Objective To explore the change of gene expression of stress activated protein kinase (SAPK) and its upstream signalregulated molecule ——mitogen activated protein kinases(MAPKs) (MKK4 and MKK7) in hypertrophic scar and autocontrol normal skin. Methods The total RNA was isolated from 8 hypertrophic scars and 8 auto-control skin, and then mRNA was purified. The gene expressions of MKK4, MKK7 and SAPK were examined with reverse transcriptionpolymerase chain reaction(RT-PCR) method. Results In hypertrophic scar, both MKK7 and SAPK genes weakly expressed. In auto-control skin, the expression of these 2 genes was significantly elevated in comparison with hypertrophic scar (Plt;0.01). The expression levelsof these 2 genes were 1.5 times and 2.6 times as long as those of hypertrophic scar, respectively. Gene expression of MKK4 had no significant difference between autocontrol skin and hypertrophic scar (Pgt;0.05). Conclusion Decreased gene expression of MKK7 and SAPK which results in reducing cell apoptosis might be one of the mechanisms for controlling the formation of hypertrophic scar.
To determine the state of fibroblast during the process of development of hypertrophic scar (HS), 40 specimens of HS in different periods were collected. The expressions of prolifrating cell nuclear antigen (PCNA) and Ag-protein in nucleolar organizer regions (Ag NORs) as well as the content of total amino acids in the tissues were examined. The hypertrophic scar of 1st and 3rd month old, the expression of PCND and Ag NORs were the highest. In the 9th and 12th month old, althrough PCNA was nearly negative, but the expression of Ag NORs was low. The content of total amino acid was increased gradually as HS developed but the increase of amount of hydroxyproline was markedly slowed down in 9 month old HS. It was suggested that: (1) in the developing process of HS the proecess of overproliferation of fibroblasts was short and limitted in 1-3 months period in the process of wound lealing; (2) the synthesis of collagen was nearly stopped at 6 months, but that of other extracellular matrix such as fibronectin and proteoglycan might be continued to aggregate after 12 months.
To investigate the inhibitory effect of Col I A1 antisense ol igodeoxyneucleotide (ASODN) transfection mediated by cationic l iposome on Col I A1 expression in human hypertrophic scar fibroblasts. Methods Scar tissue was obtained from volunteer donor. Human hypertrophic scar fibroblasts were cultured by tissue block method. The cells at passage 4 were seeded in a 6 well cell culture plate at 32.25 × 104 cells/well, and then divided into 4 groups: group A, l iposomeand Col I A1 ASODN; group B, Col I A1 ASODN; group C, l iposome; group D, blank control. At 8 hours, 1, 2, 3 and 4 days after transfection, total RNA of the cells were extracted, the expression level of Col I A1 mRNA was detected by RT-PCR, the Col I A1 protein in ECM was extracted by pepsin-digestion method, its concentration was detected by ELISA method. Results Agarose gel electrophoresis detection of ampl ified products showed clear bands without occurrence of indistinct band, obvious primer dimmer and tailing phenomenon. Relative expression level of Col I A1 mRNA: at 8 hours after transfection, group A was less than groups B, C and D (P lt; 0.05), and groups B and C were less than group D (P lt; 0.05), and no significant difference was evident between group B and group C (Pgt; 0.05); at 1 day after transfection, groups A and B were less than groups C and D (P lt; 0.05), and there was no significant difference between group A and group B, and between group C and group D (P gt; 0.05 ); at 2 days after transfection, there were significant differences among four groups (P lt; 0.05); at 3 and 4 days after transfection, group A was less than groups B, C and D (P lt; 0.05), group B was less than groups C and D (P lt; 0.05), and no significant difference was evident between group C and group D (P gt; 0.05). Concentration of Col I protein: at 8 hours after transfection, group A was less than groups B, C and D (P lt; 0.05), groups B and C were less than group D (P lt; 0.05), and no significant difference was evident between group B and group C (P gt; 0.05); at 1 day after transfection, significant differences were evident among four groups (P lt; 0.05); at 2, 3 and 4 days after tranfection, groups A and B were less than groups C and D (P lt; 0.05), and no significant difference was evident between group A and group B (P gt; 0.05). Conclusion Col I A1 ASODN can inhibit mRNA and protein expression level of Col I A1. Cationic l iposome, as the carrier, can enhance the inhibition by facil itating the entry of ASODN into cells and introducing ASODN into cell nucleus.
Objective To study the expression of heat shock protein 47 (HSP47) and its correlation to collagen deposition in pathological scar tissues. Methods The tissues of normal skin(10 cases), hypertrophic scar(19 cases), and keloid(16 cases) were obtained. The expression ofHSP47 was detected by immunohistochemistry method. The collagen fiber content was detected by Sirius red staining and polarization microscopy method. Results Compared with normal skin tissues(Mean IOD 13 050.17±4 789.41), the expression of HSP47 in hypertrophic scar(Mean IOD -521 159.50±272994.13) and keloid tissues(Mean IOD 407 440.30±295 780.63) was significantly high(Plt;0.01). And there was a direct correlation between the expression of HSP47 and the total collagen fiber content（r＝0.386，Plt;0.05）. Conclusion The HSP47 is highly expressed in pathological scartissues and it may play an important role in the collagen deposition of pathological scar tissues.
Objective To observe the differences in protein contents of three transforming growth factorbeta(TGF-β) isoforms, β1, β2, β3 andtheir receptor(I) in hypertrophic scar and normal skin and to explore their influence on scar formation. Methods Eight cases of hypertrophic scar and their corresponding normal skin were detected to compare the expression and distribution of TGF-β1, β2, β3 and receptor(I) with immunohistochemistry and common pathological methods. Results Positive signals of TGF-β1, β2, and β3 could all be deteted in normal skin, mainly in the cytoplasm and extracellular matrix of epidermal cells; in addition, those factors could also be found in interfollicular keratinocytes and sweat gland cells; and the positive particles of TGF-β R(I) were mostly located in the membrane of keratinocytes and some fibroblasts. In hypertrophic scar, TGF-β1 and β3 could be detected in epidermal basal cells; TGFβ2 chiefly distributed in epidermal cells and some fibroblast cells; the protein contents of TGF-β1 and β3 were significantly lower than that of normal skin, while the change of TGF-β2 content was undistinguished when compared withnormalskin. In two kinds of tissues, the distribution and the content of TGF-β R(I) hadno obviously difference. ConclusionThe different expression and distribution of TGF-β1, β2 andβ3 between hypertrophic scar and normal skin may beassociated with the mechanism controlling scar formation, in which the role of the TGF-βR (I) and downstream signal factors need to be further studied.
Objective To detect the expression of heat shock protein 47 mRNA in pathological scar tissue by using real-time fluorescent quantitative reversetranscription-polymerase chain reaction (RT-PCR). Methods The tissues of normal skin(n=6), hypertrophic scar(n=6) and keloid(n=6) were adopted, which were diagnosised by Pathology Department. Based on fluorescent TaqMan methodology, the real-time fluorescent quantitative RT-PCR were adopted to detect the expression ofheat shock protein 47 mRNA. Results Compared with normal skin tissue（0.019±0.021）×105, the expressions of heat shock protein47 cDNA of hypertrophic scar tissue（1.233±1.039）×105 and keloid tissue（1.222±0.707）×105 were higher， being significant differences（Plt;0.05）. Conclusion A fluorescent quantitative method was successfully applied to detecting the expression of heat shock protein 47 mRNA. Heat shock protein 47 may play an important role in promoting the formation of pathological scar tissue.
OBJECTIVE To study the influence and mechanism of gamma-IFN on fibroblasts in hypertrophic scars(HTS). METHODS The cultured fibroblastic cells were isolated from the hypertrophic scars of 10 patients. The fibroblasts were divided into two groups, one group was treated with gamma-IFN (100 U/ml, 5 days) and the other without gamma-IFN as control. The proliferative activity in both groups was investigated and compared by blood cytometer, the proportion of myofibroblast (MFB) and the ratio of apoptosis were examined and analysed between two groups by flow cytometry using alpha-smooth muscle actin (alpha-SMA) as marker. RESULTS The proliferative activity was downregulated with gamma-IFN. In gamma-IFN treated group, the differentiation of MFB were reduced and the decreasing ratio was 3.2% at the 2nd day and up to 10.5% at the 8th day, then it reduced gradually. The apoptosic ratio is 17.7% in gamma-IFN treated group, and is 10.9% in control group. The difference was statistically significant. CONCLUSION gamma-IFN could downregulate the proliferation of fibroblasts, decrease the differentiation of MFB and induce the apoptosis. It has beneficial effect in the treatment of hypertrophic scars(HTS).
OBJECTIVE: To observe the protein expression of phosphorylated form of P38 mitogen-activated protein kinase(P38MAPK) and c-Jun in hypertrophic scar skin and to explore their influences on the formation and maturation of hypertrophic scar. METHODS: The expression intensity and distribution of phosphorylated form of P38MAPK and c-Jun were examined with immunohistochemistry and pathological methods in 16 cases of hypertrophic scar skin and 8 cases of normal skin. RESULTS: In normal skin, the positive signals of phosphorylated form of P38MAPK mostly distributed in basal lamina cells of epidermis, while c-Jun was mainly located in epidermal cells and endothelial cells. The positive cellular rates of two proteins were 21.3% +/- 3.6% and 33.4% +/- 3.5% respectively. In proliferative hypertrophic scar skin, the particles of phosphorylated P38MAPK and c-Jun were mainly located in epidermal cells and some fibroblasts. The positive cellular rates of two proteins were significantly elevated to 69.5% +/- 3.3% and 59.6% +/- 4.3% respectively (P lt; 0.01). In mature hypertrophic scar, the expression of these proteins decreased but was still higher than that of normal skin. CONCLUSION: The formation and maturation of hypertrophic scar might be associated with the alteration of phosphorylated P38MAPK and c-Jun protein expression in hypertrophic scar.
Objective To explore the effect of connective tissue growth factor on the pathogenesis of hypertrophic scar and keloid tissue. Methods The content of hydroxyproline was determined and the expression of connective tissue growth factor gene was detected by the reverse transcription-polymerase chain reaction and image analysis technique in 5 normal skins, 15 hypertrophic scars and 7 keloid tissues. Results The contents of hydroxyproline in the hypertrophic scar(84.10±1.76) and keloid tissue (92.38±2.04) were significantly higher than that of normal skin tissue (26.52 ± 4.10) (P lt; 0.01). The index of connective tissue growth factor mRNA in the hypertrophic scar (0.78 ± 0.63) and keloid tissue (0.84 ± 0.04) were higher than that of normal skin tissue ( 0.09 ± 0.25) (P lt; 0.01). Conclusion Connective tissue growth factor may play an important role in promoting the fibrotic process of hypertrophic scar and keloid tissue.
Objective To identify the effect of β-endorphin in the development of paresthesia in hypertrophic scar by detecting the expression and content of β-endorphin in human normal skin and hypertrophic scar. Methods Hypertrophic scar samples were collected from 42 patients with hypertrophic scar for 1-20 years (mean, 4.5 years), including 15 males and27 females with an average age of 32.6 years (range, 16-50 years). According to the kind of paresthesia, they were divided into 3 gourps: non-pain-pruritus group (n=20), pruritus group (n=14), and pain-pruritus group (n=8). Normal skin samples (normal skin group) were harvested from 5 patients undergoing skin grafting surgery, including 3 males and 2 females with an average age of 24.6 years (range, 15-37 years). The immunofluorescence method was used to observe the expression of β-endorphin and ELISA method to detect the concentrations of β-endorphin in the tissues. Results The β-endorphin expressed in all samples, and it expressed around peri pheral nerve fibers in the dermis, fibroblasts, and monocytoid cells princi pally; and it expressed significantly ber in pruritus group and pain-pruritus group than in non-pain-pruritus group and normal skin group. The β-endorphin content was (617.401 ± 97.518) pg/mL in non-pain-pruritus group, (739.543 ± 94.149) pg/mL in pruritus group, (623.294 ± 149.613) pg/mL in pain-pruritus group, and (319.734 ± 85.301) pg/mL in normal skin group; it was significantly higher in non-pain-pruritus group, pruritus group, and pain-pruritus group than in normal skin group (P lt; 0.05); it was significantly higher in pruritus group than in non-pain-pruritus group and pain-pruritus group (P lt; 0.05); and there was no significant difference between non-pain-pruritus group and pain-pruritus group (P gt; 0.05). Conclusion The expression of β-endorphin is high in hypertrophic scar, it may paly an important role in process of pruritus in these patients.