肺动脉平滑肌细胞自噬与合成分泌功能的关系
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摘要
目的分离培养慢性阻塞性肺疾病(COPD)大鼠原代肺动脉平滑肌细胞(PASMCs),以自噬增强剂及自噬抑制剂为工具药,测定PASMCs自噬相关基因的表达量及其合成分泌功能(IL-6、血小板源性生长因子:PDGF-AB分泌量)的变化,分析PASMCs自噬与其合成分泌功能的关系。方法 (1)建立COPD大鼠模型,分离、培养原代COPD大鼠PASMCs,将第4、5代PASMCs用于后续研究;(2)实验分组:①对照组:不加入任何干预剂;②LPS组;③3-MA+LPS组;④Rapamycin+LPS组;⑤3-MA单独处理组;⑥Rapamycin单独处理组,48 h后,以上每组做4个复孔,重复实验2次。各组分别于培养48 h后,收集细胞或细胞培养上清液测定以下指标:①Western Blot检测PASMCs自噬标记蛋白LC3-Ⅰ、LC3-Ⅱ和Beclin-1的蛋白表达量;②酶联免疫吸附试验(ELISA)方法检测各组培养上清液中IL-6及PDGF的含量变化。相关分析法分别分析自噬与合成分泌功能的相关性。结果 (1)LPS诱导下大鼠PASMCs中自噬相关基因LC3-Ⅰ、LC3-Ⅱ及Beclin-1的表达量:LPS组、Rapamycin+LPS组及LPS+3-MA与正常对照组相比升高(P <0.05);Rapamycin+LPS与LPS组相比升高(P <0.05);LPS+3-MA组与LPS组相比降低(P <0.05);(2)LPS诱导下大鼠PASMCs合成分泌功能变化情况:LPS组、Rapamycin+LPS组及LPS+3-MA与正常对照组相比升高(P <0.05),Rapamycin+LPS与LPS组相比升高(P <0.05),LPS+3-MA组与LPS组相比减少(P <0.05);(3)相关性分析提示LPS诱导下大鼠PASMCs自噬与其合成分泌功能存在相关性(均R> 0.8,P <0.05)。结论以自噬调控剂为工具药,通过细胞实验,发现LPS诱导下PASMCs自噬与其合成分泌功能可能相关。
Objective Separat the primitive PASMCs,use autophagy enhancer and autophagy inhibitors as tools,detect the expression of PASMCs autophagy related gene and the change of the synthesis of secretion(IL-6,PDGF-AB),analyse the relationship between PASMCs autophagy and its synthetic secretion. Methods(1)Establish COPD rats model,then isolate and cultivate the original PASMCs in COPD rats,and the fourth and fifth generation PASMCs were used for follow-up study.(2)The experiment was divided into six groups:①the con-trol group,②LPS group,③3-MA + LPS groups,④Rapamycin + LPS group,⑤3-MA individual treatment groups⑥Rapamycin individual treatment group,after culturing for 48 hours,each group runs quadruplicate,and repeat the experiment for 2 times. After culturing for 48 hours,collect cells or cell culture supernatant of each group to de-tect the following indicators:(1)Western Blot was used to detect PASMCs autophagy marker protein LC3-Ⅰ,LC3-Ⅱ and protein expression of Beclin1. The enzyme linked immunosorbent assay(ELISA)method was used to detect the changes of IL-6,TGF-β1 and PDGF in each group. Correlation analysis was used to analyze the correlation be-tween autophagy and synthetic secretory cytokines. Results(1)The expression quantity of PASMCs autophagy related genes LC3-Ⅰ,LC3-Ⅱand Beclin 1 in rats induced by LPS were increased in LPS group,Rapamycin+LPS group and LPS + 3-MA group compared with normal control group(P < 0.05). Their expression in Rapamycin + LPS group were increased more than those in LPS group,while they were decreased in LPS + 3-MA group compared with those in LPS group;(2)Changes of synthesis secretion in rats PASMCs induced by LPS were increased in LPS group,Rapamycin + LPS group and LPS+3-MA compared with normal control group. The changes of synthesis se-cretion in The LPS + 3-MA group were decreased compared with LPS group.(3)Correlation analysis suggested that there was a correlation between PASMCs autophagy and its synthetic secretion function in rats induced by LPS(All R > 0.8,P < 0.05). Conclusions Using autophagy regulators as a tool,it is suggested that LPS-induced PASMCs autophagy was related to its synthetic secretion function in vitro study.
Objective Separat the primitive PASMCs,use autophagy enhancer and autophagy inhibitors as tools,detect the expression of PASMCs autophagy related gene and the change of the synthesis of secretion(IL-6,PDGF-AB),analyse the relationship between PASMCs autophagy and its synthetic secretion. Methods(1)Establish COPD rats model,then isolate and cultivate the original PASMCs in COPD rats,and the fourth and fifth generation PASMCs were used for follow-up study.(2)The experiment was divided into six groups:①the con-trol group,②LPS group,③3-MA + LPS groups,④Rapamycin + LPS group,⑤3-MA individual treatment groups⑥Rapamycin individual treatment group,after culturing for 48 hours,each group runs quadruplicate,and repeat the experiment for 2 times. After culturing for 48 hours,collect cells or cell culture supernatant of each group to de-tect the following indicators:(1)Western Blot was used to detect PASMCs autophagy marker protein LC3-Ⅰ,LC3-Ⅱ and protein expression of Beclin1. The enzyme linked immunosorbent assay(ELISA)method was used to detect the changes of IL-6,TGF-β1 and PDGF in each group. Correlation analysis was used to analyze the correlation be-tween autophagy and synthetic secretory cytokines. Results(1)The expression quantity of PASMCs autophagy related genes LC3-Ⅰ,LC3-Ⅱand Beclin 1 in rats induced by LPS were increased in LPS group,Rapamycin+LPS group and LPS + 3-MA group compared with normal control group(P < 0.05). Their expression in Rapamycin + LPS group were increased more than those in LPS group,while they were decreased in LPS + 3-MA group compared with those in LPS group;(2)Changes of synthesis secretion in rats PASMCs induced by LPS were increased in LPS group,Rapamycin + LPS group and LPS+3-MA compared with normal control group. The changes of synthesis se-cretion in The LPS + 3-MA group were decreased compared with LPS group.(3)Correlation analysis suggested that there was a correlation between PASMCs autophagy and its synthetic secretion function in rats induced by LPS(All R > 0.8,P < 0.05). Conclusions Using autophagy regulators as a tool,it is suggested that LPS-induced PASMCs autophagy was related to its synthetic secretion function in vitro study.
引文
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[2]LIU C F,ZHANG J,SHEN K,et al.Adventitial gene transfer of catalase attenuates angiotensin II-induced vascular remodeling[J].Mol Med Rep,2015,11:2608-2614.
[3]YAO D,SUN N L.Hyperhomocysteinemia accelerates collagen accumulation in the adventitia of balloon injured rat carotid arteries via angiotensin II type 1receptor[J].Int J Mol Sci,2014,15:19487-19498.
[4]PUSHPAKUMAR S B,KUNDU S,METREVELI N,et al.Matrix metalloproteinase inhibition mitigates renovascular remodeling in salt sensitive hypertension[J].Physiol Rep,2013,1(3):e00063.
[5]TAI S,HU X Q,PENG D Q,et al.The roles of autophagy invascular smooth muscle cells[J].Int J Cardiol,2016,211:1-6.
[6]ZHOU G F,CHEN T J,ZHOU Q U,et al.MiR-17~92 promotes hypoxia-induced pulmonary hypertension by regulating proliferation and contraction of pulmonary arterial smooth muscle cells[J].The FASEB Journal,2014,28(7):LB779.
[7]CHEN T J,ZHOU G F,ZHOU Q Y,et al.Loss of microRNA-17-92 in smooth muscle cells attenuates experimental pulmonary hypertension via induction of PDZ and LIM Domain 5[J].American Journal of Respiratory and Critical Care Medicine,2015,191(6):678-692.
[8]罗金泰,余文俊,韦安阳,等.血小板源性生长因子-BB对SD大鼠阴茎海绵体平滑肌细胞表型转化影响的初步研究[J].中华男科学杂志,2015,21(7):593-597.
[9]SALABEI J K,CUNNINS T D,SINGH M,et al.PDGF-mediated autophagy regulates vascular smooth muscle cell phenotype and resistance to oxidative stress[J].Biochem J,2013,451(3):375-388.
[10]HU H,LIU ZUO Y,et al.Dl-3n-butyphthalide suppresses PDGFBB-stimulated vascular smooth muscle cells proliferation via induction of autophagy[J].Life Sci,2016,151:182-188.
[11]DONG C,GONGORA R,SOSULSKI M L,et al.Regulation of transforming Growth factor-beta1(TGF-beta1)-induced profibrotic activities by Circadian clock gene BMAL1[J].Respir Res,2016,17:4.