降钙素基因相关肽对环状RNA介导的白细胞介素-6表达的研究
发布时间：2018-07-24 07:53
【摘要】：目的:探讨降钙素基因相关肽(Calcitonin gene related peptide,CGRP)对环状RNA(CircularRNA,circRNA)介导的巨噬细胞内白细胞介素-6(Interleukin-6,IL-6)表达的影响。材料与方法:1.细胞和试剂本实验的实验对象为RAW264.7巨噬细胞系,使用含有10%胎牛血清的高糖DMEM培养。换液前使用磷酸盐缓冲液清洗细胞。实验使用的CGRP为α-CGRP。2.CGRP最佳作用浓度和最佳作用时间的筛选实验设定CGRP浓度梯度为0.01nM、0.1nM、1nM、10nM和100nM,分别作用于接种密度相同的巨噬细胞,在设定时间点使用实时荧光定量核酸扩增检测系统(Real-time Quantitative PCR Detecting System,qPCR)来测定IL-6 mRNA的表达;实验设定CGRP时间梯度为1小时、2小时、3小时、4小时和5小时,分别以相同浓度作用于接种密度相同的巨噬细胞,在相应时间后使用qPCR来测定IL-6 mRNA的表达,通过以上两个步骤确定CGRP作用的最佳浓度和时间。3.circRNA芯片的制作和筛选将细胞以相同密度接种培养,用选定浓度的CGRP刺激RAW264.7巨噬细胞选定时间后,充分裂解实验组和空白对照组细胞,收集裂解液、制备样本并交由生物公司做circRNA芯片。根据该公司提供的芯片结果,将发生改变的circRNA按照改变倍数的大小或预测miRNA结合位点的多寡进行筛选,并通过qPCR及其产物测序进行circRNA表达的验证,采用荧光原位杂交技术(fluorescence in situ hybridization,FISH)定位和证实circRNA,最终确定进一步实验的目标circRNA。4.circRNA对IL-6表达作用的验证1)circRNA的干扰实验将细胞以相同密度接种于6孔板中。选择3种设计好的小干扰RNA(Small interferingRNA,siRNA)以相同浓度分别作用于对应实验组。刺激48小时以后,充分裂解各实验组和空白对照组细胞,使用qPCR检测各组选定circRNA的表达情况,并最终确定进一步实验所使用的siRNA。2)qPCR检测IL-6 mRNA的表达将细胞以相同密度接种于6孔板中。本部分实验分为空白对照组、CGRP组和CGRP+siRNA组。选择上一步干扰效果最好的siRNA预先处理CGRP+siRNA组的细胞48小时,然后使用选定浓度的CGRP刺激CGRP组和CGRP+siRNA组的细胞。刺激选定的最佳刺激时间后,充分裂解各组细胞并使用qPCR检测各组IL-6 mRNA的表达水平。3)Western-blot检测IL-6蛋白的表达将细胞以相同密度接种于6孔板中。本部分实验分为空白对照组、CGRP组和CGRP+siRNA组。选择干扰效果最好的siRNA预先处理CGRP+siRNA组的细胞48小时,然后使用选定浓度的CGRP刺激CGRP组和CGRP+siRNA组的细胞。刺激选定的最佳刺激时间后,充分裂解各组细胞并使用Western-blot检测各组IL-6蛋白的表达水平。5.circRNA对下游小分子RNA(microRNA,miRNA)表达作用的验证1)miRNA的筛选根据circRNA芯片结果,找到选定的circRNA对应的5个目标miRNA。根据生物信息学网站和软件预测其靶基因和参与的炎症通路,找到与IL-6相关的miRNA并进行下一步的验证。2)qPCR验证circRNA对选定miRNA表达水平的影响将细胞以相同密度接种于6孔板中。本部分实验分为空白对照组、CGRP组和CGRP+siRNA组。选择干扰效果最好的siRNA预先处理CGRP+siRNA组的细胞48小时,然后使用选定浓度的CGRP刺激CGRP组和CGRP+siRNA组的细胞。刺激选定的最佳刺激时间后,充分裂解各组细胞并使用qPCR检测各组选定miRNA的表达水平。6.目标miRNA对IL-6表达作用的验证1)qPCR检测IL-6 mRNA的表达将细胞以相同密度接种于6孔板中。本部分实验分为空白对照组、CGRP组、CGRP+mimics组和CGRP+inhibitor组。选择选定miRNA的过表达试剂(mimics)和干扰试剂(inhibitor)预先处理对应组的细胞24小时,然后使用选定浓度的CGRP刺激CGRP+mimics组和CGRP+inhibitor组的细胞。刺激选定的最佳刺激时间后,充分裂解各组细胞并使用qPCR检测各组IL-6 mRNA的表达水平。2)Western-blot检测IL-6蛋白的表达将细胞以相同密度接种于6孔板中。本部分实验分为空白对照组、CGRP组、CGRP+mimics组和CGRP+inhibitor组。选择选定miRNA的过表达试剂(mimics)和干扰试剂(inhibitor)预先处理对应组的细胞24小时,然后使用选定浓度的CGRP刺激CGRP+mimics组和CGRP+inhibitor组的细胞。刺激选定的最佳刺激时间后,充分裂解各组细胞并使用Western-blot检测各组IL-6蛋白的表达水平。7.统计学分析采用SPSS19.0统计软件对数据进行统计分析,各组数据符合正态分布,方差齐,对各组数据进行配对双侧t检验,所有实验均重复4次,结果以均值±标准差(?±s)表示,以双侧P0.05为差异有统计学意义。结果:1.细胞培养本实验的培养方法可以获得生长状态稳定良好的RAW264.7巨噬细胞。该细胞刚贴壁时呈圆形,折光度很好,镜下观察如小珍珠状散落于培养器皿底面。贴壁培养一段时间后,细胞会变为多边形,不规则,可见伪足状突起。该结果符合RAW264.7巨噬细胞系的生物学特性。2.CGRP最佳作用浓度和最佳作用时间的筛选当CGRP作用浓度为0.1nM或1nM时,IL-6 mRNA的相对表达水平与对照组的相比具有统计学差异。其中,当CGRP浓度为1nM时,IL-6 mRNA相对表达水平最高。当CGRP作用时间为2小时或3小时时,IL-6 mRNA的相对表达水平与对照组的相比具有统计学差异。其中,当CGRP作用时间为2小时时,IL-6 mRNA相对表达水平最高。3.circRNA芯片的制作和筛选芯片结果表示,相对表达值上调超过2倍的circRNA共有173个,下调超过2倍的circRNA共有179个。将这些circRNA按照改变倍数的大小排序或预测miRNA结合位点的多寡进行筛选后,通过qPCR验证,mmu_circRNA_007893的改变倍数最稳定,与芯片结果相符,产物测序与circRNA连接位点序列一致。FISH实验证明mmu_circRNA_007893确实绝大多数存在于细胞质中,与芯片结果一致。4.circRNA对IL-6表达作用的验证1)circRNA的干扰实验3种siRNA对mmu_circRNA_007893的表达都有显著抑制作用,其中siRNA3对mmu_circRNA_007893的表达抑制作用最强。2)qPCR检测IL-6 mRNA的表达CGRP组相对于空白对照组,IL-6 mRNA的相对表达值显著升高,CGRP+siRNA组相对于CGRP组,事先用siRNA处理过的细胞内IL-6 mRNA的相对表达值显著降低。3)Western-blot检测IL-6蛋白的表达CGRP组相对于空白对照组,IL-6蛋白的表达量显著升高,CGRP+siRNA组相对于CGRP组,事先用siRNA处理过的细胞内IL-6蛋白的表达量显著降低。这与qPCR的结果相一致。5.circRNA对IL-6上游miRNA作用的验证1)miRNA的筛选经过mi RDB(www.mirdb.org)、David(david.ncifcrf.gov)和Target Scan(www.targetscan.org)生物信息学网站的筛选得出,mmu-mi R-485-5p最有可能是由mmu_circRNA_007893介导的,作用于IL-6 mRNA的miRNA。mmu-mi R-485-5p在该circRNA上具有至少5个结合位点。2)qPCR验证circRNA对选定miRNA表达水平的影响CGRP组相对于空白对照组,mmu-mi R-485-5p的相对表达值显著降低,CGRP+siRNA组相对于CGRP组,事先用siRNA处理过的细胞内mmu-mi R-485-5p的相对表达值显著升高。6.目标miRNA对IL-6表达作用的验证1)qPCR检测IL-6 mRNA的表达CGRP组相对于空白对照组,IL-6 mRNA的相对表达值显著升高,CGRP+mimics组相对于CGRP组,事先用mmu-mi R-485-5p过表达试剂处理过的细胞内IL-6 mRNA的相对表达值显著降低。CGRP+inhibitor组相对于CGRP组,事先用mmu-mi R-485-5p干扰试剂处理过的细胞内IL-6 mRNA的相对表达值显著升高。2)Western-blot检测IL-6蛋白的表达CGRP组相对于空白对照组,IL-6蛋白的表达量显著升高,CGRP+mimics组相对于CGRP组,事先用mmu-mi R-485-5p过表达试剂处理过的细胞内IL-6蛋白的表达量显著降低。CGRP+inhibitor组相对于CGRP组,事先用mmu-mi R-485-5p干扰试剂处理过的细胞内IL-6蛋白的表达量显著升高。这与qPCR的结果相一致。结论1.当CGRP浓度为1nM时,IL-6 mRNA相对表达水平最高。2.当CGRP作用时间为2小时时,IL-6 mRNA相对表达水平最高。3.使用选定浓度的CGRP刺激选定时间后,mmu_circRNA_007893的表达显著升高。4.mmu_circRNA_007893主要存在于细胞质当中。5.mmu_circRNA_007893对IL-6 mRNA及蛋白的表达具有正向调节作用。6.mmu-mi R-485-5p是mmu_circRNA_007893调节的下游miRNA,且mmu_circRNA_007893对该miRNA具有负向调节的作用。7.mmu_circRNA_007893通过对mmu-mi R-485-5p表达的调控,从而影响IL-6mRNA及蛋白的表达。
[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.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.4
本文编号：2140720
[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.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.4
【参考文献】
相关期刊论文 前8条
1 慈浩粟;;降钙素基因相关肽在牙周炎患者牙龈组织中的表达[J];牙体牙髓牙周病学杂志;2014年09期
2 杨宏宇;;侵袭性牙周炎患者治疗前后血清GM-CSF、IL-2和CGRP水平检测的临床意义[J];淮海医药;2013年06期
3 乔虎;周洪;朱永进;高宇男;;大鼠正畸牙移动过程中牙周组织内CGRP的表达变化[J];上海口腔医学;2012年06期
4 李丹;任亚娜;范华骅;;巨噬细胞的分类及其调节性功能的差异[J];生命科学;2011年03期
5 李云超;管茶香;周漾;孙国瑛;施萌;邬晶;李文杰;;降钙素基因相关肽对脂多糖诱导的巨噬细胞表达髓样细胞触发受体-1的影响(英文)[J];中南大学学报(医学版);2010年02期
6 李灵敏;曹志中;沈霖德;;基于连接酶检测反应的CGRP和IL-1A基因多态与重度慢性牙周炎的相关性研究[J];牙体牙髓牙周病学杂志;2008年05期
7 刘永平;管茶香;白洪波;张敏;崔艳茹;刘惠君;张长青;;降钙素基因相关肽对LPS诱导肺泡巨噬细胞分泌MMP-9的影响[J];中国应用生理学杂志;2007年02期
8 吴军;张海鸥;陈玉丙;周芬莉;李珂静;;表达降钙素基因相关肽的单纯疱疹病毒Ⅰ型扩增子载体免疫效果观察[J];中国实验诊断学;2006年10期
，本文编号：2140720
本文链接：https://www.wllwen.com/yixuelunwen/kouq/2140720.html
最近更新
教材专著
