文章摘要
孙理想,武亦娴,曾晓宁,孔 辉,王 虹,解卫平.人原代肺动脉内皮-平滑肌接触式共培养方法的技术改进[J].南京医科大学学报,2017,(6):674~680
人原代肺动脉内皮-平滑肌接触式共培养方法的技术改进
Improvement of a direct contact co-culture model of human pulmonary artery endothelial cells and smooth muscle cells
投稿时间:2016-08-09  
DOI:10.7655/NYDXBNS20170605
中文关键词: 肺动脉内皮细胞  肺动脉平滑肌细胞  接触式共培养  Transwell小室
英文关键词: HPAECs  HPASMCs  direct contact co-culture  Transwell
基金项目: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)
作者单位
孙理想 南京医科大学第一附属医院呼吸与危重症医学科江苏 南京 210029 
武亦娴 南京医科大学第一附属医院呼吸与危重症医学科江苏 南京 210029 
曾晓宁 南京医科大学第一附属医院呼吸与危重症医学科江苏 南京 210029 
孔 辉 南京医科大学第一附属医院呼吸与危重症医学科江苏 南京 210029 
王 虹 南京医科大学第一附属医院呼吸与危重症医学科江苏 南京 210029 
解卫平 南京医科大学第一附属医院呼吸与危重症医学科江苏 南京 210029 
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中文摘要:
      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。
英文摘要:
      Objective: The current study was aimed to mimic the physiology of human pulmonary vascular using an improved contact co-culture method containing human pulmonary artery endothelial cells(HPAECs) and human pulmonary artery smooth muscle cells (HPASMCs). Methods: HPAECs and HPASMCs were seeded on the opposite sides of a porous Transwell membrane that allowed formation of cell-cell contacts. The adhesion of HPAECs was determined by inverted phase contrast microscope and Hoechst staining. Flow cytometry was used to analyze the apoptosis of HPAECs and HPASMCs in different media and serum concentrations. The markers of PAECs and PASMCs were detected separately by immunofluorescence for identifying the characterization of the two kinds of cells. Results: The data from Hoechst staining showed that gelatin-precoated Transwell membrane facilitated the adhesion of HPAECs. Moreover, the apoptosis of HPAECs was increased under the hyperosmotic pressure. Compared with DMEM and RMPI 1640 medium, the endothelial cell medium(ECM) was more suitable for the contact co-culture system. Additionally, ECM with 1% endothelial cell growth supplement(ECGs) and 2% fetal bovine serum(FBS) promoted the growth of both HPAECs and HPASMCs. Moreover, co-culture adjustment showed no significant impact on characterization of PAECs and PASMCs in the aspect of cell morphologies and specific markers. Conclusion: ECM maintaining 1% ECGs and 2% FBS is better for setting HPAECS-HPASMCs contact co-culture system to mimic the physiology of human pulmonary vascular and to investigate the interactions between the two kinds of cells.
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