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 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.
Keloids are benign skin tumors resulting from the excessive proliferation of connective tissue in wound skin. Precise prediction of keloid risk in trauma patients and timely early diagnosis are of paramount importance for in-depth keloid management and control of its progression. This study analyzed four keloid datasets in the high-throughput gene expression omnibus (GEO) database, identified diagnostic markers for keloids, and established a nomogram prediction model. Initially, 37 core protein-encoding genes were selected through weighted gene co-expression network analysis (WGCNA), differential expression analysis, and the centrality algorithm of the protein-protein interaction network. Subsequently, two machine learning algorithms including the least absolute shrinkage and selection operator (LASSO) and the support vector machine-recursive feature elimination (SVM-RFE) were used to further screen out four diagnostic markers with the highest predictive power for keloids, which included hepatocyte growth factor (HGF), syndecan-4 (SDC4), ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), and Rho family guanosine triphophatase 3 (RND3). Potential biological pathways involved were explored through gene set enrichment analysis (GSEA) of single-gene. Finally, univariate and multivariate logistic regression analyses of diagnostic markers were performed, and a nomogram prediction model was constructed. Internal and external validations revealed that the calibration curve of this model closely approximates the ideal curve, the decision curve is superior to other strategies, and the area under the receiver operating characteristic curve is higher than the control model (with optimal cutoff value of 0.588). This indicates that the model possesses high calibration, clinical benefit rate, and predictive power, and is promising to provide effective early means for clinical diagnosis.
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 replace dysfunctional Fas gene and reconstruct the blocked Fas signal by using two kinds of prepared recombinantAdenovirus which have human Fas gene. Methods After the keloids derived from fibroblasts were infected by the Adenovicus, the expressions of Fas protein before the exposure and after the exposure was compared. Then the function of the newly produced Fas protein was detected. Results The highly improve expression of Fas protein in the infected keloid derived fibroblasts was detected. Obvious apoptosis was also detected in the infected keloid derived from fibroblasts under the condition of exposing to FasMcab. Conclusion ①The recombinant Adenovirus with Fas gene can transfect the Fas gene into keloidderived fibroblasts and highly improved the expression of Fas protein. The newly expressed Fas gene can reconstruct the blocked Fas signal. ②Ad-Fas(B) has better therapeutic effect in vitro gene therapy. ③ The correlation between keloid and Fas gene was further proved and it may pave the way for further gene therapy in 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.
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.
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.
