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 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.
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 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 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 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 study the effect and mechanism of the apoptosis of hypertrophic scar fibroblasts (HSF) induced by artesunate(Art). Methods HSFs were isolated and cultured from human earlobe scars by the tissue adherence method. The 3th to 5th generation cells were harvested and divided into two groups. HSF was cultured with normal medium in control group and with medium containing60, 120 and 240 mg/L (5 ml)Art in experimental group. Apoptosis and cell cycle were identified by light microscopy, electronmicroscopy and flow cytometry. Then, HSF was cultured with normal medium in control group and with medium containing 30, 60 and 120 mg/L Art in experimental group. The changes of intracellular calcium concentration were observed. Results The primary HSF was fusiform in shape and adherent. The vimentin positive expression was analyzed by immunocytochemistry. Art could induce apoptosis of HSF in the range of 60-240 mg/L under inverted microscope. The effect was dose and timedependent. Clumping of nuclear chromatin showed margination in the experimentalgroup. And the disaggregation of the nucleolus were observed under electronmicroscopy. There were significant differences in the proportion of HSF apoptosis and HSF at G0-G1,S, G2-M stages between the two groups(P＜0.05). Apoptotic peak was shown in experimental group by flow cytometry. The peak became more evident asArt concentration increased. The intracellular calcium concentration elevated markedly in HSF with 30-120 mg/L Art treatment for 24 hours, showing significant differences between the two groups (P＜0.05). Conclusion The Art facilitates HSF cells apoptosis in vitro by the change of cell cycle. It is suggested that intracellular calcium variation may be one of the mechanisms of HSF apoptosis induced by Art.
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 investigate an effect of compressive stress on proliferation and apoptosis of human hyperplastic scar fibroblasts（HSFb） in vitro. Methods HSFb were obtained from a 20 year old female patient who developed a hyperplastic scar 3 months after operation for a largearea burn. HSFb were isolated, and were cultured in vitro with the simplified airpressure controlled cellculture instrument, and then they were randomly divided into the following 8 groups: the control group (no stress) and the 7 continuous compressive stress groups, which respectively underwent the 5, 10, 15, 25, 50, 100 and 150mmHg(1mmHg=0.133 kPa) pressure treatment for 4d ays. The absorbance (A) of the cell and the inhibition ratio (IR) of the cell proliferation were determined by the MTT assay, the cell growth cycle was determined by the flow cytometer, and the cell apoptosis was observed by the AnnexinV binding with PI labeling method. Results In the 5, 10, 15, 25, 50, 100 and 150mmHg pressure groups and the control group, the A values of the cells were 0.228±0.004, 0.226±0.003, 0.213±0.005, 0.180±0.005, 0.172±0.007, 0.165±0.004, 0.164±0.004 and 0.230±0.005, respectively; the IRs of the cell proliferation were 0.8%，2.0%，7.3%，21.7%，252%， 28.2% and 0, respectively;the ratios of the cells in G1 were 71.80％±0.44％, 72.32％±0.40％, 74.56％±1.01％, 82.82％±2.76％, 86.77％±2.06％, 88.23％±1.27％, 89.11％±1.74％ and 71.6％±0.49％,respectively; the cell apoptosis ratios were 4.22％±0.49％, 5.12％±0.74％ , 8.58％±0.79%, 19.28％±1.40%, 25.60％±1.21%, 3580％±2.39%, 36.18％±2.38% and 4.00％±0.36％, respectively. In the 5 and 10mmHggroups there were no statistically significant differences in all the above parameters when compared with those in the control group (P＞0.05); however, in the 15, 25,50, 100 and 150mmHg groups there were statistically significant differences in the above parameters when compared with those in the control group (P＜0.05). Furthermore, in the 10, 15, 25 and 50 mmHg groups, there were statistically significant differences in the Avalue of the cells and the ratios of the cells in G 1 when compared with each other (P＜0.01). By contrast, there were no statistically significant differences in the 50, 100 and 150 mmHg groups when compared witheach other (P＞0.05). In the 10, 15, 25, 50 and 100mmHg groups there werestatistically significant differences in the cell apoptosis ratio when comparedwith each other (P＜0.01). In the 100 and 150 mmHg groups there were no such statistically significant differences when compared with each other (P＞0.05).Conclusion A continuous compressive stress when given properly can have a combined effect of the proliferation inhibition and the apoptosis promotion on HSFb in vitro, and this kind of combined effects can becomeone of the important mechanisms for the pressure therapy in treating hyperplastic scar.