Screen for Coronary Artery Disease Specific Genetic Expression by Representational Differential Analysis

Objective To screen coronaryartery disease (CAD) specific expressions and clone their genes. Method Blood samples were collected from CAD and non - CAD patients at the end of coronary angiography. mRNA from samples was isolated and converted into cDNA. After ligated with specific linkers, the cDNA was amplified with complementary primers. PCR products from CAD samples were named as tester; the ones from non - CAD samples were named as driver. With different ratio of tester to driver (1 : 100,1: 1, 000, and 1: 10, 000), they were mixed, denatured, and renatured. Single strand cD-NA was eliminated by Mung bean nuclease. Double strand cDNA presented only in tester was amplified, ligated in vector pUC19 and pUC53, and transformed into E. coll DH5a. Strains with inserted cDNA fragments were picked up based on blue and white selection. Insertions were screened by endonuclease digestion and DNA sequencing. Results were compared with DNA sequences of GeneBank. Results: After the selection with representational     

广州地区中年人群血脂流行趋势的探讨

目的了解广州地区中年人群血清血脂水平的流行趋势。方法研究对象包括工人和农民。血脂测定由美国疾病控制中心标准化的实验室完成。结果1983～1994年，十年广州地区中年人群血脂水平具有上升的趋势，尤其1987～1988年农民组男性的甘油三酯升幅最大，上升达59．3％，而工人组总胆固醇的均值亦已接近200mg／dl工人组血脂水平均明显高于农民组。结论由于这些危险因素的升高而导致冠心病、脑卒中等心血管疾病的发病增多，将成为这些人群健康的潜在威胁。     

miRNA-21 regulates proatherosclerotic gene expression in macrophages

Background MicroRNAs(miRNANAs) are endogenous, small non-coding RNAs that negatively regulate gene expression in diverse cardiovascular diseases. However, the roles of miRNANAs in atherosclerogenesis needs to be elucidated. In the present study, the effect of miRNA-21 on pro-atherosclerotic genes expression was examined. Methods The pro-atherosclerotic genes including COX2, VCAM1, ICAM1, MCP1 and miRNA-21 were detected in ox-LDL-treated mouse macrophage RAW264.7 cells. ApoE knock-out(ApoEKO) mice were fed with high-fat diet for 16 weeks, and the abdominal aorta were fixed and used for miRNA-21 hybridization. Lentivirus-based vectors for enforced expression of miRNA-21 and antisense miRNA-21were prepared. The expression of proatherosclerotic genes was determined in the RAW264.7 cells with lentivirusmediated up-regulation of miRNA-21. Results COX2, VCAM1, ICAM1 and MCP1 could be up-regulated by ox-LDL treatment, and 50 μg / mL ox-LDL could significantly increase the expression of above four genes in ox-LDL EAW264.7 cells. miRNA-21 could also be markedly up-regulated in ox-LDL-induced RAW264.7cells. The result of miRNANA hybridization showed that miRNA-21 was strongly expressed in atherosclerotic plaques but not in normal aorta. Lentivirus-mediated over-expression of miRNA-21 could significantly enhance expressions of COX2, VCAM1, ICAM1 and MCP1 in RAW264.7 cells, which could be reversed by antisense miRNA-21 mediated by lentivirus vector. Conclusions miRNA-21 could be modulated by ox-LDL in macrophage RAW264.7 cells, and miRNA-21 could enhance COX2, VCAM1, ICAM1 and MCP1expressions in macrophages.     

血管紧张素转换酶2基因型与厄贝沙坦改善高血压患者左室重构及功能的关系

背景一些研究结果提示血管紧张素转换酶2(ACE2)具有调节心脏结构和功能的作用,ACE2基因多态性与左室结构和功能相关,但 ACE2基因型对降压药物改善高血压患者的左室重构和功能是否有影响,目前尚不清楚。目的观察 ACE2基因 G9570A 多态性与厄贝沙坦影响高血压患者左室结构及功能的相关性。方法选择坐位收缩压<180 mmHg 和(或)舒张压90～109 mmHg 无血缘关系的汉族原发性高血压患者395例(男性205例,女性190例),均服用厄贝沙坦治疗,起始剂量150 mg/d,治疗4周后血压未达到有效标准者药量加倍,共观察48周。应用聚合酶链反应和限制性片段长度多态性(PCR-RFLP)的方法检测 ACE2基因 G9570A 多态性,并随机抽取20份标本进行基因测序以核实基因分型。按性别分组分析降压幅度及左室结构和功能各参数的改变值与基因型的关系。结果经过48周厄贝沙坦治疗,所有患者血压明显下降(P<0.01)。不同亚组间血压下降幅度无统计学意义(P>0.05)。携带 A 等位基因患者左室质量和左室质量指数下降幅度及心脏功能改善程度显著高于其他亚组(P<0.05)。结论 ACE2基因型与左室质量指数下...     

微小RNA可在人骨髓间充质干细胞分化来源的心肌样细胞中表达

目的研究人骨髓间充质干细胞（hMSCs）分化来源的心肌样细胞中代表性微小RNA（miRNAs）的表达。方法利用FITC-偶联的CD29、CD34和CDllb抗体在流式细胞仪上检测分离的hMSCs的抗原表型。分别用5-氮胞苷（5-aza）和乳鼠心肌非接触共培养法诱导传至3代的hMSCs分化为心肌样细胞。用免疫化学法检测hMSCs分化来源的心肌样细胞中心肌特异的α-肌节辅肌动蛋白和心肌肌钙蛋白（cTnI）的表达：利用逆转录PCR和DNA测序鉴定5个代表性的心脏特异的初级微小RNA（pri-miRNAs）的表达。结果hMSC标志分子CD29表达率为98．87％．而造血细胞标志分子CD34和CDllb的表达率只是5％和0．4％。免疫化学分析显示．分别经5-aza和乳鼠心肌非接触共培养诱导的hMSC均可表达α-肌节辅肌动蛋白和cTnI,而在未分化的hMSCs中无表达。miRNA-143,-181可在5-aza诱导的hMSCs中表达,miRNA．143,-181,-206,-208可在乳鼠心肌非接触共培养的hMSCs中表达．而两种诱导方法均未能诱导miRNA-1-2在hMSCs中表达。结论利用5-aza和乳鼠心肌非接触共培养法诱导hMSCs分化的心肌样细胞中可以表达不同的心脏特异的pri-miRNAs。     

用小双链RNA抑制转化入HUVEC中绿色荧光蛋白基因的表达

目的：建立稳定表达绿色荧光蛋白(GFP)的人脐静脉血管内皮细胞（HUVECs），研究小干扰RNA(siRNA)对HUVECs中GFP表达的抑制作用。 方法： 用lipofectamine2000将编码GFP的质粒pN3-EGFP转入HUVECs中。用G418筛选并维持已转化pN3-EGFP的HUVEC(HUVEC-GFP)。利用T7 RNA转录试剂盒，制备可抑制GFP基因表达的siRNA（GFPsiRNA）和无关对照的RNA（control siRNA）。用oligofectamine将siRNA转入HUVEC-GFP中。继续培养48 h后，检测HUVEC-GFP中GFP蛋白和mRNA表达水平。 结果： 用G418筛选获得了HUVEC-GFP细胞株，可以观察到GFP的稳定表达。HUVEC-GFP转化siRNA后48 h，GFP的荧光强度明显下降，而对照组的荧光强度无明显下降。半定量RT-PCR检测显示，GFPsiRNA对GFP mRNA表达有较强的抑制作用，抑制率达40%，而control siRNA对GFP mRNA表达水平无明显的抑制作用。 结论： 利用体外转录合成的siRNA能有效地抑制HUVECs中GFP的表达。     

缺氧—复氧对培养的大鼠心肌细胞损伤与细胞凋亡的影响

目的探讨培养的大鼠心肌细胞在缺氧（缺血）和缺氧－复氧（再灌注）状态下心肌细胞损伤和细胞凋亡情况。方法采用１～３天新生ＳＤ大鼠,常规方式的培养心肌细胞,给予模拟缺氧（缺血）液、模拟复氧（再灌注）液以及干预因素（终浓度为２００Ｕ／ｍＬ的ＳＯＤ）处理,分为缺氧－复氧损伤组（Ｈ／Ｒ组）、缺氧－复氧损伤组＋ＳＯＤ（Ｈ／Ｒ＋ＳＯＤ组）、缺氧预处理组（ＨＰ组）和正常对照组（Ｃｏｎｔｒｏｌ组）,进行以下指标观察：     

TUNEL法原位检测心肌细胞凋亡的改进

目的：脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（TUNEL）是检测细胞凋亡的常用方法,但以往仍有TUNEL假阳性率高的报道。本实验目的在于探讨应用TUNEL检测心肌细胞凋亡时增强其特异性的方法。方法：缺血再灌注心肌组织的石蜡切片（2 μm）和冰冻切片（3 μm）进行TUNEL染色。分为石蜡切片HE染色组,石蜡切片常规TUNEL组,冰冻切片改良TUNEL组。石蜡切片常规TUNEL组完全按照试剂盒说明书进行,光学显微镜下观察;冰冻切片改良TUNEL组使用荧光素（FITC）标记的dUTP及4’,6-二脒基-2-苯基吲哚(DAPI）双重荧光染色,在共聚焦显微镜下观察。结果：TUNEL改良法凋亡背景清晰,阳性细胞主要分布在缺血再灌注损伤区域,其凋亡检出率与石蜡切片HE染色相比无显著差异（P>0.05）;常规TUNEL组凋亡背景清晰度较差,非缺血再灌注损伤区域亦有大量阳性细胞出现,其凋亡率明显高于石蜡切片HE染色（P<0.01）。结论：冰冻切片改良TUNEL法可能是更适宜研究SD大鼠缺血再灌注心肌细胞凋亡的方法。     

以重组CD74胞外片段抑制巨噬细胞移动抑制因子（MIF）功能域活性

目的：巨噬细胞移动抑制因子（MIF）第50-65位氨基酸残基（MIF50-65）是其生物活性功能域。MIF需与Ⅱ型穿膜蛋白CD74的胞外片段结合后引发信号传导反应。本文拟筛选能特异结合MIF50-65的CD74胞外截短体片段，并研究重组该CD74截短体对MIF50-65活性的抑制作用。方法：利用酵母双杂交实验筛选MIF50-65与CD7473-149、CD74109-149、CD74109-232、CD7473-232中特异结合的CD74截短体。利用原核表达系统，在大肠杆菌中诱导表达重组CD74截短体多肽。用GSTrap亲合柱纯化重组蛋白，通过体外血管生成实验鉴定重组CD74截短体对MIF的阻断作用。结果：只有MIF50-65与CD7473-232共转化的酵母可以在SD／-Leu-His-Trp-Ade培养基上生长，且α-x-gal底物反应为蓝色。构建了重组质粒pGEX-4T-CD7473-232，经IPTG诱导后特异表达出可溶性的重组蛋白。重组CD7473-232能特异地阻断MIF的促血管生成作用。结论：CD7473-232能与MIF50-65特异结合，并对MIF生物活性有抑制作用。