反复贴块法培养大鼠主动脉平滑肌细胞  

A repeatedly blood vessel patch-attaching method for culture of arterial smooth muscle cells

作　　者：李萍[1] 郑莲星[1] 王伶[1] 程晓曙[1] 

机构地区：[1]南昌大学第二附属医院心内科,江西省南昌市330006

出　　处：《中国组织工程研究与临床康复》2007年第46期9319-9321,共3页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：江西省卫生厅科技计划项目(0301074)~~

摘　　要：目的:原代培养鼠主动脉平滑肌细胞,传统贴块法往往由于血管片成功贴壁数少而导致失败或存在培养周期长的缺点。反复血管片贴壁法能够克服以上弊端,本实验拟进一步验证。方法:实验于2007-03/06在南昌大学第二附属医院江西省分子中心重点实验室完成。①实验方法:健康Wistar大鼠2只,断颈法处死,分离胸主动脉,清除结缔组织,纵向剖开血管去除内皮细胞,继续用力刮可见血管片分为两层,去掉外层,剩下即为所需的中膜组织,剪细置于无菌培养瓶底,组织块间距不超过0.5cm,瓶底朝上,向培养瓶内加入5.0～6.0mL含体积分数为0.15胎牛血清的培养液后,放在大平皿上使瓶口稍朝上,置于37℃孵箱,一次性培养瓶放置1h(普通培养瓶2～3h)后将其缓慢翻转平放,使组织块完全浸入培养液中。当一些组织块周围细胞生长相当密集以致失去特有的梭形形态时,即可按1∶1或1∶2进行传代。②实验评估:采用相差显微镜观察细胞的生长形态及生长规律。免疫化学法检测α-平滑肌肌动蛋白的表达。结果:①细胞生长形态:原代培养第3天,贴附良好的血管片周围细胞呈辐射状生长,形态多样。4～10d细胞数量增多,细胞生长至融汇后相互平行排列成束或放射状,血管平滑肌细胞特征性"峰"、"谷"状生长特点尤为明显。细胞生长相当密集而传代。②α-平滑肌肌动蛋白免疫组织化学检测:胞浆内出现肌丝,纵向平行排列有密区,α-平滑肌肌动蛋白免疫化学染色呈阳性(棕色)。结论:利用反复血管片贴壁法成功进行大鼠主动脉平滑肌细胞的原代培养,具有简便易行、细胞损失小、培养周期短等优点。AIM： Traditional patch-attaching method for primary culture of rat arterial smooth muscle cells always fails or takes too long cycle time due to less successful attaching. In this study, the repeatedly blood vessel patch-attaching method that may provide a solution was verified. METHODS： The experiment was carried out in the Jiangxi Molecular Central Key Laboratory, Second Affiliated Hospital of Nanchang University from March to June 2007. ①Two healthy Wistar rats were sacrificed by neck-breaking, then the thoracic aorta was separated, and connective tissue was cleared to split the blood vessel longitudinally. After the endothelial cells were scraped, the vessel tissue was shown to be two layers. When the outer layers were abscised, the remains, tunica media, were cut into small patches and put into a sterile culture flask with the space of patches was less than 0.5 cm and the bottom of flask upward. After adding 5.0-6.0 mL DMEM which contained 0.15 volume fraction fetal bovine serum, the culture flask was put on a big flat plate by keeping its orifice upward then placed into incubator at 37 %（one-time culture flask for 1 hour and common culture flask for 2-3 hours）. Then the flask was slowly turned over until the patches completely immerged into culture medium. When the cells grew so condense surrounding the patches that they lost their typical fusiform shape, they could be passaged by 1：1 or 1：2. ②Contrast phase microscope was applied to observe cell growth morphology and the regular pattern. Vascular smooth muscle cell （VSMC） α-actin was characterized by immunochemistry method. RESULTS： ①Three days after primary culture, the cells radiatively and diversified grew around the blood vessel patches that were well attached. Four to ten days later, the cells grew in number and parallelly converged into fasciculation or radiation. The specific peak or valley feature of VSMC was obvious. Cells grew condensely and passaged. ② Myofilament appeared in cytoplasm which spread parallelly and l

关 键 词：血管平滑肌细胞 原代培养 反复血管片贴壁法 

分 类 号：R556.7[医药卫生—血液循环系统疾病]