【作者】 刘清；

【导师】 石刚刚；

【作者基本信息】 汕头大学 ， 药理学， 2008， 硕士

【摘要】 碘化N-正丁基氟哌啶醇(F2)是本实验室设计合成的一种新型的氟哌啶醇季铵盐衍生物,实验表明它对心肌缺血再灌注损伤以及心肌细胞、内皮细胞缺氧复氧损伤具有保护作用,这种作用与抑制早期生长反应基因-1(egr-1)mRNA及蛋白水平的过度表达有关。缺氧可以导致血管平滑肌细胞的过度增殖,进而导致一系列血管增生性疾病。本实验以体外培养的血管平滑肌细胞(VSMCs)为研究对象,观察F2对缺氧诱导的平滑肌细胞过度增殖的作用及其与Egr-1的关系。目的研究碘化N-正丁基氟哌啶醇(F2 )对缺氧导致的血管平滑肌细胞增殖的影响,并探讨其作用机制是否与抑制egr-1高表达有关。方法1 Sprague-Dawley大鼠胸主动脉平滑肌细胞体外培养:采用组织块贴壁法。2 Sprague-Dawley大鼠胸主动脉平滑肌细胞鉴定:采用免疫组化法加以鉴定。3 Egr-1反义寡核苷酸在大鼠VSMCs中的转染。4实验分组:取3～5代VSMCs随机分为8组:对照组;缺氧组;F2组(1×10 - 6 mol/L);聚乙二醇组(1×10 - 6 mol/L);反义寡核苷酸组;正义寡核苷酸组;错义寡核苷酸组;脂质体组。除对照组外,其余各组均作缺氧模型。5取第3～5代培养的大鼠胸主动脉平滑肌细胞制作缺氧模型,应用噻唑蓝比色法(MTT)检测细胞增殖情况,流式细胞仪(FCM)观察分析VSMCs细胞周期分布特征的改变,蛋白印迹杂交方法(Western-blot方法)检测Egr-1及血小板衍生生长因子A(PDGF-A )蛋白表达情况。结果1原代培养4～7天左右可见组织块周围有细胞长出,2～3周达亚融合,传代后细胞“峰、谷”状生长明显。2α- SM actin免疫组化染色阳性。3 FITC标记的反义寡核苷酸在488nm的激发光源下呈现绿色,荧光显微镜下可见细胞核及细胞浆中有绿色荧光,发出绿色荧光的位置与同一视野下细胞所在位置相一致,说明反义寡核苷酸成功转入平滑肌细胞。4缺氧诱导的血管平滑肌细胞中Egr-1蛋白的表达在缺氧1 h后明显升高,2 h达高峰,此后逐渐下降,直到20 h仍然高于相应的对照组。5与Control组相比,缺氧组、正义组、错义组、聚乙二醇组以及脂质体组的比色吸光度A值、细胞周期S+ G2 /M期比例、Egr-1及PDGF-A蛋白表达水平均明显增高,G0/ G1期比例明显下降;与缺氧组比较, F2组及反义组比色吸光度A值、细胞周期S+ G2 /M期比例、Egr-1及PDGF-A蛋白表达水平均明显降低,G0/ G1期比例明显升高。结论F2可通过抑制Egr-1蛋白高表达来抑制缺氧导致的VSMCs的过度增殖。更多还原

【Abstract】 N-n-butyl haloperidol iodide (F2) is a new quaternary ammonium salt derivative of Haloperidol, which was synthesized by our laboratory.Our previous studies have shown its protective effects on myocardial ischemia/reperfusion (I/R) injury and hypoxia/reoxygenation(H/R) injury of cardiocytes and endotheliocytes,which is associated with inhibiting egr-1 mRNA transcription and protein overexpression .Hypoxia can induce overproliferation of vascular smooth muscle cells(VSMCs),which further induces a series of vascular proliferous diseases. The purpose of this experiment is to investigate effects of F2 on hypoxia induced proliferation of vascular smooth muscle cells and to explore its relationship with Egr-1.ObjectiveTo investigate the effect and mechanism of F2 on hypoxia-induced proliferation of rat thoracic aortic smooth muscle cells in vitro and show the relationship between the effect of F2 and the overexpression of early growth response gene-1(egr-1) .Methods1 Culture of rat thoracic aortic smooth muscle cells in vitro: cultured by the method of adhesion to wall and observed under the microscope.2 Detection of rat thoracic aortic smooth muscle cells in vitro:detected by the immunohistochemistryα-SM-actin antibody.3 Transfection of egr-1 antisense oligodeoxynucleotides to cultured VSMCs in rat.4 Groups:Passage 3~5 of cultured VSMCs were randomly divided into one of eight groups:control group,hypoxia group, F2 group(1×10 - 6 mol/L), PEG group (1×10 - 6 mol/L),AS-ODN group,S-ODN group,Sc-ODN group and LIP group.5 Passages 3 to 5 of cultured rat thoracic aortic SMCs were used to establish hypoxia models. The viability of cultured VSMCs was detected by MTT assay. The cell cycle of cultured VSMCs was analyzed by flow cytometry(FCM). The expression levels of Egr-1 and PDGF-A proteins were examined by Western blot.Results1 A few of cells grew out of tissue-block about 4 to 7 days late and approached confluence approximately 2 to 3 weeks.A typical“hill-valley”growth pattern was displayed after cells were passaged.2 98% of the cultured smooth muscle cells were positive forα- SM actin.3 FITC labeled antisense oligodeoxynucleotides emited green fluorescence. The location of fluorescence is coincidented with the area of cells under the inverted phasecontrast microscope, which indicated that antisense oligodeoxynucleotides had transfected VSMCs successfully.4 Hypoxia injured VSMCs caused a time-dependent induction of Egr-1 protein, being evident at 1 h, peaking at 2 h, and declining thereafter.5 Compared with the control group, the absorption value,proliferation index and the levels of Egr-1 and PDGF-A of hypoxia group, PEG group,S-ODN group,Sc-ODN group and LIP group were all increased.Compared with the hypoxia group,all these above items of antisense oligodeoxynucleotide group and F2 group were all decreased.ConclusionsF2 inhibts hypoxia-induced proliferation of VSMCs in vitro,which is mediated by Egr-1.更多还原