慢病毒介导microRNA-126基因敲减治疗高血压病的可行性研究

发布时间：2018-04-13 17:41

本文选题：原发性高血压 + microRNA-126　；参考：《首都医科大学》2017年博士论文

【摘要】：【目的】1.通过基因芯片检测人类多种micro RNA,发现micro RNA与高血压病的相关性;2.通过构建micro RNA-126基因敲减慢病毒载体,探讨micro RNA-126作为基因靶点治疗高血压病的可行性;3.阐述micro RNA-126在原发性高血压中发生发展的作用机制。【方法】收集年龄、性别、体重等相匹配的6例原发性高血压病患者和4例健康人血液标本,委托上海康成生物公司micro RNA芯片检测两组样本micro RNA表达谱。采用人类免疫缺陷病毒-1(HIV-1)为来源的慢病毒载体,构建mi R-126基因敲减慢病毒载体(lenti-mi R-126-KD)及干扰对照慢病毒载体(lenti-scramblemir)。自发性高血压大鼠(SHR)分为高剂量组(1*108,n=5)、低剂量组(1*107,n=6)、干扰对照组(5*107,n=6)、空白对照组(PBS 350ul,n=6),经尾静脉注射后,连续观察8周应用尾袖法采集收缩压、舒张压、平均压及心率,8周后计算心体比值(Hw/Bw),留取心、肾、脑、肝、肺组织标本进行冰冻切片,并在荧光显微镜下观察慢病毒载体在体内的分布情况,应用天狼星红苦味酸染色及HE染色,观察高血压靶器官损害情况;应用ELISA法测定大鼠血清一氧化氮(NO)含量及各种重要生化指标。【结果】1.micro RNA-126的3’末端(1.410 vs 0.838,P=0.03)以及5’末端(1.799 vs0.997,P=0.03)在高血压病患者中呈高表达,在健康人中呈低表达,且两组间有显著差异,P0.05;2.慢病毒载体构建及表达:慢病毒载体构建成功。在给药后8周,各组织冰冻切片均可观测到绿色荧光蛋白表达;3.血压、心率:给药前,各组无统一学差异(P0.05),给药后,每周观察血压、心率变化情况,各组间均无统计学差异(P0.05);4.心体比值(mg/g):高剂量组(4.39±0.52),低剂量组(3.98±0.31),干扰对照组(3.96±0.19),空白对照组(4.28±0.39),P0.05,无统计学差异;5.天狼星红苦味酸染色及HE染色:各组间组织形态差异不明显;6.一氧化氮(umol/L):高剂量组(8.30±5.01),低剂量组(8.08±6.35),干扰对照组(13.87±9.56),空白对照组(4.52±2.12),P0.05,无统计学差异;【结论】1.在人群中证实,micro RNA-126在高血压病患者中呈高表达,在健康人中呈低表达,micro RNA-126与原发性高血压存在相关性;2.成功构建micro RNA-126基因敲减慢病毒载体;3.micro RNA-126作为基因治疗靶点治疗原发性高血压无明显降压效果,对靶器官无明显保护作用;4.micro RNA-126在高血压患者中表达增高可能为一种代偿机制。
[Abstract]:[objective] 1.The gene microarray was used to detect various micro RNAs in human. The relationship between micro RNA and hypertension was found to be 2. 2%.By constructing micro RNA-126 gene knockdown virus vector, the feasibility of treating hypertension with micro RNA-126 as gene target was discussed.The mechanism of micro RNA-126 in the occurrence and development of essential hypertension was described. [methods] Blood samples from 6 patients with essential hypertension and 4 healthy persons matched by age, sex and body weight were collected.Shanghai Kangcheng Biological Company micro RNA chip was commissioned to detect the micro RNA expression profile in two groups of samples.Using the lentivirus vector of human immunodeficiency virus (HIV-1) as the source, we constructed the vector of mil R-126 gene knockdown virus (Lenti-mi R-126-KDV) and the interfering control lenti-scramblemirin (Lenti-scramblemiririn).Spontaneously hypertensive rats were divided into two groups: high dose group (1: 108), low dose group (n = 10 7), interference control group (n = 5) and blank control group (n = 350). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were collected by tail cuff method for 8 weeks.After 8 weeks of mean pressure and heart rate, the heart body ratio was calculated and the heart, kidney, brain, liver and lung were taken for frozen sections, and the distribution of lentivirus vector in vivo was observed under fluorescence microscope.Sirius red picric acid staining and HE staining were used to observe the damage of target organs in hypertension.The content of nitric oxide (no) in serum of rats was determined by ELISA method, and various important biochemical indexes were determined. [results] the 3'end of 1.micro RNA-126 was 1.410 vs 0.838% (P0. 03), and the expression of 5 '-terminus was 1. 799 vs 0. 997P0. 03) in patients with hypertension and low expression in healthy persons.There was significant difference between the two groups (P 0.05).Construction and expression of lentivirus vector: lentivirus vector was successfully constructed.After 8 weeks of administration, the expression of green fluorescent protein (GFP) was observed in frozen sections of all tissues.Blood pressure, heart rate: before administration, there was no uniform difference in blood pressure and heart rate. After administration, the changes of blood pressure and heart rate were observed weekly. There was no significant difference in blood pressure and heart rate between each group (P 0.05).The ratio of heart to body was 4.39 卤0.52g in high dose group, 3.98 卤0.31 in low dose group, 3.96 卤0.19 in interfering control group and 4.28 卤0.39 in blank control group. There was no significant difference between the two groups.Sirius red picric acid staining and HE staining: there was no significant difference in histomorphology among the groups.There was no statistical difference between high dose group (8.30 卤5.01), low dose group (8.08 卤6.35), interfering control group (13.87 卤9.56), blank control group (4.52 卤2.12) (P 0.05); [conclusion] 1.The high expression of micro RNA-126 in patients with hypertension and the low expression of micro micro RNA-126 in healthy people were confirmed in the population. There was a correlation between micro micro RNA-126 and essential hypertension.Successful construction of micro RNA-126 gene knockdown virus vector 3.micro RNA-126 as a target for gene therapy for essential hypertension has no obvious antihypertensive effect. 4. The increase of micro RNA-126 expression in hypertensive patients may be a compensatory mechanism.
【学位授予单位】：首都医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R544.1

【相似文献】