体外培养前脂肪细胞与生物衍生材料小肠黏膜下层复合的黏附状态  

Combination and adherence of in vitro cultured preadipocyte and small intestinal submucosa

作　　者：兰海[1] 黄富国[1] 杨志明[1] 罗静聪[1] 李秀群[1] 

机构地区：[1]四川大学货栈医院骨科,四川省成都市610041

出　　处：《中国组织工程研究与临床康复》2008年第10期1801-1805,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金(30270398);教育部博士点基金(20020620084)~~

摘　　要：背景：细胞与支架的成功复合是组织工程重要的技术环节,小肠黏膜下层作为一种生物衍生材料具有良好的细胞相容性,同时促血管化能力较强。目的：观察前脂肪细胞与小肠黏膜下层的体外复合情况及其黏附状态。设计、时间及地点：2004-07/2005-07在四川大学华西医院组织工程实验室完成的随机对照细胞观察。材料：前脂肪细胞来源于腰椎创伤前路手术的成年女性腹部皮下脂肪组织。小肠黏膜下层来源于新鲜猪大肠,为白色半透明膜,由四川大学华西医院组织工程实验室制备。方法：将脂肪组织剪碎成颗粒状,采用酶消化法获得原代前脂肪细胞,传代后加入含有地塞米松、胰岛素、IBMX、吲哚美辛的诱导培养基进行扩增。将小肠黏膜下层预湿,分3种方式,即分别用磷酸盐缓冲液、10%胎牛血清、10%胎牛血清＋DMEM浸泡。取第3代前脂肪细胞制备细胞悬液,分两种方法接种在小肠黏膜下层：浸渍法是直接向置有小肠黏膜下层的孔内加入1mL细胞悬液;沉淀法是向每个小肠黏膜下层的表面均匀滴入按所需接种密度高浓缩的细胞悬液20μL。主要观察指标：MTT法检测小肠黏膜下层经不同预湿和接种方法对黏附细胞数的影响。苏木精-伊红染色和电镜观察前脂肪细胞在小肠黏膜下层的黏附状态。结果：相同接种方法下,经3种方式预湿的小肠黏膜下层细胞吸光度值基本相似（P〉0.05）。相同预湿方式下,沉淀法接种细胞黏附率显著高于浸渍法（P〈0.05）。采用沉淀法接种复合,接种密度为（2.5～7.5）×10^4/cm^2时黏附细胞数逐渐增多,呈正性相关（r=0.93,P〈0.05）,升高至1×10^5/cm^2后黏附细胞数不再增加。接种24h后,前脂肪细胞能够在小肠黏膜下层表面生长,并有突起长出。结论：①小肠黏膜下层给予不同的预湿方式对前脂肪细胞黏附状态无影响,但沉淀法接种复合效果优于浸渍BACKGROUND： Successful combination of seed cells and scaffold is very important for tissue engineering, Small intestinal submucosa （SIS）, as bio-derived material, has good biocompatibility and vascularized effect, OBJECTIVE： To observe in vitro combination and adherence condition of preadipocyte and SIS, DESIGN, TIME AND SETTING： A randomized controlled cell observation was performed at Tissue Engineering Laboratory of West China Hospital, Sichuan University between July 2004 and July 2005, MATERIALS： Preadipocytes were from abdominal subcutaneous fatty tissue of adult female undergoing anterior approach operation of lumbar fractures. SIS was white translucent membrane from fresh swine large intestine, which was prepared in Medical Science Tissue Engineering Experiment Center of West China University, METHODS： Adipose tissue was cut into granules. Primary preadipocytes were collected by enzyme digestion. When the cells were subcultured, medium containing Dexamethasone, insulin, IBMX, and indomethacin was added for inducing culture. SIS was wetted by 3 different solutions： PBS, 10% fetal bovine serum （FBS）, and 10% FBS＋DMEM, respectively, The 3rd passage of preadipocyte suspension was seeded on SIS by 2 different ways： pickling process, in which 1 mL cell suspension was added into hole of plate with SIS; precipitation method： 20 μ L cell suspension at high density was dropped on the surface of SIS, MAIN OUTCOME MEASURES： Influence of different prewetting and seeding methods on number of adherent cells by MTT essay. Adherence and growth of preadipocyte on SIS by HE straining and electron microscope. RESULTS： Under the same seeding method, absorbance of cells by three different pre-wetting methods was similar （P 〉 0.05）. Under the same pre-wetting method, cell adherence by precipitation method was significantly higher than by pickling process （P 〈 0.05）. When cells were seeded by precipitation method at density of （2.5-7.5）×10^4/cm^2, adherent cell number was gradually

关 键 词：前脂肪细胞 小肠黏膜下层 组织工程 细胞分化 细胞增殖 

分 类 号：R318[医药卫生—生物医学工程]