本文選題：ApoE基因敲除小鼠 + 微小RNA�。� 參考：《第二軍醫(yī)大學》2017年碩士論文

【摘要】：一、研究背景微泡主要存在于血小板中,內部含有多種micro RNAs、蛋白質等生物活性物質,參與細胞之間的信號轉導。大量研究表明,在動脈粥樣硬化及其并發(fā)癥的患者的血液中,微泡的含量明顯增多。Micro RNAs是目前廣泛研究的基因表達調控因子,其結構為長約22~25核苷酸的單鏈RNA分子。近年來,miRNAs在心血管系統(tǒng)中的重要調控作用不斷得以闡明,取得了令人矚目的研究成果。我們前期研究發(fā)現(xiàn),血小板在被激活以后,其分泌的微泡中多種miRNAs的含量發(fā)生改變,且可以被內皮細胞吸收,從而影響其增殖、遷移、凋亡、分泌。在這當中,miRNA-223的含量改變最為顯著。隨著研究的進展,miRNA-223在動脈粥樣硬化過程中發(fā)揮的作用越來越受到重視,作用機制也越來越明確,有可能成為診斷和治療動脈粥樣硬化的新型生物靶點。二、研究目的1、以C57BL/6純系Apo E基因敲除小鼠為基本研究對象,建立適當?shù)膭游飳嶒災Ｐ筒⑦M行分組,為探索PMP中miRNA-223在動脈粥樣硬化內皮損傷修復機制提供可靠有力的動物實驗基礎。2、檢測對比各組小鼠之間血脂水平以及血小板來源的微泡中miRNA-223含量的差異。3、分析各組小鼠動脈粥樣硬化病變程度,初步探索動脈粥樣硬化病變程度與血小板來源微泡中miRNA-223含量之間的關系。三、研究方法1、以6周齡左右的C57BL/6純系背景的載脂蛋白E基因敲除(Apo E-/-)雄性小鼠12只以及同周齡的C57BL/6普通雄性小鼠6只為研究對象,在相同的飼養(yǎng)條件下分成三組,即給予高脂飲食,尾靜脈注射miRNA-223 antagomir抑制miRNA-223轉錄表達的Apo E-/-小鼠組(AG組);給予高脂飲食,經(jīng)尾靜脈注入同劑量生理鹽水的Apo E-/-小鼠組(AE組);給予SPF全價營養(yǎng)鼠料飼養(yǎng),經(jīng)尾靜脈注入同劑量生理鹽水的普通小鼠組(對照組)。2、飼養(yǎng)至12周后對三組小鼠進行稱重,處死,取血,離心取上清液,使用全自動生化分析儀檢測各組小鼠體內血脂水平;提取小鼠血液中的血小板微泡,實時定量PCR測定PMP中miRNA-223的含量。3、對各組小鼠主動脈粥樣硬化情況進行標本大體觀察,HE染色后光學顯微鏡下病理觀察以及使用醫(yī)學數(shù)碼圖像分析系統(tǒng)對產(chǎn)生斑塊的厚度、面積進行定量分析。四、研究結果1、小鼠自購入經(jīng)喂養(yǎng)、處置至處死前,18只實驗小鼠均成功存活,生命跡象良好,無死亡、疾病、厭食等不良狀況。對三組小鼠分別進行稱重,AG組為(27.21±2.05)g,AE組為(27.72±2.59)g,對照組為(23.51±1.55)g。稱重結果顯示:AG組與AE組小鼠體重無明顯統(tǒng)計學差異(P0.05),AG組與對照組、AE組與對照組體重有統(tǒng)計學差異(P0.05,P0.05),AG、AE兩組小鼠體重水平均高于對照組小鼠。2、通過梯度離心、超速離心、逆轉錄以及定量PCR等方法對三組小鼠體內PMP中miRNA-223相對水平進行檢測對比,結果顯示:AG組小鼠體內PMP中miRNA-223的含量較其它兩組明顯下降,而AE組含量較對照組升高。折換成倍數(shù)顯示:AG組與對照組為(0.14±0.06vs1.02±0.09,P0.05);AG與AE組為(0.14±0.06vs2.71±0.44,P0.05),AE組與對照組為(2.71±0.44vs1.02±0.09,P0.05)。對各組小鼠體內的血脂水平檢測結果顯示,三組小鼠的TG、TC、LDL-C、HDL-C水平分別為AG組(0.60±0.08mmol/L,11.03±1.29 mmol/L,5.06±0.86 mmol/L,0.75±0.99 mmol/L),AE組(0.61±0.10 mmol/L,11.07±1.53 mmol/L,5.13±1.13 mmol/L,0.70±0.99 mmol/L),對照組(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組與AE組小鼠TG、TC以及LDL-C和HDL-C水平均無明顯統(tǒng)計學差異(P0.05),與對照組相比AG組與AE組的TG、TC以及LDL-C水平均明顯升高且有統(tǒng)計學差異(P0.05,P0.05),HDL-C水平則較對照組明顯降低且差異有統(tǒng)計學意義(P0.05,P0.05)。3、對小鼠主動脈動脈粥樣硬化病變大體觀察以及光學顯微鏡下觀察病理改變,發(fā)現(xiàn)AG組與AE組小鼠幾乎都出現(xiàn)不同程度的動脈粥樣硬化,而對照組小鼠幾乎無動脈粥樣硬化改變。使用醫(yī)學數(shù)碼圖像分析系統(tǒng)對AG組、AE組小鼠動脈粥樣硬化斑塊的厚度以及斑塊面積進行定量分析顯示,兩組小鼠斑塊厚度和面積分別為:AG組:(0.095±0.013)mm,(0.075±0.015)mm2;AE組:(0.104±0.015)mm,(0.080±0.014)mm2。AG組與AE組小鼠動脈粥樣硬化斑塊的厚度與斑塊面積差異均無統(tǒng)計學意義(P0.05,P0.05)。將AG組與AE組所有小鼠(n=12)的動脈粥樣硬化斑塊面積與體內相應的PMP中miRNA-223的相對含量進行相關性比較,結果顯示:AG組與AE組間比較兩者之間相關性(r=0.22,P0.05),AG組組內比較(r=0.36,P0.05),AE組組內比較(r=0.06,P0.05)。PMP中miRNA-223的相對水平與AS斑塊面積無明顯相關性。五、結論1、三組實驗小鼠均可以成功存活,且AG組與AE組通過高脂飼養(yǎng)體內已出現(xiàn)了不同程度的動脈粥樣硬化改變。2、AE組動脈粥樣硬化小鼠體內的PMP中miRNA-223的含量較正常對照小鼠升高;miRNA-223的轉錄合成可以被miRNA-223 antagomir所抑制,AG組小鼠體內PMP中miRNA-223的含量明顯低于AE組和對照組小鼠。3、AG組與AE組小鼠在體重、TC、TG、LDL-C、動脈粥樣硬化病變程度上均高于對照組,HDL-C低于對照組;AG組與AE組小鼠之間體重、血脂水平以及動脈粥樣硬化病變程度均無統(tǒng)計學差異。Apo E上可能存在著PMP中miRNA-223對血脂水平調節(jié)的作用靶點。PMP中miRNA-223可能主要參與動脈粥樣硬化的啟動環(huán)節(jié),在已經(jīng)發(fā)生動脈粥樣硬化改變的AG組和AE組小鼠體內,動脈粥樣硬化的病變程度與PMP中miRNA-223的含量無明顯相關性。
[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.

【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R543.5

【參考文獻】

相關期刊論文 前2條

1 Amitesh Aggarwal;Saurabh Srivastava;M Velmurugan;;Newer perspectives of coronary artery disease in young[J];World Journal of Cardiology;2016年12期

2 Heng-Song Tian;Qing-Guo Zhou;Fang Shao;;Relationship between arterial atheromatous plaque morphology and platelet-associated miR-126 and miR-223 expressions[J];Asian Pacific Journal of Tropical Medicine;2015年04期

，

本文編號：1788765