严重烧伤患者血清对人脐带MSCs生物学特性的影响  

EFFECT OF SERUM FROM SEVERE BURN PATIENTS ON BIOLOGY CHARACTERISTICS OF HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS

作　　者：刘玲英[1] 柴家科[1] 侯玉森[1] 段红杰[1] 郁永辉[1] 尹会男[1] 胡泉[1] 樊军[1] 杜俊东[1] 

机构地区：[1]解放军总医院第一附属医院烧伤整形科,北京100048

出　　处：《中国修复重建外科杂志》2013年第7期769-774,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家科技支撑计划资助项目(2009BAI87B03);解放军总医院第一附属医院院管课题资助项目(QN201207)~~

摘　　要：目的通过观察严重烧伤患者血清对体外培养的人脐带MSCs(human umbilical cord MSCs,hUCMSCs)生物学行为的影响,探讨hUCMSCs移植治疗严重烧伤的可行性。方法取足月剖腹产手术的健康胎儿脐带组织分离培养hUCMSCs,将第3代hUCMSCs用于实验。根据培养血清不同将实验分为3组,分别为10%FBS组(A组)、10%正常血清组(B组)和10%烧伤血清组(C组)。于培养24 h、72 h和6 d,采用倒置显微镜观察各组细胞形态及融合情况,MTT法测定细胞增殖;培养后第6天采用碘化丙啶染色、流式细胞仪检测细胞周期,吖啶橙/溴化乙锭染色法检测细胞凋亡,β-半乳糖苷酶(β-galactosidase,β-gal)染色检测细胞衰老,ELISA法测定B、C组血清中TNF-α、IL-1、PDGF及IGF-1含量。结果各时间点各组细胞形态均呈长梭形,部分呈多角形;但C组细胞融合速度显著快于A、B组。细胞增殖曲线显示,培养2～6 d,C组增殖细胞数高于A、B组,差异有统计学意义(P<0.05)。流式细胞仪检测示,培养6 d A、B、C组hUCMSCs增殖期(S期)比例分别为9.21%±1.02%、11.79%±1.87%和20.54%±2.03%,C组显著高于A、B组(P<0.05)。各时间点各组细胞形态和结构正常,未见细胞核着橘红色荧光的凋亡细胞或死亡细胞。培养6 d A、B、C组β-gal染色阳性细胞百分率分别为4.8%±0.2%、3.8%±0.4%和2.6%±0.1%,C组显著低于A、B组(P<0.05)。ELISA法检测C组血清中TNF-α、IL-1、PDGF及IGF-1含量显著高于B组,差异有统计学意义(P<0.05)。结论严重烧伤患者血清中含有较高水平的TNF-α、IL-1、PDGF和IGF-1等细胞因子,可显著刺激hUCMSCs快速增殖,不引起细胞凋亡,能减少细胞衰老,将hUCMSCs移植治疗严重烧伤可行。Objective To investigate the effect of the serum from severe burn patients on the biology characteristics of human umbilical cord mesenchymal stem cells （hUCMSCs） in vitro, so as to explore the feasibility of hUCMSCs transplantation for treating severe burn. Methods The 3rd passage of hUCMSCs were randomly divided into 3 groups： 10% fetal bovine serum group （group A）, 10% normal serum group （group B）, and 10% burn serum group （group C）. At 24 hours, 72 hours, and 6 days after culture, the cell morphology and density were observed by inverted microscope; the cell proliferation was assessed by MTT; after 6 days of culture, the cell cycle by propidium iodide staining and flow cytometry, the apoptosis by acridine orange/ethidium bromide staining, and the cell senescence by β-galactosidase staining; the levels of tumor necrosis factor α （TNF-α）, interleukin 1 （IL-1）, platelet-derived growth factor （PDGF）, and insulin-like growth factor 1 （IGF-1） in serum were detected by a double-antibody sandwich ELISA kit. Results hUCMSCs were long spindle/polygon in 3 groups. The cell fusion of group C was obviously faster than that in group A and group B. The cell proliferation curves showed that the velocity and number of cell proliferation in group C were significantly higher than those in group A and group B at 2-6 days after culture （P 〈 0.05）. The rates of proliferation period （S） of hUCMSCs were 9.21% ± 1.02%, 11.79% ± 1.87%, and 20.54% ± 2.03%, respectively in groups A, B, and C at 6 days, and group C was significantly higher than that of group A and group B （P 〈 0.05）. The hUCMSCs showed normal morphology and structure in 3 groups, and no apoptosis cells was observed. The positive cells percentage of group C （2.6% ± 0.1%） was significantly lower than that of group A （4.8% ± 0.2%） and group B （3.8% ± 0.4%） （P 〈 0.05）. The levels of TNF-α, IL-1, PDGF, and IGF-1 in group C were significantly higher than those in group B （P 〈 0.05）. Conclusion

关 键 词：人脐带MSCs 烧伤患者血清 细胞增殖 细胞衰老 细胞治疗 

分 类 号：R644[医药卫生—外科学]