| Objective: The present study was performed to: (1) study the influence of Lipopolysaccharide(LPS) on the biological characteristics (cell proliferation and collagen synthesis) of normal skin fibroblasts so as to elucidate the influence of LPS on skin wound healing in the initial stage. (2) observe pathobiology, biological characteristics and gene expression profiles change of normal skin fibroblasts after stimulated by definite concentration of LPS and continuous passage, fibroblasts from hypertrophic scar tissue obtained from the same patients were used control, investigate its influence on hypertrophic scar formation in the later stage and its possible mechanism.Methods: 20 patients with hypertrophic scar in proliferative stage were selected randomly, the primary cultured fibroblasts derived from their hypertrophic scar tissue and normal skin. Normal skin fibroblasts of passage 3 were stimulated with different concentrations of LPS, stimulus concentrations were divided into seven groups as follows: (1) normal skin fibroblasts group. (2) 0.005ug/ml LPS group. (3) 0.010ug/ml LPS group. (4) 0.050ug/ml LPS group. (5)0.100ug/ml LPS group. (6) 0.500ug/ml LPS group. (7) 1.000ug/ml LPS group. The following methods were used to detect the biological characters ( cell proliferation and collagen synthesis ) of fibroblasts: the proliferation of fibroblasts were determined with the colorirneteric thiazolylblue (MTT) assay, cell counting and the cell cycle analyze; collagen synthesis of fibroblasts in culture medium was measured with the method of pepsin digestion after incorporation of 3H-proline into stable, single-layered, confluent fibroblasts. The result is that 0.100ug/ml LPS has the significant effect, continuous passage of these fibroblasts, observe the variance of the ultrastructure with transmission electronic microscope at passage (4, 6, 8, 10), the fibroblasts from passage 8 had similar ultrastructure withfibroblasts from hypertrophic scar tissue, the same as the result detected by HE and immunohistochemistry staining. Therefore, fibroblasts of passage 8 were used for experiment and divided into three groups as follows: (1) hypertrophic scar tissue fibroblasts group (positive control group). (2) normal skin fibroblasts group (negative control group). (3) 0.1ug/ml LPS group. The following methods were used to detect the characters of cell proliferation and secretion of fibroblasts: the proliferation of fibroblasts were determined with MTT assay; collagen synthesis of fibroblasts in culture medium was measured with the method of pepsin digestion after incorporation of 3H-proline into stable, single-layered, confluent fibroblasts; the amount of type I , III collagen in the cultured supernatant were measured by ELISA. The following methods were used to detect the gene expression of fibroblasts: the expression of procollagen type I , III and collagenase mRNAs of fibroblasts were tested by reverse transcription polymerase chain reaction (RT-PCR), taking β- actin as internal standard, the gene expression profiles of fibroblasts were detected by cDNA microarray, select the different genes related to the formation of hypertrophic scar between hypertrophic scar tissue fibroblasts and normal skin fibroblasts stimulated with 0.01ug/ml LPS, and validated using RT-PCR analysis. Except above-mentioned index, we adopted ELISA to measure the amount of TGF- β1 and IFN-γ of the cultured supernatant of fibroblasts to propose the possible mechanism of LPS on fibroblasts.Results: 1, The influence of lipopolysaccharide on the proliferation and collagen synthesis of normal skin fibroblasts in the initial stage: LPS could promote the proliferation and collagen synthesis of fibroblasts within a certain extent of concentrations(0.005μg/ml～0.5μg/ml)( P < 0.05 ), the effect reached the peak when LPS concentration was 0.1μg/ml, while over-high concentrations of LPS ( 1.0μg/ml) inhibites the proliferation and collagen synthesis of fibroblasts ( P < 0.05 ) . 2, The pathology change of normal skin fibroblasts after stimulated by LPS of 0.1 μg/ml and continuous passage: the fibroblasts from passage 8 hadsimilar ultrastructure with fibroblasts from hypertrophic scar tissue, the same as the result detected by HE and immunohistochemistry staining. 3, The proliferation change of normal skin fibroblasts after stimulated by LPS of 0.1 μg/ml and continuous passage: the proliferation rate of normal skin fibroblasts increased after stimulated by LPS of 0.1 μg/ml and continuous passage, and the proliferation rate were similar to that of positive control group (P>0.05) . 4, The collagen synthesis and the secretion of type I and III collagen change of normal skin fibroblasts after stimulated by LPS of 0.1μg/ml and continuous passage: the collagen synthesis and the secretion of type I and III collagen enhanced, the ratio of type I and III collagen upregulated of normal skin fibroblasts after stimulated by LPS of 0.1μg/ml and continuous passage, and the collagen synthesis and the secretion of type I and III collagen and the ratio of type I and III collagen were similar to that of positive control group (P > 0.05) . 5, The expression of procollagen type I , III and collagenase mRNAs change of normal skin fibroblasts after stimulated by LPS of 0.1μg/ml and continuous passage: the expression of procollagen type I and type III mRNAs markedly increased, but the expression of collagenase mRNAs significantly decreased after stimulated by LPS of 0.1 μg/ml and continuous passage, the expression of procollagen type I , III and collagenase mRNAs were similar to that of positive control group (P> 0.05) .6, The variance of gene expression profiles of normal skin fibroblasts after stimulated by LPS of 0.1 μg/ml and continuous passage: the expression of the procollagen I , c-myc and TGF-β1 which related to the collagen metabolism were up-regulated, the expression of procollagen I , c-myc and TGF-β1 mRNAs were similar to that of positive control group (P>0.05 ) . 7, The secretion of TGF-β1 and IFN-γ change of normal skin fibroblasts after stimulated by LPS of 0.1 μg/ml and continuous passage: the secretion of TGF-β1 were enhanced and the secretion of IFN-γ were ihibited after stimulated by LPS of 0.1μg/ml and continuous passage, and the amount of TGF-β1, IFN-γ were similar to that of positive control group (P>0.05) .Conclusions: 1, Stimulated by Lipopolysaccharide(LPS) in proper concentration can change the biological characteristics of normal skin fibroblasts, it can enhance the proliferation and collagen synthesis of fibroblasts in vitro, while high doses of LPS has opposite effect. The results suggests that appropriate concentration of LPS may have no effect or be beneficial to skin wound healing, excessive concentration of LPS may be delay the time of wound healing. 2, After stimulation with definite concentration of LPS and continuous passage, normal skin fibroblasts will have similar morphology, and some biological characteristics with fibriblasts from hypertrophic scar of the same patients: for example, the ultrastructure, morphology and phenotype were similar to that of hypertrophic scar tissue fibroblasts, cell proliferation, collagen synthesis, the secretion of type I and III collagen the ratio of type I and III collagen increased, the mRNAs level of procollagen I and procollagen III up-regulated, while the mRNAs level of collagenase down-regulated. Stimulated by 0.1ug/ml LPS and continuous passage, the gene expression profiles of normal skin fibroblasts changed, the genes expression related to the formation of hypertrophic scar were similar to hypertrophic scar tissue fibroblasts. The results suggested that LPS might convert normal skin fibroblasts to hypertrophic scar tissue fibrolasts and participate in the formation of hypertrophic scar. The possible mechanism of LPS on fibroblasts may be it can enhance the secretion of TGF-β1 and inhibit the secretion of IFN-γ.
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