人原代肺动脉内皮-平滑肌接触式共培养方法的技术改进

发布时间：2019-03-17 13:33

【摘要】：目的:改进人肺动脉内皮细胞(human pulmonary artery endothelial cells,HPAECs)-人肺动脉平滑肌细胞(human pulmonary artery smooth muscle cells,HPASMCs)接触式共培养方法 ,为更好模拟体内两种细胞间相互作用提供研究平台。方法:采用微孔聚碳酸酯膜为载体,将HPAECs和HPASMCs接种于微孔膜两侧,建立HPAECs-HPASMCs联合培养模型以模拟正常血管壁两种细胞的结构关系,通过调整细胞种植密度、培养时间、培养液体系、培养基种类、血清浓度等,倒置相差显微镜、Hoechst染色及流式细胞术比较不同条件下接触式共培养模型中细胞贴壁、生长和凋亡的差异,免疫荧光染色观察优化共培养条件后细胞标记物的表达变化。结果:(1)明胶预包被Transwell膜可促进内皮细胞的黏附和生长;(2)渗透压升高增加内皮细胞凋亡;(3)内皮细胞培养基较DMEM、RPMI1640更有利于接触式共培养模型的建立;(4)内皮细胞生长因子为1%、胎牛血清为2%时两种细胞生长稳定;(5)优化共培养条件对两种细胞的自身特性无显著影响。结论:当培养体系为内皮细胞培养基、维持1%内皮细胞生长因子及2%胎牛血清组分条件,可建立稳定的HPAECs-HPASMCs接触式共培养模型,为体外模拟人肺动脉血管壁结构功能和研究两种细胞间相互作用构建了良好平台。
[Abstract]:Aim: to improve the contact co-culture method of human pulmonary artery endothelial cells (human pulmonary artery endothelial cells,HPAECs) and human pulmonary artery smooth muscle cells (human pulmonary artery smooth muscle cells,HPASMCs) in order to provide a research platform for better simulating the interaction between two kinds of cells in vivo. Methods: microporous polycarbonate membrane was used as carrier, HPAECs and HPASMCs were inoculated on both sides of microporous membrane. HPAECs-HPASMCs co-culture model was established to simulate the structural relationship between the two kinds of cells in normal vascular wall. The differences of cell adhesion, growth and apoptosis in contact co-culture model under different conditions were compared by inverted phase contrast microscope, Hoechst staining and flow cytometry, including culture medium system, type of culture medium, serum concentration, inverted phase contrast microscope, flow cytometry and so on. Immunofluorescence staining was used to observe the expression of cell markers after optimizing the co-culture conditions. Results: (1) Transwell membrane precoated with gelatin could promote the adhesion and growth of endothelial cells, (2) the increase of osmotic pressure increased the apoptosis of endothelial cells, (3) the culture medium of endothelial cells was more favorable to the establishment of contact co-culture model than that of DMEM,RPMI1640. (4) when the endothelial growth factor was 1% and the fetal bovine serum was 2%, the growth of the two kinds of cells was stable, and (5) the optimal co-culture conditions had no significant effect on the self-characteristics of the two kinds of cells. Conclusion: stable HPAECs-HPASMCs contact co-culture model can be established by maintaining 1% endothelial growth factor and 2% fetal bovine serum components in the medium of endothelial cell culture. A good platform was constructed for simulating the structure and function of human pulmonary artery wall in vitro and studying the interaction between two kinds of cells.
【作者单位】： 南京医科大学第一附属医院呼吸与危重症医学科;
【基金】：国家自然科学基金(81273571) 江苏高校优势学科建设工程资助项目(JX10231802)
【分类号】：R563
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本文编号：2442350