牛磺酸对大鼠肺动脉平滑肌细胞凋亡的影响

发布时间：2018-01-01 00:12

本文关键词：牛磺酸对大鼠肺动脉平滑肌细胞凋亡的影响　出处：《哈尔滨商业大学》2011年硕士论文　论文类型：学位论文

【摘要】：肺动脉高压是指静息时肺动脉平均压3.33kPa(25mmHg)或运动时4kPa(30mmHg)者。肺动脉平滑肌细胞是肺血管的主要成分,肺动脉平滑肌细胞的增殖与凋亡失衡会引起肺血管官腔狭窄、闭塞,肺血管阻力增加,最终导致肺动脉高压。肺动脉高压的两个重要的的病理生理改变,即肺动脉血管收缩和肺动脉血管的重塑。而由于重构主要是由于破坏细胞的动态平衡,主要包括抑制细胞的凋亡或促进细胞的增殖两个方面。而且,肺动脉平滑肌细胞,作为肺动脉血管的主要构成成分,是肺动脉高压血管重构的主要执行者。因此研究调控PASMCs的增殖与凋亡能为预防和治疗肺动脉高压寻找治疗的方法提供理论基础。细胞凋亡是细胞的一种基本生物学现象,在多细胞生物去除不需要的或异常的细胞中起着必要的作用。它在生物体的进化、内环境的稳定以及多个系统的发育中起着重要的作用。细胞凋亡不仅是一种特殊的细胞死亡类型,而且具有重要的生物学意义及复杂的分子生物学机制。牛磺酸(Tau)几乎存在于所有动物细胞中,大多以游离形式存在,它是机体一种内源性抗损伤物质,Tau具有很多生物学效应,例如保持内钙稳态,稳定细胞膜,清除体内自由基等。有研究表明Tau在某些细胞中也具有诱导细胞凋亡的作用。我们选用大鼠肺动脉平滑肌细胞为研究对象,观察Tau对它的作用。目的：观察Tau对肺动脉平滑肌细胞(PASMCs)凋亡作用的影响,并探讨其机制是否通过线粒体途径和死亡受体途径。方法：取Wistar雄性大鼠肺动脉平滑肌细胞培养；分为正常组(control)、凋亡组(SD)、给药组(Tau高、中、低剂量)。给药前,按需要将所有组用低糖无血清培养基饥饿24h,使细胞处于相同生长状态,按需要给药24h或48h后,MTT法测定96孔板细胞的生存率；用吖啶橙(AO)法,在荧光显微镜下观察6孔板细胞核形态学变化；用线粒体膜电位试剂盒测定细胞膜电位的变化；用Western-blot法分别测定Bax、Bcl-2、Procaspase-9、Procaspase-3和Fas的蛋白表达变化。结果：MTT测定细胞生存率发现,和正常组相比,加药组生存率明显低于正常组,并和凋亡组的趋势相同,可以明显看出呈剂量依赖性诱导肺动脉平滑肌细胞凋亡,且40mmol/L的Tau给药量为最佳浓度。吖啶橙法三组结果可以看出,凋亡组和Tau给药组的细胞核皱缩,形成凋亡小体。线粒体膜电位法测定,发现凋亡组和Tau组相对正常组,膜电位降低,图中出现较多绿色荧光区域。(?)Vestern-blot法测定发现Tau升高Bax和Fas蛋白表达,降低Procaspase-3、Procaspase-9和Bcl-2的表达。结论：Tau诱导大鼠PASMCs凋亡,其作用机制可能与调节线粒体途径和死亡受体途径有关。
[Abstract]:Pulmonary hypertension refers to resting pulmonary arterial pressure (25mmHg) or 3.33kPa 4kPa (30mmHg) during exercise. Pulmonary artery smooth muscle cells is the main component of pulmonary vascular, imbalance of proliferation and apoptosis of pulmonary artery smooth muscle cells can cause pulmonary vascular lumens stenosis, occlusion, increased pulmonary vascular resistance, resulting in pulmonary hypertension pulmonary hypertension. Two important pathophysiological changes, namely pulmonary vasoconstriction and pulmonary vascular remodeling. Because the reconstruction is mainly due to the dynamic balance of destruction of cells, including inhibition of cell apoptosis and promote cell proliferation in two aspects. Moreover, pulmonary artery smooth muscle cells, as the main form the pulmonary artery is the main component of high pressure to perform remodeling of the pulmonary arteries. Therefore study on the regulation of proliferation and apoptosis of PASMCs can provide the theory for the prevention and treatment of pulmonary arterial hypertension for treatment Basics.
Apoptosis is a basic biological phenomenon of cells, plays a necessary role in multicellular organisms to remove unwanted or abnormal cells. It is in the evolution of organisms, plays an important role in the stability of the environment and the development of multiple systems. Apoptosis is not only a kind of special types of cell death. It also has important biological significance and complex molecular mechanisms.
Taurine (Tau) exists in almost all animal cells, mostly in the free form, it is a endogenous anti injury substance, Tau has many biological effects, such as maintaining calcium homeostasis, cell membrane stability, scavenging free radicals in vivo. Studies have shown that Tau has induced apoptosis in certain cells. We use the rat pulmonary artery smooth muscle cells as the research object, to study the effect of Tau on it.
Objective: To observe the effect of Tau on the apoptosis of pulmonary artery smooth muscle cells (PASMCs) and to explore whether its mechanism is through mitochondrial pathway and death receptor pathway.
Methods: Wistar male rat pulmonary artery smooth muscle cells; divided into normal group (control group), apoptosis (SD), drug group (Tau high, low dose). Before administration, according to the needs of all groups with low sugar starvation in serum-free medium 24h, the cells in the same growth according to the state Administration of 24h or 48h, the survival rate was measured in 96 hole plate cell MTT; using acridine orange (AO), to observe the morphological changes of the cell nucleus in 6 hole plate under a fluorescence microscope; Determination of cell membrane potential changes in mitochondrial membrane potential kit; Bax were measured by Western-blot method, Bcl-2. Procaspase-9, the expression of Procaspase-3 and Fas protein.
Results: MTT determination of cell survival rate was found, compared with the normal group, the survival rate of treatment group was significantly lower than that in normal group, and apoptosis groups the same trend, evident dose dependently induced pulmonary artery smooth muscle cell apoptosis, 40mmol/L and Tau dosage was the best concentration of acridine orange method. The results of the three groups can be seen apoptosis group and Tau treatment group to cell shrinkage and formation of apoptotic bodies. Determination of mitochondrial membrane potential, apoptosis group and Tau group compared with normal group, the membrane potential decreased, more green fluorescent areas appear in the picture. (?) indicated that the increased expression of Tau, Bax and Fas Vestern-blot protein by the method of reduced expression of Procaspase-9 and Procaspase-3. Bcl-2.
Conclusion: Tau induces apoptosis of PASMCs in rats, and its mechanism may be related to the regulation of mitochondrial pathway and death receptor pathway.

【学位授予单位】：哈尔滨商业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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