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.
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.
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.
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 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.
【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 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 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.