大鼠远端肺动脉平滑肌细胞原代培养及其生物学特性和功能的研究

摘要

Objective Toinvestigatethesimilaritiesanddifferencesofbiologicalcharacteristicsandfunctionsbetween chronic obstructive pulmonary disease ( COPD ) model rats and normal rats distal pulmonary arterial smooth muscle cells (PASMCs)byusingtissueadherentculturedPASMCs.Methods 12SPFSDratswererandomlydividedintotwogroups:the control group was given saline injection on the 1st and 14th day,without exposure to cigarette smoke;The COPD model group was given intratracheal injection of lipopolysaccharide( LPS)solution,200μg/time,as well as cigarette smoke exposure of 1 h/d for a total of eight weeks. After the model was established,tissue adherent method was used to culture distal PASMCs. Cell mor-phology and growth characteristics were observed under inverted phase contrast microscope. Cell immunohistochemistry and elec-tron microscopy was used to identify PASMCs,and CCK-8 and fluorescence flow cytometry was used to detect cell prolifera-tion.Results Underinvertedphasecontrastmicroscope,PASMCsshapedlikelongspindleorfishtail,typically"peak-val-ley" style growth;Cell immunohistochemistry and electron microscopy identified that 97% of the cultured cells were PASMCs. The PASMCs absorbance of the COPD model group(1. 079±0. 119)was higher than that of the control group(0. 871±0. 041)(t=4. 039,P<0. 05). The PASMCs proliferation of COPD model group(1. 315±0. 203)was stronger than that of thecontrolgroup(1.023±0.057)(t=3.387,P<0.05).℅onclusion TheprimaryculturedPASMCshavesimilarmorphol-ogy in the control group and COPD model group,but have different biological characteristics and functionality.%目的：采用组织块贴壁法体外培养原代大鼠远端肺动脉平滑肌细胞（ PASMCs），探讨慢性阻塞性肺疾病（COPD）模型大鼠与正常大鼠远端PASMCs的生物学特性及功能的异同。方法 SPF级SD雄性大鼠12只，按随机数字表法分为2组：对照组大鼠第1、14天经气道内注入0.9%氯化钠溶液，无香烟烟雾暴露。COPD模型组大鼠于实验第1、14天经气道内注入脂多糖（ LPS）溶液，200μg/次，香烟烟雾暴露1 h/d，共8周。模型建立后，组织块贴壁法提取并原代培养各组大鼠远端PASMCs。倒置相差显微镜下观察细胞形态及生长特点；细胞免疫组化染色、透射电镜鉴定PASMCs；CCK-8和细胞荧光流式细胞仪检测各组细胞增殖情况。结果倒置相差显微镜下PASMCs为长梭形或鱼尾状，为典型的“峰-谷”样生长；细胞免疫组化染色、透射电镜鉴定所培养的细胞97%以上为PASMCs。COPD模型组PASMCs吸光度（1.079±0.119）高于对照组（0.871±0.041）（t=4.039，P<0.05）。COPD模型组PASMCs增殖（1.315±0.203）强于对照组（1.023±0.057）（t=3.387，P<0.05）。结论对照组与COPD模型组原代培养的PASMCs形态学相似，但生物学特性不同，功能亦存在差异。