本文選題：心肌成纖維細(xì)胞 + 肌成纖維細(xì)胞��； 參考：《第四軍醫(yī)大學(xué)》2010年碩士論文

【摘要】： 【研究背景】 心肌成纖維細(xì)胞(CFs)過度增殖、轉(zhuǎn)分化為肌成纖維細(xì)胞(MFB)而表達(dá)α-平滑肌肌動蛋白(α-smooth muscle actin,α-SMA)導(dǎo)致膠原合成分泌過多是心肌纖維化的重要病理基礎(chǔ)。轉(zhuǎn)化生長因子-β1 (TGF-β1)具有促進(jìn)CFs向MFB轉(zhuǎn)分化,上調(diào)α-SMA和促進(jìn)膠原合成等作用,是重要的促心肌纖維化細(xì)胞因子。心臟中腎素-血管緊張素系統(tǒng)( rennin-angiotensin system, RAS)的主要產(chǎn)物血管緊張素Ⅱ( angiotensinⅡ,AngⅡ)也能促進(jìn)心肌纖維化的形成。Notch信號通路通過受體與配體相互作用來精確調(diào)控細(xì)胞分化,在心血管發(fā)育生理、病理過程中起重要作用,已證實其參與調(diào)節(jié)了成纖維細(xì)胞向MFB的分化,但其在CFs向MFB轉(zhuǎn)分化過程中作用如何尚缺乏明確報道。 【研究目的】 1.觀察基礎(chǔ)狀態(tài)下CFs表達(dá)Notch各受體情況以及DAPT對CFs的α-SMA,HYP的作用,初步闡明Notch通路是否參與心肌纖維化。 2.明確TGF-β1誘導(dǎo)CFs的α-SMA、HYP變化過程中Notch各受體的變化情況。 3.明確AngⅡ誘導(dǎo)CFs的α-SMA、HYP變化過程中Notch各受體的變化情況。 【研究內(nèi)容】 1.檢測基礎(chǔ)狀態(tài)下CFs表達(dá)Notch各受體情況以及分別使用DAPT不同作用濃度和不同作用時間對CFs的α-SMA、HYP的影響:分別采用實時定量PCR和蛋白印跡的方法檢測Notch各受體的基因和蛋白水平;采用免疫熒光細(xì)胞化學(xué)和蛋白印跡的方法檢測α-SMA的表達(dá);采用消化法測定HYP含量。 2. TGF-β1不同作用時間和作用濃度對CFs的α-SMA、HYP的影響以及在TGF-β1誘導(dǎo)CFs向MFB轉(zhuǎn)分化中Notch各受體的表達(dá)變化:采用免疫熒光細(xì)胞化學(xué)和蛋白印跡的方法檢測α-SMA的表達(dá);采用消化法測定HYP含量;采用實時熒光定量PCR和蛋白印跡的方法檢測Notch各受體的基因和蛋白水平。 3. AngⅡ不同作用時間和作用濃度對CFs的α-SMA、HYP的影響以及在AngⅡ誘導(dǎo)CFs向MFB轉(zhuǎn)分化中Notch各受體的表達(dá)變化:采用免疫熒光細(xì)胞化學(xué)和蛋白印跡的方法檢測α-SMA的表達(dá);采用消化法測定HYP含量;采用實時熒光定量PCR和蛋白印跡的方法檢測Notch各受體的基因和蛋白水平。 【研究結(jié)果】 1.基礎(chǔ)狀態(tài)下CFs有Notch各受體基因和蛋白的表達(dá);隨著DAPT作用濃度的增加和作用時間的延長CFs的α-SMA、HYP均呈遞增趨勢。 2. TGF-β1可以上調(diào)CFs的α-SMA、HYP表達(dá)量,隨著TGF-β1作用濃度的增加和作用時間的延長α-SMA、HYP均呈遞增趨勢。但Notch1、Notch3、Notch4隨著TGF-β1作用時間的延長呈遞減趨勢,Notch2則無明顯變化。 3. AngⅡ可以上調(diào)CFs的α-SMA、HYP表達(dá)量,隨著AngⅡ作用濃度的增加和作用時間的延長α-SMA、HYP均呈遞增趨勢。但Notch1、Notch3、Notch4隨著AngⅡ作用時間的延長呈遞減趨勢,Notch2表達(dá)則無明顯變化。 【研究結(jié)論】 1. Notch各受體基因和蛋白在基礎(chǔ)狀態(tài)下CFs上均有表達(dá)。DAPT阻斷Notch通路后,可以引起α-SMA、HYP的增加,導(dǎo)致CFs向MFB發(fā)生轉(zhuǎn)分化。Notch通路在CFs向MFB轉(zhuǎn)分化過程中可能具有重要作用。 2. TGF-β1能夠顯著增加CFsα-SMA、HYP的表達(dá)量,并且呈時間和劑量依賴效應(yīng),可以誘導(dǎo)CFs向MFB轉(zhuǎn)分化。在TGF-β1誘導(dǎo)CFs向MFB轉(zhuǎn)分化過程中Notch1、Notch3、Notch4呈遞減趨勢,Notch2則無明顯變化。 3. AngⅡ能夠顯著增加CFsα-SMA、HYP的表達(dá)量,而且呈時間和劑量依賴效應(yīng),可以誘導(dǎo)CFs向MFB轉(zhuǎn)分化。在AngⅡ誘導(dǎo)CFs向MFB轉(zhuǎn)分化過程中Notch1、Notch3、Notch4逐漸降低,Notch2變化不顯著。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R363

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