本文選題：黏菌素 + 自噬��； 參考：《東北農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：黏菌素(colistin)又稱多黏菌素E,是堿性環(huán)狀的多肽類抗生素,臨床常用其硫酸鹽,被認(rèn)為是治療多重耐藥革蘭氏陰性菌感染的有效藥物,但是由于黏菌素潛在的神經(jīng)毒性和腎毒性,限制了它在臨床的廣泛應(yīng)用。本課題組前期已經(jīng)證實(shí)黏菌素的神經(jīng)毒性,并發(fā)現(xiàn)其可引起大鼠腎上腺嗜鉻細(xì)胞瘤細(xì)胞系(PC12細(xì)胞)凋亡和自噬,并在近期實(shí)驗(yàn)中得知黏菌素誘導(dǎo)的自噬發(fā)生于凋亡之前,并且該自噬具備凋亡的負(fù)調(diào)控能力;同時(shí)發(fā)現(xiàn)p53基因可參與黏菌素誘導(dǎo)的神經(jīng)細(xì)胞自噬和凋亡。由于凋亡和自噬的相互作用關(guān)系一直是該研究領(lǐng)域的熱點(diǎn)問題,明確兩者之間的基因(p53)及信號通路(JNK)的作用及相關(guān)機(jī)制,將從分子水平上豐富該藥的毒理學(xué)資料,并可為尋找和開發(fā)對抗黏菌素致神經(jīng)毒性的藥物或預(yù)防措施提供新路徑。本次試驗(yàn)仍以PC12細(xì)胞為研究對象,分別運(yùn)用JNK信號通路抑制劑(SP600125)及活化劑(anisomycin)調(diào)節(jié)JNK在黏菌素誘導(dǎo)的PC12細(xì)胞中的活性。通過MTT法確定SP600125和anisomycin在本試驗(yàn)中的最適濃度分別為20μM和4μM,PC12細(xì)胞經(jīng)20μM SP600125和4μM anisomycin預(yù)處理1 h后,給予125μg·m L-1黏菌素作用12 h,運(yùn)用real-time PCR、Western blot、透射電鏡、免疫熒光和Hoechst 33258染色等方法檢測細(xì)胞自噬和凋亡相關(guān)指標(biāo)及形態(tài)學(xué)變化,以探討JNK信號通路與黏菌素誘導(dǎo)的細(xì)胞自噬的關(guān)系;通過si RNA干擾技術(shù)使p53蛋白達(dá)有效沉默的目的,運(yùn)用Western blot法鑒定p53沉默效率,然后采用4μM anisomycin預(yù)處理沉默p53基因的PC12細(xì)胞,通過測定p53、p-JNK表達(dá)量及ROS含量,探討p53、JNK及ROS三者之間關(guān)系。試驗(yàn)結(jié)果表明:(1)與空白對照組相比,黏菌素可誘導(dǎo)PC12細(xì)胞中JNK的活化,同時(shí)伴有Bcl2的活化,并且12 h時(shí)間點(diǎn)表達(dá)量最高,Bax表達(dá)量增加開始于12 h,并且呈現(xiàn)時(shí)間依賴性。(2)與陰性對照組相比(colistin組),SP600125預(yù)處理可使自噬相關(guān)基因和蛋白(Beclin1、和LC3)表達(dá)量顯著降低(p0.01),p62表達(dá)量增加(p0.01),電鏡觀察到自噬小體明顯減少;anisomycin預(yù)處理可致自噬相關(guān)基因和蛋白表達(dá)量增加(p0.01),電鏡下可見自噬小體明顯增多,LC3免疫熒光點(diǎn)增加;并且伴隨凋亡基因及活化蛋白的表達(dá)量增加(p0.01),細(xì)胞核固縮、偏移現(xiàn)象更加顯著。(3)與陰性對照組相比,黏菌素誘導(dǎo)的PC12細(xì)胞通過anisomycin活化JNK,自噬相關(guān)蛋白表達(dá)量增加,同時(shí)伴有Bcl2的活化,并且在6 h時(shí)間點(diǎn)時(shí)表達(dá)量最高;同時(shí)伴有Bax表達(dá)量的增加,凋亡相關(guān)蛋白(caspase3、PARP)活化開始于6 h,并且呈現(xiàn)時(shí)間依賴性。(4)與陰性對照組相比(colistin+sip53),anisomycin預(yù)處理導(dǎo)致ROS含量顯著增加,同時(shí)p-JNK蛋白表達(dá)量顯著增加(p0.01)。PC12細(xì)胞轉(zhuǎn)染p53 si RNA使p53蛋白有效沉默,以及轉(zhuǎn)染p53過表達(dá)質(zhì)粒使p53蛋白過表達(dá),運(yùn)用Western blot法鑒定p53干擾效率,并通過p53免疫熒光確定p53蛋白的亞細(xì)胞定位。將黏菌素作用于PC12細(xì)胞12 h或24 h,試驗(yàn)設(shè)立空白組、colistin(12 h、24 h)組、colistin+negative control(NC)(12 h、24 h)組、colistin+sip53(12 h、24 h)組、colistin+pc DNA3.1(12 h、24 h)組和colistin+pc DNA3.1+p53(12 h、24 h)組,采用上述實(shí)驗(yàn)方法檢測細(xì)胞自噬和凋亡的相關(guān)基因和蛋白表達(dá)量變化,結(jié)合形態(tài)學(xué)變化和一系列生化指標(biāo)改變,分析并探討p53基因沉默及過表達(dá)在黏菌素誘導(dǎo)自噬過程中的調(diào)控機(jī)制;再分別使用100 n M溶酶體抑制劑(BFA)和5 mM自噬抑制劑(3-MA)分別將細(xì)胞做預(yù)處理,檢測自噬和凋亡的標(biāo)志性蛋白表達(dá)的指標(biāo)及細(xì)胞形態(tài)學(xué)變化,以分析和評定p53基因表達(dá)量變化對自噬功能的影響。試驗(yàn)結(jié)果表明:(1)與陰性對照組相比(colistin+NC;colistin+pc DNA3.1),Western blot和p53免疫熒光的檢測結(jié)果顯示,p53 si RNA和pc DNA3.1+p53能有效地沉默和過表達(dá)細(xì)胞質(zhì)中的p53基因。(2)與陰性對照組相比(colistin+NC),在黏菌素誘導(dǎo)的p53基因沉默組PC12細(xì)胞中,12 h時(shí)間點(diǎn)自噬相關(guān)基因和蛋白表達(dá)量明顯降低(p0.01),LC3免疫熒光點(diǎn)減弱,電鏡下觀察自噬小體減少;24 h時(shí)間點(diǎn)自噬相關(guān)基因和蛋白表達(dá)量顯著增加(p0.01),LC3免疫熒光點(diǎn)增加,電鏡下觀察自噬小體增多。(3)與colistin+sip53組相比,colistin+sip53+BFA組自噬通量指標(biāo)無顯著變化;colistin+sip53+3-MA組凋亡相關(guān)活性蛋白表達(dá)量顯著降低(p0.01),細(xì)胞核固縮、偏移現(xiàn)象減少。(4)與陰性對照組相比(colistin+pc DNA3.1),在黏菌素誘導(dǎo)的p53基因過表達(dá)組PC12細(xì)胞中,自噬相關(guān)基因和蛋白表達(dá)量明顯降低(p0.01)并呈現(xiàn)時(shí)間依賴性,LC3免疫熒光點(diǎn)減弱,電鏡下觀察自噬小體明顯減少;凋亡相關(guān)活性蛋白及基因的表達(dá)量顯著降低(p0.01),細(xì)胞核固縮、偏移現(xiàn)象明顯。綜上所述,本試驗(yàn)通過對JNK信號通路在黏菌素誘導(dǎo)自噬機(jī)制的初探,發(fā)現(xiàn)JNK參與黏菌素誘導(dǎo)的自噬,JNK-Bcl2-Bax信號通路調(diào)節(jié)黏菌素誘導(dǎo)的細(xì)胞自噬與凋亡,加速了黏菌素誘導(dǎo)的自噬,并提前了凋亡的發(fā)生,同時(shí)有ROS-JNK-p53活化環(huán)路參與在其中;p53蛋白沉默使得黏菌素誘導(dǎo)的PC12細(xì)胞自噬現(xiàn)象在12 h時(shí)下調(diào)、24 h上調(diào),并且上調(diào)的自噬促進(jìn)黏菌素誘導(dǎo)的細(xì)胞凋亡;p53蛋白的過表達(dá)抑制黏菌素誘導(dǎo)的自噬并加速了細(xì)胞凋亡現(xiàn)象的發(fā)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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R96

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本文編號：1952105