胰岛素样生长因子1保护心肌细胞免于凋亡机制的探讨

发布时间：2018-06-05 07:59

本文选题：心肌细胞凋亡 + IGF-1　；参考：《广州医学院》2010年博士论文

【摘要】：【目的】随着年龄的增长,人的心脏功能呈衰退趋势,心肌细胞逐渐减少,心肌细胞凋亡是心肌细胞减少的主要原因。IGF-1可以保护心肌细胞免于凋亡,IGF-1对于心肌细胞的保护能力和在对抗心肌细胞凋亡方面的卓越表现,使其成为一个非常有吸引力的和临床应用潜力的心脏保护类药物,但是对于其作用机制认识的不足是IGF-1作为心脏保护类药物应用于临床的主要障碍。本研究的目的在于揭示IGF-1保护心肌细胞免于凋亡的新的作用机制,为IGF-1作为心脏保护类药物应用于临床打下基础。【方法】采用基因芯片的方法观察了IGF-1作用于大鼠心肌细胞后的基因表达谱,通过Affymetrix、Genespring和Cyber T三种统计学方法对基因芯片数据进行处理,对处理后得到的表达下降的基因分别进行了文献检索,对于新发现的从未被报道与凋亡相关的基因进一步文献检索,通过RT-PCR和Western-blot方法观察了大鼠心肌细胞中IGF-1对BTEB和CYP1A1基因表达的调控。利用BTEB和CYP1A1的特异性siRNA下调这两个基因的表达水平以后,以DNA梯状带、Caspase3活性观察大鼠心肌细胞系H9C2在血清饥饿的条件下的凋亡情况,以台酚蓝染色法观察了细胞存活情况。【结果】通过对基因芯片数据的三种统计学处理,我们一共得到了31个表达下降的基因。我们将这31个基因分别进行了文献检索,发现了一个新的从未被报道与凋亡相关的基因-基础转录元件结合蛋白BTEB(Basic transcription element binding protein)。进一步研究发现IGF-1可以通过下调BTEB进而下调CYP1A1的基因表达,而且BTEB在心肌细胞中也同样可以调节CYP1A1的基因表达。我们利用BTEB和CYP1A1的特异性siRNA下调这两个基因的表达水平以后,大鼠心肌细胞系H9C2在血清饥饿的条件下,凋亡情况有了一定的改善,与IGF-1的作用情况相似。【结论】发现了一个未被报道过的IGF-1抑制心肌细胞凋亡的信号传导通路--BTEB-CYP1A1通路,这一机制提示了线粒体也同样参与到IGF-1抑制心肌细胞凋亡的过程当中。【创新点】本研究的创新点主要体现在以下几个方面: 1.利用基因芯片技术观察了IGF-1作用于大鼠心肌细胞后的基因表达情况,发现了一条未被报道过的IGF-1保护心肌细胞免于凋亡的通路—BTEB-CYP1A1通路。这一通路首次提示了IGF-1可能通过细胞色素c参与的线粒体机制抑制心肌细胞凋亡,该发现丰富了我们对于IGF-1保护心肌细胞免于凋亡的认识,为IGF-1作为心脏保护药物最终应用于临床打下了基础。 2.在同一组中利用5块芯片进行平行实验,最大可能地减少了假阳性结果。由于基因芯片实验成本较高,所以有些研究人员往往只采用一对芯片进行实验,通过比较荧光强度倍数的差异来确定基因表达的差异,这样往往得到大量的假阳性结果,我们采用同一样品5块芯片平行实验,利用组间t检验代替荧光强度倍数,使我们结果的可靠性大为增加。 3.相同的基因芯片数据采用不同的统计学处理方法往往得到不同的实验结果,但是对于大多数医学尤其是临床医学研究人员来说一般并不具备足够的统计学知识,所以选取一个合适的统计学处理软件是一个切实可行的办法。本研究比较了目前常用的三种基因芯片数据处理的统计学软件在同一实验中的处理结果,我们发现Affymetrix公司自带的软件在基因芯片数据处理中误差较大,而Genespring和CyberT误差较小,相较于Genespring,CyberT处理基因芯片数据后可信度更高。这一结果为其他从事基因芯片的研究的临床医务工作者提供了可贵的参考。
[Abstract]:[Objective] with the growth of age, the heart function of the human is declining, the myocardial cells gradually decrease, and the apoptosis of cardiac myocytes is the main reason for the reduction of cardiac myocytes..IGF-1 can protect the cardiomyocytes from apoptosis. The protective ability of IGF-1 to myocardial cells and the excellent performance in the resistance to cardiomyocyte apoptosis make it a one. Very attractive and clinical potential cardiac protective drugs, but the lack of understanding of its mechanism is the main obstacle to the clinical application of IGF-1 as a cardioprotective drug. The purpose of this study is to reveal the new mechanism that IGF-1 protects cardiomyocytes from apoptosis and for IGF-1 as a protective drug for the heart. It is applied to the clinic to lay the foundation.
[Methods] gene chip was used to observe the gene expression profiles of IGF-1 in rat cardiac myocytes. The gene chip data were processed by three statistical methods of Affymetrix, Genespring and Cyber T. The genes that were reduced after treatment were retrieved, and the new discoveries were never reported. In further literature retrieval related to apoptosis related genes, the regulation of BTEB and CYP1A1 gene expression by IGF-1 in rat cardiac myocytes was observed by RT-PCR and Western-blot methods. After the expression level of these two genes was down regulated by BTEB and CYP1A1 specific siRNA, the DNA ladder like zone was used to observe the H9C2 in the blood of the rat cardiac myocytes in the Caspase3 activity. Apoptosis was observed under starvation condition, and the survival rate was observed by using phenol blue staining.
[results] through three statistical processing of gene chip data, we got 31 genes that expressed decreased expression. We searched the 31 genes and found a new gene that was never reported to be associated with apoptosis combined with the Basic transcription element binding prot (transcription element binding prot) EIN). Further studies have found that IGF-1 can down regulate the gene expression of CYP1A1 by downregulating BTEB, and BTEB can also regulate the gene expression of CYP1A1 in cardiac myocytes. We use the specific siRNA down regulation of BTEB and CYP1A1 to reduce the expression level of these two genes, and the rat cardiac myocyte line H9C2 is withered under the condition of serum starvation. The situation of death has improved somewhat, which is similar to that of IGF-1.
[Conclusion] an unknown signal transduction pathway --BTEB-CYP1A1 pathway for IGF-1 inhibition of cardiomyocyte apoptosis is found. This mechanism suggests that mitochondria are also involved in the process of IGF-1 inhibition of cardiomyocyte apoptosis.
[innovation] the innovations of this study are mainly reflected in the following aspects:
1. a gene chip technique was used to observe the gene expression of IGF-1 after the action of the rat cardiac myocytes. An unknown IGF-1 pathway, the BTEB-CYP1A1 pathway, was found to protect cardiomyocytes from apoptosis. This pathway was the first indication that IGF-1 may inhibit cardiomyocyte apoptosis through the mitochondrial mechanism involved in cytochrome c. It has enriched our understanding of IGF-1 for protecting cardiomyocytes from apoptosis, and laid the foundation for IGF-1 as a cardioprotective drug and ultimately applied in clinic.
2. parallel experiments in the same group using 5 chips can minimize false positive results. Because of the high cost of the gene chip experiment, some researchers tend to use only one pair of chips to test the difference of the fluorescence intensity multiplier to determine the difference in gene expression, which often results in a lot of false positive. As a result, we used the parallel test of 5 chips of the same sample and used the t test to replace the fluorescence intensity multiplier, which greatly increased the reliability of our results.
3. the same genetic chip data using different statistical processing methods often get different experimental results, but for most medical researchers, especially clinical researchers do not have enough statistical knowledge, so selecting a suitable statistical processing software is a practical way. Compared with the results of the data processing software of three commonly used gene chip data processing in the same experiment, we found that the Affymetrix company's own software has a large error in the data processing of gene chip, but the error of Genespring and CyberT is small. Compared with Genespring, CyberT has higher reliability after processing gene chip data. The results provide valuable references for other clinical medical workers engaged in gene chip research.
【学位授予单位】：广州医学院
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R346

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