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

Primary Culture of Rat Distal Pulmonary Arterial Smooth Muscle Cells and Their Biological Characteristics and Function

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。

Objective To investigate the similarities and differences of biological characteristics and functions between chronic obstructive pulmonary disease（ COPD） model rats and normal rats distal pulmonary arterial smooth muscle cells（ PASMCs） by using tissue adherent cultured PASMCs.Methods 12 SPF SD rats were randomly divided into two groups： the control group was given saline injection on the 1st and 14 th day,without exposure to cigarette smoke; The COPD model group was given intratracheal injection of lipopolysaccharide（ LPS） solution,200 μg / 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 morphology and growth characteristics were observed under inverted phase contrast microscope.Cell immunohistochemistry and electron microscopy was used to identify PASMCs,and CCK- 8 and fluorescence flow cytometry was used to detect cell proliferation.Results Under inverted phase contrast microscope,PASMCs shaped like long spindle or fishtail,typically ＂ peak- valley＂ 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 the control group（ 1.023 ± 0.057）（ t = 3.387,P 〈0.05）.Conclusion The primary cultured PASMCs have similar morphology in the control group and COPD model group,but have different biological characteristics and functionality.