中华烧伤杂志
中華燒傷雜誌
중화소상잡지
16
2010年
2期
133-137
,共5页
白晓智%胡大海%张万福%张战凤%石继红%蔡维霞%朱华宇%朱雄翔%汤朝武
白曉智%鬍大海%張萬福%張戰鳳%石繼紅%蔡維霞%硃華宇%硃雄翔%湯朝武
백효지%호대해%장만복%장전봉%석계홍%채유하%주화우%주웅상%탕조무
烧伤%成纤维细胞%细胞凋亡%细胞转分化%角质形成细胞
燒傷%成纖維細胞%細胞凋亡%細胞轉分化%角質形成細胞
소상%성섬유세포%세포조망%세포전분화%각질형성세포
Burns%Fibroblasts%Apoptosis%Cell transdifferentiation%Keratinocyte
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.
目的 瞭解KC熱損傷後培養上清液對真皮Fb生物學行為的影響.方法 分離培養人真皮Fb,另建立KC(選用HaCaT細胞)熱損傷模型.收集正常堵養及熱損傷後12 h的KC培養上清液,用無血清DMEM以1:1體積比稀釋,分彆製成正常KC和熱損傷KC 2種條件培養液.將Fb分彆用無血清DMEM和2種條件培養液培養:(1)于培養12、24、36、48 h時,採用噻唑藍法檢測Fb增殖活性;(2)于培養12 h時,用流式細胞術檢測Fb的凋亡情況(Fb預先緻熱損傷,併增設Fb傷後無處理對照);(3)培養24 h時,用免疫熒光法檢測Fb胞質α平滑肌肌動蛋白(α-SMA)的錶達情況,併用實時定量PCR法測定Fb α-SMA mRNA的錶達.對實驗結果進行單因素方差分析,用LSD-t檢驗行組間兩兩比較.結果 (1)Fb增殖活性:Fb經熱損傷KC條件培養液培養12、24、36、48 h時,其吸光度值均高于同時相點用無血清DMEM培養的Fb(t值分彆為1.89、2.35、2.02、1.94,P值均小于0.01);其中12、24、48 h時與用正常KC條件培養液培養的Fb比較,差異有統計學意義(12 h時t=1.83,P<0.01;24 h時t=2.91,P<0.05;48 h時t=1.83,P<0.05).(2)Fb凋亡檢測:熱損傷KC條件培養液培養的Fb與正常KC條件培養液培養以及傷後無處理的Fb比較,凋亡率均明顯下降(t=3.31,P<0.05;t=1.47,P<0.01).(3)Fb胞質α-SMA錶達:熒光顯微鏡下可見,用無血清DMEM或正常KC條件培養液培養的Fb中,α-SMA暘性錶達(紅色熒光)細胞較少;熱損傷KC條件培養液培養的Fb中,α-SMA暘性錶達細胞明顯增多.(4)α-SMA mRNA錶達:熱損傷KC條件培養液培養的Fb α-SMA mRNA相對錶達量為1.32±0.06,高于正常KC條件培養液培養的Fb(1.14±0.07,t=2.51,P<0.05)和無血清DMEM培養的Fb(1.00±0.09,t=1.77,P<0.05).結論 KC熱損傷後12 h培養上清液可明顯促進Fb增殖、抑製Fb凋亡、促進Fb嚮肌成纖維細胞轉分化.
목적 료해KC열손상후배양상청액대진피Fb생물학행위적영향.방법 분리배양인진피Fb,령건립KC(선용HaCaT세포)열손상모형.수집정상도양급열손상후12 h적KC배양상청액,용무혈청DMEM이1:1체적비희석,분별제성정상KC화열손상KC 2충조건배양액.장Fb분별용무혈청DMEM화2충조건배양액배양:(1)우배양12、24、36、48 h시,채용새서람법검측Fb증식활성;(2)우배양12 h시,용류식세포술검측Fb적조망정황(Fb예선치열손상,병증설Fb상후무처리대조);(3)배양24 h시,용면역형광법검측Fb포질α평활기기동단백(α-SMA)적표체정황,병용실시정량PCR법측정Fb α-SMA mRNA적표체.대실험결과진행단인소방차분석,용LSD-t검험행조간량량비교.결과 (1)Fb증식활성:Fb경열손상KC조건배양액배양12、24、36、48 h시,기흡광도치균고우동시상점용무혈청DMEM배양적Fb(t치분별위1.89、2.35、2.02、1.94,P치균소우0.01);기중12、24、48 h시여용정상KC조건배양액배양적Fb비교,차이유통계학의의(12 h시t=1.83,P<0.01;24 h시t=2.91,P<0.05;48 h시t=1.83,P<0.05).(2)Fb조망검측:열손상KC조건배양액배양적Fb여정상KC조건배양액배양이급상후무처리적Fb비교,조망솔균명현하강(t=3.31,P<0.05;t=1.47,P<0.01).(3)Fb포질α-SMA표체:형광현미경하가견,용무혈청DMEM혹정상KC조건배양액배양적Fb중,α-SMA양성표체(홍색형광)세포교소;열손상KC조건배양액배양적Fb중,α-SMA양성표체세포명현증다.(4)α-SMA mRNA표체:열손상KC조건배양액배양적Fb α-SMA mRNA상대표체량위1.32±0.06,고우정상KC조건배양액배양적Fb(1.14±0.07,t=2.51,P<0.05)화무혈청DMEM배양적Fb(1.00±0.09,t=1.77,P<0.05).결론 KC열손상후12 h배양상청액가명현촉진Fb증식、억제Fb조망、촉진Fb향기성섬유세포전분화.
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 obviously increased in Fb cultured with HKCM. (4) The relative expression amount of a-SMA mRNA in Fb cultured with HKCM was 1.32 ±0.06, which was higher than that both in Fb cultured with NKCM (1. 14 ± 0. 07, t = 2. 51 , P < 0. 05 ) and in Fb cultured with non-serum DMEM (1.00 ± 0. 09 , t =1.77, P < 0. 05 ). Conclusions The supernatant of KC 12 hours after heat injury can obviously promote the proliferation of Fb, inhibit its apoptosis and accelerate transdifferentiation of Fb to myofibroblasts.
