热损伤角质形成细胞培养上清液对成纤维细胞生物学行为的影响  

Effect of heat injured keratinocytes supernatant on biological behavior of fibroblasts

作　　者：白晓智[1] 胡大海[1] 张万福[1] 张战凤[1] 石继红[1] 蔡维霞[1] 朱华宇[1] 朱雄翔[1] 汤朝武[1] 

机构地区：[1]第四军医大学西京医院烧伤与皮肤外科,西安710032

出　　处：《中华烧伤杂志》2010年第2期133-137,共5页Chinese Journal of Burns

基　　金：国家自然科学基金（30772249）

摘　　要：目的 了解KC热损伤后培养上清液对真皮Fb生物学行为的影响.方法 分离培养人真皮Fb,另建立KC（选用HaCaT细胞）热损伤模型.收集正常堵养及热损伤后12 h的KC培养上清液,用无血清DMEM以1：1体积比稀释,分别制成正常KC和热损伤KC 2种条件培养液.将Fb分别用无血清DMEM和2种条件培养液培养：（1）于培养12、24、36、48 h时,采用噻唑蓝法检测Fb增殖活性;（2）于培养12 h时,用流式细胞术检测Fb的凋亡情况（Fb预先致热损伤,并增设Fb伤后无处理对照）;（3）培养24 h时,用免疫荧光法检测Fb胞质α平滑肌肌动蛋白（α-SMA）的表达情况,并用实时定量PCR法测定Fb α-SMA mRNA的表达.对实验结果进行单因素方差分析,用LSD-t检验行组间两两比较.结果 （1）Fb增殖活性：Fb经热损伤KC条件培养液培养12、24、36、48 h时,其吸光度值均高于同时相点用无血清DMEM培养的Fb（t值分别为1.89、2.35、2.02、1.94,P值均小于0.01）;其中12、24、48 h时与用正常KC条件培养液培养的Fb比较,差异有统计学意义（12 h时t=1.83,P〈0.01;24 h时t=2.91,P〈0.05;48 h时t=1.83,P〈0.05）.（2）Fb凋亡检测：热损伤KC条件培养液培养的Fb与正常KC条件培养液培养以及伤后无处理的Fb比较,凋亡率均明显下降（t=3.31,P〈0.05;t=1.47,P〈0.01）.（3）Fb胞质α-SMA表达：荧光显微镜下可见,用无血清DMEM或正常KC条件培养液培养的Fb中,α-SMA阳性表达（红色荧光）细胞较少;热损伤KC条件培养液培养的Fb中,α-SMA阳性表达细胞明显增多.（4）α-SMA mRNA表达：热损伤KC条件培养液培养的Fb α-SMA mRNA相对表达量为1.32±0.06,高于正常KC条件培养液培养的Fb（1.14±0.07,t=2.51,P〈0.05）和无血清DMEM培养的Fb（1.00±0.09,t=1.77,P〈0.05）.结论 KC热损伤后12 h培养上清液可明显促进Fb增殖、抑制Fb凋亡、促进Fb向肌成纤维细胞转分化.Objective To observe the effect of the supernatant of heat injured keratinocytes （KC） on biological behavior of the dermal fibroblasts （ Fb）. Methods Human dermal Fb were isolated and cultured. A model of heat injured KC （ HaCaT） was reproduced in vitro. Supernatant of normal KC and the supernatant of KC culture 12 hours after heat injury were collected and diluted with non-serum DMEM in 1：1 volume ratio to make normal KC conditioned medium （ NKCM ） and heat injury KC conditioned medium （ HKCM） respectively. Fb was respectively treated with non-serum DMEM and 2 kinds of conditioned medium. （1） The proliferation of Fb was detected with MTT method at post culture hour （PCH） 12, 24, 36, 48. （2） The apoptosis of Fb was determined by flow cytometry at PCH 12 （ Fb were heat injured in advance; Fb without heat treatment was used as control）. （3） At PCH 24, expression of a-SMA in Fb cytoplasm was determined with immunofluorescence method; expression of a-SMA mRNA in Fb was determined with real-time quantified PCR. Data were processed with one-way analysis of variance, and pairwise comparison among groups with LSD-t test. Results （1） The proliferation of Fb： the absorbance value of Fb cultured with HKCM at PCH 12, 24, 36, 48 wa9 respectively higher than that of Fb cultured with non-serum DMEM （ with t value respectively 1. 89, 2. 35 , 2.02, 1.94, and P values all below 0. 01）. There were significant statistical differences between the absorbance values of Fb cultured with HKCM and those of Fb cultured with 1MKCM at PCH 12, 24, and 48 （at PCH 12, t = 1. 83, P 〈0. 01; at PCH 24,t =2.91, P 〈 0. 05 ; at PCH 48 , t = 1. 83 , P 〈 0.05）. （2 ） Apoptosis of Fb cultured with HKCM was diminished as compared with that of Fb cultured with NKCM and of Fb without treatment （ t =3.31 , P 〈0.05; t = 1.47, P 〈 0.01）. （3） The expression of α-SMA （red fluorescence） in Fb cultured with non-serum DMEM or NKCM was less as seen under fluorescence scope, and it was

关 键 词：烧伤 成纤维细胞 细胞凋亡 细胞转分化 角质形成细胞 

分 类 号：R644[医药卫生—外科学]