摘要
目的:应用231培养基培养成人大隐静脉平滑肌细胞探讨其有效方法。方法:实验于2004-11/2005-3在首都医科大学附属北京安贞医院北京心肺血管疾病研究所中心实验室完成。①平滑肌细胞分离:40例冠状动脉旁路移植术患者(年龄43～74岁)术中废弃的大隐静脉,经患者同意无菌取2.0～3.0cm。2.5g/L胰蛋白酶溶液吹打消化15min,去除内皮细胞,同时松动平滑肌细胞,便于细胞游离出组织块生长。将血管剪成2.0～3.0mm2的小块贴到培养瓶底,加入完全231培养液3mL,直立放入37℃、体积分数为0.05的CO2、饱和湿度的培养箱中,24h后将培养瓶放平使培养液浸没组织块继续培养。②平滑肌细胞传代:当细胞延组织块周围密集成片生长时,即可传代。③平滑肌细胞形态观察:应用倒置显微镜观察平滑肌细胞生长状况,记录生长曲线,观察各生长时期变化。④平滑肌细胞鉴定:应用透射电镜和免疫组织化学方法鉴定平滑肌细胞。结果:①大隐静脉平滑肌细胞形态观察结果:大隐静脉平滑肌细胞原代培养沿组织块长出时间为2～14d,经过7d潜伏期细胞进入对数生长期,细胞呈“峰谷”长势。原代细胞培养第(25±2.1)天传第1代,传代24h后进入对数生长期,细胞传10代以上仍保持良好的形态结构。培养成功率100%。培养过程中未见杂细胞或微生物污染。②大隐静脉平滑肌细胞鉴定结果:电镜下观察,胞浆内富含肌丝,纵向平行排列,胞内有密体,具有平滑肌细胞特征;抗α平滑肌肌动蛋白免疫组织化学染色后可见胞浆内染成褐色呈细丝网状,所有细胞均呈阳性染色。证实培养细胞为平滑肌细胞。结论:利用消化贴块方法分离人大隐静脉平滑肌细胞,应用231培养基进行培养,不仅培养成功率高,并且可产生高纯度及多数量的平滑肌细胞。技术具有操作简便,污染机会少,为心血管疾病研究和组织工程学研究及应用提供可靠的细胞模型。
AIM： To investigate the suitable method to cultivate the adult human smooth muscle cells （HSMCs）from great saphenous vein using 231 media.
METHODS：Tbe experiment was performed in the Central Experiment Unit, Beijing Institute of Cardiopulmenary and Vascular Diseases, Beijing Anzhen Hospital Affiliated to Capital University of Medical Sciences from November 2004 to March 2005. ①Separation of HSMCs： 40 samples small discarded segments about 2-3 cm of human great saphenous vein of patients （43-74 years old） undergoing coronary artery bypass surgery under the sterile condition with patients con sent were obtained and digested with 2.5 g/L trypsin, in order to get rid of endothelial cells and loose the HSMCs so as to assure that HSMCs emigrated easily firom the tissue explants. Then the tissue of vein was sliced into block with the size of 2.0-3.0 mm^2 and directly stuck to the wall of culture flask incubated at 37℃, adding 3 mL completely 231 culture medium, CO2 with the volume fraction of 0.05. Incubator of saturation wetness for 24 hours, and then the culture flask was hid flatly so as to immerse tissue blocks for continuous culture. ②HSMCs passage： The cells could be transferred when they grew full around the tissue explants. ③Forms of HSMCs： Growth status of HSMCs was observed under inverted phase oontmst microscope. The growth curve of HSMCs was drawn to observe the changes at each period of growth. ④Identiflcation d HSMCs： The cell was confirmed to be HSMCs by immunohistochemical method and transmissi6n electron microscope. RESULTS： ① Form observation result of HSMCs of great saphenous vein： The HSMCs emigrated from the tissue explants about 2-14 days after primary culture and after 7 days latent adaptive phase it entered the logarithm grow period. The growth of cells formed a typical “hill” and “valley” pattern. The 40 samples HSMCs were passed first generation after primary culture （25±2.1） days, after 24 hours cells entered the logarithm grow period. HSMCs grew well over 10 generations. Successive rate of 40 samples culture reached 100%, but no other type cell and microbe contamination. ②Identiflcation result of HSMCs of great saphenous vein： HSMCs was confirmed under electron microscope, there were myofilamfnts in cytoplasm, the ceils showed parallel array vertically with dense areas, they had the characteristics of smooth muscle cells; Anti-a-aetin antibodies after immunohistochemical staining, lots of stained brown filament-shape bundles were observed in cell cytoplasm, all cells were stained and positive. It was confirmed to be HSMCs.
CONCLUSION： HSMCs are isolated by collagenase and adherent method and cultured with 231 mediums. It can produce plenty of smooth muscle cells of high purity. The culture technique of method has the advantages of easy to operate, fewer occasion of contamination; it can supply reliable cell models for study on tissue engineering and cardiovascular diseases.
出处
《中国临床康复》
CSCD
北大核心
2006年第29期83-85,i0003,共4页
Chinese Journal of Clinical Rehabilitation
基金
首都医学发展科研基金资助项目(306)~~
