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.
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 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 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.
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 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.
OBJECTIVE To study the influence and mechanism of gamma-IFN on fibroblasts in hypertrophic scars(HTS). METHODS The cultured fibroblastic cells were isolated from the hypertrophic scars of 10 patients. The fibroblasts were divided into two groups, one group was treated with gamma-IFN (100 U/ml, 5 days) and the other without gamma-IFN as control. The proliferative activity in both groups was investigated and compared by blood cytometer, the proportion of myofibroblast (MFB) and the ratio of apoptosis were examined and analysed between two groups by flow cytometry using alpha-smooth muscle actin (alpha-SMA) as marker. RESULTS The proliferative activity was downregulated with gamma-IFN. In gamma-IFN treated group, the differentiation of MFB were reduced and the decreasing ratio was 3.2% at the 2nd day and up to 10.5% at the 8th day, then it reduced gradually. The apoptosic ratio is 17.7% in gamma-IFN treated group, and is 10.9% in control group. The difference was statistically significant. CONCLUSION gamma-IFN could downregulate the proliferation of fibroblasts, decrease the differentiation of MFB and induce the apoptosis. It has beneficial effect in the treatment of hypertrophic scars(HTS).
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.
【摘要】 目的 原肌球蛋白是肌球蛋白主要相關蛋白之一，在細胞骨架與運動中起著重要的作用。探討原肌球蛋白在增生性瘢痕中的作用，有助于揭示瘢痕攣縮的產生機制。 方法 收集2006年3月-2008年7月48例患者不同時期增生與非增生瘢痕組織標本，利用基因芯片篩選出的瘢痕相關基因——原肌球蛋白基因特異片段，制備成寡核苷酸探針與瘢痕組織切片進行原位雜交。同時，將各標本進行原代細胞培養，制備成纖維細胞爬片，進行原位雜交。 結果 原肌球蛋白基因在3、6個月的增生性瘢痕中表達均明顯強于9、12個月增生性瘢痕及非增生性瘢痕，陽性細胞比例也高于9、12個月增生性瘢痕及非增生性瘢痕。 結論 瘢痕增生攣縮與細胞骨架運動的相關基因存在密切關系，而原肌球蛋白可能在其中起著重要作用。【Abstract】 Objective Tropomyosin is one of the proteins of cytoskeleton and cell movement. The aim of this study is to investigate the effect of gene expression of fibroblast tropomyosin on the formation and contraction of hypertrophic scar. Methods According to the results of differently expressed genes, in hypertrophic scar by gene microarray, topomyosin, one of the most important genes, was selected and made into oligonucleotide probe. Twenty-four hypertrophic scars and 24 non-hypertrophic scars and 12 normal skins were used and these scar were taken on 3, 6, 9, and 12 months after burned between March 2006 and July 2008. Frozen section and cultured fibroblasts were made to detect the expression of the gene by in situ hybridization. Results Expression of tropomyosin was defected in scar tissue, but those in the hypertrophic scar on 3 and 6 months after burn were significantly ber than those in the hypertrophic scar on 9 and 12 months after burn and non-hypertrophic scar. Conclusion Overexpression of cytoskeletal relative genes causes the contraction of scar and tropomyosin acts leading and key functions.