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 investigate the relationship between keloid proliferation and destruction of skin appendages(SAs). Methods Pathological biopsies of keloids were derived from 17 patients whounderwent scar resection. All samples were divided into 4 groups: infiltrating growth locus of keloids（K-I,n=9），proliferative keloids (K-P,n=17), atrophic keloids (K-A,n=10), and edging normal skin (K-N,n=6). Normal skin derived from thorax of patients was used as control (NS, n=6). The density of SAs and the expressive characteristics of pan-cytokeratin (CKp), cytokeratin19 (CK19), secretory component of glandular epithelium(SC), proliferating cell nuclear antigen(PCNA), and apoptosis related proteins (Bcl-2 and Bax) were observed with immunohistochemical method. Results Compared with K-N and NS, the density of SAs expressing CKP and SC in keloids was apparently decreased, and remnant of CKp protein was observed after the disappearance of SAs structures. Protein expression of Bax was increased in epithelial cellsof most SAs. SAs containing postive immunostaining signals of Bcl-2, PCNA and CK19 exhibited squamous epithelization and abnormal structure. The structure of SAs underwent 3 morphological stages: infiltrating, proliferating, and maturing.In correspondence to each stage, SAs underwent proliferation, structural destruction, and fibrosis which were caused by cellular migration, nflammatory reaction, and vascular occlusion respectively. Conclusion Abnormal proliferation of epithelial cells and their structural destruction of SAs may beassociated with tissue fibrosis in keloid lesion.
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.
OBJECTIVE: To investigate the expression and distribution of platelet derived growth factor receptor-beta(PDGFR-beta) in normal skin and keloid and to discuss its biological function in keloid formation. METHODS: 1. To detect the expression and distribution of PDGFR-beta in normal skin and keloid tissue by immunohistochemistry; 2. To detect the receptor expression in vitro by Flow cytometry (FCM); 3. To detect the subcellular distribution of receptor by Laser confocal microscope. RESULTS: 1. Immunohistochemistry showed that normal skin and keloid tissue were almost the same in expression but different in distribution of PDGFR-beta; 2. There was more expression of PDGFR-beta in normal fibroblasts than that in keloid fibroblasts in vitro by FCM; 3. Laser confocal microscope revealed that the PDGFR-beta concentrated on the surface of cell membrane in keloid fibroblasts, but in normal skin fibroblasts, the receptors were coagulated on the nuclear membrane and intranucleus. CONCLUSION: Compared with the fibroblasts in vivo, there was a difference of the PDGFR-beta expression in fibroblasts in vitro, more expression of PDGFR-beta in normal fibroblast than that in keloid fibroblast in vitro; and the subcellular distribution of PDGFR-beta was different in normal skin and keloid fibroblasts. The characteristics of the expression and distribution of PDGFR-beta in keloid may contribute to the formation of keloid.
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.
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 curative effects of keloid by operation combined with postoperative β radiation and silicone gel sheeting. Methods From 1996 to 2002, 598 patients with keloid(243 males, 355 females, aging 15-55 years with an average of 28.6 years) were treated by integrated therapy. Their disease courses were from 6 months to 6 years. The keloid area ranged from 1.0 cm×1.5 cm~8.0 cm×15 cm. First, keloid was removed by operation, and then the wounds weresutured directly(group suture) or covered with skin graft(group graft). In groupsuture, the operational sites were managed by β ray radiotherapy 24-48 hours after operation. The total doses of radiation were 12-15 Gy, 5 times 1 week(group suture A) and 10 times 2 weeks (group suture B). Radiotherapy was not taken until stitches were taken out in group graft, and then the same methods were adopted as group suture B. After radiotherapy, silicone gel sheeting was used in 325 cases for 3-6 months. Results All patients were followed up for 12-18 months. (1) The overall efficacy was 91.3% in group suture A(n=196), and 95.8% in group suture B (n=383), respectively. There was significant difference between the two groups(Plt;0.01). (2) Radiotherapy was of no effect in 6 cases of group graft(n=19). (3) Silicone gel sheeting had effectivenessin 185 cases. Silicone gel sheeting had no obvious effect on the overall efficacy, but it could improve the quality of texture and color of skin. Conclusion By use of integrated methods to treat keloid, if the wound can be sutured directly, skin grafting should not be adopted. The results in group suture B are better than those in group suture A; silicone gel sheeting should be used as possible.
Objective To detect gene mutations of Fas gene death domain （exons 7-9） in 2 Chinese keloid pedigrees and to investigatethe significance of Fas gene mutations in the keloid formation.Methods The samples were selected from keloid pedigrees A and B in 2005. The polymerase chainreaction and DNA sequencing analysis technique were used to detect the sequenceof exons 7-9 of Fas gene from keloid tissues of 2 male patients in pedigree A,their peripheral vein blood and their surrounding normal skin served as their own contrast, their spouses’ peripheral vein blood served as normal contrast, the peripheral vein blood of 2 patients in pedigree B served as a contrast between different keloid pedigrees.Results No gene mutations and single nucleotidepolymorphism in Fas gene exons 7, 8 were found in all samples from pedigrees A and B. But point mutations and single nucleotide polymorphism in Fas gene exon 9were identified in 11 bp and 53 bpin 2 keloid tissue samples from Chinese keloid pedigree A.Conclusion Fas gene point mutations maybe indicate some relations in Fas protein function and keloid formation.
Objective To observe the effect of gene expression of p53 and the polymorphism of p53 gene codon 72 on cl inical phenotype of keloids. Methods The tissue and blood samples were taken from 35 patients with keloids, 19 males and 16 females, and the course of disease was from 4 months to 8 years. Meanwhile, autologous peripheral blood was collected for genotype analysis. According to the observing scope, the tissue samples of the keloids were divided into 2 groups: the central group involving the central part of the keloids (the central area within two-thirds of the radius) and the peripheral group involving the peripheral part of the keloids (the peripheral area within one-third of the radius). According to the largest diameter of the keloids, the two groups were divided into 3 subgroups: the small size group with 5 patients (lt; 1 cm), the medium size group with 21 patients (1-3 cm) and the large size group with 9 patients (gt; 3 cm). DNA of the tissue and blood samples were extracted, and the PCR followed by DNA sequencing was used to detect the polymorphism of p53 gene codon 72. The expression change of P53 was detected by immunohistochemical staining. The fibroblast apoptosis in keloid tissues was detected by TUNEL method. Results The genetic genotype of p53 gene codon 72 in keloids included Arg/Arg in 7 cases, Pro/Arg in 21 cases, Pro/ Pro in 7 cases. The significant correlation was found between genotype and cl inical phenotype (P lt; 0.05). Immunohistochemical staining revealed that P53 was detectable in peripheral and central groups of small-medium size keloids and central groups keloids, and detectable in few cells in peripheral groups of large size keloids. The absorbency value was 3 439.359 8 ± 538.527 5 in Arg/Arg genotype, 3 273.186 2 ± 375.213 9 in Arg/Pro genotype, 1 691.372 9 ± 98.989 3 in Pro/Pro genotype. There weresignificant differences among the three genotypes (P lt; 0.05). The fibroblast apoptosis was detected by TUNEL, and the apoptotic cells were evenly distributed. The apoptosis index was 31.000 0 ± 3.266 0 in peripheral group of large size keloids, 42.300 0 ± 4.354 8 in peripheral group of medium size keloids, 44.600 0 ± 5.253 6 in peripheral group of small size keloids. There were significant differences among the three groups (P lt; 0.05). Conclusion There is close relationshi p between the cl inical phenotype of keloids and the expression of P53. The polymorphism variation of p53 gene codon 2 is beneficial for apoptosis of fibroblasts in keloids.