三维及二维培养大鼠胰腺导管来源 干细胞的体外对比实验研究# ** 5  摘要：目的  观察在不同培养模式下大鼠胰腺导管来源干细胞（PDSC）的生物学特性。方 法 以大鼠胰腺组织为材料，采用动力性三维培养及传统二维培养方法进行细胞培养，获得 原代 PDSC，并连续传代，纯化 PDSC，比较动力性三维培养及传统二维培养细胞间连接及 细胞凋亡周期的差异。结果 动力性三维培养的大鼠 PDSC 在培养第 2 天即可沿三维支架 10 贴壁生长，在第 7 天即可形成细胞团，并沿三维支架多向贴壁生长； 二维培养的 PDSC 在 培养第 5 天贴壁生长。三维培养与二维培养相比，二维培养的细胞间连接少见 , 三维培 养的细胞连接结构丰富。三维培养与二维培养相比，三维培养的 G1 期细胞增多，G2 期细 胞相对减少（P ＜0.01 ），三维培养早期自发凋亡细胞比例相对减少（P ＜0.05 ），晚期 自发凋亡与坏死细胞比例两种培养方式间的差异无统计学意义（P ＞0.05 ）。结论动力性 15 20 三维细胞培养较传统二维细胞培养，在细胞贴壁时间、细胞连接以及细胞凋亡方面具有明显 的优势。 关键词：三维细胞培养；二维细胞培养；胰腺导管来源干细胞 Culture of Pancreatic Duct-Derived Stem Cells of Rats in The Three-Dimension and Two-dimension Cell Culture Chen Xuchun, Yang Lei, Cheng Ying, Li Hong, Liu Yongfeng (Department of Organ Transplantation Surgery and General Surgery, The First Af fi liated Hospital of China Medical University, ShenYang 110001) Abstract: Objective To identify pancreatic duct-derived stem cells （PDSCs ） from the 25 30 35 pancreatic duct of rats, and culture in three-dimension cell culture system or two-dimension cell culture, with characterization on their morphol-ogy and phenotype. Methods Adult male Wistar rats underwent perfusion with collagenase Ⅴvia the pancreatic duct, the pancreas was surgically procured, digested, followed by discontinued density gradient centrifuge to isolate islets from acinar and ductal tissue. The acinar and ductal tissues were cultivated in serum-containing medium in the three-dimen-sion or two-dimension cell culture seperatedly to obtain adherent cells, as PDSCs, which were expanded by consecutive passages. The cell junction, cell cycle, and cell apoptosis rate between the two-dimension and three-dimision cell culture were compared by means of microscope and fl ow cytometry. Results PDSCs of rats showed direct adherence culture in three-dimension and two-dimision cell culture systerm. PDSCs grew in many layers in three-dimension cell culture system on 2 days after culture, and possessed capacity of high proliferation on 7 days.