摘要
目的研究冻存对内皮生长晕细胞增殖能力和成血管能力的影响,探讨内皮生长晕细胞冻存和复苏的可行性。方法分离脐血中单个核细胞,采用贴壁培养法培养扩增内皮生长晕细胞,免疫组织化学染色和荧光染色法鉴定其内皮细胞特性。扩增后的细胞采用浓度为650μmol/L(体积比为50mL/L)和1040μmol/L(体积比为80mL/L)二甲基亚砜的培养基冻存至液氮中,24h后复苏并观察冻存细胞的复苏率、复苏后细胞的增殖能力和成血管能力的改变。结果采用贴壁法培养的细胞具有多种内皮细胞特性。细胞采用含不同浓度二甲基亚砜(50mL/L和80mL/L)的培养基冻存后,其复苏率差异无统计学意义,细胞的增殖能力在复苏后36h内50mL/L、80mL/L二甲基亚砜组均较未冻存组减弱(P<0.05),72h后三组间比较差异无统计学意义。而复苏后6h内成血管速率较未冻存组减弱(P<0.05),但30h后三组间比较差异无统计学意义。结论内皮生长晕细胞可以进行冻存和复苏。
Aim To investigate the effect of cryopreservation on the proliferative abilitiy and vasculogenesis activity of endothelial outgrowth cells（EOC）,and to explore the possibility of its resuscitation.Methods The mononuclear cells were harvested from umbilical cord blood by density gradient centrifugation,induced into EOC and expanded in vitro.The endothelial characteristics of EOC were identified by immunostaining and fluorescent staining.Then EOC were cryopreserved at-20℃ for 4 h,-75℃ overnight and then cryopreserved into liquid nitrogen using culture medium containing 50 mL/L and 80 mL/L dimethyl sulfoxide（DMSO）. After 24 h cryopreserved EOC were resuscitated, and the resuscitative rate, proliferative ability and vasculogencsis activity were measured. Results EOC possessed many endothelial characteristics. Immunostaining showed that surface antigens Factor Ⅷ, CD34 and Flk-1 were postive; and fluorescent staining experiment revealed that EOC both bound to FITC-UEA-1 and uptook DiI-ac-LDL. There were no statistical differences between 50 mL/L and 80 mL/L DMSO group in resuscitative rate （79%±6% vs88%±4%, P〉0.05）. During early peried after resuscitation （within36 h）, two cryoprcservated groups （ 50 mL/L and 80 mL/L DMSO group） both showed decrease in proliferative abilitiy compared with non-cryoprcservated group（ P 〈 0.05）. 72 h later there were no significant differenccs btween 50 mL/L, 80 mL/L DMSO group and non-cryopreservated group in the proliferative ability（ P 〉 0.05）. In contrast, vasculogenesis activity analysis revealed attenuation in vasculogenesis activity within 6 h （ P 〈 0.05 ） while 30 h later such attenuation became insignificant （ P 〉 0.05 ）. Conclusions EOC can be cryopreservated and resuscitated. The cryoprcservated cell shows no significant decrease in proliferative abilitiy and vasculogenesis activity after resuscitation.
出处
《中国动脉硬化杂志》
CAS
CSCD
2007年第11期819-822,共4页
Chinese Journal of Arteriosclerosis
基金
浙江省自然科学基金(303648)
关键词
细胞生物学
内皮祖细胞
内皮生长晕细胞
冻存
复苏
增殖能力
成血管能力
Endothelial Progenitor Cell
Endothelial Outgrowth Cell
Cryopreservation
Resuscitation
Proliferative Ability
Vasculogenesis Activity
