The ultrastructures of 14 keloids and 7 hypertrophic scars were examined by electron micrascopy.Both lesions were found to be comprised of fibroblasts, macrophages, microfi brils of collagen andmicrovessels which were partly or completely obliterated. Most fibroblasts were of active cell types.They contained abundant coarse endoplasmic reticulum and prominent Golgi complexes. The fibrils inthe lesions were irtegularly arranged. Meanwhile myofibroblasts were often seen in the keloid.In the cytoplasm of the myofibroblasts, in addition to coarse endoplasmic reticulum and Golgi complexes, many fine myofilaments, dense bodies, dense patches and distrupted basal lamina were present. These characteristic features might help to differentiate keloid from hypertrophic sacr.
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 compare gene express difference ofkeloid and normal skin tissues by using the suppression subtractive hybridization (SSH) so asto find the differential express gene in keloid. Methods mRNA extracted fromkeloid and normal skin tissues was used as the template to synthesis cDNA of keoid and normal skin. The cDNA of keloid served as a tester, the cDNA of normal skin as a driver. cDNA was digested with RsaⅠ. Adaptor-ligated tester cDNA was prepared. Then first hybridization, second hybridization and PCR amplificationwere done. Differentially expressed cDNA was selectively amplified during thesereactions. After SSH, the PCR mixture was ligated with T-vector. The positive clones were selected and the insert gene fragments were analyzed. Southern hybridization identified the keloid differential express genes. The positive clones ofSouthern hybridization were selected, and these sequences were analyzed. The results were compared with that of GeneBank. Results Thirteen differential genes were found in keloid, of which 11 gene clones have been known their function, and 2 clones have not known their function. 〖WTHZ〗Conclusion Keloid differentially expressed gene was screened successfully by SSH.
Objective To build animal models of keloid by method of tissue engineering and to discuss the feasibility of using it in clinical and lab researches. Methods Fibroblasts(FB) were isolated from keloids and cultured. The seventh and eighth generation of the cultured FBs were inoculated into the copolymers of polylactic acid and polyglycolic PLGA. After being cultured in rotatory cell culture system （RCCS）for 1 week,the FB was transplanted into athymic mice. The specimens were obtained 4 weeks and 8 weeks and examined histologically. Results All mice survived.The collagen patterns of all keloids were pressed in every specimen obtained 8 weeks. Fibrocytes andFB were observed in specimens by electronic microscope. There were abundent rough endoplasmic reticulum (RER) in FB， which indicated that FB’s capability of synthesizing and secreting collagen was preserved and the cellular characteristicwas remained. Conclusion There is a good affinity between PLGAand FB. The composition of PLGA and FB can form keloids in athymic mice,so that it deserves further researching and developing.
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.
Objective To clarify the correlation of p53 codon 72 polymorphism in peripheral blood with keloid susceptibility in Chinese population. Methods All the literatures of case-control research on the correlation between p53 codon 72 polymorphism in peripheral blood and keloid in Chinese population were searched in PubMed, EBSCO, CNKI, CBM, and WanFang Data from their establishment to August 2010. Meta-analyses were performed to detect whether there were differences between the keloid group and the control group about the distribution of genotypes of p53 codon 72 in peripheral blood, such as, Pro/Pro vs. Arg/Arg, Pro/Pro vs. Pro/Arg, and alleles Pro vs. Arg. Results Five studies involving 328 keloid patients and 420 patients in the control group were included. The results of meta-analyses showed that the population having the genotype Pro/Pro presented no increased keloid risk compared to that with the genotypes Arg/Arg (OR=2.17, 95%CI 0.86 to 5.47) or Pro/Arg (OR=1.90, 95%CI 0.92 to 3.93), while the allele Pro showed significant association with increased keloid risk compared to the allele Arg (OR=1.86, 95%CI 1.03 to 3.35). Conclusion The allele Pro of p53 codon 72 in peripheral blood of Chinese population is significantly associated with increased keloid risk.
ObjectiveTo investigate the expression and significance of peroxisome proliferator activated receptor γ(PPAR-γ) in human keloid. MethodsTwenty-three keloid samples were harvested from the patients undergoing keloid and auto-skin grafting operation as the experimental group (keloid group), and the residual normal skin after auto-skin grafting operation was collected as the control group. The expression of PPAR-γ protein was examined by immunohistochemistry staining in both keloid and normal skin. Referring to Shimizu immunohistochemical standard, the result was graded; the positive rate of samples and the rate of positive cells were calculated. ResultsImmunohistochemistry staining showed that PPAR-γ protein was expressed in both keloid and normal skin. In keloid, it located in the pricle cell layer, and granular layer of epidermis, and the dermal vessel; the degree of dyeing was very light. However, in normal skin, it located in the base layer of epidermis, dermal vessel walls, sweat glands and sebaceous glands; the dyeing degree was deeper. Immunohistochemical staining score in the keloid group (2.65±0.78) was significantly lower than that in the control group (3.65±1.19) (t=5.030, P=0.000). The positive rate of samples in the keloid group (52.17%, 12/23) was significantly lower than that in the control group (82.61%, 19/23) (χ2=4.847, P=0.028). The rate of positive cells was 46.04%±8.61% in the keloid group, which was significantly lower than that in the control group (59.39%±11.26%) (t=5.974, P=0.000). ConclusionCompared with normal skin, the expression of PPAR-γ protein in keloid is down-regulated in in human keloid, indicating that PPAR-γ may be related to the formation of keloid.
ObjectiveTo explore if Smad7 protein can inhibit growth of keloids by observing the gene and protein expressions of Smad7, collagen type Ⅰ, and collagen type Ⅲ and cell proliferation after over-expression vectors of Smad7 transfecting keloid fibroblasts (KFb). MethodsFibroblasts were acquired from 10 male patient with keloids at the age of 20 to 25 years. After in vitro culture, KFb were divided into 3 groups: untransfected group (group A), pcDNA3.1 (-) transfected group (group B), and pcDNA3.1 (-)-smad7 transfected group (group C). The mRNA and protein expression levels of Smad7, collagen type Ⅰ, and collagen type Ⅲ were detected by real-time fluorescence quantitative PCR and Western blot at 48 hours after transfection. The cell proliferation ability was detected by MTT assay at 24 hours after transfection. ResultsThe relative expression levels of mRNA and protein of Smad7 in group C were significantly higher than those in group A and group B (P < 0.01). The relative expression levels of mRNA and protein of collagen type Ⅰ and collagen type Ⅲ in group C were significantly lower than those in group A and group B (P < 0.01). The relative expression levels of mRNA of collagen type Ⅰ and collagen type Ⅲ in group B were significantly higher than those in group A (P < 0.01); and the relative expression levels of proteins of Smad7, collagen type Ⅰ, and collagen type Ⅲ were significantly lower than those in group A (P < 0.01). The cell proliferation ability in group C was significantly lower than that in group A and group B at each time point by MTT assay (P < 0.05), but no difference was found between group A and group B (P>0.05). ConclusionGene expressions of collagen type Ⅰ, and collagen type Ⅲ and cell proliferation will be inhibited after KFb are transfected by over-expression vector of Smad7.
Objective To evaluated the role of wt-P53 protein in telomerase regulation in keloid fibroblasts(KFBs). Methods The fibroblasts were derived from humankeloid tissue which was proved by pathological diagnosis. KFBs were divided into 2 groups, the transfection group and the untransfection group. wt-p53 gene was transfected into the fibroblasts by adenovirus vectors in the transfection group. The KFBs untransfected with wt-p53 gene served as control (untransfection group). After 48 hours of transfection, the expression of wt-P53 protein was analyzed by both Western blotting and immunofluorescence method, respectively. The telomerase activity was evaluated by TRAP-ELISA after 1-7 days of transfection. Results All the KFBs from 2 groups expressed wt-P53 protein. But the expression level of wt-P53 protein in the transfection group was significantly higher than that in the untransfection group.At the same time of high expression of wt-P53 protein, the telomeraseactivity of KFBs in transfection group was significantly lower than that in theuntransfection group(P＜0.05). Conclusion High level expression of wt-P53 protein can transiently inhibit the telomerase activity of KFBs.
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.