摘要
目的通过改变细胞培养基中血清的质量和浓度,探索构建小鼠肠干细胞群更合适的血清及浓度。方法取孕17 d昆明系小白鼠的胚胎鼠肠组织,予肠干细胞的培养,将获得的细胞溶于8组细胞培养液,种于24孔板中。细胞培养液共有8组,采用DMEM培养基和新生牛血清或胎牛血清配制,血清的浓度分别为5%、10%、15%及20%,除此以外还含有胰岛素、谷铵酰胺、表皮生长因子以及双抗(青霉素、链霉素)等成分。最后进行角蛋白抗体及波形蛋白抗体免疫组化染色鉴定细胞来源。结果 24~48 h后,细胞开始贴壁生长。显微镜下可见,除采用10%新生牛血清的组外,其他组细胞均含有大量的间质细胞,上皮细胞所占比例不足10%。而如采用含10%的新生牛血清培养基培养,则贴壁生长的细胞绝大部分为上皮细胞。经传代后,所培养出的肠干细胞群角蛋白18亚型染色阳性。而其他组的细胞,则基本上为间质细胞。结论构建肠干细胞群应选用10%的新生牛血清。
Objective To culture the mouse intestinal stem cells and search for the best serium and its concentration. Methods The intestines were got out from the embryo of the 17-day pregnant KunMing mouse. Then the tissue was cut out after the mesentery was split. The constitution was digested with alidase and XI collagenase. The digestion was temainate by centrifugation 20 minutes later. After that the cells were sedimentate several times to remove the fibroblasts and the smooth muscle cells. After centrifugation, the cells were admixed with the culture medium which contains DMEM, serum, insuline, Glutamyl transpeptidase and epidermal growth factor. The serum in the culture medium was fetal bovine serum or newborn bovin serum and its concentration changed with 5%, 10%, 15%, 20%. Implant the cells in the 24-pore plate. The culture medium was exchanged after 48 hours then it was exchanged every 72 hours. The cells were indentified by the immunohistochemistry methods with anti-cytokeratin and anti-vimentin. Results After 48 hours, cells began to adhere. Cells cultured with 10% newborn bovin serum appeared 30% of the clones found to be lined with a homogeneous population of large, undifferentiated epithelial-like cells, and 20% of the clones were lined with a layer of flattened epithelial cells. The remaining clones （50%）, appeared to be lined with a more heterogeneous epithelium containing both large and flattened. Cells ofthe No. 3 and No. 4 generation were the most active. Atier this stage, the number of stem cells descents, and the cells apoptosised in the end. Several generations later, the cells were indentified by anti- cytokeratin- 18 peptide, and the result was masccline. But other cells was indentified to be mesenchymal cells.Conclusions The better serum for culturing the mouse intestinal stem cells is new born bovine serum and the concentration is 10%.
作者
刘冠琳
程跃
谢国海
严泽军
袁鹤胜
LIU Guanlin;CHENG Yue;XIE Guohai;YAN Zejun;YUAN Hesheng(The First Hospital of Ningbo,Ningbo 315010,Zhejiang,China)
出处
《现代实用医学》
2018年第10期1266-1268,1419,共4页
Modern Practical Medicine
基金
国家自然科学基金资助项目(30371423)
关键词
肠干细胞
表皮生长因子
新生牛血清
角蛋白
Intestinal stem cell
Epidermal growth factor
Newborn bovine serum
Cytoketarin
