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 Col I A1 antisense oligodeoxyneucleotide (ASODN) has inhibitory effect on collagen synthesis in cultured human hypertrophic scar fibroblasts. To investigate the effects of intralesional injection of Col I A1 ASODN on collagen synthesis in human hypertrophic scar transplanted nude mouse model. Methods The animal model of humanhypertrophic scar transplantation was established in the 60 BALB/c-nunu nude mice (specific pathogen free grade, weighing about 20 g, and aged 6-8 weeks) by transplanting hypertrophic scar without epidermis donated by the patients into the interscapular subcutaneous region on the back, with 1 piece each mouse. Fifty-eight succeed models mice were randomly divided into 3 groups in accordance with the contents of injection. In group A (n=20): 5 μL Col I A1 ASODN (3 mmol/L), 3 μL l iposome, and 92 μL Opti-MEM I; in group B (n=20): 3 μL l iposome and 97 μL Opti-MEM I; in group C (n=18): only 100 μL Opti-MEM I. The injection was every day in the first 2 weeks and once every other day thereafter. The scar specimens were harvested at 2, 4, and 6 weeks after injection, respectively and the hardness of the scar tissue was measured. The collagens type I and III in the scar were observed under polarized l ight microscope after sirius red staining. The ultrastructures of the scar tissues were also observed under transmission electronic microscope (TEM). Additionally, the Col I A1 mRNAs expression was determined by RT-PCR and the concentrations of Col I A1 protein were measured with ELISA method. Results Seventeen mice died after intralesional injection. Totally 40 specimens out of 41 mice were suitable for nucleic acid and protein study, including 14 in group A, 13 in group B, and 14 in group C. The hardness of scars showed no significant difference (P gt; 0.05) among 3 groups at 2 weeks after injection, whereas the hardness of scars in group A was significantly lower than those in groups B and C at 4 and 6 weeks (P lt; 0.05), and there was no significant difference between groups B and C (P gt; 0.05). The collagen staining showed the increase of collagentype III in all groups, especially in group A with a regular arrangement of collagen type I fibers. TEM observation indicated that there was degeneration of fibroblasts and better organization of collagen fibers in group A, and the structures of collagen fibers in all groups became orderly with time. The relative expressions of Col I A1 mRNA and the concentrations of Col I A1 protein at 2 and 4 weeks after injection were significant difference among 3 groups (P lt; 0.05), and they were significantly lower in group A than in groups B and C (P lt; 0.05) at 6 weeks after injection, but no significant difference was found between groups B and C (P gt; 0.05). Conclusion Intralesional injection of Col I A1 ASODN in the nude mice model with human hypertrophic scars can inhibit the expression of Col I A1 mRNA and collagen type I, which enhances the mature and softening of the scar tissue. In this process, l iposome shows some assistant effect.
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.
【Abstract】 Objective To investigate the effects on forming of hypertrophic scar after BMSCs infected with adenovirus carrying TGF-β3c2s2 were transplanted into the wound of animal scar model. Methods The third passage of rabbit’ s BMSCs were infected with 150 mutiple infection, and were cultured 24 hours. The concentration of the BMSCs infected with recombinant adenovirus containing the TGF-β3c2s2 gene was 1×105cell/mL. The purified and evaporated recombinant adenovirus grains containing the TGF-β3c2s2 gene were diluted by DMEM/F12 (without FBS) to 1×108 pfu/mL. The animal scar model of the standard Japanese big ear rabbit was establ ished. Eighty wounds were generated on the gastroside of ear and were randomized to 4 groups in each rabbit, which were divided into 3 control groups (A: control, B: Ad-TGF-β3c2s2, C: BMSCs) and 1 experimental group (D: BMSCs/Ad-TGF-β3c2s2). Then the wounds were tranplanted with cells. On 45 days and 90 days after wounded, thicknessand hardness of scars were measured with color ultrasound diagnostic unit and especial measurement for skin and scar hardness. On 21, 45 and 90 days, three specimens were harvested respectively for further histological study. Results The wound of groups A, B, C gradually formed the different degree scars after epithel ial ization. The hyperplasty of scars reached peak on 45 days after wounded and lasted about 90 days. There was no prominent scar formed in group D during the whole observed procedure. Thickness and hardness of scar of group D and group E were approximate on 45 days and 90 days. Thickness and hardness of scar of groups A, B and C were lower than those of group D (P lt; 0.01), and group B showed more lower than group A and group C (P lt; 0.01). Disorder structure and overlapping arrangement, enlargement collagen fibers were showed in the HE histological sections of the scars of groups A, C. The structure of the scars of groups B, C were similar to Group E. The constitutionsof groups A, B, C, D on 90 days resembled to each one on 45 days. In section of immunohistochemistry after wounded on21 days and 45 days, positive expressions of BrdU in nucleus of Groups C, D were observed. Negative expressions of BrdU in Groups A, B, E were showed. Conclusion BMSCs with Ad-TGF-β3c2s2 gene transplanted into wound could inhibit the forming of hypertrophic scar.
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.
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 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.
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 study the effect of myofibroblast on the development of pathological scar. Methods From 1998 to 2000， 14 cases of keloid(k), 13 cases of hypertrophic scar(HS), and 7 cases of scar were studied through immunohistochemistry and electronical microscope. Results Myofibroblasts were often observed in the hypertrophic HS by electronical microscope, but no myofibroblast was observed in the K and NS. αSMactin was expressed in fibroblast of HS, but was not expressed in K and NS. Conclusion Myofibroblast may play a role in the development of hypertrophic scar. The difference between the absence of myofibroblast in keloid and the invasion of keloid deserves further study.