血小板来源微泡中miRNA-223参与小鼠动脉粥样硬化的机制研究

发布时间：2018-04-22 20:22

本文选题：ApoE基因敲除小鼠 + 微小RNA　；参考：《第二军医大学》2017年硕士论文

【摘要】：一、研究背景微泡主要存在于血小板中,内部含有多种micro RNAs、蛋白质等生物活性物质,参与细胞之间的信号转导。大量研究表明,在动脉粥样硬化及其并发症的患者的血液中,微泡的含量明显增多。Micro RNAs是目前广泛研究的基因表达调控因子,其结构为长约22~25核苷酸的单链RNA分子。近年来,miRNAs在心血管系统中的重要调控作用不断得以阐明,取得了令人瞩目的研究成果。我们前期研究发现,血小板在被激活以后,其分泌的微泡中多种miRNAs的含量发生改变,且可以被内皮细胞吸收,从而影响其增殖、迁移、凋亡、分泌。在这当中,miRNA-223的含量改变最为显著。随着研究的进展,miRNA-223在动脉粥样硬化过程中发挥的作用越来越受到重视,作用机制也越来越明确,有可能成为诊断和治疗动脉粥样硬化的新型生物靶点。二、研究目的1、以C57BL/6纯系Apo E基因敲除小鼠为基本研究对象,建立适当的动物实验模型并进行分组,为探索PMP中miRNA-223在动脉粥样硬化内皮损伤修复机制提供可靠有力的动物实验基础。2、检测对比各组小鼠之间血脂水平以及血小板来源的微泡中miRNA-223含量的差异。3、分析各组小鼠动脉粥样硬化病变程度,初步探索动脉粥样硬化病变程度与血小板来源微泡中miRNA-223含量之间的关系。三、研究方法1、以6周龄左右的C57BL/6纯系背景的载脂蛋白E基因敲除(Apo E-/-)雄性小鼠12只以及同周龄的C57BL/6普通雄性小鼠6只为研究对象,在相同的饲养条件下分成三组,即给予高脂饮食,尾静脉注射miRNA-223 antagomir抑制miRNA-223转录表达的Apo E-/-小鼠组(AG组);给予高脂饮食,经尾静脉注入同剂量生理盐水的Apo E-/-小鼠组(AE组);给予SPF全价营养鼠料饲养,经尾静脉注入同剂量生理盐水的普通小鼠组(对照组)。2、饲养至12周后对三组小鼠进行称重,处死,取血,离心取上清液,使用全自动生化分析仪检测各组小鼠体内血脂水平;提取小鼠血液中的血小板微泡,实时定量PCR测定PMP中miRNA-223的含量。3、对各组小鼠主动脉粥样硬化情况进行标本大体观察,HE染色后光学显微镜下病理观察以及使用医学数码图像分析系统对产生斑块的厚度、面积进行定量分析。四、研究结果1、小鼠自购入经喂养、处置至处死前,18只实验小鼠均成功存活,生命迹象良好,无死亡、疾病、厌食等不良状况。对三组小鼠分别进行称重,AG组为(27.21±2.05)g,AE组为(27.72±2.59)g,对照组为(23.51±1.55)g。称重结果显示:AG组与AE组小鼠体重无明显统计学差异(P0.05),AG组与对照组、AE组与对照组体重有统计学差异(P0.05,P0.05),AG、AE两组小鼠体重水平均高于对照组小鼠。2、通过梯度离心、超速离心、逆转录以及定量PCR等方法对三组小鼠体内PMP中miRNA-223相对水平进行检测对比,结果显示:AG组小鼠体内PMP中miRNA-223的含量较其它两组明显下降,而AE组含量较对照组升高。折换成倍数显示:AG组与对照组为(0.14±0.06vs1.02±0.09,P0.05);AG与AE组为(0.14±0.06vs2.71±0.44,P0.05),AE组与对照组为(2.71±0.44vs1.02±0.09,P0.05)。对各组小鼠体内的血脂水平检测结果显示,三组小鼠的TG、TC、LDL-C、HDL-C水平分别为AG组(0.60±0.08mmol/L,11.03±1.29 mmol/L,5.06±0.86 mmol/L,0.75±0.99 mmol/L),AE组(0.61±0.10 mmol/L,11.07±1.53 mmol/L,5.13±1.13 mmol/L,0.70±0.99 mmol/L),对照组(0.34±0.70 mmol/L,3.18±0.54 mmol/L,1.59±0.36 mmol/L,2.28±0.37 mmol/L)。AG组与AE组小鼠TG、TC以及LDL-C和HDL-C水平均无明显统计学差异(P0.05),与对照组相比AG组与AE组的TG、TC以及LDL-C水平均明显升高且有统计学差异(P0.05,P0.05),HDL-C水平则较对照组明显降低且差异有统计学意义(P0.05,P0.05)。3、对小鼠主动脉动脉粥样硬化病变大体观察以及光学显微镜下观察病理改变,发现AG组与AE组小鼠几乎都出现不同程度的动脉粥样硬化,而对照组小鼠几乎无动脉粥样硬化改变。使用医学数码图像分析系统对AG组、AE组小鼠动脉粥样硬化斑块的厚度以及斑块面积进行定量分析显示,两组小鼠斑块厚度和面积分别为:AG组:(0.095±0.013)mm,(0.075±0.015)mm2;AE组:(0.104±0.015)mm,(0.080±0.014)mm2。AG组与AE组小鼠动脉粥样硬化斑块的厚度与斑块面积差异均无统计学意义(P0.05,P0.05)。将AG组与AE组所有小鼠(n=12)的动脉粥样硬化斑块面积与体内相应的PMP中miRNA-223的相对含量进行相关性比较,结果显示:AG组与AE组间比较两者之间相关性(r=0.22,P0.05),AG组组内比较(r=0.36,P0.05),AE组组内比较(r=0.06,P0.05)。PMP中miRNA-223的相对水平与AS斑块面积无明显相关性。五、结论1、三组实验小鼠均可以成功存活,且AG组与AE组通过高脂饲养体内已出现了不同程度的动脉粥样硬化改变。2、AE组动脉粥样硬化小鼠体内的PMP中miRNA-223的含量较正常对照小鼠升高;miRNA-223的转录合成可以被miRNA-223 antagomir所抑制,AG组小鼠体内PMP中miRNA-223的含量明显低于AE组和对照组小鼠。3、AG组与AE组小鼠在体重、TC、TG、LDL-C、动脉粥样硬化病变程度上均高于对照组,HDL-C低于对照组;AG组与AE组小鼠之间体重、血脂水平以及动脉粥样硬化病变程度均无统计学差异。Apo E上可能存在着PMP中miRNA-223对血脂水平调节的作用靶点。PMP中miRNA-223可能主要参与动脉粥样硬化的启动环节,在已经发生动脉粥样硬化改变的AG组和AE组小鼠体内,动脉粥样硬化的病变程度与PMP中miRNA-223的含量无明显相关性。
[Abstract]:First, the background microbubbles mainly exist in the platelets, which contain a variety of micro RNAs, protein and other bioactive substances, and participate in signal transduction between cells. A large number of studies have shown that in the blood of patients with atherosclerosis and its complications, the content of microbubbles is significantly increased.Micro RNAs is a widely studied gene expression. The structure is a single strand RNA molecule with a length of about 22~25 nucleotides. In recent years, the important regulatory role of miRNAs in the cardiovascular system has been clarified, and remarkable research results have been obtained. The skin cells are absorbed, which affect their proliferation, migration, apoptosis, and secretion. In this, the miRNA-223 content changes most significantly. With the progress of the study, the role of miRNA-223 in atherosclerosis is becoming more and more important, the mechanism of action is becoming more and more clear, and it may become a new type of diagnosis and treatment of atherosclerosis. Target target. Two, research objective 1, using C57BL/6 pure Apo E gene knockout mice as the basic research object, establish appropriate animal experimental model and group, in order to explore the mechanism of miRNA-223 in the atherosclerosis of atherosclerotic endothelial injury to provide a reliable and powerful animal experimental basis for animal experiment.2, test and compare the levels of blood lipids among mice in each group. The difference of miRNA-223 content in the microbubbles from the platelets was.3, and the degree of atherosclerotic lesions in the mice was analyzed. The relationship between the degree of atherosclerotic lesions and the miRNA-223 content in the platelet derived microbubbles was preliminarily explored. Three, the study method 1, the apolipoprotein E gene knockout (Apo E-/-) in the C57BL/6 system background of 6 weeks old. 12 male mice and 6 C57BL/6 male mice of the same age were divided into three groups, which were divided into three groups under the same feeding condition, that is, high fat diet was given, and the tail vein was injected with miRNA-223 antagomir to inhibit the miRNA-223 transcriptional expression of Apo E-/- mice group (group AG); the high fat diet was given, and the same dose of the normal saline was injected into the Apo E-/ through the tail vein. The mice group (group AE) was fed with SPF total nutritive rats and injected into the normal mice group (control group) with the same dose of saline by the tail vein (control group).2. After feeding to 12 weeks, the three groups of mice were weighed, killed, taken blood, and centrifuged to take the supernatant, and the blood lipid levels were detected by the automatic biochemical analyzer, and the blood of mice was extracted. Plate microbubbles, real-time quantitative PCR determination of the content of miRNA-223 in PMP,.3. The aorta atherosclerosis in each group of mice was observed. After HE staining optical microscope pathological observation and the use of medical digital image analysis system to produce plaque thickness, area for quantitative analysis. Four, study results 1, mice bought from the feed Before the treatment, 18 experimental mice were successfully survived, with good signs of life, good signs of life, no death, disease, anorexia and other adverse conditions. Three groups of mice were weighed, AG group was (27.21 + 2.05) g, AE group was (27.72 + 2.59) g, and the control group was (23.51 + 1.55) g. weight results showed no significant difference between group AG and AE group (P0.05), AG (P0.05), AG The weight of group AE and control group was statistically different (P0.05, P0.05), AG, AG, AE two groups were higher than the control group of mice.2, through gradient centrifugation, overspeed centrifugation, reverse transcription and quantitative PCR and other methods to compare the miRNA-223 relative water level in the PMP of the three groups of mice, the results showed that AG group mice in PMP miRN. The content of A-223 was significantly lower than that of the other two groups, but the content of AE group was higher than that of the control group. The fold change multiple showed that the AG group and the control group were (0.14 + 0.06vs1.02 + 0.09, P0.05), AG and AE group were (0.14 + 0.06vs2.71 + 0.44, P0.05), AE and control groups were (2.71 + 0.44vs1.02 + 0.09, P0.05). The levels of TG, TC, LDL-C, and HDL-C in the three groups were AG (0.60 + 0.08mmol/L, 11.03 + 1.29 mmol/L, 5.06 + 0.86 mmol/L, 0.75 + 0.99 mmol/L), AE group (0.61 + 0.10 mmol/L, 11.07 + 1.53 mmol/L, 5.13 +) There was no significant difference in the level of TG, TC, LDL-C and HDL-C in the group of mice (P0.05). Compared with the control group, the level of TG, TC and LDL-C in the AG group and AE group increased significantly and had statistical differences (P0.05, P0.05), and the level was significantly lower than the control group and the difference has statistical significance. General observation of pathological changes and observation of pathological changes under optical microscope showed that almost all the atherosclerotic changes were found in group AG and AE mice, while the control group had almost no atherosclerosis. The thickness of atherosclerotic plaque and the area of atherosclerotic plaques in group AG, AE group and group of mice were treated with medical digital image analysis system. Quantitative analysis showed that the thickness and area of plaque in the two groups were: group AG: (0.095 + 0.013) mm, (0.075 + 0.015) mm2, AE group: (0.104 + 0.015) mm, (0.080 + 0.014) mm2.AG and AE group, the thickness of atherosclerotic plaque in group AE was not statistically significant (P0.05, P0.05). The artery of AG group and AE group (n=12) artery The correlation between the area of atherosclerotic plaque and the relative content of miRNA-223 in PMP was compared. The results showed that the correlation between the AG group and the AE group (r=0.22, P0.05), the comparison of the AG group (r=0.36, P0.05), and the relative level of the AE group (r=0.06, P0.05) had no significant correlation with the area of the plaque. Five, Conclusion 1, three groups of experimental mice can survive successfully, and the AG and AE groups have different degrees of atherosclerotic changes in.2 through high fat feeding, and the content of miRNA-223 in PMP in the AE group of atherosclerotic mice is higher than that in the normal control mice; miRNA-223 transcriptional synthesis can be inhibited by miRNA-223 antagomir, AG group. The content of miRNA-223 in PMP in mice was significantly lower than that of group AE and control group.3. The weight, TC, TG, LDL-C, and atherosclerotic lesions of group AG and AE mice were higher than those of the control group, and HDL-C was lower than that of the control group. There was no statistical difference between the AG group and the AE group mice. The effect of miRNA-223 on the regulation of blood lipid levels in PMP may be the target of miRNA-223, which may be mainly involved in the initiation of atherosclerosis, and there is no significant correlation between the degree of atherosclerosis and the content of miRNA-223 in PMP in the AG and AE mice that have undergone atherosclerotic changes.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R543.5

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