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

Culture of Pancreatic Duct-Derived Stem Cells of Rats in The Three-Dimension and Two-dimension Cell Culture

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。

Objective To identify pancreatic duct-derived stem cells （PDSCs） from the 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 V via 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 flow 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. Cells were adherent to the bottom of flask on 5 days after culture in two-dimision cell culture. Cell junctions were more obvious in three-dimension eel1 culture system. Compared with the two-dimision cell culture, the cells of G1 phase increased [ （56.46±1.17） % vs. （37.33 ± 1.21） %], and the cells of G2 phase decreased [ （14.62±0. 15） % vs. （32. 40± 1.21） %] in three-dimension cell culture system （P〈0. 01）. The early period apoptosis rate was lower in three-dimension cells culture system than that in two-dimision cell culture [ （1.48±0. 39） % vs. （5.24±1.33） %, P〈0.05]. The late period apoptosis rate was no statistically significant difference between the three-dimension and two-dimension cell culture （P〉0. 05）. ConclusionsPDSCs of rats could be obtained in the three-dimension cell culture system. There is cell junction ture mode, which shows a significant enhancement on intercellular interaction, and the biological between the three-dimension and two-dimension cell culture. in three-dimension cul- characters are different