小鼠脂肪来源间充质干细胞培养上清液对热损伤引起的角质形成细胞凋亡的影响  

Effects of mouse adipose-derived stem cell conditioned medium on the apoptosis of keratinocytes in- duced by thermal injury in vitro

作　　者：贾文斌[1] 胡大海[1] 王洪涛[1] 陈冬冬[2] 白晓智[1] 李娜[1] 韩夫[1] 方小兵[1] 杨龙龙[1] 

机构地区：[1]第四军医大学西京医院全军烧伤中心,烧伤与皮肤外科,西安710032 [2]解放军第四五一医院神经内科二区

出　　处：《中华烧伤杂志》2014年第2期102-108,共7页Chinese Journal of Burns

摘　　要：目的探讨小鼠脂肪来源问充质干细胞培养上清液（ADSC-CM）对热损伤引起KC（人表皮细胞株HaCaT）凋亡的影响。方法（1）取5只健康BALB／c小鼠双侧腹股沟脂肪组织，采用胶原酶消化法分离纯化脂肪来源间充质干细胞（ADSC）。取第3代细胞进行形态学观察，采用流式细胞仪检测间充质十细胞表面标志物CD3l、CD34、CD45、CD90、CDl05的表达，并行成脂成骨诱导分化鉴定。（2）利用51．5℃水浴孵育HaCaT细胞35S建立KC热损伤模型，流式细胞仪检测热损伤后即刻细胞凋亡率。（3）另取热损伤后HaCaT细胞按照随机数字表法分为常规培养组、无血清培养组、50％ADSC-CM组、100％ADSC-CM组，分别以含体积分数10％FBS的DMEM培养液、无血清的DMEM培养液、含体积分数50％ADSC．CM的DMEM培养液、体积分数100％ADSC-CM培养24h。采用吖啶橙-溴化乙啶（AO—EB）染色法观察各组细胞的凋亡情况，流式细胞仪检测各组细胞凋亡率。实时荧光定量RT—PCR和蛋白质印迹法检测各组细胞Bcl-2、半胱氨酸天冬氨酸蛋白酶3（easpase-3）的mRNA和蛋白表达水平（蛋白表达结果以灰度值比值表示）。流式细胞仪检测各组细胞周期分布。以上实验均重复测定3次。对数据行单因素方差分析和LSD．t检验。结果（1）分离培养的细胞形态规则，与Fb类似，CD3l、CD34、CD45阳性表达率低于10．0％，CD90、CDl05阳性表达率均高于90．0％，经诱导后可分化成脂肪细胞、骨细胞，鉴定为ADSC。（2）热损伤后即刻，HaCaT细胞的凋亡率为（9．8±0．4）％。（3）AO—EB染色结果显示，无血清培养组HaCaT细胞凋亡数量明显多于其他3组。无血清培养组细胞凋亡率为（8．1±1．2）％，明显高于50％ADSC-CM组的（6．0±0．8）％、常规培养组的（4．6±0．8）％和100％ADSC-CM组的（3．1±0．4）％（t值分别为3．02、4．96、6．60，P值均小于0．01）Objective To investigate the effects of mouse adipose-derived stem cell conditioned me- dium （ADSC-CM） on apoptosis of keratinocytes （human epithelial cell line HaCaT） induced by thermal in- jury in vitro. Methods （ 1 ） Adipose-derived stem cells （ADSCs） from inguinal adipose tissue of 5 healthy BALB/c mice were isolated, euhured, and purified by collagenase digestion in vitro. The 3rd pas- sage of cells were collected for morphologic observation, detection of expressions of surface markers CD31 , CD34, CD45, CD90, and CD105 with flow cytometer, and identification of adipogenie and osteogenic differ- entiation. （2） HaCaT cells were incubated in water at 51.5 ℃ for 35 seconds to reproduce thermal injury model, and then the apoptosis rate was detected immediately after injury by flow eytometer. （3） Thermally injured HaCaT cells were divided into routine culture group （ RC, cultured with DMEM containing 10% FBS）, serum-free group （cultured with serum-free DMEM） , 50% ADSC-CM group （cultured with DMEM containing 50% ADSC-CM） , and 100% ADSC-CM group （cultured with 100% ADSC-CM） according to the random number table. After 24 hours, apoptosis of HaCaT cells was observed by acridine orange-ethidium bromide （AO-EB） staining; apoptotie rate was determined by flow cytometer; the mRNA and protein levels of Bel-2 and easpase-3 were respectively determined by real-time fluorescent quantitative RT-PCR technique and Western blotting （protein level was denoted as gray value） ; the cell cycles were determined by flow ey- tometer. All above experiments were repeated for 3 times. Data were processed with one-way analysis of vari- ance and LSD- t test. Results （ 1 ） The 3rd passage of cells proliferated well showing fusiform shape simi- lar to fibroblasts. The positive expression rates of CD31, CD34, and CD45 were less than 10.0% , while those of CD90 and CD105 were above 90.0%. The cells could differentiate into adipocytes and osteoblasts. They were identified as ADSCs. （2）

关 键 词：间质干细胞 细胞凋亡 热损伤 角质形成细胞 

分 类 号：R594.1[医药卫生—内科学]