人脐静脉间充质干细胞原代培养换液频度与细胞增殖生长的关系

Relationship between medium change frequency and cell proliferation in mesenchymal stem cells from human umbilical vein during primary culture

目的:体外分离培养人脐静脉内皮及内皮下间充质干细胞,观察采用不同的换液频度与细胞增殖和生长的关系。方法:实验于2006-03/11在辽宁医学院解剖学实验室完成。①选取正常健康产妇顺产或剖宫产的新生儿脐带,由辽宁医学院附属第一医院提供,产妇及其家属均知情同意。②根据更换细胞培养液时间的不同,分为接种后每24h,48h,72h更换培养液组。③无菌条件下将脐带用预热的磷酸盐缓冲液充分洗涤去血渍,从脐静脉一端插入留置针,用预热的磷酸盐缓冲液冲净静脉腔血,止血钳夹闭另一端,采用胶原酶消化法分离人脐静脉内皮及内皮下细胞,取消化30min的细胞悬液进行贴壁培养,以1×108L-1密度接种于24孔培养板中,每孔的细胞数和培养液量相同,按实验设计分组的时间方法更换培养液。④各组自培养24h开始,每天分别取各组细胞4孔,以噻唑蓝比色法测定生长曲线。并采用免疫细胞化学方法对分离的细胞进行表面抗原鉴定。结果:①间充质干细胞的形态学观察:原代培养1周,细胞以梭形细胞为主。传代培养2h细胞开始贴壁变形,24h基本完成贴壁,48h可见部分已贴壁的细胞开始分裂增殖,细胞形态多样,呈椭圆形、梭形、三角形等。5～7d贴壁细胞可见多核的成纤维细胞样。在细胞生长增生过程中,接种后每24h更换培养液组细胞增殖明显,生长状态均最好;接种后每48h更换培养液组次之;接种后每72h更换培养液组细胞增殖较慢,细胞生长状态也较差。②生长曲线:接种后每24h更换培养液组的细胞生长增殖较快,比接种后每48h及72h更换培养液组达到的同样细胞数平均提前2～3d(P<0.05);接种后每48h及72h更换培养液组之间差异无显著性意义(P>0.05)。③间充质干细胞表面抗原特性:免疫细胞化学分析显示CD166呈阳性,vWF呈阴性。结论:胶原酶消化法体外分离获得的人脐静脉内皮及内皮下细胞数量高、活性佳。更换培养液的频度不同,细胞的增殖生长过程和形态变化时间不同,接种后每24h更换培养液更有利于间充质干细胞的增殖生长和纯化。

AIM： To isolate and culture mesenchymal stem cells （MSCs） derived from endothelial and subendothelial cells of human umbilical vein （hUV） in vitro and investigate the relation of medium change frequency with cell proliferation and growth. METHODS： The experiment was performed in the Department of Anatomy, Liaoning Medical University between March and November 2006. （1）The neonatal umbilical cords provided by First Affiliated Hospital of Liaoning Medical College were obtained from healthy lying-in women with spontaneous labor or caesarean birth after each mother and their family numbers signed the informed consent. （2）According to the difference of the medium change time, they wore divided into every 24 hours, 48 hours and 72 hours medium change groups after inoculation. （3）The hUV was catheterized and washed sterilely with warm phosphate buffer solution （PBS） and indwelling needle was inserted in umbilical vein that was washed with warm PBS and the distal end was clamped with hemostatic forceps. Endothelial and subendothelial cells wore isolated from hUV by collagenase digestion method. The cells wore adherently cultured and plated onto 24-woll culture plate cultured at the density of 1×10^8 L^-1 after 30-minute digestion. The cells and medium quantities wore the same and the medium was changed according to the above experiment design. （4）Cells from 4 wells of each group wore harvested every day and then collected from the 24th hour. The growth curves of the cells wore measured by MTT colorimetric assay. Surface antigen of isolated cells was determined by immunocytochemical method. RESULTS： （1）Morphological observation of MSCs： After cultivation for 1 week, numerous spindle-shape cells appeared. After passage cultivation, the cells began to metamorphose at hour 2, almost adhered to wall at hour 24, appeared to divide and showed spindle-shape and triangle at hour 48. Coenocytic fibroblast-like cells wore observed at days 5-7. The cells proliferated obviously in the group of medium change every 24 hours after inoculation with the best growth, followed by the group of medium change every 48 hours after inoculation and the cells proliferated slowly in the group of medium change every 72 hours with bad growth. （2）Growth curve： The cells in the group of medium change every 24 hours proliferated rapidly, averagely 2-3 days ahead of schedule as compared with the group of medium change every 48 hours and the group of medium change every 72 hours （P 〈 0.05）. There was rio significant difference between the group of medium change every 48 hours and the group of medium change every 72 hours （P 〉 0.05）. （3）Surface antigen characteristic of MSCs： Immunocytochemistry analysis showed that the cells were negative for vWF but expressed CD166. CONCLUSION： The isolating cells from endothelial and subendothelial cells of hUV exhibits the capability of stable passage and good cell viability by collagenase digestion method. MSCs derived from hUV are capable to be different proliferation process and the different morphology change according to the frequency of medium change. MSCs with medium change every 24 hours are more advantageous in purification and proliferation.