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.
Objective To explore the expression characteristics of chaperone interacting protein (CHIP) in normal, scar and chronic ulcer tissues and its relationship with wound healing. Methods Twenty biopsies including scar tissues(n=8), chronic ulcer tissues(n=4) and normal tissues(n=8)were used in this study. The immunohistochemical staining (power visionTMtwo-step histostaining reagent) was used to explore the amount and expression characteristics of such protein.Results The positive expression of CHIP was observed in fibroblasts, endothelial cells and epidermal cells in dermis and epidermis. It was not seen ininflammatory cells. The expression amount of CHIP in scar tissues, chronic ulcer tissues and normal tissues was 89%, 83% and 17% respectively. Conclusion Although the function of CHIP is not fully understood at present, the fact that this protein is expressed only at the mitogenic cells indicates that it may be involved in mitogenic regulation during wound healing.
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 study the variations of l ipid peroxidation products and copper, zinc-superoxide dismutase(CuZn-SOD) in pathological scars (hypertrophic scars and keloids). Methods The specimens were gained from patients of voluntary contributions from May 2005 to August 2005. The tissues of hypertrophic scar (10 cases, aged 16-35 years, the mean course of disease was 2.2 years), keloid (10 cases, aged 17-32 years, the mean course of disease was 8 months) and normal skin (8 cases, aged 16-34 years) were obtained. The content of malonaldehyde (MDA)and CuZn-SOD activity were detected by spectrophotometric method. The expression of CuZn-SOD was evaluated by immunohistochemistry technique. Results The contents of MDA and CuZn-SOD activity were significantly higher in hypertrophic scars[MDA (1.139 0 ± 0.106 7)nmoL/mg prot, CuZn-SOD (31.65 ± 2.21)U/mg prot, (P lt; 0.05)]and keloids[MDA (1.190 0 ± 0.074 8)nmoL/ mg prot, CuZn-SOD (34.36 ± 5.01)U/mg prot (P lt; 0.05)] than those of normal skin tissues [MDA (0.821 3 ± 0.086 4)nmoL/mg prot, CuZn-SOD (20.60 ± 5.56)U/mg prot]. Immunohistochemical studies indicated that the brown particles were CuZn-SOD positive signals, which mainly located cytoplasm in normal skin tissues, hypertrophic scars as well as keloids epidermal keratinocytes and dermal fibroblasts. CuZn-SOD expression evaluation in hypertrophic scars (4.14 ± 0.90, P lt; 0.05) and keloids epidermal keratinocytes (4.43 ± 0.79, P lt; 0.05) markedly increased when compared with normal skin tissues (2.20 ± 0.45). The expression of CuZn-SODin hypertrophic scars (4.00 ± 0.82, P lt; 0.05) and keloids dermal fibroblasts (4.43 ± 0.53, P lt; 0.05) were significantly higher than that of normal skin tissues (1.60 ± 0.89). There were no differences in the content of MDA, CuZn-SOD activity and expression evaluation between hypertrophic scars and keloids (P gt; 0.05). Conclusion In pathological scars, the contents of MDA and CuZn-SOD activity increase and the expressions of CuZn-SOD are enlarged.
【Abstract】 Objective To investigate the angiogenesis in hypertropic scar tissue of rabbit ears at different periods and to explore a new method to prevent hyperplastic scar. Methods Nineteen Japanese white rabbits(weigthing 2.0-2.5 kg) were made animal models of hypertropic scar of ear. At 10th, 30th, 60th and 90 days, after epithel ization, the microvessel and microcirculation in hyperplastic scar of 8 rabbits were studied by microcirculation microscope and laser Doppler flowmetry. The other 11 rabbits’ right or left ears were randomly chosen into experimental group and control group. At 10 days after epithel ization,40 μL of adenovirus extracellular protein with metalloprotease and thrombospondin 1 domains (Ad-METH1) was injected into tissue of scar along the perimeter of the scar in experimental group. The same volume of empty adenovirus was injected in control group. After 30 days of injection, the gross appearance of 10 rabbits’ ears scar was recorded, the number of microvessel in scarwas counted and HE stainning of scar tissue was performed in experimental and control groups. One additional rabbit was used to evaluate the mRNA and protein expression of METH1 by RT-PCR and Western blot after 3 days of injection. R e sults The average number of microvessel at 10, 30, 60 and 90 days after epithel ization was 42.37 ± 3.89, 49.46 ± 4.13, 33.12± 4.34 and 13.24 ±2.31, respectively; the average value of microcirculatory perfusion at 10, 30, 60 and 90 days after epithetl ization was (37.75 ±2.11), (59.87 ± 6.46), (44.53 ± 6.14) and (29.21 ± 1.84)PU; the density of microvessels and perfusion of microcirculation in scar tissues during prol iferative stage (from 10 to 60 days after epithel ization) were markedly higher than that during mature period (90 days after epithel ization, P lt; 0.05).At 10 to 30 days after epithel ization, the histol igical features of scar showed early stage of prol iferation and prol iferative stage appearance; at 60 days after epithel ization, it is still in prol iferative stage, while some of scars were in mature phase; at 90 days after epithel ization, the histol igical features of scar were mature period appearance. At 3 days after Ad-METH1 injection, METH1 gene was successfully expressed at both mRNA and protein levels in experimental group, but not in control group. At 30 days after injection, the gross appearanceobservation showed that scars in experimental group were flat and soft with the color close to normal, but scars incontrol group were obvious and hard. The number of microvessel of scar tissue was 12.38±2.56 in experimental group and 48.12±6.46 in control group, showing statistically significant difference between two groups(P lt; 0.01). In experimental group, HE staining shows that the density of microvessel and the number of fibroblasts were greatly decreased and collagen fibers arranged regularly. In control group, plenty of fibroblasts and abundant microvessels were observed. Thick and tight collagen fibers were seen in the outer layer of dermis with a irregular arrangement. Conclusion Theanti-angiogenesis by Ad-METH1 may have a promising appl ication in the prevention of human hyperthropic scar.
OBJECTIVE: To localize the distribution of basic fibroblast growth factor (bFGF) and transforming growth factor-beta(TGF-beta) in tissues from dermal chronic ulcer and hypertrophic scar and to explore their effects on tissue repair. METHODS: Twenty-one cases were detected to localize the distribution of bFGF and TGF-beta, among them, there were 8 cases with dermal chronic ulcers, 8 cases with hypertrophic scars, and 5 cases of normal skin. RESULTS: Positive signal of bFGF and TGF-beta could be found in normal skin, mainly in the keratinocytes. In dermal chronic ulcers, positive signal of bFGF and TGF-beta could be found in granulation tissues. bFGF was localized mainly in fibroblasts cells and endothelial cells and TGF-beta mainly in inflammatory cells. In hypertrophic scar, the localization and signal density of bFGF was similar with those in granulation tissues, but the staining of TGF-beta was negative. CONCLUSION: The different distribution of bFGF and TGF-beta in dermal chronic ulcer and hypertrophic scar may be the reason of different results of tissue repair. The pathogenesis of wound healing delay in a condition of high concentration of growth factors may come from the binding disorder of growth factors and their receptors. bFGF may be involved in all process of formation of hypertrophic scar, but TGF-beta may only play roles in the early stage.
OBJECTIVE: To explore the expression of alpha-smooth muscle actin (alpha-SMA) induced by transforming growth factor beta 1 (TGF-beta 1). METHODS: Five samples of hypertrophic scars and three samples of normal mature scars were collected as the experimental and control groups respectively. The fibroblasts were isolated from scars, and cultured in 2-dimension or 3-dimension culture system. The immunohistochemical staining method of LSAB were used to investigate the expression of alpha-SMA in fibroblasts in the different concentration of TGF-beta 1. RESULTS: The expression of alpha-SMA in 3-dimension culture system were markedly lower than those in 2-dimension culture system with respect to the fibroblasts in the experimental group. The expression of alpha-SMA in fibroblasts were different in response to various TGF-beta 1 concentration, it was more effective at the concentration of 5 ng/ml. The expression of alpha-SMA in the fibroblasts from hypertrophic scars seemed to be more sensitive to TGF-beta 1 compared to that of the normal mature scars. CONCLUSION: There are concentration-dependent in the expression of alpha-SMA induced by TGF-beta 1 in scar fibroblasts in vitro. The biological characteristics of the fibroblasts from hypertrophic scars and normal mature scars and their sensitivity to the inducement of TGF-beta 1 were different. The inducement of TGF-beta 1 may be depressed by extracellular matrix components and that may decrease the expression of alpha-SMA.
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.
In order to study the biological properties of fibroblasts isolated from different tissues. The fibroblasts from normal skin, hypertrophic scar and keloid were cultured, respectively, in vitro, and their morphologies and growth kinetics were compared. The results revealed that although fibroblasts in keloid were irregularly arranged, crisscross and overlapping with loss of polarization, there was no significant difference in the 3 groups so far the cellular morphology of fibroblast itself, cellular growth curve, cellular mitotic index, cloning efficiency and DNA content provided those cultures were in the same cellular density and culture conditions. It was concluded that fibroblasts isolated from culture of normal skin, hypertrophic scar and keloid in vitro showed no significant difference in morphology and growth kinetics, on the contrary, their biological behaviors were quite similar.