组织工程瓣膜的种子细胞:犬隐静脉间质细胞体外钙化实验  

Seed cells of tissue-engineered valve: calcification of canine saphenous vein interstitial cells in vitro

作　　者：宿学家[1] 刘维永[1] 顾春虎[1] 欧阳辉[1] 卫向阳[1] 

机构地区：[1]解放军第四军医大学西京医院全军心血管病研究所心血管外科,陕西省西安市710032

出　　处：《中国临床康复》2004年第33期7430-7432,F003,共4页Chinese Journal of Clinical Rehabilitation

基　　金：国家自然科学基金资助项目(30271292)~~

摘　　要：目的:观察作为组织工程瓣膜种子细胞的隐静脉间质细胞的体外钙化机制。方法:实验于2003-05/2004-08在解放军第四军医大学全军心血管外科研究所实验室完成。分离培养并鉴定犬后肢隐静脉间质细胞,取3～6代细胞,分为钙化组和对照组。钙化组在钙化条件培养液中培养,对照组在标准培养液中培养。15d后,行vonkossa染色、钙沉积、碱性磷酸酶及上清液骨钙素测定。结果:犬隐静脉间质细胞经Ⅷ因子、α-SMA、Venmintin免疫组化染色鉴定,主要有平滑肌细胞、成纤维细胞和肌纤维母细胞组成。培养15d后,钙化组vonkossa染色呈强阳性,对照组呈弱阳性。钙化组钙沉积比对照组高27倍(5.384±0.443,0.192±0.024,P<0.01),碱性磷酸酶高2.13倍(9.611±0.639,3.069±0.178,P<0.01),骨钙素于培养第6天开始升高,至第12天到达高峰,然后有轻度下降。结论:体外培养的犬隐静脉间质细胞具有向成骨细胞表型转化的潜能,在钙化条件培养下具有成骨细胞的表型,有明显的钙化现象发生。AIM:To observe the mechanism of calcification of saphenous vein interstitial cells(SVICs) as the seed cells of tissue engineered valve in vitro.METHODS:The trial was completed in the Department of Cardiovascular Surgery,Xijing Hospital,Fourth Military Medical University of Chinese PLA during May 2003 to August 2004.Canine SVICs of the hindlimbs were isolated,cultured and identified.Cells of passages 3 to 6 were divided into calcification group and control group.Cells in the calcification group were cultured in the calcific conditioned medium,while those in the control group were cultured in the standard medium.After cultured for 15 days,von kossa staining, calcium deposition and determinations of alkaline phospatase(ALP) and osteocalcin(OC) in the cultured supernatant were performed.RESULTS:SVICs were mainly composed of smooth muscle cell,fibroblast and myofibroblast,which were identified by the immunocytochemical staining of Ⅷfactor,α-SMA and Venmintin.Fifteen days after cultivation,the von kossa staining was intensely positive in the calcification group but weakly positive in the control group.The calcium deposition in the calcification group was 27 times higher than that in the control group(5.384±0.443 vs 0.192±0.024,P< 0.01).The level of AKP was 2.13 times higher in the calcification group than in the control group(9.611±0.639 vs 3.069±0.178,P< 0.01).OC in cultured supernatant began to increase on the 6th day after cultivation and reached the peak value on the 12th day,and then was mildly decreased.CONCLUSION:The cultured canine SVICs in vitro have the potential of osteoblast-like phenotype,and they would switch to osteoblast phenotype when cultured in calcified medium,there is occurrence of obvious calcification.

关 键 词：隐静脉 间质细胞 生物医学工程 种子 细胞 

分 类 号：R318[医药卫生—生物医学工程]