【Abstract】 Objective To summarize the recent progress in related research on transforming growth factor β1 （TGF-β1）/Smad3 signal transduction pathway and post-traumatic scar formation. Methods Recent related literature at home and abroad on TGF-β1/Smad3 signal transduction pathway and post-traumatic scar formation was reviewed and summarized. Results TGF-β1 is an important influence factor of fibrotic diseases, and it plays biological effects by TGF-β1/Smad3 signal transduction pathway. The pathway is regulated by many factors and has crosstalk with other signal pathways at cellular and molecular levels. The pathway is involved in the early post-traumatic inflammatory response, wound healing, and late pathological scar formation. Intervening the transduction pathway at the molecular level can influence the process of fibrosis and extracellular matrix deposition. Conclusion TGF-β1/Smad3 signal transduction pathway is an important way to affect post-traumatic scar formation and extracellular matrix deposition. The further study on the pathway will provide a theoretical basis for promotion of wound healing, as well as prevention and treatment of pathological scar formation.
Objective To observe the protein expression of c-Jun amino-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) in normal skin and keloid and to explore their influences on the formation of kloid. Methods Keloid tissues and normal skin tissues were collected from 16 keloid resection patients (experimental group) and 10 voluntary plastic surgery patients (control group). In the experimental group, the keloid formation time ranged from 8 months to 10 years; the keloid tissues were collected from the chest in 6 cases, the ear lobe in 4 cases, the perineum in 2 cases, the shoulder in 3 cases, and the abdomen in 1 case; and all keloid tissues were confirmed by pathological examination. In the control group, normal skin tissues were collected from the abdomen in 4 cases, the thighs in 3 cases, the shoulder in 2 cases, and the back in 1 case. Two-step l ine of Envision immunohistochemical staining was performed to observe the expressions of nonphosphorylated and phosphorylated JNK and ERK; Image Pro Plus 4.5 image analysis system was used to measure the integrated absorbance (IA) and to observe the positive staining strength. Results The immunohistochemical staining showed that no obvious expressions of phosphorylated and non-phosphorylated ERK, JNK were observed in the fibroblasts of the control group, and the expressions of phosphorylated JNK and ERK proteins were significantly higher in the experimental group than in the control group (P lt; 0.05). There was no significant difference in the expressions of non-phosphorylated JNK and ERK proteins between 2 groups (P gt; 0.05). Conclusion Activation of ERK and JNK pathways might be involved in formation of keloid.
Objective To seek for a method of constructing the tissue microarray which contains keloid, skin around keloid, and normal skin. Methods The specimens were gained from patients of voluntary donation between March and May2009, including the tissues of keloid (27 cases), skin around keloid (13 cases), and normal skin (27 cases). The specimens were imbedded by paraffin as donor blocks. The traditional method of constructing the tissue microarray and section were modified according to the histological characteristics of the keloid and skin tissue and the experimental requirement. The tissue cores were drilled from donor blocks and attached securely on the adhesive platform which was prepared. The adhesive platform with tissue cores in situ was placed into an imbedding mold, which then was preheated briefly. Paraffin at approximately 70℃ was injected to fill the mold and then cooled to room temperature. Then HE staining, immunohistochemistry staining were performed and the results were observed by microscope. Results The constructed tissue microarray block contained 67 cores as designed and displayed smooth surface with no crack. All the cores distributed regularly, had no disintegration or manifest shift. HE staining of tissue microarray section showed that all cores had equal thickness, distinct layer, manifest contradistinction, well-defined edge, and consistent with original pathological diagnosis. Immunohistochemistry staining results demonstrated that all cores contained enough tissue dose to apply group comparison. However, in tissue microarray which was made as traditional method, many cores missed and a few cores shifted obviously. Conclusion Applying modified method can successfully construct tissue microarray which is composed of keloid, skin around keloid, and normal skin. This tissue microarray will become an effective tool of researching the pathogenesis of keloid.
【Abstract】 Objective To summarize the effectiveness of surgical removal combined with adjuvant therapy onthe aural region keloid. Methods From January 2000 to December 2005, 42 patients (71 side ears) with keloid at the auralregion were treated. There were 8 males and 34 females, aged 16 to 50 years (mean 26.2 years). The course of diseaseranged from 6 months to 4 years. The causes of disease included earhole piercing (n=32), ear trauma(n=7), and postoperativehyperplasia(n=3); the sizes of keloids ranged from 0.3 cm × 0.3 cm× 0.2 cm to 6.0 cm × 4.0 cm × 1.0 cm with globular, dumb-bell,nodular shapes. According to the different sizes and the range of keloids, different operations to remove the keloids and repairthe defect tissue were chosen. Wounds were exposed to the electron beam at first 24 hours after operation, once a day at 2 Gyeach time for 10 days. An immediate local injection for the keloid with hormones anti-scar drugs, which was a mixture of Betamethasone(Diprospan) and 2% Lidocaine with a proportion of 1 ∶ 3, was given to the patients who had recurrence trend 3 times,every 3 weeks. Results After operation, all the wounds healed by first intention. And 37 cases(64 lateral ears) were followedup for 1 year, and all achieved cl inical cure. Five cases (7 lateral ears) had the trend of recurrence 3-6 months after operation andwere cured after the immediate local injection for the keloid with hormones anti-scar drugs. According to LIU Wenge’s curativecriterion, 37cases were cured and 5 cases responded to treatment. Conclusion Surgical removal combined with local radiationand hormones infiltrated individually as early as possible can effectively treat aural region keloids. And it is an optimal method.
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 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.
Objective To study the mutations at 1 573 fragment of TNF receptor II (TNFR-II) gene in patients with keloid. Methods The tissue DNA was extracted from 22 samples of keloids donated by 22 patients (6 males and 16 females, aged 18-53 years), and all keloids were examined and classified by pathologist. The peri pheral blood DNA was extracted from the same patients as the control. PCR was used to ampl ify the 1 573 fragment of TNFR-II gene from the keloid tissue DNA and peripheral blood DNA. The PCR products were sequenced directly and then compared with the GeneBankdata. Results All the concentration of the extracted DNA in trial were higher than 0.50 μg/μL and the purity (A260/A280) ofthe extracted DNA were higher than 1.5. It closed to the magnitude of the design DNA fragment by agarose gel electrophoresis examining, and corresponded with the test requirement. Mutations at 1 573 fragment of TNFR-II gene were detected in 13 out of 22 keloids. The mutation incidence was 59.1%. Among them, 9 had point mutation at codon 1 663, accounting 40.9%. No TNFR-II gene mutation was detected in all peripheral blood samples. There were significant difference between keloids DNA and peripheral blood DNA (P lt;0.01). The mutations involved point mutation, deletion and insertion as well as multisite and multitype. Conclusion There is a correlation between the mutation at 1 573 fragment of TNFR-II gene and keloid.
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 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 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.