体外修复血管平滑肌细胞的方法学研究  

Methodological study on the in vitro repair of vascular smooth muscle cells

作　　者：王家宏[1] 黄永麟[1] 赵敏[1] 尤棋[2] 

机构地区：[1]哈尔滨医科大学附属第一医院心内科,黑龙江省哈尔滨市150000 [2]哈尔滨医科大学附属第一医院妇产科,黑龙江省哈尔滨市150000

出　　处：《中国临床康复》2006年第9期102-105,共4页Chinese Journal of Clinical Rehabilitation

基　　金：国家自然科学基金资助项目(30170368);哈尔滨市科技攻关计划项目(2005AA9CS116-28)~~

摘　　要：目的:采用不同方法体外培养人脐动脉平滑肌细胞,选择以获得体外修复受损平滑肌细胞的最佳方法。方法:实验于2004-12/2005-10在哈尔滨医科大学附属第一医院实验中心卫生部细胞移植重点实验室进行。①取新生儿脐带约5cm长(家属志愿提供)原代分离血管平滑肌细胞;分别用贴块法和胰酶/胶原酶酶解法分离、培养血管平滑肌细胞。②贴块法:将血管平滑肌细胞组织块20%M199完全培养基(200mL/L胎牛血清+10mL/L双抗+5mL/LL-谷氨酰胺)贴壁24h后,补足完全培养基培养;胰酶/胶原酶酶解法:第1步胰酶消化:将脐动脉剪成1mm×1mm大小。装入含2.5g/L胰酶培养瓶中,消化15～30min,加入20%M199完全培养基终止消化。第2步胶原酶消化:将上述处理过的组织块放入含1g/LⅠ型胶原酶的无血清M199培养基中,过夜,离心,收集细胞,以1×104个细胞/cm2密度接种培养皿中,补足20%M199完全培养基培养。③对两种方法培养的细胞生长形态、数量及平滑肌а-肌动蛋白表达情况进行观察和检测。结果:①贴块法:组织块种植后2周时,多数细胞已贴壁伸展。4～6周细胞达80%融合时,镜下成“峰与谷”样,可传代培养,传代间隔期一般是两三周;胰酶/胶原酶酶解法:细胞多为伴有胞质突起的多角形大细胞,达到生长融合时细胞密度低。2周左右细胞融合,可传代培养。传代间隔期一般是一两周。②贴块法获得的细胞初期生长速度慢,但达到融合前细胞数量多,细胞得率高;胰酶/胶原酶酶解法获得的细胞初期生长速度快,群体倍增时间短,但达到融合前细胞数量少于贴块法,细胞得率低。③胰酶/胶原酶法α-肌动蛋白阳性细胞率高于贴块法(93.1%,71.4%,χ2=4.626,P<0.05)。结论:两种方法均可成功培养人血管平滑肌细胞;贴块法合成表型细胞比例大,胰酶/胶原酶酶解法收缩表型细胞比例大,后者所获得的细胞更适合用作修复血管平滑肌细�AIM：To compare the two different isolation methods of human vascular smooth muscle cells（VSMC） cultured in vitro and of gain the suitable way to repair the damaged vascular smooth muscle cells in vitro. METHODS： This study was conducted at the Key Laboratory for Cell Transplantation, Department of Health of Experimental Center, First Hospital Affiliated to Harbin Medical University from December 2004 to October 2005. ①Vascular smooth muscle cells which were isolated from human umbilical cord which was provided by volunteer family members with tissue explant and pancreatin/collagen digestion method respectively. ②Tissue explant method： Vascular smooth tissue was cultivated in 20% M199 complete medium （200 mL/L fetal bovine serum ＋10 mL/L penicilin/streptomycin＋5 mL/L L-glutamine） after adhesion to wall for 24 hours. Complete culture medium was supplemented. Pancreatin/collagen digestion method： The 1^st step of pancreatin digestion： The isolated tissue of VSMC in 1 mm×1mm was digested in 2.5 g/L pancreatin for 15-30 minutes, then stopped by 20%M199 complete medium. The 2^nd step of collagen digestion： The above-handling tissue was incubated in 1 g/L collagen type Ⅰ and 20% M199 medium together overnight. Collected cells after centrifugalization and inoculated culture in 1×10^4/cm^2 in 20% M199 medium.③The morphology , proliferation and expression of smooth muscle α-actin of the cultured cells between the two groups were observed and detected. RESULTS： ①Tissue explant method： Most cells had been extended after inoculated for 2 weeks, At 4-6 weeks, 80% cells presented confluence, while showed ＂peak-valley＂ under the microscope, cultured VSMC could be serially subcultivated with the passage period of 2-3 weeks, Pancreatin/collagen digestion method： VSMCs by pancreatin/collagen digestion method showed cytoplasmic enation or polygon large cells usually, The cell density was lower than that in the tissue explant method. Confluence appeared at about 2 weeks and passage could

关 键 词：生物学 肌细胞 平滑肌 细胞培养 人 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]