摘要
目的探讨主动脉夹层(AD)血管干细胞(VSC)向平滑肌细胞(SMC)分化过程中是否存在失调,并验证Notch3通路在该过程中的作用。方法获取南方医科大学附属广东省人民医院心脏大血管外科接受主动脉血管置换术的AD患者以及心脏移植供体的主动脉组织,用酶消化法以及c-kit免疫磁珠分选出VSC,将细胞分为正常供体来源的VSC组(Ctrl-VSC组)和AD来源的VSC组(AD-VSC组),用免疫组化染色法检测主动脉外膜VSC的存在,用干细胞功能鉴定试剂盒鉴定VSC。使用转化生长因子-β1(10μg/L)诱导分化7 d体外建立的VSC向SMC分化模型,并分为正常供体VSC来源的SMC组(Ctrl-VSC-SMC组)、AD的VSC来源SMC组(AD-VSC-SMC组)和AD的VSC来源SMC+Notch3抑制剂DAPT组(AD-VSC-SMC+DAPT组,诱导分化过程中加入DAPT 20μmol/L),用免疫荧光染色法检测主动脉中膜和VSC来源的SMC中的收缩型标志物钙调理蛋白1(CNN1)的表达,用蛋白质免疫印迹试验检测主动脉中膜和VSC来源的SMC中收缩型标志物α-平滑肌肌动蛋白(α-SMA)、CNN1以及Notch3细胞内结构域(NICD3)的蛋白表达。结果免疫组化染色显示,主动脉血管外膜存在一群c-kit阳性的VSC,且无论是正常供体还是AD患者的VSC都具有向脂肪细胞和软骨细胞分化的能力。与正常供体血管组织相比,AD血管中膜收缩型SMC标志物α-SMA、CNN1表达下调(α-SMA/β-actin:0.40±0.12比1.00±0.11,CNN1/β-actin:0.78±0.07比1.00±0.14,均P<0.05),而NICD3蛋白表达上调(NICD3/GAPDH:2.22±0.57比1.00±0.15,P<0.05)。与Ctrl-VSC-SMC组相比,AD-VSC-SMC组收缩型SMC标志物α-SMA、CNN1表达下调(α-SMA/β-actin:0.35±0.13比1.00±0.20,CNN1/β-actin:0.78±0.06比1.00±0.07,均P<0.05),而NICD3蛋白表达上调(NICD3/GAPDH:22.32±1.22比1.00±0.06,P<0.01)。与AD-VSC-SMC组相比,AD-VSC-SMC+DAPT组收缩型SMC标志物α-SMA、CNN1表达上调(α-SMA/β-actin:1.70±0.07比1.00±0.15,CNN1/β-actin:1.62±0.03比1.00±0.02,均P<0.05)。结论AD的VSC向SMC分化过�
Objective To explore whether the differentiation of vascular stem cells(VSC)into smooth muscle cells(SMC)in aortic dissection(AD)is dysregulated,and to verify the role of Notch3 pathway in this process.Methods Aortic tissues were obtained from AD patients undergoing aortic vascular replacement and heart transplant donors at Department of Cardiovascular Surgery,Guangdong Provincial People's Hospital Affiliated to Southern Medical University.VSC were isolated by enzymatic digestion and c-kit immunomagnetic beads.The cells were divided into normal donor-derived VSC group(Ctrl-VSC group)and AD-derived VSC group(AD-VSC group).The presence of VSC in the aortic adventitia was detected by immunohistochemical staining,and VSC was identified by stem cell function identification kit.The differentiation model of VSC into SMC established in vitro was induced by transforming growth factor-β1(10μg/L)for 7 days.They were divided into normal donor VSC-SMC group(Ctrl-VSC-SMC group),AD VSC-SMC group(AD-VSC-SMC group)and AD VSC-SMC+Notch3 inhibitor DAPT group(AD-VSC-SMC+DAPT group,DAPT 20μmol/L was added during differentiation induction).The expression of contractile marker Calponin 1(CNN1)in SMC derived from aortic media and VSC were detected by immunofluorescence staining.The protein expressions of contractile markersα-smooth muscle actin(α-SMA),CNN1 as well as Notch3 intracellular domain(NICD3)in SMC derived from aortic media and VSC were detected by Western blotting.Results Immunohistochemical staining showed there was a population of c-kit-positive VSC in the adventitia of aortic vessels,and VSC from both normal donors and AD patients had the ability to differentiate into adipocytes and chondrocytes.Compared with normal donor vascular tissue,the expressions of SMC markersα-SMA and CNN1 of tunica media contraction in AD were down-regulated(α-SMA/β-actin:0.40±0.12 vs.1.00±0.11,CNN1/β-actin:0.78±0.07 vs.1.00±0.14,both P<0.05),while the protein expression of NICD3 was up-regulated(NICD3/GAPDH:2.22±0.57 vs.1.00±0.15,P
作者
韩依池
何海威
李欣
Han Yichi;He Haiwei;Li Xin(Department of Emergency Medicine,Guangdong Provincial People's Hospital(Guangdong Academy of Medical Sciences),Southern Medical University,Guangzhou 510080,Guangdong,China)
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2023年第5期503-508,共6页
Chinese Critical Care Medicine
基金
国家自然科学基金(82102312)。
