摘要
背景:肺微血管内皮细胞的分离和培养国外多采用消化培养法,对培养基和其他实验条件要求较高。国内大部分采用组织块贴壁培养法。目的:比较消化法和组织块法培养肺微血管内皮细胞的差异,并进行改进,以获取大量纯化的肺微血管内皮细胞。设计、时间及地点:细胞水平的观察对比实验,于2006-09/2007-09在解放军第四军医大学分子生物与生化实验室完成。材料:选用Wistar大鼠30只,按随机数字表法分为2组,组织块法培养组及消化法培养组各15只。方法:分别采用组织块法与消化法进行肺微血管内皮细胞原代培养,并在培养过程中进行如下改进:①加大灌注大鼠肺脏的磷酸盐缓冲液液体量并改进灌注方法。②改进大鼠肺脏脏层胸膜的去除方法。主要观察指标:通过形态学观察、免疫组织化学染色、扫描电镜、描绘生长曲线等方法对培养的细胞进行鉴定。结果:①组织块法培养的肺微血管内皮细胞在细胞活力、增殖速度方面明显优于消化法(P<0.01)。②组织块法获得的肺微血管内皮细胞倒置显微镜下呈典型铺路石样排列,AgⅧ相关抗原免疫组化染色呈阳性,扫描电镜下观察可见细胞表面有丰富的微绒毛。结论:相对于消化法,组织块法培养的肺微血管内皮细胞更具活力,增殖速度也更快。
BACKGROUND： People often isolate and culture of pulmonary microvascular endothelial cells using enzyme digestion culture method abroad, which has higher requirements to medium and other conditions, while people at home often use tissue culture method.
OBJECTIVE： To compare the difference of enzyme digestion culture method and tissue culture method in culturing pulmonary microvascular endothelial cells and to improve the methods to obtain a large quantity of purified pulmonary microvascular endothelial cells.
DESIGN, TIME AND SETTING： Contrast observation at cellular level was conducted at Laboratory of Biochemistry and Molecular Biology, the Fourth Military Medical University between September 2006 and September 2007.
MATERIALS： Thirty Wistar rats were randomized into enzyme digestion culture group and tissue culture group by random digits table, 15 rats in each group.
METHODS： The pulmonary microvascular endothelial cells were cultured with enzyme digestion culture method and tissue culture method, respectively. And the culture methods were improved as follows： increase the volume of phosphate buffer that infused rat lung and improve the infusion method; improve the removal method of visceral pleura of the rat lung.
MAIN OUTCOME MEASURES： We identified the cultured cells through cell morphology, immunohistochemistical staining, scanning electron microscopy and growth curve.
RESULTS： The vitality and growth rate of pulmonary microvascular endothelial ceils cultured by tissue culture method were higher than that cultured by the enzyme digestion culture method （P 〈 0.01）. Under inverted microscope, the pulmonary microvascular endothelial cells cultured by tissue culture method presented a typical cobblestone-like appearance, immunohistochemistry staining of factor Ⅷ-associated antigen was positive; we could see rich microvilli on the surface of the pulmonary microvascular endothelial cells under scanning electron microscope.
CONCLUSION： The vitality and growth rate of pulmonary microvascular endothelial cells cultured by tissue culture method were higher than that cultured by the enzyme digestion culture method.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第31期6141-6144,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
