Ad-IRE1α腺病毒载体的构建及对软骨细胞分化与凋亡的效应研究
发布时间：2018-11-25 14:15
【摘要】：背景：内质网应激(Endoplasmic reticulum stress,ERS)激活的一系列信号通路统称未折叠蛋白反应(Unfolded Protein Response,UPR)。 UPR信号通路的三个组成部分(inositol requiring enzyme1, IRE1; PKR-like ER resistant kinase, PERK and activating transcription factor6, ATF6)帮助细胞减少未折叠或错误折叠的蛋白质水平,促进细胞的存活。其中IRE1包括两个同源体IRE1α和IRE1β。IRE1α/XBP1是目前研究最多的一条信号通路,IRE1α是一种内质网上固有的跨膜蛋白,含有感应ER应激的内质网腔结构域和具有激酶和核糖核酸内切酶活性的胞浆结构域。IRElα在内质网应激中起核心作用并且是许多内质网应激相关疾病的潜在治疗靶点。 软骨细胞是关节软骨内存在的唯一细胞成分,对软骨生长,关节软骨机械支持和关节连接具有重要作用。由于软骨细胞位于关节连接处,没有血管和神经的支配,软骨细胞在分化过程中会经历各种应激状态,如渗透性应激,氧化应激和机械应激,内质网应激可能是软骨细胞的蛋白在内质网合成和运输阶段的正常反应。软骨细胞的生存状态和软骨基质代谢的平衡状态,直接影响着关节疾病的发生和发展。近年来研究认为,内质网应激参与一些软骨疾病的发生和发展,如假性软骨发育不良,多发性骨骺发育不良和骨关节炎等。越来越多的研究表明,不同因素诱导的内质网应激参与软骨细胞的病理过程,与软骨疾病的发病机制具有一定关系。 目的：ATDC5细胞株来源于小鼠畸胎瘤细胞,是一个具有软骨细胞典型特点的细胞株。它具有类似软骨细胞不同阶段的分化过程,从而成为研究软骨内骨形成分子机制的一个良好的细胞模型。本研究利用该细胞株,探索重组腺病毒介导的IRE1α及其核心功能域截短体R+K对软骨细胞分化和凋亡的影响。为进一步研究内质网应激对软骨细胞增殖、分化和凋亡的影响提供实验依据。 第一部分：Ad-IRE1α腺病毒载体构建 方法：应用pAdEasyTM腺病毒载体系统,构建得到IRE1(?)基因全长和Rnase+Kinase核心结构域的重组穿梭质粒pAdTrack-IRE1α和pAdTrack-R+K,通过电转法分别同腺病毒骨架质粒pAdEasy-1进行同源重组,获得重组腺病毒pAd-IRE1α、pAd-R+K。随后在HEK-293细胞中包装并扩增重组腺病毒颗粒Ad-IRE1α、Ad-R+K。用直接PCR法鉴定重组腺病毒。 结果：酶切鉴定和PCR证实成功构建了携带IRE1α基因全长及其核心功能域重组腺病毒Ad-R+K, 结论：重组腺病毒质粒pAd-IRE1α、pAd-R+K构建成功。 第二部分：Ad-IRE1α腺病毒载体对软骨细胞分化的影响 方法：将成功构建的重组腺病毒质粒体外感染软骨干细胞ATDC5,观察绿色荧光蛋白(GFP)感染效率。高密度培养ATDC5细胞,分别在1、3、5、7、9天各时间点,采用Real-time PCR法检测COL Ⅱ和COL X的表达,免疫印迹法检测COL X和COMP的表达,设立BMP2为阳性对照组。 结果：Real-time PCR结果显示：重组腺病毒质粒Ad-IRE1α同BMP2一样,促进ATDC5细胞COL Ⅱ和COL X的表达。免疫印迹法检测结果表明：重组腺病毒质粒Ad-IRE1α促进COL X和COMP的表达。 结论：含IRE1α基因的重组腺病毒,促进ATDC5细胞的分化。 第三部分：IRElα对软骨细胞凋亡的影响 方法：应用siRNA技术下调软骨细胞中IRElα的蛋白表达水平,并采用流式细胞仪(FCM)在Tm诱导的ERS状态下,检测si-IRE1α对单层培养的C28I2细胞的凋亡情况；同时,采用免疫印迹法分别观察IRE1α重组腺病毒对ATDC5细胞中CHOP蛋白表达的影响及转染si-IRE1α的C28I2细胞中caspase-3的表达情况。 结果：在ER Stress状态下,FCM检测结果表明,与对照组相比,干扰质粒pS1和pS2组C28I2细胞凋亡率明显增加,分别为48.96%、49.88%；同时,免疫印迹结果显示,过表达IRE1α抑制ATDC5细胞中CHOP的表达,而下调C28I2软骨细胞中IRE1α的表达,可促进caspase-3蛋白的表达。 结论：siRNA沉默方法结合FCM和免疫印迹检测结果说明,IRE1α抑制软骨细胞的凋亡。
[Abstract]:Background: A series of signal pathways activated by endoplasmic reticulum stress (ERS) are collectively referred to as Unfold Protein Response (UPR). The three components of the UPR signal pathway (IRE1; PKR-like ER resistant kinase, PERK and activating transcription factor 6, ATF6) help the cells to reduce the level of protein that is not folded or incorrectly folded and to promote the survival of the cells. wherein IRE1 comprises two homologues IRE1 and IRE1. IRE1-1/ XBP1 is one of the most current signal pathways in the study, and the IRE1 gene is an inherent transmembrane protein in the endoplasmic reticulum, the endoplasmic reticulum cavity domain containing the ER stress, and the cytoplasmic domain with the enzyme activity of the kinase and the ribonucleic acid. IRELl plays a central role in endoplasmic reticulum stress and is a potential therapeutic target for many endoplasmic reticulum stress-related diseases. Chondrocytes are the only cellular components present in the articular cartilage, which are important for the growth of the cartilage, the mechanical support of the articular cartilage and the joint connection. in that proces of differentiation, the chondrocytes experience various physiological states, such as osmotic stress, oxidative stress, and mechanical, because the cartilage cells are located at the joint of the joints without the control of blood vessels and nerves. Stress and endoplasmic reticulum stress may be the normal protein of the chondrocytes in the endoplasmic reticulum synthesis and transport phase. The survival state of the chondrocytes and the equilibrium state of the cartilage matrix metabolism directly affect the occurrence and the occurrence of the joint diseases. Development. In recent years, it has been found that endoplasmic reticulum stress is involved in the occurrence and development of some of the cartilage diseases, such as pseudochondrodysplasia, polyosteogenesis and osteoarthrosis. More and more studies have shown that the endoplasmic reticulum stress induced by different factors is involved in the pathological process of the chondrocyte, and the pathogenesis of the cartilage disease has a certain effect. Objective: The ATDC5 cell line is derived from the mouse teratoma and is a typical chondrocyte. The cell line of the point. It has a differentiation process similar to the different stages of the chondrocyte, thus becoming a good mechanism for studying the formation of the bone in the cartilage. In this study, the cell line was used to explore the recombinant adenovirus-mediated IRE1 gene and its core functional domain truncated R + K to differentiate the chondrocyte. The effects of endoplasmic reticulum stress on proliferation, differentiation and apoptosis of chondrocytes were studied. For experimental basis. Part 1: Ad-IRE 1. Construction method of adenovirus vector: using pAdEasy.TM. adenovirus carrier system Series and constructing the recombinant shuttle plasmid pAdTrack-IRE1 and pAdTrack-R + K of the total length of the IRE1 (?) gene and the Rnase + Kinase core domain, and homologous recombination with the adenovirus skeleton plasmid pAdEasy-1 by an electric rotation method, so as to obtain the recombinant adenovirus pAd-IR. E1, pAd-R + K. The recombinant adenovirus particles Ad-I were then packaged and amplified in HEK-293 cells RE1, Ad-R + K. Direct The recombinant adenovirus was identified by PCR. The results showed that the full length of the IRE1 gene and its core were successfully constructed by enzyme digestion and PCR. Functional domain recombinant adenovirus Ad-R + K, Conclusion: The recombinant adenovirus plasmid pAd-IRE1, pAd-R + K build success. Part 2: Ad The effect of the-IRE1 adenovirus vector on the differentiation of cartilage cells: the successfully constructed recombinant adenovirus plasmid is successfully constructed to infect the chondrocytes in vitro ATDC5 was used to observe the infection efficiency of green fluorescent protein (GFP). High-density culture of ATDC5 cells was performed at 1, 3, 5, 7 and 9 days, and the expression of COL 鈪，
本文编号：2356401
[Abstract]:Background: A series of signal pathways activated by endoplasmic reticulum stress (ERS) are collectively referred to as Unfold Protein Response (UPR). The three components of the UPR signal pathway (IRE1; PKR-like ER resistant kinase, PERK and activating transcription factor 6, ATF6) help the cells to reduce the level of protein that is not folded or incorrectly folded and to promote the survival of the cells. wherein IRE1 comprises two homologues IRE1 and IRE1. IRE1-1/ XBP1 is one of the most current signal pathways in the study, and the IRE1 gene is an inherent transmembrane protein in the endoplasmic reticulum, the endoplasmic reticulum cavity domain containing the ER stress, and the cytoplasmic domain with the enzyme activity of the kinase and the ribonucleic acid. IRELl plays a central role in endoplasmic reticulum stress and is a potential therapeutic target for many endoplasmic reticulum stress-related diseases. Chondrocytes are the only cellular components present in the articular cartilage, which are important for the growth of the cartilage, the mechanical support of the articular cartilage and the joint connection. in that proces of differentiation, the chondrocytes experience various physiological states, such as osmotic stress, oxidative stress, and mechanical, because the cartilage cells are located at the joint of the joints without the control of blood vessels and nerves. Stress and endoplasmic reticulum stress may be the normal protein of the chondrocytes in the endoplasmic reticulum synthesis and transport phase. The survival state of the chondrocytes and the equilibrium state of the cartilage matrix metabolism directly affect the occurrence and the occurrence of the joint diseases. Development. In recent years, it has been found that endoplasmic reticulum stress is involved in the occurrence and development of some of the cartilage diseases, such as pseudochondrodysplasia, polyosteogenesis and osteoarthrosis. More and more studies have shown that the endoplasmic reticulum stress induced by different factors is involved in the pathological process of the chondrocyte, and the pathogenesis of the cartilage disease has a certain effect. Objective: The ATDC5 cell line is derived from the mouse teratoma and is a typical chondrocyte. The cell line of the point. It has a differentiation process similar to the different stages of the chondrocyte, thus becoming a good mechanism for studying the formation of the bone in the cartilage. In this study, the cell line was used to explore the recombinant adenovirus-mediated IRE1 gene and its core functional domain truncated R + K to differentiate the chondrocyte. The effects of endoplasmic reticulum stress on proliferation, differentiation and apoptosis of chondrocytes were studied. For experimental basis. Part 1: Ad-IRE 1. Construction method of adenovirus vector: using pAdEasy.TM. adenovirus carrier system Series and constructing the recombinant shuttle plasmid pAdTrack-IRE1 and pAdTrack-R + K of the total length of the IRE1 (?) gene and the Rnase + Kinase core domain, and homologous recombination with the adenovirus skeleton plasmid pAdEasy-1 by an electric rotation method, so as to obtain the recombinant adenovirus pAd-IR. E1, pAd-R + K. The recombinant adenovirus particles Ad-I were then packaged and amplified in HEK-293 cells RE1, Ad-R + K. Direct The recombinant adenovirus was identified by PCR. The results showed that the full length of the IRE1 gene and its core were successfully constructed by enzyme digestion and PCR. Functional domain recombinant adenovirus Ad-R + K, Conclusion: The recombinant adenovirus plasmid pAd-IRE1, pAd-R + K build success. Part 2: Ad The effect of the-IRE1 adenovirus vector on the differentiation of cartilage cells: the successfully constructed recombinant adenovirus plasmid is successfully constructed to infect the chondrocytes in vitro ATDC5 was used to observe the infection efficiency of green fluorescent protein (GFP). High-density culture of ATDC5 cells was performed at 1, 3, 5, 7 and 9 days, and the expression of COL 鈪，
本文编号：2356401
本文链接：https://www.wllwen.com/xiyixuelunwen/2356401.html
最近更新
教材专著
