Notch信号通路在心肌成纤维细胞向肌成纤维细胞转分化中的作用

发布时间：2018-05-13 10:15

  本文选题：心肌成纤维细胞 + 肌成纤维细胞　； 参考：《第四军医大学》2010年硕士论文

【摘要】： 【研究背景】 心肌成纤维细胞(CFs)过度增殖、转分化为肌成纤维细胞(MFB)而表达α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)导致胶原合成分泌过多是心肌纤维化的重要病理基础。转化生长因子-β1 (TGF-β1)具有促进CFs向MFB转分化,上调α-SMA和促进胶原合成等作用,是重要的促心肌纤维化细胞因子。心脏中肾素-血管紧张素系统( rennin-angiotensin system, RAS)的主要产物血管紧张素Ⅱ( angiotensinⅡ,AngⅡ)也能促进心肌纤维化的形成。Notch信号通路通过受体与配体相互作用来精确调控细胞分化,在心血管发育生理、病理过程中起重要作用,已证实其参与调节了成纤维细胞向MFB的分化,但其在CFs向MFB转分化过程中作用如何尚缺乏明确报道。 【研究目的】 1.观察基础状态下CFs表达Notch各受体情况以及DAPT对CFs的α-SMA,HYP的作用,初步阐明Notch通路是否参与心肌纤维化。 2.明确TGF-β1诱导CFs的α-SMA、HYP变化过程中Notch各受体的变化情况。 3.明确AngⅡ诱导CFs的α-SMA、HYP变化过程中Notch各受体的变化情况。 【研究内容】 1.检测基础状态下CFs表达Notch各受体情况以及分别使用DAPT不同作用浓度和不同作用时间对CFs的α-SMA、HYP的影响:分别采用实时定量PCR和蛋白印迹的方法检测Notch各受体的基因和蛋白水平;采用免疫荧光细胞化学和蛋白印迹的方法检测α-SMA的表达;采用消化法测定HYP含量。 2. TGF-β1不同作用时间和作用浓度对CFs的α-SMA、HYP的影响以及在TGF-β1诱导CFs向MFB转分化中Notch各受体的表达变化:采用免疫荧光细胞化学和蛋白印迹的方法检测α-SMA的表达;采用消化法测定HYP含量;采用实时荧光定量PCR和蛋白印迹的方法检测Notch各受体的基因和蛋白水平。 3. AngⅡ不同作用时间和作用浓度对CFs的α-SMA、HYP的影响以及在AngⅡ诱导CFs向MFB转分化中Notch各受体的表达变化:采用免疫荧光细胞化学和蛋白印迹的方法检测α-SMA的表达;采用消化法测定HYP含量;采用实时荧光定量PCR和蛋白印迹的方法检测Notch各受体的基因和蛋白水平。 【研究结果】 1.基础状态下CFs有Notch各受体基因和蛋白的表达;随着DAPT作用浓度的增加和作用时间的延长CFs的α-SMA、HYP均呈递增趋势。 2. TGF-β1可以上调CFs的α-SMA、HYP表达量,随着TGF-β1作用浓度的增加和作用时间的延长α-SMA、HYP均呈递增趋势。但Notch1、Notch3、Notch4随着TGF-β1作用时间的延长呈递减趋势,Notch2则无明显变化。 3. AngⅡ可以上调CFs的α-SMA、HYP表达量,随着AngⅡ作用浓度的增加和作用时间的延长α-SMA、HYP均呈递增趋势。但Notch1、Notch3、Notch4随着AngⅡ作用时间的延长呈递减趋势,Notch2表达则无明显变化。 【研究结论】 1. Notch各受体基因和蛋白在基础状态下CFs上均有表达。DAPT阻断Notch通路后,可以引起α-SMA、HYP的增加,导致CFs向MFB发生转分化。Notch通路在CFs向MFB转分化过程中可能具有重要作用。 2. TGF-β1能够显著增加CFsα-SMA、HYP的表达量,并且呈时间和剂量依赖效应,可以诱导CFs向MFB转分化。在TGF-β1诱导CFs向MFB转分化过程中Notch1、Notch3、Notch4呈递减趋势,Notch2则无明显变化。 3. AngⅡ能够显著增加CFsα-SMA、HYP的表达量,而且呈时间和剂量依赖效应,可以诱导CFs向MFB转分化。在AngⅡ诱导CFs向MFB转分化过程中Notch1、Notch3、Notch4逐渐降低,Notch2变化不显著。
[Abstract]:[research background]
Myofibroblast (CFs) is overproliferating and transdifferentiated into myofibroblast (MFB) and the expression of alpha smooth muscle actin (alpha -smooth muscle actin, alpha -SMA) is an important pathological basis for myocardial fibrosis. Transforming growth factor beta 1 (TGF- beta 1) can promote CFs to MFB transdifferentiation, up regulation of alpha -SMA and promote collagen Synthesis and so on, it is an important factor in promoting myocardial fibrosis. The main product of rennin-angiotensin system (RAS) in the heart, angiotensin II (angiotensin II, Ang II) also promotes the formation of myocardial fibrosis, the.Notch signaling pathway can accurately regulate the cell division through the interaction of the receptor with the ligand. It plays an important role in the physiological and pathological process of cardiovascular development. It has been proved to be involved in regulating the differentiation of fibroblasts to MFB, but the role in the process of CFs to MFB transdifferentiation is not clearly reported.
[purpose]
1. to observe the expression of Notch receptors in CFs under basic state and the role of DAPT on CFs -SMA, HYP, and to clarify whether Notch pathway is involved in myocardial fibrosis.
2. to clarify the changes of Notch receptors in CFs induced by TGF- beta 1 during the change of -SMA and HYP.
3. to clarify the changes of Notch receptors in the CFs -SMA and HYP induced by Ang II.
[research content]
1. the CFs expression of Notch receptor in the basic state and the effects of different concentrations of DAPT and different action time on the alpha -SMA and HYP of CFs were detected respectively. The gene and protein levels of Notch receptors were detected by real-time quantitative PCR and Western blotting, and the methods of immunofluorescence cytochemistry and Western blotting were used to detect the genes and protein levels of Notch receptors respectively. The expression of alpha -SMA; the content of HYP was determined by digestion.
2. TGF- beta 1 different action time and action concentration on the CFs alpha -SMA, HYP and the changes in the expression of Notch receptors in TGF- beta 1 induced CFs to MFB transdifferentiation: using immunofluorescence cytochemistry and Western blotting to detect the expression of alpha -SMA; use the digestion method to determine the content of HYP; the use of real-time fluorescent quantitative PCR and Western blot. The gene and protein level of each Notch receptor were detected by the method.
The effects of different time and concentration of 3. Ang II on CFs's alpha -SMA, HYP and the changes in the expression of Notch receptors in MFB transdifferentiation induced by Ang II: detection of the expression of alpha -SMA by immunofluorescence cytochemistry and Western blotting; the determination of HYP content by digestion, and the use of real-time fluorescent quantitative PCR and Western blotting. The gene and protein level of each Notch receptor were detected by the method.
[results]
The expression of Notch receptor genes and proteins in CFs was 1. under the basic state, and HYP increased with the increase of DAPT concentration and the prolongation of the action time of CFs.
2. TGF- beta 1 could increase the CFs expression of alpha -SMA and HYP. With the increase of the concentration of TGF- beta 1 and the prolongation of the action time, HYP increased. But Notch1, Notch3 and Notch4 decreased with the prolongation of the action time of TGF- beta 1, and there was no obvious change in Notch2.
3. Ang II can increase the CFs expression of alpha -SMA and HYP. With the increase of the concentration of Ang II and the prolongation of the action time, the HYP increases. But Notch1, Notch3, Notch4 are decreasing with the prolongation of the Ang II action time, and there is no obvious change in Notch2 expression.
[Conclusion]
1. Notch receptor genes and proteins express.DAPT blocking the Notch pathway on the base of CFs, which can cause the increase of alpha -SMA and HYP, which leads to the.Notch pathway of CFs to MFB, which may play an important role in CFs to MFB transdifferentiation.
2. TGF- beta 1 can significantly increase the expression of CFs alpha -SMA, HYP, and the time and dose dependence effect, which can induce the transformation of CFs to MFB. In the course of TGF- beta 1 induced CFs to MFB transdifferentiation, Notch1, Notch3, Notch4 presents a decreasing trend, but there is no obvious change.
3. Ang II can significantly increase the expression of CFs alpha -SMA, HYP, and the time and dose dependence effect, which can induce the transformation of CFs to MFB. In Ang II induced CFs to MFB transdifferentiation, Notch1, Notch3, Notch4 gradually decreases.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

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