中国血液净化

中国血液净化 ›› 2019, Vol. 18 ›› Issue (01): 35-41.doi: 10.3969/j.issn.1671-4091.2019.01.008

• 基础研究 • 上一篇    下一篇

尿毒症血清活化Notch 信号通路调控血管平滑肌细胞表型转化

刘方圆1,黄凤璋1,刘日光1,傅君舟1,梁鸣1   

  1. 1. 广州医科大学附属市一人民医院肾内科
  • 收稿日期:2018-07-16 修回日期:2018-09-24 出版日期:2019-01-12 发布日期:2018-12-25
  • 通讯作者: 梁鸣liangming1972@126.com E-mail:liangming1972@126.com
  • 基金资助:

    国家自然科学基金(81570689) 广东省自然科学基金(2017A030313566)

Uremic serum regulates the phenotype transformation of vascular smooth muscle cells through activating the Notch signaling pathway

  • Received:2018-07-16 Revised:2018-09-24 Online:2019-01-12 Published:2018-12-25

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摘要： 【摘要】目的研究尿毒症血清对血管平滑肌细胞(vascular smooth muscle cell，VSMC)表型转化影响，探讨动静脉内瘘(arteriovenous fistula，AVF)狭窄及失功的机制。方法①弹性纤维染色(elastic-Van Gieson staining，EVG)比较内膜厚度，免疫组化法观察α-SMA、FSP-1、PCNA 的表达；②培养人主动脉平滑肌细胞株(human aortic smooth muscle cells，HASMCs)，分为无血清组(control，Ctl)、10%正常血清组(normal serum group，NS)、5%～20%尿毒症血清组(uremia serum group，US)，检测平滑肌细胞α-SMA、SMMHC、SM22α、FSP-1、PCNA 的表达；③Western blot 检测各组平滑肌细胞N1ICD 蛋白的表达，QPCR 检测细胞Notch1-3、Hes1、Hes5 的mRNA 表达，并比较Notch 信号抑制剂DAPT 预处理24h 对细胞SM22α、SMMHC、FSP-1、PCNA、Hes1 的mRNA 表达的影响；④免疫组化染色检测Jagged1 的表达，ELISA法检测人脐静脉内皮细胞(human umbilical vein endothelial cells，HUVECs)Jagged1 的表达，并用Western blot 法检测HASMCs 的Jagged1 蛋白表达。结果① 内瘘内膜显著增厚，增生内膜α-SMA 呈阳性表达；与正常血管比较，增生内膜FSP-1、PCNA 阳性表达增加，差异均有统计学意义(FSP-1：Ctl-A：P=0.024，Ctl-V：P=0.011；PCNA：Ctl-A：P=0.002；Ctl-V：P=0.005)；②与10%NS 组相比，10%US 组细胞α-SMA、SM22α蛋白表达下降，而FSP-1、PCNA 蛋白表达增加，差异均有统计学意义(P=0.002；P=0.001；P=0.001；P＜0.001)，且上述指标与尿毒症血清呈浓度依赖性；10%US 组细胞SM22α、SMMHC 的mRNA 表达下降，而FSP-1、PCNA 的mRNA 表达增加，差异有统计学意义(P=0.014；P=0.003；P=0.045；P=0.001)；③与10%NS 组比较，10%US 组细胞N1/CD 蛋白的表达显著增加，差异有统计学意义(P＜0.001)；10%US 组细胞Notch 1,3、Hes 1、Hes 5 的mRNA 表达增加，差异有统计学意义(P 值分别为＜0.001，0.025，＜0.001，0.035)，Notch2 的表达无统计学差异(P=0.907)；DAPT 预处理可上调SM22α、SMMHC 的mRNA 表达，下调FSP-1、PCNA及Hes1 的mRNA 表达，差异均有统计学意义(P=0.003；P=0.020；P=0.036；P=0.009；P＜0.001)；④内瘘组织Jagged1 的表达增加，主要位于增生内膜；与10%NS 组相比，10%US 组HASMCs 及HUVEC 的Jagged1 蛋白表达均增加，差异均有统计学意义(P 值分别为0.001，0.005)。结论尿毒症血清通过上调配体Jagged1 的表达，激活Notch信号通路，促进VSMC 表型由收缩型向合成型转变，可能是AVF 内膜增生的机制之一。

关键词: 尿毒症血清, 血管平滑肌细胞, 动静脉内瘘, 表型转化

Abstract: 【Abstract】Objective To study the effect of uremic serum on phenotype transformation of vascular smooth muscle cells and to correlate the results to the mechanism of arteriovenous fistula (AVF) stenosis and dysfunction. Methods ①The intimal thickness of AVF was compared with normal vessles by elastic Van Gieson staining, and the expressions of α-SMA, FSP-1 and PCNA were evaluated by immunohistochemical staining. ②Human aortic smooth muscle cells (HASMCs) were cultured in vitro and divided into 3 groups: serum-free group (Ctrl group), 10% normal serum group (NS group), and 5～20% uremia serum group (US group). The expressions of α-SMA, SM22α, FSP-1 and PCNA proteins and mRNAs were assessed by Western blot and quantitative PCR. ③The expression of N1ICD protein in different groups was assayed by Western blot; the expressions of Notch1-3, Hes1 and Hes5 mRNAs were detected by quantitative PCR. HASMCs were pre-treated with the Notch signal inhibitor DAPT for 24h, and then the expressions of SM22α, SMMHC, FSP-1, PCNA and Hes1 mRNAs were measured by quantitative PCR. ④The expression of Jagged1, a ligand of Notch, was detected by immunohistochemical staining. Jagged1 protein expressed by human umbilical vein endothelial cells (HUVECs) was assayed by ELISA; Jagged1 expression in HASMCs was measured by Western blot. Results ①In AVF, intimal thickness increased significantly, the expression of α-SMA in neointima was positive, and the positive rates of FSP-1 and PCNA were higher than those in normal vascular tissue (P＜0.05). ②In US groups, the expressions of α-SMA and SM22α proteins decreased significantly (P=0.002 and 0.001, respectively), while the expressions of FSP- 1 and PCNA proteins increased significantly (P=0.001 and ＜0.001, respectively), as compared with those in NS group. These changes showed a tendency of uremia serum concentration dependence. In US group, the expressions of SM22α and SMMHC mRNAs decreased significantly (P=0.014 and 0.003, respectively), while the expressions of FSP-1 and PCNA mRNAs increased significantly (P=0.045 and 0.001, respectively). ③In US group, the expression of N1ICD protein increased significantly (P＜0.001), the expressions of Notch1, Notch3, Hes1 and Hes5 mRNAs increased significantly (P＜0.001, 0.025, ＜0.001 and 0.035, respectively), and Notch2 mRNA had no significant difference (P=0.907), as compared with those in NS group. Pre-treatment of DAPT up-regulated the expressions of SM22α and SMMHC mRNAs (P=0.003 and 0.020, respectively) and down-regulated the expressions of FSP-1, PCNA and Hes1 mRNAs (P=0.036, 0.009 and ＜0.001, respectively). ④In AVF, the expression of Jagged1 increased mainly in neointima. In US group, the expression of Jagged1 protein in HASMCs and HUVECs increased significantly (P=0.001 and 0.005, respectively), as compared with those in NS group. Conclusion Uremic serum promoted the phenotype transformation of vascular smooth muscle cells from deflating type to proliferation type through the up-regulation of Notch ligand Jagged1 and then activation of Notch signaling
pathway, probably being the mechanism of neointimal hyperplasia in AVF in uremic patients.

Key words: Uremia Serum, Vascular smooth muscle cell, Arteriovenous fistula, Phenotype transformation