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1、上海交通大学硕士学位论文反义Smad3阻断TGF-1信号传导对血管平滑肌细胞增生的影响姓名：程智慧申请学位级别：硕士专业：内科学（老年医学）指导教师：盛净20090501上海交通大学医学院 2006 级硕士研究生学位论文 6第一部分 损伤后 VSMCs 的培养和鉴定 第一部分 损伤后 VSMCs 的培养和鉴定 目的目的：建立大鼠颈总动脉急性球囊损伤模型，取损伤后血管组织培养增生型VSMCs,为体外研究增生型 VSMCs 的生物学行为及探索治疗增生性血管病变的措施提供理想的实验材料。 方法方法：采用球囊导管来回拖拉剥脱血管内膜的方法构建大鼠颈总动脉急性球囊损伤模型，建模后 3 天、7 天取损伤血
2、管组织 HE 染色，未损伤血管组织作为对照组，光镜下观察管壁形态学变化以证明建模成功。建立模型后 3 天、7 天和 14 天，取损伤血管组织，采用组织块贴壁法培养增生型 VSMCs,免疫细胞化学鉴定细胞表型标志物 SMactin， 及四氮唑盐(MTT)法测定增殖能力,未损伤血管组织取材培养收缩型VSMCs 作为对照组, 组间比较采用配对 t 检验。 结果结果：模型组血管壁随时间延长失去正常形态结构且明显增厚,管腔逐渐狭窄，HE 染色见大量细胞浸润。取损伤后血管用组织块贴壁法培养的细胞经免疫细胞化学鉴定其表型标志物 SMactin 证明是 VSMCs， 经 MTT 法测定增殖能力并且与收缩型VS
3、MCs 对比显示其增殖能力显著增强（P6.3E+12pfu/mL。 结论: 结论: 本实验方法成功构建反义 Smad3 腺病毒载体。 第三部分第三部分 反义反义 Smad3 阻断阻断 TGF-1 信号传导信号传导 对增生型对增生型 VSMCs 增殖能力的影响增殖能力的影响 目的目的：观察反义 Smad3 的腺病毒载体转染增生型 VSMCs 后， 通过阻断内源性 Smad3基因表达,从而阻断 TGF1 的信号在胞内的进一步传导，进而对增殖型 VSMCs 增上海交通大学医学院 2006 级硕士研究生学位论文 8生能力的影响。 方 法方 法：当 细 胞 生 长 到 70-80% 融 合 时 , 按
4、感 染 复 数 (multiplicity of infection,MOI) 为 100 空斑形成单位(pfu) /cell 转染 VSMCs。荧光显微镜下观察转染是否成功,并在转染后收集细胞抽提 RNA 行 RT-PCR 检测 Smad3 表达变化，空载病毒组作为阴性对照组,无血清 DMEM 培养液组作为空白对照组。并进一步采用四氮唑盐(MTT)法分别检测转染组， 阴性对照组和空白对照组增生型 VSMCs 在转染后 2 天、3 天、5 天和 7 天的增殖能力变化，组间变化差异比较采用单因素方差分析。 结果: 结果: 荧光显微镜下见转染组细胞内有绿色荧光颗粒，空白对照组呈阴性，提示腺病毒载体
5、可以转染增殖型 VSMCs。RT-PCR 检测 Smad3 基因表达，琼脂糖凝胶电泳后 Smad3 条带,转染组较阴性对照组,空白对照组 Smad3 表达显著减少。转染后 2 天、3 天、5 天,转染组增生型 VSMCs 的增殖能力较阴性对照组和空白对照组的显著降低(P0.05） 。 结论结论: 腺病毒载体可以成功转染增殖型 VSMCs，并且携带的目的基因可以进一步发挥其生物学效应。反义 Smad3 阻断 TGF-1 信号传导在一定程度上能抑制增生型VSMCs 增殖。 关键词：关键词：血管平滑肌细胞，转化生长因子，增生，信号转导，抑制，反义 上海交通大学医学院 2006 级硕士研究生学位论文
6、9THE INFLUENCE ON THE PROLIFERATION OF VASCULAR SMOOTH MUSCLE CELLS OF INTERRUPTING TRANSFORMING GROWTH FACTER-1s SIGNAL TRANSDUCTION BY ANTI-SMD3 ABSTRACT THE INFLUENCE ON THE PROLIFERATION OF VASCULAR SMOOTH MUSCLE CELLS OF INTERRUPTING TRANSFORMING GROWTH FACTER-1s SIGNAL TRANSDUCTION BY ANTI-SMD
7、3 ABSTRACT Proliferative vascular diseases such as Hypertention,Atherosclerosis(AS) Restenosis after percutaneous transluminal coronary angioplasty(PTCA),Diabetic angiopathies,and transplanted angiopathies,are characterized by lumina stenosis even obliteration along with intima thickening.The experi
8、mental analysis of neointima demonstrate that the main component of the neointima is vascular smooth muscle cells(VSMCs) and extra cellular matrix(ECM).In the process of their pathology,the activation,migration,proliferation,secreting ECM is their core.The activated VSMCs is not only the main source
9、 of many cytokines,but also is the target cell of numerous inflammatory factors.So we can inhibit the proliferation of neointima to prevent luminal stenosis through regulating the growth ,differentiation,secretion of VSMCs. Among the numerous cytokines which regulate the biological behaviour of VSMC
10、s,the transforming growth factor 1(TGF-1) is the focus of reseach for the past few years,many reseaches show that TGF-1 play an important role in the whole process of the occurence and development of neointima proliferating.This conclusion promote us to prevent neointima proliferating by interruptin
11、g the signal transduction of TGF-1. 上海交通大学医学院 2006 级硕士研究生学位论文 10Part I Cultivation and identification of vascular smooth muscle cell(VSMCs) after injury Objective: Objective: To establish acute carotid artery injury model in rat and to culture proliferative vascular smooth muscle cells using vascula
12、r tissue after injury for studying the biological behaviour of proliferative vacular smooth muscle cells(VSMCs) and provide ideal experimental subject for exploring the therapeutic measure of proliferative vascular diseases. Methods: Methods: Firstly,Construct acute carotid artery injury model in ra
13、t through stripping tunica intima vasorum by hauling the balloon catheter back and forth. To prove whether the model-constructing is success or not through observing the morphologic change of the vessel wall under microscopy after HE staining in injury blood vessel after 3 days and 7 days of congstr
14、ucting model.Then,after 3 days,7days and 14 days of injury,culture proliferative vascular smooth muscle cells by tissue adherence method using injured vessel tissue.At the same time, identify cellular phenotypic marker smooth muscle -actin by immunocytochemistry and evaluate proliferative activity b
15、y MTT,the uninjured vessel tissue as control group,then make a comparison among these groups by paired T test. Result: Result: The vessel wall of model group is losing nomal morphosis and is thickening gradually along the time,the lumen of the vessel is narrowing.There are much more cells in vessel
16、wall after HE staining in injury group than in control group.It turns out that the cell which is from the vessel tissue of model group is vascular smooth muscle cell by immunocytochemistry.The ability to proliferate of model group is stronger than control group(P0.05 vs N Group 各组细胞生长曲线00.20.40.60.8T1T3T5TIME（DAY）OD(570nm)D3 D7 D14 N图 5：各组细胞的生长曲线图 Picture 5：Growth curve of every group 注：D3 为损伤后 3 天，D7 为损伤后 7 天，D14 为损伤后 14 天，N 为正常组 讨 论 讨
