摘要
目的:探讨SD大鼠冠状动脉平滑肌细胞(coronary artery smooth muscle cells,CASMCs)原代培养方法,并建立CASMCs内质网应激(endoplasmic reticulum stress,ERS)模型。方法:用组织块贴壁法培养CASMCs,光学显微镜观察细胞形态。免疫荧光技术检测CASMCs的标志分子α-SMA和SM-MHC的表达。采用Western blot检测ERS发生的标志物BiP和CHOP的蛋白表达水平。结果:冠状动脉组织块贴壁培养6 d后,细胞从组织块边缘爬出,呈长梭型,9~10 d细胞生长汇合后表现出平滑肌细胞典型的"峰-谷"状。免疫荧光技术鉴定结果显示,α-SMA和SM-MHC表达呈阳性。不同浓度(0.5、1和2μmol/L)的毒胡萝卜素(thapsigargin,TG)处理CASMCs 24 h后,Western blot结果显示,TG(1和2μmol/L)处理组的BiP和CHOP蛋白表达与对照组相比增加,差异具有统计学意义。与对照组相比较,1μmol/L TG处理CASMCs 24和48 h后BiP和CHOP蛋白表达显著性增加。结论:采用组织块贴壁法可以成功培养CASMCs。利用1μmol/L TG诱导24 h可建立CASMCs的ERS模型。
AIM：To investigate the primary culture method for coronary artery smooth muscle cells（CASMCs）,and to establish the endoplasmic reticulum stress（ERS）model in CASMCs of SD rats. METHODS：CASMCs were cultured by tissue explant method. The morphological characteristics were observed under optical microscope. The marker proteins of CASMCs,including α-SMA and SM-MHC,were identified by immunofluorescence technique. The protein expression levels of BiP and CHOP,the marker molecules of ERS,were determined by Western blot.RESULTS：The spindle-shaped CASMCs climbed out from the edge of coronary artery tissues after 6 d,and formed the typical ＂ hill and valley＂ growth pattern of CASMCs at 9 ~ 10 d. The result of immunofluorescence technique showed that α-SMA and SM-MHC were positively expressed. The results of Western blot showed that the protein expression of BiP and CHOP in TG（1 and 2 μmol/L）treatment groups was increased compared with control group. Compared with control group,the protein expression of BiP and CHOP was significantly increased after 1 μmol/LTG treatment for 24 and 48 h.CONCLUSION：CASMCs can be successfully cultured by tissue explant method. ERS model of CASMCs was established by 1 μmol/LTG treatment for 24 h.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2017年第1期128-132,共5页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81273516
No.81470440)
关键词
冠状动脉平滑肌细胞
组织块贴壁法
内质网应激
Coronary artery smooth muscle cells
Tissue explant method
Endoplasmic reticulum stress
