JNK信号通路及p53基因调控黏菌素诱导的PC12细胞自噬作用研究

发布时间：2018-05-29 19:29

本文选题：黏菌素 + 自噬　；参考：《东北农业大学》2017年博士论文

【摘要】：黏菌素(colistin)又称多黏菌素E,是碱性环状的多肽类抗生素,临床常用其硫酸盐,被认为是治疗多重耐药革兰氏阴性菌感染的有效药物,但是由于黏菌素潜在的神经毒性和肾毒性,限制了它在临床的广泛应用。本课题组前期已经证实黏菌素的神经毒性,并发现其可引起大鼠肾上腺嗜铬细胞瘤细胞系(PC12细胞)凋亡和自噬,并在近期实验中得知黏菌素诱导的自噬发生于凋亡之前,并且该自噬具备凋亡的负调控能力;同时发现p53基因可参与黏菌素诱导的神经细胞自噬和凋亡。由于凋亡和自噬的相互作用关系一直是该研究领域的热点问题,明确两者之间的基因(p53)及信号通路(JNK)的作用及相关机制,将从分子水平上丰富该药的毒理学资料,并可为寻找和开发对抗黏菌素致神经毒性的药物或预防措施提供新路径。本次试验仍以PC12细胞为研究对象,分别运用JNK信号通路抑制剂(SP600125)及活化剂(anisomycin)调节JNK在黏菌素诱导的PC12细胞中的活性。通过MTT法确定SP600125和anisomycin在本试验中的最适浓度分别为20μM和4μM,PC12细胞经20μM SP600125和4μM anisomycin预处理1 h后,给予125μg·m L-1黏菌素作用12 h,运用real-time PCR、Western blot、透射电镜、免疫荧光和Hoechst 33258染色等方法检测细胞自噬和凋亡相关指标及形态学变化,以探讨JNK信号通路与黏菌素诱导的细胞自噬的关系;通过si RNA干扰技术使p53蛋白达有效沉默的目的,运用Western blot法鉴定p53沉默效率,然后采用4μM anisomycin预处理沉默p53基因的PC12细胞,通过测定p53、p-JNK表达量及ROS含量,探讨p53、JNK及ROS三者之间关系。试验结果表明:(1)与空白对照组相比,黏菌素可诱导PC12细胞中JNK的活化,同时伴有Bcl2的活化,并且12 h时间点表达量最高,Bax表达量增加开始于12 h,并且呈现时间依赖性。(2)与阴性对照组相比(colistin组),SP600125预处理可使自噬相关基因和蛋白(Beclin1、和LC3)表达量显著降低(p0.01),p62表达量增加(p0.01),电镜观察到自噬小体明显减少;anisomycin预处理可致自噬相关基因和蛋白表达量增加(p0.01),电镜下可见自噬小体明显增多,LC3免疫荧光点增加;并且伴随凋亡基因及活化蛋白的表达量增加(p0.01),细胞核固缩、偏移现象更加显著。(3)与阴性对照组相比,黏菌素诱导的PC12细胞通过anisomycin活化JNK,自噬相关蛋白表达量增加,同时伴有Bcl2的活化,并且在6 h时间点时表达量最高;同时伴有Bax表达量的增加,凋亡相关蛋白(caspase3、PARP)活化开始于6 h,并且呈现时间依赖性。(4)与阴性对照组相比(colistin+sip53),anisomycin预处理导致ROS含量显著增加,同时p-JNK蛋白表达量显著增加(p0.01)。PC12细胞转染p53 si RNA使p53蛋白有效沉默,以及转染p53过表达质粒使p53蛋白过表达,运用Western blot法鉴定p53干扰效率,并通过p53免疫荧光确定p53蛋白的亚细胞定位。将黏菌素作用于PC12细胞12 h或24 h,试验设立空白组、colistin(12 h、24 h)组、colistin+negative control(NC)(12 h、24 h)组、colistin+sip53(12 h、24 h)组、colistin+pc DNA3.1(12 h、24 h)组和colistin+pc DNA3.1+p53(12 h、24 h)组,采用上述实验方法检测细胞自噬和凋亡的相关基因和蛋白表达量变化,结合形态学变化和一系列生化指标改变,分析并探讨p53基因沉默及过表达在黏菌素诱导自噬过程中的调控机制;再分别使用100 n M溶酶体抑制剂(BFA)和5 mM自噬抑制剂(3-MA)分别将细胞做预处理,检测自噬和凋亡的标志性蛋白表达的指标及细胞形态学变化,以分析和评定p53基因表达量变化对自噬功能的影响。试验结果表明:(1)与阴性对照组相比(colistin+NC;colistin+pc DNA3.1),Western blot和p53免疫荧光的检测结果显示,p53 si RNA和pc DNA3.1+p53能有效地沉默和过表达细胞质中的p53基因。(2)与阴性对照组相比(colistin+NC),在黏菌素诱导的p53基因沉默组PC12细胞中,12 h时间点自噬相关基因和蛋白表达量明显降低(p0.01),LC3免疫荧光点减弱,电镜下观察自噬小体减少;24 h时间点自噬相关基因和蛋白表达量显著增加(p0.01),LC3免疫荧光点增加,电镜下观察自噬小体增多。(3)与colistin+sip53组相比,colistin+sip53+BFA组自噬通量指标无显著变化;colistin+sip53+3-MA组凋亡相关活性蛋白表达量显著降低(p0.01),细胞核固缩、偏移现象减少。(4)与阴性对照组相比(colistin+pc DNA3.1),在黏菌素诱导的p53基因过表达组PC12细胞中,自噬相关基因和蛋白表达量明显降低(p0.01)并呈现时间依赖性,LC3免疫荧光点减弱,电镜下观察自噬小体明显减少;凋亡相关活性蛋白及基因的表达量显著降低(p0.01),细胞核固缩、偏移现象明显。综上所述,本试验通过对JNK信号通路在黏菌素诱导自噬机制的初探,发现JNK参与黏菌素诱导的自噬,JNK-Bcl2-Bax信号通路调节黏菌素诱导的细胞自噬与凋亡,加速了黏菌素诱导的自噬,并提前了凋亡的发生,同时有ROS-JNK-p53活化环路参与在其中;p53蛋白沉默使得黏菌素诱导的PC12细胞自噬现象在12 h时下调、24 h上调,并且上调的自噬促进黏菌素诱导的细胞凋亡;p53蛋白的过表达抑制黏菌素诱导的自噬并加速了细胞凋亡现象的发生。
[Abstract]:Colistin, also known as polymyxin E, is an alkaline cyclic polypeptide antibiotic. It is commonly used as an effective drug for the treatment of multidrug-resistant gram-negative bacteria. However, the potential neurotoxicity and nephrotoxicity of the bacteria limit its extensive clinical application. The neurotoxicity of the hormone is found to cause apoptosis and autophagy in the rat adrenal pheochromocytoma cell line (PC12 cell). In the recent experiment, it was found that the autophagy induced autophagy occurred before apoptosis and the autophagy has the ability to regulate the apoptosis. At the same time, the p53 gene was found to be involved in the autophagy and withering of the nyphysin induced neurons. The interaction of apoptosis and autophagy has been a hot issue in this field. It is possible to enrich the toxicological data of the drug and to provide the drug or preventive measures for the development of the neurotoxicity induced by the p53 and the signal pathway (JNK). The test still took PC12 cells as the research object, using JNK signal pathway inhibitor (SP600125) and activator (anisomycin) to regulate the activity of JNK in PC12 cells induced by clay bacteria. The optimum concentration of SP600125 and anisomycin in this experiment was 20 mu M and 4 mu M by MTT method. The PC12 cells were 20 mu and 4. After 1 h pretreatment of 1 h, 125 g. M L-1 was given to 12 h, and real-time PCR, Western blot, transmission electron microscopy, immunofluorescence and Hoechst 33258 staining were used to detect the autophagy and apoptosis related indexes and morphological changes, so as to explore the relationship between the signaling pathway and the cell autophagy induced by clay bacteria. Western blot method was used to identify the p53 silencing efficiency by using the interfering technique to effectively silence the p53 protein. Then the PC12 cells that were pretreated with 4 mu M anisomycin were used to determine the p53, p-JNK expression and ROS content, and the relationship between the p53, p-JNK and the three were investigated. The activation of JNK in PC12 cells was accompanied by activation of Bcl2, and the expression of the 12 h time point was highest, the expression of Bax increased at 12 h and showed time dependence. (2) compared with the negative control group (colistin group), SP600125 preconditioning could reduce the expression of autophagy related genes and proteins (Beclin1, LC3) significantly (P0.01) and p62 expression amount. Increasing (P0.01), the autophagic corpuscle was significantly reduced by electron microscopy, and anisomycin preconditioning could increase the expression of autophagy related genes and proteins (P0.01). Under electron microscopy, autophagic bodies were significantly increased, LC3 immunofluorescence points increased, and the expression of apoptotic and activated proteins increased (P0.01), and nuclear condensation and migration were more significant. (3) compared with the negative control group, the PC12 cells induced by colyzine activated JNK through anisomycin, the expression of autophagy related proteins increased, accompanied by the activation of Bcl2, and the highest expression at the time of 6 h. At the same time, the activation of apoptosis related protein (Caspase3, PARP) began to be 6 h and showed time dependence with the increase of Bax expression. (4 Compared with the negative control group (colistin+sip53), anisomycin preconditioning resulted in a significant increase in the content of ROS, while the expression of p-JNK protein increased significantly (P0.01).PC12 cells transfected with p53 Si RNA to effectively silence the p53 protein, and the transfection of p53 overexpressed plasmids to make the p53 protein overexpressed. Determine the subcellular location of p53 protein by fluorescence, and act on the 12 h or 24 h of PC12 cells, and set up a blank group, colistin (12 h, 24 h) group, colistin+negative control (NC) (12 h, 24 h) group, 12, 24, 24, 12, 24, and adopt the above experimental method The changes in the related genes and protein expression of autophagy and apoptosis were detected, combined with morphological changes and a series of biochemical changes, the regulation mechanism of p53 gene silencing and overexpression in the process of autophagy induced by clay was analyzed, and the cells were respectively used 100 N M lysosome inhibitor (BFA) and 5 mM autophagy inhibitor (3-MA) respectively. The markers of the expression of autophagy and apoptosis and the morphological changes of cell morphology were detected to analyze and evaluate the effect of p53 gene expression changes on autophagy. The results showed: (1) compared with the negative control group (colistin+NC; colistin+pc DNA3.1), the results of Western blot and p53 immunofluorescence showed p53 Si RN A and PC DNA3.1+p53 could effectively silence and overexpress the p53 gene in the cytoplasm. (2) compared with the negative control group (colistin+NC), the expression of autophagy related genes and proteins at the time point of 12 h decreased significantly (P0.01), the immunofluorescence point of LC3 weakened, and the autophagic corpuscle was observed under the electron microscope, and the 24 h was observed in the p53 gene silencing group PC12 cells induced by the clay. The expression of autophagy related genes and proteins increased significantly (P0.01), LC3 immunofluorescence point increased, and autophagic bodies increased under electron microscope. (3) there was no significant change in the autophagy flux index in the colistin+sip53+BFA group compared with the colistin+sip53 group; the expression of apoptosis related active protein in the colistin+sip53+3-MA group decreased significantly (P0.01), and the nucleus retraction was found. (4) compared with the negative control group (colistin+pc DNA3.1), the expression of autophagy related genes and proteins decreased significantly (P0.01) in the PC12 cells of p53 gene overexpression induced by clay bacteria (P0.01) and showed time dependence, the immunofluorescence point of LC3 decreased, and the autophagic corpuscle was obviously reduced under the electric microscope; apoptosis related active proteins and bases were observed. In this experiment, we found that JNK participates in the autophagy induced by the colysin, the JNK-Bcl2-Bax signaling pathway regulates the autophagy induced by the clay bacteria, and the JNK-Bcl2-Bax signaling pathway regulates the autophagy induced cell autophagy induced by the clay bacteria and accelerates the autophagy induced by the clay bacteria. At the same time, apoptosis occurred in advance, and ROS-JNK-p53 activation loop was involved in it. P53 protein silencing made the autophagy induced PC12 cell autophagy down at 12 h, up up by 24 h, and up up autophagy promoted the apoptosis induced by clay bacteria; the overexpression of p53 protein inhibited the autophagy induced by clay and accelerated the apoptosis. The occurrence of a phenomenon.
【学位授予单位】：东北农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R96

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