【摘要】：目的:探討降鈣素基因相關(guān)肽(Calcitonin gene related peptide,CGRP)對(duì)環(huán)狀RNA(CircularRNA,circRNA)介導(dǎo)的巨噬細(xì)胞內(nèi)白細(xì)胞介素-6(Interleukin-6,IL-6)表達(dá)的影響。材料與方法:1.細(xì)胞和試劑本實(shí)驗(yàn)的實(shí)驗(yàn)對(duì)象為RAW264.7巨噬細(xì)胞系,使用含有10%胎牛血清的高糖DMEM培養(yǎng)。換液前使用磷酸鹽緩沖液清洗細(xì)胞。實(shí)驗(yàn)使用的CGRP為α-CGRP。2.CGRP最佳作用濃度和最佳作用時(shí)間的篩選實(shí)驗(yàn)設(shè)定CGRP濃度梯度為0.01nM、0.1nM、1nM、10nM和100nM,分別作用于接種密度相同的巨噬細(xì)胞,在設(shè)定時(shí)間點(diǎn)使用實(shí)時(shí)熒光定量核酸擴(kuò)增檢測(cè)系統(tǒng)(Real-time Quantitative PCR Detecting System,qPCR)來測(cè)定IL-6 mRNA的表達(dá);實(shí)驗(yàn)設(shè)定CGRP時(shí)間梯度為1小時(shí)、2小時(shí)、3小時(shí)、4小時(shí)和5小時(shí),分別以相同濃度作用于接種密度相同的巨噬細(xì)胞,在相應(yīng)時(shí)間后使用qPCR來測(cè)定IL-6 mRNA的表達(dá),通過以上兩個(gè)步驟確定CGRP作用的最佳濃度和時(shí)間。3.circRNA芯片的制作和篩選將細(xì)胞以相同密度接種培養(yǎng),用選定濃度的CGRP刺激RAW264.7巨噬細(xì)胞選定時(shí)間后,充分裂解實(shí)驗(yàn)組和空白對(duì)照組細(xì)胞,收集裂解液、制備樣本并交由生物公司做circRNA芯片。根據(jù)該公司提供的芯片結(jié)果,將發(fā)生改變的circRNA按照改變倍數(shù)的大小或預(yù)測(cè)miRNA結(jié)合位點(diǎn)的多寡進(jìn)行篩選,并通過qPCR及其產(chǎn)物測(cè)序進(jìn)行circRNA表達(dá)的驗(yàn)證,采用熒光原位雜交技術(shù)(fluorescence in situ hybridization,FISH)定位和證實(shí)circRNA,最終確定進(jìn)一步實(shí)驗(yàn)的目標(biāo)circRNA。4.circRNA對(duì)IL-6表達(dá)作用的驗(yàn)證1)circRNA的干擾實(shí)驗(yàn)將細(xì)胞以相同密度接種于6孔板中。選擇3種設(shè)計(jì)好的小干擾RNA(Small interferingRNA,siRNA)以相同濃度分別作用于對(duì)應(yīng)實(shí)驗(yàn)組。刺激48小時(shí)以后,充分裂解各實(shí)驗(yàn)組和空白對(duì)照組細(xì)胞,使用qPCR檢測(cè)各組選定circRNA的表達(dá)情況,并最終確定進(jìn)一步實(shí)驗(yàn)所使用的siRNA。2)qPCR檢測(cè)IL-6 mRNA的表達(dá)將細(xì)胞以相同密度接種于6孔板中。本部分實(shí)驗(yàn)分為空白對(duì)照組、CGRP組和CGRP+siRNA組。選擇上一步干擾效果最好的siRNA預(yù)先處理CGRP+siRNA組的細(xì)胞48小時(shí),然后使用選定濃度的CGRP刺激CGRP組和CGRP+siRNA組的細(xì)胞。刺激選定的最佳刺激時(shí)間后,充分裂解各組細(xì)胞并使用qPCR檢測(cè)各組IL-6 mRNA的表達(dá)水平。3)Western-blot檢測(cè)IL-6蛋白的表達(dá)將細(xì)胞以相同密度接種于6孔板中。本部分實(shí)驗(yàn)分為空白對(duì)照組、CGRP組和CGRP+siRNA組。選擇干擾效果最好的siRNA預(yù)先處理CGRP+siRNA組的細(xì)胞48小時(shí),然后使用選定濃度的CGRP刺激CGRP組和CGRP+siRNA組的細(xì)胞。刺激選定的最佳刺激時(shí)間后,充分裂解各組細(xì)胞并使用Western-blot檢測(cè)各組IL-6蛋白的表達(dá)水平。5.circRNA對(duì)下游小分子RNA(microRNA,miRNA)表達(dá)作用的驗(yàn)證1)miRNA的篩選根據(jù)circRNA芯片結(jié)果,找到選定的circRNA對(duì)應(yīng)的5個(gè)目標(biāo)miRNA。根據(jù)生物信息學(xué)網(wǎng)站和軟件預(yù)測(cè)其靶基因和參與的炎癥通路,找到與IL-6相關(guān)的miRNA并進(jìn)行下一步的驗(yàn)證。2)qPCR驗(yàn)證circRNA對(duì)選定miRNA表達(dá)水平的影響將細(xì)胞以相同密度接種于6孔板中。本部分實(shí)驗(yàn)分為空白對(duì)照組、CGRP組和CGRP+siRNA組。選擇干擾效果最好的siRNA預(yù)先處理CGRP+siRNA組的細(xì)胞48小時(shí),然后使用選定濃度的CGRP刺激CGRP組和CGRP+siRNA組的細(xì)胞。刺激選定的最佳刺激時(shí)間后,充分裂解各組細(xì)胞并使用qPCR檢測(cè)各組選定miRNA的表達(dá)水平。6.目標(biāo)miRNA對(duì)IL-6表達(dá)作用的驗(yàn)證1)qPCR檢測(cè)IL-6 mRNA的表達(dá)將細(xì)胞以相同密度接種于6孔板中。本部分實(shí)驗(yàn)分為空白對(duì)照組、CGRP組、CGRP+mimics組和CGRP+inhibitor組。選擇選定miRNA的過表達(dá)試劑(mimics)和干擾試劑(inhibitor)預(yù)先處理對(duì)應(yīng)組的細(xì)胞24小時(shí),然后使用選定濃度的CGRP刺激CGRP+mimics組和CGRP+inhibitor組的細(xì)胞。刺激選定的最佳刺激時(shí)間后,充分裂解各組細(xì)胞并使用qPCR檢測(cè)各組IL-6 mRNA的表達(dá)水平。2)Western-blot檢測(cè)IL-6蛋白的表達(dá)將細(xì)胞以相同密度接種于6孔板中。本部分實(shí)驗(yàn)分為空白對(duì)照組、CGRP組、CGRP+mimics組和CGRP+inhibitor組。選擇選定miRNA的過表達(dá)試劑(mimics)和干擾試劑(inhibitor)預(yù)先處理對(duì)應(yīng)組的細(xì)胞24小時(shí),然后使用選定濃度的CGRP刺激CGRP+mimics組和CGRP+inhibitor組的細(xì)胞。刺激選定的最佳刺激時(shí)間后,充分裂解各組細(xì)胞并使用Western-blot檢測(cè)各組IL-6蛋白的表達(dá)水平。7.統(tǒng)計(jì)學(xué)分析采用SPSS19.0統(tǒng)計(jì)軟件對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析,各組數(shù)據(jù)符合正態(tài)分布,方差齊,對(duì)各組數(shù)據(jù)進(jìn)行配對(duì)雙側(cè)t檢驗(yàn),所有實(shí)驗(yàn)均重復(fù)4次,結(jié)果以均值±標(biāo)準(zhǔn)差(?±s)表示,以雙側(cè)P0.05為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果:1.細(xì)胞培養(yǎng)本實(shí)驗(yàn)的培養(yǎng)方法可以獲得生長(zhǎng)狀態(tài)穩(wěn)定良好的RAW264.7巨噬細(xì)胞。該細(xì)胞剛貼壁時(shí)呈圓形,折光度很好,鏡下觀察如小珍珠狀散落于培養(yǎng)器皿底面。貼壁培養(yǎng)一段時(shí)間后,細(xì)胞會(huì)變?yōu)槎噙呅?不規(guī)則,可見偽足狀突起。該結(jié)果符合RAW264.7巨噬細(xì)胞系的生物學(xué)特性。2.CGRP最佳作用濃度和最佳作用時(shí)間的篩選當(dāng)CGRP作用濃度為0.1nM或1nM時(shí),IL-6 mRNA的相對(duì)表達(dá)水平與對(duì)照組的相比具有統(tǒng)計(jì)學(xué)差異。其中,當(dāng)CGRP濃度為1nM時(shí),IL-6 mRNA相對(duì)表達(dá)水平最高。當(dāng)CGRP作用時(shí)間為2小時(shí)或3小時(shí)時(shí),IL-6 mRNA的相對(duì)表達(dá)水平與對(duì)照組的相比具有統(tǒng)計(jì)學(xué)差異。其中,當(dāng)CGRP作用時(shí)間為2小時(shí)時(shí),IL-6 mRNA相對(duì)表達(dá)水平最高。3.circRNA芯片的制作和篩選芯片結(jié)果表示,相對(duì)表達(dá)值上調(diào)超過2倍的circRNA共有173個(gè),下調(diào)超過2倍的circRNA共有179個(gè)。將這些circRNA按照改變倍數(shù)的大小排序或預(yù)測(cè)miRNA結(jié)合位點(diǎn)的多寡進(jìn)行篩選后,通過qPCR驗(yàn)證,mmu_circRNA_007893的改變倍數(shù)最穩(wěn)定,與芯片結(jié)果相符,產(chǎn)物測(cè)序與circRNA連接位點(diǎn)序列一致。FISH實(shí)驗(yàn)證明mmu_circRNA_007893確實(shí)絕大多數(shù)存在于細(xì)胞質(zhì)中,與芯片結(jié)果一致。4.circRNA對(duì)IL-6表達(dá)作用的驗(yàn)證1)circRNA的干擾實(shí)驗(yàn)3種siRNA對(duì)mmu_circRNA_007893的表達(dá)都有顯著抑制作用,其中siRNA3對(duì)mmu_circRNA_007893的表達(dá)抑制作用最強(qiáng)。2)qPCR檢測(cè)IL-6 mRNA的表達(dá)CGRP組相對(duì)于空白對(duì)照組,IL-6 mRNA的相對(duì)表達(dá)值顯著升高,CGRP+siRNA組相對(duì)于CGRP組,事先用siRNA處理過的細(xì)胞內(nèi)IL-6 mRNA的相對(duì)表達(dá)值顯著降低。3)Western-blot檢測(cè)IL-6蛋白的表達(dá)CGRP組相對(duì)于空白對(duì)照組,IL-6蛋白的表達(dá)量顯著升高,CGRP+siRNA組相對(duì)于CGRP組,事先用siRNA處理過的細(xì)胞內(nèi)IL-6蛋白的表達(dá)量顯著降低。這與qPCR的結(jié)果相一致。5.circRNA對(duì)IL-6上游miRNA作用的驗(yàn)證1)miRNA的篩選經(jīng)過mi RDB(www.mirdb.org)、David(david.ncifcrf.gov)和Target Scan(www.targetscan.org)生物信息學(xué)網(wǎng)站的篩選得出,mmu-mi R-485-5p最有可能是由mmu_circRNA_007893介導(dǎo)的,作用于IL-6 mRNA的miRNA。mmu-mi R-485-5p在該circRNA上具有至少5個(gè)結(jié)合位點(diǎn)。2)qPCR驗(yàn)證circRNA對(duì)選定miRNA表達(dá)水平的影響CGRP組相對(duì)于空白對(duì)照組,mmu-mi R-485-5p的相對(duì)表達(dá)值顯著降低,CGRP+siRNA組相對(duì)于CGRP組,事先用siRNA處理過的細(xì)胞內(nèi)mmu-mi R-485-5p的相對(duì)表達(dá)值顯著升高。6.目標(biāo)miRNA對(duì)IL-6表達(dá)作用的驗(yàn)證1)qPCR檢測(cè)IL-6 mRNA的表達(dá)CGRP組相對(duì)于空白對(duì)照組,IL-6 mRNA的相對(duì)表達(dá)值顯著升高,CGRP+mimics組相對(duì)于CGRP組,事先用mmu-mi R-485-5p過表達(dá)試劑處理過的細(xì)胞內(nèi)IL-6 mRNA的相對(duì)表達(dá)值顯著降低。CGRP+inhibitor組相對(duì)于CGRP組,事先用mmu-mi R-485-5p干擾試劑處理過的細(xì)胞內(nèi)IL-6 mRNA的相對(duì)表達(dá)值顯著升高。2)Western-blot檢測(cè)IL-6蛋白的表達(dá)CGRP組相對(duì)于空白對(duì)照組,IL-6蛋白的表達(dá)量顯著升高,CGRP+mimics組相對(duì)于CGRP組,事先用mmu-mi R-485-5p過表達(dá)試劑處理過的細(xì)胞內(nèi)IL-6蛋白的表達(dá)量顯著降低。CGRP+inhibitor組相對(duì)于CGRP組,事先用mmu-mi R-485-5p干擾試劑處理過的細(xì)胞內(nèi)IL-6蛋白的表達(dá)量顯著升高。這與qPCR的結(jié)果相一致。結(jié)論1.當(dāng)CGRP濃度為1nM時(shí),IL-6 mRNA相對(duì)表達(dá)水平最高。2.當(dāng)CGRP作用時(shí)間為2小時(shí)時(shí),IL-6 mRNA相對(duì)表達(dá)水平最高。3.使用選定濃度的CGRP刺激選定時(shí)間后,mmu_circRNA_007893的表達(dá)顯著升高。4.mmu_circRNA_007893主要存在于細(xì)胞質(zhì)當(dāng)中。5.mmu_circRNA_007893對(duì)IL-6 mRNA及蛋白的表達(dá)具有正向調(diào)節(jié)作用。6.mmu-mi R-485-5p是mmu_circRNA_007893調(diào)節(jié)的下游miRNA,且mmu_circRNA_007893對(duì)該miRNA具有負(fù)向調(diào)節(jié)的作用。7.mmu_circRNA_007893通過對(duì)mmu-mi R-485-5p表達(dá)的調(diào)控,從而影響IL-6mRNA及蛋白的表達(dá)。
[Abstract]:Objective: To investigate the effect of Calcitonin gene related peptide (CGRP) on the expression of interleukin -6 (Interleukin-6, IL-6) in macrophages mediated by RNA (CircularRNA, circRNA). Materials and methods: 1. cells and reagents were tested for RAW264.7 macrophage, using 10% fetal bovine serum. High glucose DMEM culture. Cleaning cells using phosphate buffer solution before changing liquid. The screening experiments using CGRP as the optimal concentration of alpha -CGRP.2.CGRP and the best action time set CGRP concentration gradient for 0.01nM, 0.1nM, 1nM, 10nM and 100nM respectively, acting on the macrophages with the same density of inoculation, and using real time fluorescence quantitation at the set time point. The nucleic acid amplification detection system (Real-time Quantitative PCR Detecting System, qPCR) was used to determine the expression of IL-6 mRNA; the experiment set the CGRP time gradient for 1 hours, 2 hours, 3 hours, 4 hours and 5 hours, acting on the same density of macrophages at the same concentration with the same concentration, and using qPCR to determine the expression of IL-6 mRNA at the corresponding time. After two steps above, the best concentration and time of CGRP were determined and the.3.circRNA chip was made and screened to inoculate the cells at the same density. After the selected concentration of CGRP was used to stimulate the selected time of RAW264.7 macrophages, the experimental group and the blank control group were fully cracked, the lysate was collected, and the samples were prepared and made by the biological company for circR. NA chip. According to the results provided by the company, the changed circRNA is screened according to the size of the change multiple or the number of miRNA binding sites, and the qPCR and its products are sequenced to verify the circRNA expression, and the fluorescence in situ hybridization (fluorescence in situ hybridization, FISH) is used to locate and verify circRNA. Finally determine the target circRNA.4.circRNA for further experiment to verify the role of IL-6 expression 1) circRNA interference experiment to inoculate the cells in the 6 orifice with the same density. Select 3 kinds of well designed small interference RNA (Small interferingRNA, siRNA) acting on the experimental group at the same concentration, respectively. After the stimulation for 48 hours, it fully cleavage each solid. The cells of the test group and the blank control group were used to detect the expression of the selected circRNA by qPCR, and finally determined the siRNA.2 qPCR used by the further experiment. The expression of IL-6 mRNA was inoculated in the 6 orifice with the same density. This part of the experiment was divided into blank control group, CGRP group and CGRP+siRNA group. The best interference effect was the best. SiRNA pretreated the cells of group CGRP+siRNA for 48 hours, and then used the selected concentration of CGRP to stimulate the cells of group CGRP and CGRP+siRNA group. After the selected optimum stimulation time, the cells were fully cracked and qPCR was used to detect the expression level.3 of IL-6 mRNA in each group. The expression of IL-6 protein was detected by the same density of the cells by the same density. The experiment was divided into 6 orifice plates. This part of the experiment was divided into blank control group, CGRP group and CGRP+siRNA group. The best siRNA with the best interference effect was pre treated for 48 hours in the CGRP+siRNA group. Then the selected concentration CGRP was used to stimulate the cells of the CGRP group and the CGRP+siRNA group. After the selected optimum stimulation time, the cells were fully cracked and Wester was used. N-blot detection of the expression level of IL-6 protein in each group.5.circRNA on the expression of RNA (microRNA, miRNA) in the downstream small molecules 1) miRNA was screened based on the results of circRNA chip and found the 5 target miRNA. of the selected circRNA, which was based on the bioinformatics website and software to predict the target gene and the inflammatory pathway involved, and found the correlation with IL-6. The miRNA and the next validation.2) qPCR verified the effect of circRNA on the selected miRNA expression level. The cells were inoculated in the 6 orifice with the same density. This part of the experiment was divided into blank control group, CGRP group and CGRP+siRNA group. The best siRNA with the best interference effect was pre treated with the cells of the CGRP+siRNA group for 48 hours, and then the selected concentration of CG was used. RP stimulated the cells of group CGRP and CGRP+siRNA. After the selected optimum stimulation time, the cells were fully cracked and qPCR was used to detect the expression level of miRNA in each group.6. target miRNA for IL-6 expression. 1) qPCR detection IL-6 mRNA was inoculated with the same density in the 6 orifice. This part of the experiment was divided into blank pairs. Group CGRP, group CGRP, group CGRP+mimics and group CGRP+inhibitor. Select the selected miRNA overexpression reagents (mimics) and interference reagent (inhibitor) to pretreat the cells of the corresponding group for 24 hours, and then use the selected concentration of CGRP to stimulate the cells of the CGRP+mimics group and the CGRP+inhibitor group. The expression level of IL-6 mRNA in each group was detected by qPCR) and the expression of IL-6 protein was detected by Western-blot. The cells were inoculated with the same density in 6 orifice plates at the same density. This part of the experiment was divided into blank control group, CGRP group, CGRP+mimics group and CGRP+inhibitor group. Selected miRNA overexpressed agent (mimics) and interference reagent (inhibitor) were pretreated. The cell of the corresponding group was 24 hours, and then the selected concentration of CGRP was used to stimulate the cells of group CGRP+mimics and CGRP+inhibitor. After the selected optimum stimulation time, the cells were fully cracked and the expression level of IL-6 protein in each group was detected by Western-blot, and the statistical analysis of the data was analyzed by SPSS19.0 statistics software. The data of each group were consistent with normal distribution and homogeneity of variance. The data of each group were paired bilateral t test. All the experiments were repeated 4 times. The results were expressed with mean mean standard deviation (? + s). The difference of bilateral P0.05 was statistically significant. Results: the culture method of the 1. cell culture experiment could be used to obtain the stable RAW264.7 macrophages with stable growth state. The cells were just round, and the refractive index was very good. Under the microscope, the cells were observed as small pearls scattered on the bottom of the culture utensils. After a period of time, the cells became polygons, irregular, and the pseudo foot shaped protuberances were observed. The results were in accordance with the biological characteristics of the RAW264.7 macrophage system, the optimum concentration of.2.CGRP and the optimum time of action of C. When the concentration of GRP is 0.1nM or 1nM, the relative expression level of IL-6 mRNA has a statistically significant difference compared with that of the control group. The relative expression level of IL-6 mRNA is the highest when CGRP concentration is 1nM. When CGRP action time is 2 hours or 3 hours, the relative expression level of IL-6 mRNA is statistically different from that of the control group. When the time of P action is 2 hours, the highest relative expression level of IL-6 mRNA is the highest.3.circRNA chip production and screening chip results, and the relative expression value up to 2 times of circRNA has 173, and the downregulation of more than 2 times a total of 179 circRNA, the circRNA is sorted or predicted by the size of the miRNA binding site according to the size of the change multiplier. After selection, by qPCR verification, the mmu_circRNA_007893 change multiple is the most stable and consistent with the chip results. The product sequencing and the circRNA connection site sequence consistent.FISH experiment proved that mmu_circRNA_007893 really exists in the cytoplasm, and the results of the chip are consistent with the results of.4.circRNA for IL-6 expression 1) circRNA interference experiment 3 Si. The expression of RNA has a significant inhibitory effect on the expression of mmu_circRNA_007893, in which the expression of siRNA3 has the strongest inhibitory effect on the expression of mmu_circRNA_007893. The expression of IL-6 mRNA by qPCR is relative to the blank control group, and the relative expression of IL-6 mRNA is significantly higher than that of the IL-6 mRNA. The expression of the relative expression value was significantly reduced by.3) Western-blot detection of IL-6 protein expression in the CGRP group compared with the blank control group, the expression of IL-6 protein increased significantly, and the CGRP+siRNA group compared with the CGRP group, the expression of IL-6 protein in the cells treated with siRNA in advance decreased significantly. This is consistent with the results of qPCR.5.circRNA on upstream IL-6. Validation 1) miRNA screening through the screening of the MI RDB (www.mirdb.org), David (david.ncifcrf.gov) and Target Scan (www.targetscan.org) bioinformatics web site, the mmu-mi R-485-5p is most likely to be mediated by mmu_circRNA_007893, which has at least 5 binding sites on it. The effect of circRNA on the level of selected miRNA expression in CGRP group relative to the blank control group, the relative expression value of mmu-mi R-485-5p decreased significantly, the CGRP+siRNA group relative to the CGRP group, the relative expression value of mmu-mi R-485-5p in the cells treated with siRNA in advance was significantly higher than that of.6. target miRNA. The relative expression value of IL-6 mRNA in the CGRP group was significantly higher than that in the blank control group. The relative expression value of IL-6 mRNA in the cells treated with mmu-mi R-485-5p overexpression reagents in advance was significantly lower than that in the CGRP group compared with the CGRP group, and the.CGRP+inhibitor group was compared with the CGRP group. The relative expression value of intracellular IL-6 mRNA increased significantly by.2) Western-blot detection of IL-6 protein expression in CGRP group compared with blank control group, the expression of IL-6 protein increased significantly. CGRP+mimics group compared with CGRP group, the expression of IL-6 protein in cells treated with mmu-mi R-485-5p overexpression reagents decreased significantly. For the CGRP group, the expression of IL-6 protein in cells treated with mmu-mi R-485-5p interfering reagent increased significantly. This is in accordance with the results of qPCR. Conclusion 1. when CGRP concentration is 1nM, the highest level of IL-6 mRNA relative expression level is 2 hours when CGRP action time, IL-6 facies is the highest expression level with selected concentration After the selection of time, the expression of mmu_circRNA_007893 increased significantly,.4.mmu_circRNA_007893 mainly existed in the cytoplasm,.5.mmu_circRNA_007893 had a positive regulation on the expression of IL-6 mRNA and protein,.6.mmu-mi R-485-5p was the downstream miRNA of mmu_circRNA_007893 regulation, and mmu_circRNA_007893 was negatively regulated for miRNA. .7.mmu_circRNA_007893 regulates the expression of mmu-mi and R-485-5p, thereby affecting the expression of IL-6mRNA and protein.
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R781.4

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