[Effect of heat injured keratinocytes supernatant on biological behavior of fibroblasts].
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 was 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 NKCM 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 alpha-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 alpha-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.
Bai
Xiao-zhi
XZ
Department of Burns and Cutaneous Surgery, Xijing Hospital, the Fourth Military Medical University, Xi'an 710032, China.
Hu
Da-hai
DH
Zhang
Wan-fu
WF
Zhang
Zhan-feng
ZF
Shi
Ji-hong
JH
Cai
Wei-xia
WX
Zhu
Hua-yu
HY
Zhu
Xiong-xiang
XX
Tang
Chao-wu
CW
D000199:Actins / Q000378:metabolism; D017209:Apoptosis; D002454:Cell Differentiation; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D018882:Heat Stress Disorders; D006358:Hot Temperature / Q000009:adverse effects; D006801:Humans; D015603:Keratinocytes / Q000166:cytology*; D012333:RNA, Messenger / Q000235:genetics
C541116:ACTA2 protein, human; D000199:Actins; D017077:Culture Media, Conditioned; D012333:RNA, Messenger