Objective To investigate the effects of asiaticoside onthe proliferation and the Smad signal pathway of the hypertrophic scar fibroblasts.Methods The hypertrophic scar fibroblasts were cultured with tissue culture method. The expressions of Smad2 and Smad7 mRNA after asiaticoside treatment were determined by reverse transcriptionpolymerase chain reaction 48 hours later. Thecell cycle, the cell proliferation, the cell apoptosis and the expression of phosphorylated Smad2 and Smad7 with(experimental group) or without(control group) asiaticoside were detected with flow cytometry, immunocytochemistry and Western blot. Results Asiaticoside inhibited the hypertrophic scar fibroblasts from phase S to phase M. The Smad7 content and the expression of Smad7 mRNA were (1.33±1.26)% and (50.80±22.40)% in experimental group, and (9.15±3.36)% and (32.18±17.84)% in control group; there were significant differences between two groups (P＜0.05). While the content and the mRNA expression of Smad2 had no significant difference between two groups. Conclusion Asiaticoside inhibits the scar formation through Smad signal pathway.
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.
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.
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.
【摘要】 目的 建立兔耳中期瘢痕動物模型，尋找兔耳瘢痕形成的最佳位點。 方法 選用日本大耳白兔20只，在兔耳腹側選定6個位點，作直徑1 cm直達軟骨表面的皮膚全層及軟組織缺損240個。創面暴露，于傷后7 d去除軟骨上面的肉芽及血漿痂殼一次。術后連續3個月觀察創面自然愈合及瘢痕增生情況;用HE及苦味酸-天狼星紅染色觀察瘢痕形成及膠原分布情況;用計算機圖像分析系統測定膠原含量。 結果 兔耳腹側可制作類似人的增生性瘢痕模型，瘢痕的發生率42.5%~56.7%，瘢痕增生的高峰在造創后30~50 d。不同位點瘢痕增生程度不同，膠原含量也不同。 結論 兔耳腹側可建立中期瘢痕動物模型，兔耳腹側的中分和耳尖外側部分是制作兔耳增生性瘢痕的理想位點。【Abstract】 Objective To establish an animal model of intermediate stage hypertrophic scar on the rabbit ears and to find out the best sites of scar formation. Methods A total of 240 full-thickness skin and tissue defect directing access to the cartilage surface was created on the ventral side in 20 Japan white rabbits and each ear contain 6 defect sites.The wound was treated by exposure method.On the 7th day after operation, the granulation tissue and plasma shell were removed on the cartilage.Wound healing and scar proliferation under natural condition were observed continuously for 3 months.The scar formation and collagen distribution were observed by HE and Sirius red staining, and the collagen content was analyzed by using computer image analysis system. Results The ventral wound of rabbit’s ears produced hypertrophic scar similar to human hypertrophic scar, the incidence of scar was between 42.5% to 56.7%.The peak of scar proliferation was in 30 days to 50 days after operation.The degree of scar proliferation and collagen content varied at different sites. Conclusion The ventral wound of rabbit’s ears can produce intermediate stage hypertrophic scar model, the middle sites and the lateral ear tip are ideal site for madding animal model of hypertrophic scar on the rabbit ears.
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.
ObjectiveTo explore the reaction of normal skin fibroblasts from different sites of human body to cyclic stretch. MethodsThe normal skin tissues from scapular upper back and medial side of upper arm of 3 patients were cultured in vitro. Fibroblasts of experimental group were loaded by cyclic stretch with 10% amplitude for 24, 36, and 48 hours respectively. Fibroblasts of control group were cultured without cyclic stretch. The morphologic changes were observed using inverted microscope. CCK-8 method was used to detect the proliferation of the fibroblasts. The expressions of integrin β1 mRNA, p130Crk-associated substance (P130Cas) mRNA, transform growth factor β1 (TGF-β1) mRNA, and collagen type Ⅰ α1 chain (COL1A1) mRNA were detected by real-time quantitative PCR. The protein levels of collagen type Ⅰ and TGF-β1 were detected by ELISA. ResultsThe cultured cells showed a significantly increased cell proliferation ability, and apparent orientation after the applied strain. The proliferation activity, mRNA expression levels of integrin β1, P130Cas, and TGF-β1, protein levels of TGF-β1 in back skin were significantly higher than those in arm skin (P<0.05) when the fibroblasts were loaded for 36 and 48 hours, but no significant difference between back skin and arm skin at 24 hours (P>0.05). There was no significant difference in mRNA expression level of COL1A1 and protein level of collagen type Ⅰ between back skin and arm skin at 24, 36, and 48 hours (P>0.05). There was no significant difference in all above indexes between back skin and arm skin in control group (P>0.05). ConclusionFibroblasts from scapular upper back and medial side of upper arm display different reactions to cyclic stretch, which indicates that there exists site difference in the reactions of fibroblasts to cyclic stretch. It might be related with the incidence of hypertrophic scar in different sites of the body.
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.
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 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.