钙离子依赖的内质网应激信号通路在压力刺激下大鼠髁突软骨病理变化过程中的作用及其机制研究
发布时间：2019-03-19 18:21
【摘要】：第一部分体外实验钙离子依赖的内质网应激信号通路在大鼠髁突软骨细胞中的体外研究[目的]研究钙离子依赖的ERS信号通路在体外培养大鼠髁突软骨细胞中的作用。[方法]选取3周龄的SD大鼠,取颞下颌关节髁突软骨进行原代培养,采用第三代髁突软骨细胞进行实验。用常见的ERS诱导剂4 μg/ml衣霉素(Tunicamycin, Tm)处理大鼠髁突软骨细胞,或用钙离子通道受体阻断剂2-APB、Rya,2-APB+Rya预处理30min后再加入4 μg/ml Tm,处理24h后收集细胞。实验分为：空白对照组、Tm组、Tm+2APB组、Tm+Rya组、Tm+2APB+Rya组。采用Fura-2/AM荧光染料进行胞浆内钙离子染色,然后用荧光光谱仪检测细胞内钙离子浓度的变化；流式细胞仪检测细胞凋亡率；采用RT-qPCR和Western Blot检测GRP78.GRP94、 CHOP、Caspase-12内质网应激标志分子在nRNA和蛋白水平的表达改变。[结果]1.Tm处理大鼠髁突软骨细胞24小时后,细胞钙离子浓度显著升高(P0.05)。经钙离子通道受体阻断剂预处理后,细胞内钙离子浓度显著降低(Tm+2APB+Rya组Tm+Rya组Tm+2APB组),但仍高于空白对照组。而单纯用钙离子通道受体阻断剂处理后与空白对照组无显著差异。2.Tm处理大鼠髁突软骨细胞24小时后,细胞有明显凋亡增加(P0.01),而经钙离子通道受体阻断剂预处理后,凋亡显著减少(P0.01),与空白对照组无显著差异。3.Tm处理大鼠髁突软骨细胞24小时后,ERS标志分子GRP78、GRP94、CHOP、 Caspase-12 mRNA及其蛋白水平上表达升高。钙离子通道受体阻断剂可以抑制Tm诱导的大鼠髁突软骨细胞(GRP78、GRP94、CHOP、Caspase-12 mRNA及其蛋白水平上表达升高,除Caspase-12,均以2APB+Rya效果佳。[结论]1.Tm可以诱导大鼠髁突软骨细胞发生ERS,导致细胞凋亡。2.Tm可以使大鼠髁突软骨细胞内钙离子浓度升高。3.钙离子通道受体阻断剂可以抑制大鼠髁突软骨细胞发生ERS,降低相应的内质网应激蛋白分子表达,从而对Tm诱导的大鼠髁突软骨细胞的凋亡起保护作用。第二部分体内实验钙离子依赖的内质网应激信号通路在压力刺激下大鼠髁突软骨病理变化过程中的作用及其机制研究[目的]观察钙离子阻断剂及压力作用下大鼠髁突软骨组织形态学、超微结构以及凋亡活性变化,以及ERS相关蛋白GRP78、GRP94、CHOP、Caspase-12表达的改变,探讨钙离子信号依赖的内质网信号通路在大鼠髁突软骨病理变化过程中的作用及其机制。[方法]选择6周龄SD雄性大鼠90只,用本课题组自主设计的颞下颌关节压应力加载的动物模型装置(专利号：201120210396.4),对大鼠髁突软骨施加向上、向后的80g/侧超负荷压应力刺激诱导大鼠髁突软骨发生内质网应激,设为空白对照组、MF(mechanical force)组、MF+2APB组、MF+Rya组、MF+2APB+Rya组,加力时间为3天和7天组。采用GENMED组织钙离子浓度比色法定量检测大鼠髁突软骨组织钙离子浓度的变化；苏木素-伊红(HE)染色观察大鼠髁突软骨层厚度及形态学的变化；透射电镜观察大鼠髁突软骨细胞内质网超微结构的变化；TUNEL荧光染色观察大鼠髁突软骨细胞凋亡的变化；免疫组化(immunohistochemistry, IHC)检测内质网应激标志分子GRP78、GRP94. Caspase-12的表达改变。[结果]压力加载后大鼠髁突软骨中的钙离子浓度升高,随时间的延长,浓度逐渐增加。在重力加载下,HE切片染色显示大鼠髁突软骨层厚度变薄,髁突软骨细胞出现内质网扩张和空泡性改变；细胞凋亡在3天组出现增加。IHC结果显示大鼠髁突软骨细胞内内质网应激标志分子GRP78、GRP94, Caspase-12的表达明显升高。而当关节腔注射钙离子通道受体阻断剂后,可以抑制上述情况的发生,且2-APB+Rya组要依次优于Rya组和2-APB组,单纯加药组与空白对照在上述各项中无显著差异。[结论]钙离子依赖的内质网应激信号通路在大鼠髁突软骨病理变化过程起着重要作用,钙离子通道受体阻断剂可以降低髁突软骨组织钙离子浓度,缓解大鼠髁突软骨的变薄效应,抑制大鼠髁突软骨细胞发生凋亡。钙离子通道受体阻断剂可以通过降低大鼠髁突细胞内ERS标志分子的表达来缓解持续的内质网应激反应,从而对大鼠髁突软骨起着保护作用。
[Abstract]:The role of the calcium-ion-dependent ERS signal pathway in the in vitro culture of the condylar cartilage cells was studied in the first part in vitro. [Methods] Three-week-old SD rats were selected for primary culture of the mandibular joint condylar cartilage, and the third-generation condylar chondrocytes were used for the experiment. The cells of the condylar cartilage of the rat were treated with 4. mu. g/ ml of tunicamycin (Tm), or 4. m u.g/ ml of Tm was added after the pretreatment with the calcium channel receptor blocker 2-APB, Rya,2-APB + Rya for 30 min, and the cells were collected after 24 h treatment. The experiment was divided into two groups: blank control group, Tm group, Tm + 2APB group, Tm + Rya group, Tm + 2APB + Rya group. Intracellular calcium ion was stained with a Fura-2/ AM fluorescent dye, then the change of intracellular calcium ion concentration was detected by a fluorescence spectrometer, and the cell apoptosis rate was detected by flow cytometry. RT-qPCR and Western Blot were used to detect the changes in the expression of nRNA and protein in GRP78. GRP94, CHOP and Caspase-12 endoplasmic reticulum stress marker. [Results] 1. After 24 hours of treatment of the condylar cartilage cells of the rat, the concentration of calcium in the cells increased significantly (P0.05). After the pretreatment of the calcium channel receptor blocker, the intracellular calcium ion concentration decreased significantly (Tm + 2APB + Rya group, Tm + Rya group, Tm + 2APB group), but was still higher than that of the blank control group. 2. After 24 hours of treatment of the condylar cartilage cells of the rat, the apoptosis of the cells increased significantly (P0.01), and the apoptosis was significantly reduced after the pretreatment with the calcium channel receptor blocker (P0.01). There was no significant difference between the control group and the blank control group. After 24 hours of treatment of the condylar cartilage cells of the rat, the expression of the ERS marker molecules GRP78, GRP94, CHOP, Caspase-12 mRNA and its protein level was increased. The calcium channel receptor blocking agent can inhibit the expression of the expression of the mRNA and its protein of the condylar cartilage of the rat (GRP78, GRP94, CHOP, Caspase-12) induced by the Tm, except the Caspase-12, which has the best effect of 2 APB + Rya. [Conclusion] 1. Tm can induce the formation of ERS of the condylar cartilage cells of the rat, resulting in the apoptosis of the cells. The calcium channel receptor blocking agent can inhibit the generation of ERS of the condylar cartilage cells of the rat, and reduce the expression of the corresponding endoplasmic reticulum stress protein so as to protect the apoptosis of the Tm-induced rat condylar cartilage cells. In the second part, the effect of calcium ion-dependent endoplasmic reticulum stress signal pathway on the pathological changes of the condylar cartilage of the rat under pressure stimulation and its mechanism were studied.[Objective] To observe the morphology of the condylar cartilage of the rat under the action of calcium ion blocking agent and pressure. The changes of ultrastructure and apoptosis activity, as well as the changes of the expression of ERS-related protein GRP78, GRP94, CHOP, and Caspase-12, discussed the role of endoplasmic reticulum signaling pathway dependent on calcium ion signal in the pathological changes of condylar cartilage in rats and its mechanism. [Methods] 90 male rats of 6-week-old SD were selected, and the animal model of the mandibular joint stress loading (Patent No.: 201120210396.4), which was designed by the research group, was applied to the condylar cartilage of the rat. The endoplasmic reticulum stress in the condylar cartilage of the rat was induced by stress stimulation of 80 g/ side, which was set as the blank control group, the MF (mechanical force) group, the MF + 2 APB group, the MF + Rya group, the MF + 2 APB + Rya group, and the time of application was 3 days and 7 days. The changes of the calcium ion concentration in the condylar cartilage of the rat were measured by using the GENMED tissue calcium ion concentration colorimetric method. The changes of the thickness and morphology of the condylar cartilage were observed by hematoxylin-eosin (HE) staining, and the ultrastructure of the endoplasmic reticulum of the condylar cartilage was observed by transmission electron microscopy. TUNEL (TUNEL) fluorescence staining was used to observe the changes of the apoptosis of the condylar cartilage cells in the rat. The expression of the endoplasmic reticulum stress marker (GRP78, GRP94) was detected by immunohistochemistry (IHC). The expression of Caspase-12 was changed. [Results] The concentration of calcium in the condylar cartilage of the rat after pressure loading was increased, and the concentration was gradually increased with the time. Under gravity loading, HE section staining showed that the thickness of the condylar cartilage layer of the rat was thin, the endoplasmic reticulum expansion and the vacuolation of the condylar cartilage cells were changed, and the apoptosis of the cells increased in the 3-day group. IHC results show that the expression of endoplasmic reticulum stress marker molecule GRP78, GRP94 and Caspase-12 in the condylar cartilage cell of the rat is obviously increased. The 2-APB + Rya group was superior to the Rya group and the 2-APB group, and the simple treatment group and the blank control group had no significant difference in the above-mentioned items. [Conclusion] The calcium ion-dependent endoplasmic reticulum stress signal pathway plays an important role in the process of the pathological changes of the condylar cartilage of the rat, and the calcium channel receptor blocking agent can reduce the calcium ion concentration of the condylar cartilage tissue and relieve the thinning effect of the condylar cartilage of the rat. So as to inhibit the apoptosis of the condylar cartilage cells of the rat. The calcium channel receptor blocking agent can relieve the sustained endoplasmic reticulum stress response by reducing the expression of the ERS marker molecules in the condylar cells of the rat, thereby protecting the condylar cartilage of the rat.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R782
本文编号：2443775
[Abstract]:The role of the calcium-ion-dependent ERS signal pathway in the in vitro culture of the condylar cartilage cells was studied in the first part in vitro. [Methods] Three-week-old SD rats were selected for primary culture of the mandibular joint condylar cartilage, and the third-generation condylar chondrocytes were used for the experiment. The cells of the condylar cartilage of the rat were treated with 4. mu. g/ ml of tunicamycin (Tm), or 4. m u.g/ ml of Tm was added after the pretreatment with the calcium channel receptor blocker 2-APB, Rya,2-APB + Rya for 30 min, and the cells were collected after 24 h treatment. The experiment was divided into two groups: blank control group, Tm group, Tm + 2APB group, Tm + Rya group, Tm + 2APB + Rya group. Intracellular calcium ion was stained with a Fura-2/ AM fluorescent dye, then the change of intracellular calcium ion concentration was detected by a fluorescence spectrometer, and the cell apoptosis rate was detected by flow cytometry. RT-qPCR and Western Blot were used to detect the changes in the expression of nRNA and protein in GRP78. GRP94, CHOP and Caspase-12 endoplasmic reticulum stress marker. [Results] 1. After 24 hours of treatment of the condylar cartilage cells of the rat, the concentration of calcium in the cells increased significantly (P0.05). After the pretreatment of the calcium channel receptor blocker, the intracellular calcium ion concentration decreased significantly (Tm + 2APB + Rya group, Tm + Rya group, Tm + 2APB group), but was still higher than that of the blank control group. 2. After 24 hours of treatment of the condylar cartilage cells of the rat, the apoptosis of the cells increased significantly (P0.01), and the apoptosis was significantly reduced after the pretreatment with the calcium channel receptor blocker (P0.01). There was no significant difference between the control group and the blank control group. After 24 hours of treatment of the condylar cartilage cells of the rat, the expression of the ERS marker molecules GRP78, GRP94, CHOP, Caspase-12 mRNA and its protein level was increased. The calcium channel receptor blocking agent can inhibit the expression of the expression of the mRNA and its protein of the condylar cartilage of the rat (GRP78, GRP94, CHOP, Caspase-12) induced by the Tm, except the Caspase-12, which has the best effect of 2 APB + Rya. [Conclusion] 1. Tm can induce the formation of ERS of the condylar cartilage cells of the rat, resulting in the apoptosis of the cells. The calcium channel receptor blocking agent can inhibit the generation of ERS of the condylar cartilage cells of the rat, and reduce the expression of the corresponding endoplasmic reticulum stress protein so as to protect the apoptosis of the Tm-induced rat condylar cartilage cells. In the second part, the effect of calcium ion-dependent endoplasmic reticulum stress signal pathway on the pathological changes of the condylar cartilage of the rat under pressure stimulation and its mechanism were studied.[Objective] To observe the morphology of the condylar cartilage of the rat under the action of calcium ion blocking agent and pressure. The changes of ultrastructure and apoptosis activity, as well as the changes of the expression of ERS-related protein GRP78, GRP94, CHOP, and Caspase-12, discussed the role of endoplasmic reticulum signaling pathway dependent on calcium ion signal in the pathological changes of condylar cartilage in rats and its mechanism. [Methods] 90 male rats of 6-week-old SD were selected, and the animal model of the mandibular joint stress loading (Patent No.: 201120210396.4), which was designed by the research group, was applied to the condylar cartilage of the rat. The endoplasmic reticulum stress in the condylar cartilage of the rat was induced by stress stimulation of 80 g/ side, which was set as the blank control group, the MF (mechanical force) group, the MF + 2 APB group, the MF + Rya group, the MF + 2 APB + Rya group, and the time of application was 3 days and 7 days. The changes of the calcium ion concentration in the condylar cartilage of the rat were measured by using the GENMED tissue calcium ion concentration colorimetric method. The changes of the thickness and morphology of the condylar cartilage were observed by hematoxylin-eosin (HE) staining, and the ultrastructure of the endoplasmic reticulum of the condylar cartilage was observed by transmission electron microscopy. TUNEL (TUNEL) fluorescence staining was used to observe the changes of the apoptosis of the condylar cartilage cells in the rat. The expression of the endoplasmic reticulum stress marker (GRP78, GRP94) was detected by immunohistochemistry (IHC). The expression of Caspase-12 was changed. [Results] The concentration of calcium in the condylar cartilage of the rat after pressure loading was increased, and the concentration was gradually increased with the time. Under gravity loading, HE section staining showed that the thickness of the condylar cartilage layer of the rat was thin, the endoplasmic reticulum expansion and the vacuolation of the condylar cartilage cells were changed, and the apoptosis of the cells increased in the 3-day group. IHC results show that the expression of endoplasmic reticulum stress marker molecule GRP78, GRP94 and Caspase-12 in the condylar cartilage cell of the rat is obviously increased. The 2-APB + Rya group was superior to the Rya group and the 2-APB group, and the simple treatment group and the blank control group had no significant difference in the above-mentioned items. [Conclusion] The calcium ion-dependent endoplasmic reticulum stress signal pathway plays an important role in the process of the pathological changes of the condylar cartilage of the rat, and the calcium channel receptor blocking agent can reduce the calcium ion concentration of the condylar cartilage tissue and relieve the thinning effect of the condylar cartilage of the rat. So as to inhibit the apoptosis of the condylar cartilage cells of the rat. The calcium channel receptor blocking agent can relieve the sustained endoplasmic reticulum stress response by reducing the expression of the ERS marker molecules in the condylar cells of the rat, thereby protecting the condylar cartilage of the rat.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R782
【参考文献】
相关期刊论文 前1条
1 胡畔;李涛;丁晓莉;刘良明;;GRP78和CHOP蛋白表达增加与脓毒性休克大鼠肺血管通透性改变的关系[J];第三军医大学学报;2013年09期
，本文编号：2443775
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