【作者】 郭悦劼；

【导师】 李胜；

【作者基本信息】 大连医科大学 ， 神经病学， 2011， 硕士

【摘要】 研究背景与目的:动脉粥样硬化(atherosclerosis)是缺血性脑卒中的重要病因,但是近年的研究显示颅内、外动脉粥样硬化(intra- and extracranial atherosclerosis)可能具有不同的危险因素和发病机制。血管平滑肌细胞(vascular smooth muscle cells,VSMCs)是血管中膜的主要细胞成分,当血管损伤后,VSMCs可发生表型转化(phenotypic modulation),即从静息态的收缩表型(contractile phenotype)转变为增生态的合成表型(synthetic phenotype),重新获得向内膜迁移并增殖的能力。VSMCs的表型转化引发的细胞增殖、迁移、黏附等细胞功能的变化是动脉粥样硬化、血管成形术后再狭窄等血管增生性疾病共同的病理学特征。因此,VSMCs的体外培养技术为研究其生物学行为以及相关疾病的发病机制及防治策略提供了一条重要途径。目前颅外动脉VSMCs的培养已经得到广泛应用,但有关颅内脑动脉VSMCs培养的报道少见,且多取材于牛、猪、犬、兔等动物较粗大的脑血管。以往报道的酶解离法培养VSMCs因操作繁琐,而限制了其应用。本研究旨在对原有的酶消化法进行改良,建立大鼠颅内、外脑动脉VSMCs的体外培养方法,为颅内、外动脉粥样硬化等脑血管疾病机制及治疗的研究提供一个良好的体外实验模型。方法:1、VSMCs的原代培养:(1)大鼠脑基底动脉VSMCs:取2只健康雄性Sprague-Dawley(SD)大鼠(体重250~300 g) ,断颈处死后以75%酒精全身湿润消毒。无菌条件下分离大鼠脑基底动脉,去除血管外膜后剪成约0.2 mm的小段, 37℃分别用0.1%Ⅰ型胶原酶消化5 h,0.125%胰蛋白酶消化10 min。将消化后得到的细胞用含20%胎牛血清的DMEM/F12培养基进行培养。(2)大鼠颈动脉VSMCs:1只SD大鼠取仰卧位固定,于颈部正中切一2 cm长切口。无菌条件下暴露左侧颈总动脉,切取长约1.5 cm的血管段。将其去除血管外膜及内膜后剪成约0.2 mm的小段,然后按照前述方法行酶消化和培养。2、VSMCs的传代培养:当细胞生长达80% ~90%汇合时,采用0.25%胰蛋白酶消化和传代培养。3、细胞纯化:采用人工刮除法及差速贴壁法纯化细胞。4、VSMCs鉴定:平滑肌细胞通过观察其形态学及生长方式来鉴定。α-平滑肌肌动蛋白是分化型VSMCs的特异性标志物,因此同时采用α-平滑肌肌动蛋白免疫细胞化学方法鉴定。一抗为小鼠抗大鼠α-平滑肌肌动蛋白抗体(1:300),二抗为生物素化山羊抗小鼠IgG,然后采用SABC试剂盒说明行免疫组化染色。5、细胞存活率测定:取第5代细胞,采用血球计数器和台盼蓝排斥实验来计算细胞存活率。结果:原代培养3 d后,来自大鼠基底动脉或颈总动脉的细胞开始贴壁,2周后细胞呈梭形,汇合后具有平滑肌细胞典型的“峰-谷”样生长特点。传代培养的细胞保持上述特征,第5代细胞经α-平滑肌肌动蛋白表达鉴定,基底动脉VSMCs和颈总动脉VSMCs的纯度分别达97%和98%以上。台盼蓝排斥实验检测大鼠基底动脉VSMCs或颈总动脉VSMCs存活率达95%或96%以上。结论:这种方法操作简单、结果可靠、成本低廉,可为颅内、外动脉粥样硬化、再狭窄等脑血管疾病机制和治疗的研究提供适宜的体外培养细胞模型。更多还原

【Abstract】 Background and Objective: Atherosclerosis is one of the major causes of the ischemic stroke, but the risk factors and the mechanisms seem to be different between intra- and extracranial atherosclerosis. Vascular smooth muscle cells (VSMCs) are the major cellular component of the tunica media. After vascular injury, VSMCs characteristically exhibit phenotypic modulation, change from the quiescent "contractile" phenotype to the active "synthetic" phenotype, that can migrate and proliferate from media to the intima, thereby contribute to the progression of many vascular diseases such as atherosclerosis and restenosis. Therefore, primary culture of VSMCs in vitro can provide an important way to study the biological behavior of VSMCs and the pathogenesis and prevention strategy of the related vascular diseases. At present, the culture of extracranial arterial VSMCs has been widely used. In contrast, the culture of intracranial arterial VSMCs has rarely been reported, most of which are obtained from the bovines, pigs, dogs, rabbits with the relatively large diameter cerebral arteries. Moreover, the use of traditional enzymatic dispersion methods is limited because of their complex operations. The aim of the present study was to culture VSMCs derived from rat intracranial and extracranial arteries by using a modified enzymatic dispersion method of, providing a good in vivo model system for studying the molecular mechanisms and treatment of cerebrovascular diseases including intracranial and extracranial atherosclerosis.Methods:1. Primary culture of VSMCs: (1) Rat basilar artery VSMCs:Two healthy male Sprague-Dawley (SD) rats (250~300g) were sacrificed by cervical dislocation and decontaminated with 75% ethanol thoroughly. The basilar arteries were surgically isolated from the rat brain under sterile conditions. After removal of the adventitia, they were cut into approximately 0.2 mm rings and then digested with 0.1% typeⅠcollagenase for 5 hours and with 0.125% trypsin for another 10 minutes at 37℃, respectively. After digestion, cells were cultured in DMEM/F12 supplemented with 20% fetal calf serum. (2) Rat carotid artery VSMCs:A SD rat was placed in dorsal recumbency and a 2-cm longitudinal midline incision was made in the neck. The left common carotid artery was exposed and then excised (about 1.5 cm long) under sterile conditions quickly. After removal of both the adventitia and the intima, it was cut into approximately 0.2 mm rings and then digested and cultured according to the methods as mentioned above. 2. Subculture of VSMCs:When cultures reached 80-90% confluence, cells were subcultured using 0.25% trypsin for dissociation. 3. Purification of cells:The cells were purified by using a combination of manual scraping and differential attachment techniques. 4. Identification of VSMCs:VSMCs were identified by the morphological feature and growth pattern. In addition, since smooth muscleα-actin is considered as a specific and well-known differentiation marker of VSMCs, immunocytochemical staining of smooth muscleα-actin was also performed. A mouse monoclonal antibody against smooth muscleα-actin (1:300) was applied as the primary antibody to identify VSMCs in the culture. A goat anti-mouse biotinylated immunoglobulin conjugated with avidin-biotinylated horseradish peroxidase was used as the secondary antibody, followed by streptavidin biotin peroxidase complex (SABC) staining according to the manufacturer’s instructions. 5. Cell viability:At passage 5, the number and viability of cultured cells were determined using a hemocytometer and the trypan blue (0.4%) dye exclusion assay.Results:After 3 days of incubation, primary cultures of cells isolated from the basilar artery or from the common carotid artery began to attach to the wall of the incubation dishes. After 2 weeks, cells were exhibited a spindle-shaped morphology with a classic“hill-and-valley”growth pattern at confluence. These features remained unchanged through five passages. The purity of fifth passaged VSMCs from the basilar artery or from the common carotid artery was greater than 97% to 98% as confirmed by their expression of smooth muscleα-actin. The viability of fifth passaged VSMCs from the basilar artery or the common carotid artery, measured by trypan blue dye exclusion, was more than 95% to 96%.Conclusion:The method described here is a relatively simple, reliable and inexpensive for establishing an in vitro cell culture model, which is suitable for studying the mechanism and treatment of cerebrovascular diseases such as intra- and extracranial atherosclerosis, and restenosis.更多还原