1.Interaction between fibroblasts and keratinocytes in the wound edge skin tissue of a diabetic foot patient and the mechanism
Qiongfang RUAN ; Siyu ZHANG ; Maomao XI ; Jingjing RUAN ; Shuhua LIU ; Binghui LI ; Weiguo XIE
Chinese Journal of Burns 2024;40(8):762-771
Objective:To investigate the interaction between fibroblasts (Fb) and keratinocytes (KC) in the wound edge skin tissue of a diabetic foot patient and the mechanism.Methods:This was an experimental research. The wound edge skin tissue from a diabetic foot patient (male and 33 years old) admitted to the Department of Wound Repair of Liyuan Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology in August 2021 and from an acute foot injury patient (male and 50 years old) admitted to the Department of Hand Surgery of the hospital in September 2021 was collected. The single-cell transcriptome sequencing was performed to analyze the interaction between chemokine ligands of Fb subgroup and chemokine receptors of KC subgroup. The supernatant was collected after human foreskin fibroblast (HFF) was cultured routinely and with high concentration of glucose for 7 days as normal conditioned medium (CM) and high glucose CM, respectively. HaCaT cells were collected and divided into normal CM group cultured with normal CM and high glucose CM group cultured with high glucose CM, the scratch test was performed to calculate the cell migration rates at 24 and 48 h after scratch ( n=3). The content of cytokines in the two kinds of CM was detected by liquid suspension chip ( n=5). HFF was collected and divided into normal group cultured routinely and high glucose group cultured with high concentration of glucose for 7 days, and the mRNA expressions of C-X-C motif chemokine ligand 1 (CXCL1), CXCL2, CXCL8, and CXCL12 were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction ( n=6). HaCaT cells in normal CM group and high glucose CM group were collected to detect the protein expressions of C-X-C motif chemokine receptor 4 (CXCR4) in cells cultured for 48 h by Western blotting ( n=3). HaCaT cells were collected and divided into normal CM group, high glucose CM group, normal CM+CXCL12 group, and high glucose CM+CXCL12 group. The first two groups of cells were treated as before, and the latter two groups of cells were cultured with normal CM and high glucose CM containing recombinant human CXCL12, respectively. Scratch test was performed, and cell migration rates were calculated at 24 and 48 h after scratch ( n=3); the protein expression of CXCR4 in cells cultured for 48 h was detected by Western blotting ( n=3). Results:Compared with those in the wound edge skin tissue of acute foot injury, the interactions between chemokine ligands (CXCL1, CXCL2, CXCL3, CXCL8, and CXCL12) of Fb subgroup and chemokine receptors (CXCR2 and CXCR4) of KC subgroup were significantly weakened in the wound edge skin tissue of diabetic foot. At 24 and 48 h after scratch, the migration rates of HaCaT cells in high glucose CM group were significantly lower than those in normal CM group (with t values of 23.50 and 15.65, respectively, P<0.05). Compared with that in normal CM, the content of CXCL1 in high glucose CM was significantly increased ( P<0.05), and the content of CXCL12 was significantly decreased ( P<0.05). After 7 days of culture, compared with those in normal group, the mRNA expressions of CXCL1, CXCL2, and CXCL8 in HFF in high glucose group were significantly increased (with t values of 4.25, 4.98, and 10.04, respectively, P<0.05), while the mRNA expression of CXCL12 was significantly decreased ( t=4.10, P<0.05). After 48 h of culture, the CXCR4 protein expression in HaCaT cells in high glucose CM group was significantly lower than that in normal CM group ( t= 5.13, P<0.05). At 24 and 48 h after scratch, the migration rates of HaCaT cells in high glucose CM group were significantly lower than those in normal CM group and high glucose CM+CXCL12 group (with P values all <0.05); at 24 h after scratch, the migration rate of HaCaT cells in normal CM+CXCL12 group was significantly lower than that in normal CM group ( P<0.05); at 48 h after scratch, the migration rate of HaCaT cells in normal CM+CXCL12 group was significantly higher than that in high glucose CM+CXCL12 group ( P<0.05). At 48 h of culture, the CXCR4 protein expression of HaCaT cells in high glucose CM+CXCL12 group was 0.446±0.050, which was significantly higher than 0.247±0.010 in high glucose CM group ( P<0.05) and similar to 0.522±0.082 in normal CM+CXCL12 group ( P>0.05); the CXCR4 protein expression in HaCaT cells in normal CM group was 0.509±0.055, which was significantly higher than that in high glucose CM group ( P<0.05). Conclusions:The interactions between chemokine ligands of Fb subgroup and chemokine receptors of KC subgroup were significantly weakened in the wound edge skin tissue of diabetic foot. High glucose can inhibit CXCL12 secretion of HFF, and the stimulation of its cell culture supernatant can decrease HaCaT cell migration ability and CXCR4 expression. Exogenous CXCL12 protein can increase the CXCR4 protein expression in HaCaT cells and enhance the cell migration ability.
2.Construction of a Continuous Nursing Quality Evaluation Indicator System for Inflammatory Bowel Disease Patients Based on Donabedian's Three-Dimensional Quality Structure
Gairong MA ; Xinxian ZHAO ; Huiqin XI ; Ying JU ; Chunfeng RUAN ; Mei XU ; Jingjing WANG
Acta Academiae Medicinae Sinicae 2024;46(5):692-699
Objective To construct a continuous nursing quality evaluation indicator system for inflam-matory bowel disease patients and provide a basis for the evaluation of continuous nursing quality.Methods On the basis of Donabedian's three-dimensional(structure,process,and outcome)quality structure,we employed liter-ature review,qualitative interview,Delphi method,and hierarchical analysis to determine the content and weights of indicators of continuous nursing quality for the patients with inflammatory bowel disease.Results A total of 15 experts completed 2 rounds of consultation,which had the questionnaire recovery rates of 100%,the expert authority coefficients of 0.930 and 0.919,and the Kendall harmony coefficients of 0.149 and 0.177(both P<0.001),respectively.The established nursing quality evaluation indicator system included 3 first-level indicators,10 sec-ond-level indicators,and 39 third-level indicators.Conclusion The continuous nursing quality evaluation indi-cator system for the patients with inflammatory bowel disease that was constructed in this study was reasonable,reliable,and practical,providing reference for evaluating the continuous nursing quality for the patients with in-flammatory bowel disease.
3.Determination of four organotin compounds in human whole blood by high performance liquid chromatography-inductively coupled plasma-mass spectrometry
Anping MA ; Banghua WU ; Ming DONG ; Xiaolin RUAN ; Jingjing QIU ; Weifeng RONG ; Jiaheng HE
China Occupational Medicine 2023;50(6):712-716
{L-End}Objective To establish a method for the simultaneous determination of dimethyltin (DMT), trimethyltin (TMT), diethyltin (DET), and triethyltin (TET) in human whole blood using high performance liquid chromatography-inductively coupled plasma-mass spectrometry (ICP-MS). {L-End}Methods The 1.0 mL of blood was added with 4.0 mL 65% aqueous solution (containing 6% acetic acid), extracted and separated by C4 column (150 mm×3 mm×3 μm) using a mobile phase of methanol and 4% acetic acid aqueous solution (containing 0.25 mmol/L tropolone) at a volume ratio of 35∶65, and detected by ICP-MS. {L-End}Results The linear range of DMT, TMT, DET, and TET was 30.60-550.80, 29.00-522.00, 46.10-829.80, and 34.05-612.90 μg/L, respectively. All correlation coefficients were 0.999. The detection limit of DMT, TMT, DET and TET was 21.40, 20.30, 32.27 and 23.80 μg/L, respectively. The recovery rate was 81.9%-104.9%. The within-run and between-run relative standard deviation was 1.6%-6.9% and 0.1%-10.0%, respectively. The samples can be stored at -20 ℃ and 4 ℃ for at least three days. {L-End}Conclusion This method can be used for trace analysis of DMT, TMT, DET, and TET in whole blood.
4.Pan-cancer analysis of ubiquitin-specific protease 7 and its expression changes in the carcinogenesis of scar ulcer
Siyu ZHANG ; Jingjing RUAN ; Dongmei JIN ; Nuo CHEN ; Weiguo XIE ; Qiongfang RUAN
Chinese Journal of Burns 2023;39(6):518-526
Objective:To explore the biological role and clinical significance of ubiquitin-specific protease 7 (USP7) in the carcinogenesis of scar ulcer.Methods:A retrospective observational study combined with bioinformatics analysis was used. The RNA expression profile data of USP7 in tumor and/or its corresponding paracancular normal tissue were obtained from The Cancer Genome Atlas (TCGA) database and the Gene Expression Omnibus database, and the RNA sequencing data were transformed by log 2. The variations of USP7 gene were analyzed by cBioPortal database. The USP7 mRNA expression in tumor and adjacent normal tissue in TCGA database were obtained by using the "Gene_DE" module in TIMER 2.0 database. The survival rates of patients with high and low USP7 expression in cutaneous melanoma (SKCM), cervical squamous cell carcinoma (CESC), lung squamous cell carcinoma (LUSC), and head and neck squamous cell carcinoma (HNSC) were analyzed using the Gene Expression Profile Interactive Analysis 2 (GEPIA2) database, and the Kaplan-Meier survival curves were drawn. Sangerbox database was used to analyze the correlation of USP7 expression in pan-cancer with microsatellite instability (MSI) or tumor mutation burden (TMB) pan-cancer. Through the "correlation analysis" module in the GEPIA2 database, the correlation of USP7 expression in pan-cancer with the expression levels of five DNA mismatch repair genes ( MLH1, MSH2, MSH6, PMS2, and EPCAM) and three essential DNA methyltransferases (DNMT)--DNMT1, DNMT3A, and DNMT3B were evaluated. The USP7 expression in CESC, HNSC, LUSC, and SKCM and its correlation with infiltration of immune cells (B cells, CD4 + T cells, CD8 + T cells, neutrophils, macrophages, and dendritic cells) were analyzed by the "Immune-Gene" module in TIMER 2.0 database. The "Similar Genes Detection" module of GEPIA2 database was used to obtain the top 100 protein sets with similar expression patterns to USP7. Intersection analysis was performed between the aforementioned protein sets and the top 50 protein sets that were directly physically bound to USP7 obtained by using the STRING database. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis were performed for the two protein sets mentioned above using the DAVID database. The samples of normal skin, hypertrophic scar, scar ulcer, and scar carcinoma with corresponding clinicopathologic features were collected from the Department of Pathology of Tongren Hospital of Wuhan University & Wuhan Third Hospital from October 2018 to October 2022, and the USP7 expression in tissue was detected by immunohistochemical method, with the number of samples of 6. Data were statistically analyzed with Log-rank test, one-way analysis of variance, and Bonferroni test. Results:In pan-cancer, the main gene variations of USP7 were mutation and amplification, and the top 3 tumors with the highest variation frequency (>6%) were bladder urothelial carcinoma, SKCM, and endometrial carcinoma. The main mutation of USP7 gene in pan-cancer was missense mutation. In SKCM with the highest mutation frequency, the main type of mutation was missense mutation in USP7_ICP0_bdg domain. USP7 mRNA expression in breast invasive carcinoma, bile duct carcinoma, colon carcinoma, esophageal carcinoma, HNSC, renal chromophobe cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, LUSC, prostate carcinoma, and gastric carcinoma was significantly higher than that in corresponding paracancer normal tissue ( P<0.05). USP7 mRNA expression in glioblastoma multiforme, renal clear cell carcinoma, renal papillary cell carcinoma, and thyroid carcinoma was significantly lower than that in corresponding paracancular normal tissue ( P<0.05). In addition, USP7 mRNA expression in SKCM metastases was much higher than that in primary tumor tissue ( P<0.05). Survival curves showed no significant difference in survival rate between patients with high USP7 expression and patients with low USP7 expression in CESC, HNSC, LUSC, and SKCM (Log-rank P>0.05, with hazard ratios of 1.00, 0.99, 1.00, and 1.30, respectively). USP7 expression in colon cancer, colorectal cancer, thymic cancer, and thyroid cancer was negatively correlated with TMB (with Pearson correlation coefficients of -0.26, -0.19, -0.19, and 0.11, respectively, P<0.05). USP7 expression in glioma, CESC, lung adenocarcinoma, mixed renal carcinoma, and LUSC was positively correlated with MSI expression (with Pearson correlation coefficients of 0.22, 0.14, 0.15, 0.08, and 0.14, respectively, P<0.05), and USP7 expression in colon cancer, colorectal cancer, invasive breast cancer, prostate cancer, HNSC, thyroid cancer, and diffuse large B-cell lymphoma were significantly negatively correlated with MSI expression (with Pearson correlation coefficients of -0.31, -0.27, -0.13, -0.19, -0.16, -0.18, and -0.53, respectively, P<0.05). The expression of USP7 in CESC was positively correlated with that of both MSH2 and MSH6 (with Spearman correlation coefficients of 0.51 and 0.44, respectively, P<0.05), and the expression of USP7 in HNSC was positively correlated with the expression of EPCAM, MLH1, MSH2, MSH6, and PMS2 (with Spearman correlation coefficients of 0.39, 0.14, 0.49, 0.54, and 0.41, respectively, P<0.05), and the expression of USP7 in LUSC was positively correlated with the expression of EPCAM, MSH2, MSH6, and PMS2 (with Spearman correlation coefficients of 0.20, 0.36, 0.40, and 0.34, respectively, P<0.05), and the expression of USP7 in SKCM was positively correlated with the expression of EPCAM, MLH1, MSH2, MSH6, and PMS2 (with Spearman correlation coefficients of 0.11, 0.33, 0.42, 0.55, and 0.34, respectively, P<0.05). The expression of USP7 in CESC, HNSC, LUSC, and SKCM was significantly positively correlated with the expression of DNMT1, DNMT3A, and DNMT3B (with Spearman correlation coefficients of 0.42, 0.34, 0.22, 0.45, 0.52, 0.22, 0.36, 0.36, 0.22, 0.38, 0.46, and 0.21, respectively, P<0.05). The expression of USP7 in CESC, HNSC, LUSC, and SKCM was positively correlated with CD4 + T cell infiltration (with Partial correlation coefficients of 0.14, 0.22, 0.13, and 0.16, respectively, P<0.05). Being similar to the pattern of USP7 expression and ranked among top 100 protein sets, the top 5 proteins were C16orf72, BCLAF1, UBN, GSPT1, ERI2 (with Spearman correlation coefficients of 0.83, 0.74, 0.73, and 0.72, respectively, all P values<0.05). The top 50 protein sets that directly physically bind to USP7 overlapped with the aforementioned protein set by only one protein, thyroid hormone receptor interaction factor 12. KEGG enrichment analysis showed that USP7 related genes were involved in cell cycle, spliceosome, cell senescence, and p53 signal pathway. GO enrichment analysis showed that USP7 related genes were involved in transcriptional regulation, protein ubiquitination, DNA repair, and cytoplasmic pattern recognition receptor signal pathways. Analysis of clinical samples showed that USP7 expression was significantly higher in hypertrophic scars (0.35±0.05), scar ulcers (0.43±0.04), and scar cancers (0.61±0.03) than in normal skin (0.18±0.04), P<0.05. Conclusions:USP7 may be a clinical biomarker for the progression of cicatricial ulcer cancer.
5.Prospective study on the effects of resistance training with elastic band at home on muscle function and walking ability of severely burned children
Ting HUO ; Jingjing RUAN ; Meijun JIANG ; Fang LEI ; Wei HUANG ; Wenqian TANG ; Weiguo XIE ; Xiangyang XU ; Song WANG ; Shuhua LIU
Chinese Journal of Burns 2023;39(12):1131-1139
Objective:To explore the effects of resistance training with elastic band at home on muscle function and walking ability of severely burned children.Methods:A prospective non-randomized controlled study was conducted. From January 2022 to April 2023, 40 children with severe burns who met the inclusion criteria were admitted to Tongren Hospital of Wuhan University & Wuhan Third Hospital. According to the willingness of the children or their families, the children were assigned to conventional rehabilitation group and combined rehabilitation group. During the study, 8 children dropped out of the study, 17 children were finally included in the conventional rehabilitation group with 6 males and 11 females, aged (8.5±2.4) years, and 15 children were included in the combined rehabilitation group with 5 males and 10 females, aged (9.6±2.5) years. The children in the 2 groups received conventional burn rehabilitation treatment in the hospital, including active and passive activity training, scar massage, and pressure therapy. The children in combined rehabilitation group received resistance training with elastic band of 3 to 5 times per week after discharge, and the children in conventional rehabilitation group received daily activity ability training after discharge. Before home rehabilitation training (1 week before discharge) and 12 weeks after home rehabilitation training, the grip strength was measured using a handheld grip dynamometer, the muscle strengths of the upper and lower limbs were measured using a portable dynamometer for muscle strength, lean body mass was measured by bioelectrical impedance measuring instrument, and the 6-min walking distance was measured. Data were statistically analyzed with independent sample t test, paired sample t test, Mann-Whitney U test, or Fisher's exact probability test. Results:After 12 weeks of home rehabilitation training, the grip strengths of children in combined rehabilitation group and conventional rehabilitation group were (15±4) and (11±4) kg, respectively, which were significantly higher than (10±4) and (9±4) kg before home rehabilitation training (with t values of -9.99 and -11.89, respectively, P values all <0.05); the grip strength of children in combined rehabilitation group was significantly higher than that in conventional rehabilitation group ( t=3.24, P<0.05). After 12 weeks of home rehabilitation training, the muscle strengths of upper and lower limbs of children in combined rehabilitation group (with t values of -11.39 and -3.40, respectively, P<0.05) and the muscle strengths of upper and lower limbs of children in conventional rehabilitation group (with t values of -7.59 and -6.69, respectively, P<0.05) were significantly higher than those before home rehabilitation training, and the muscle strengths of upper and lower limbs of children in combined rehabilitation group were significantly higher than those in conventional rehabilitation group (with t values of 3.80 and 7.87, respectively, P<0.05). After 12 weeks of home rehabilitation training, the lean body mass of children in combined rehabilitation group was significantly higher than that before home rehabilitation training ( t=0.21, P<0.05). After 12 weeks of home rehabilitation training, the 6-min walking distances of children in conventional rehabilitation group and combined rehabilitation group were significantly longer than those before home rehabilitation training (with t values of -5.33 and -3.40, respectively, P<0.05), and the 6-min walking distance of children in combined rehabilitation group was significantly longer than that in conventional rehabilitation group ( t=3.81, P<0.05). Conclusions:Conventional burn rehabilitation treatment in hospital and home resistance training with elastic band for 12 weeks after discharge can significantly improve the muscle function and walking ability of severely burned children.
6.Value of 18F-FDG PET/CT in evaluating the activity of Takayasu arteritis
Jingjing ZHANG ; Xin WEN ; Qiao RUAN ; Xingmin HAN
Chinese Journal of Nuclear Medicine and Molecular Imaging 2022;42(8):457-461
Objective:To investigate the value of 18F-FDG PET/CT in qualitative evaluation of disease activity in patients with Takayasu arteritis (TA), and discuss the relationship between PET vascular activity score (PETVAS) and disease activity. Methods:A total of 84 patients (12 males, 72 females, age: (30.0±7.3) years) with TA, who underwent 18F-FDG PET/CT imaging in the First Affiliated Hospital of Zhengzhou University between February 2016 and August 2021, were retrospectively reviewed. All patients were divided into active ( n=68) and inactive groups ( n=16) according to Kerr score. PET/CT images were semiquantitatively evaluated according to the visual score and PETVAS was calculated. Mann-Whitney U test was used to compare the differences of PETVAS and inflammatory indexes (erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP)) between two groups. Spearman rank correlation analysis was used to evaluate the relationships among PETVAS, Kerr score and inflammatory indexes. ROC curve analysis was employed to compare the diagnostic efficiencies of PETVAS, ESR and CRP for disease activity. Results:The sensitivity, specificity and accuracy of 18F-FDG PET/CT in evaluating the activity of TA by visual score (≥2) were 86.8%(59/68), 15/16 and 88.1%(74/84), respectively. PETVAS, ESR(mm/1 h) and CRP(mg/L) in active group were higher than those in inactive group (11.0 (6.0, 18.0) vs 0.0 (0.0, 3.0), 67.0 (25.3, 104.0) vs 10.1 (7.1, 20.8) and 32.3 (7.1, 72.1) vs 1.8 (1.0, 5.3), respectively; z values: 4.53-5.23, all P<0.001). PETVAS was positively correlated with Kerr score ( rs=0.65, P<0.001), ESR ( rs=0.57, P<0.001) and CRP ( rs=0.56, P<0.001). The correlation coefficient between Kerr score and PETVAS was higher than that between Kerr score and CRP ( rs=0.55, P<0.001) and ESR ( rs=0.55, P<0.001). ROC AUC and Youden Index (YI) of PETVAS (AUC=0.92, YI=0.77) were higher than those of CRP (AUC=0.87, YI=0.65) and ESR ( AUC=0.87, YI=0.66). Conclusions:18F-FDG PET/CT is an effective tool for qualitative evaluation of TA disease activity. PETVAS is helpful in evaluating disease activity of TA, with a better efficacy than inflammatory indexes.
7.Analysis of genomic DNA methylation level in foxtail millet by Methylation Sensitive Amplified Polymorphism.
Yuxin ZHANG ; Yanan RUAN ; Chen ZHAO ; Minmin XUE ; Bo LI ; Jingjing WANG ; Yang LIU ; Kaixi WANG ; Hongyan WANG
Chinese Journal of Biotechnology 2019;35(2):263-269
DNA methylation is an important type of epigenetic modification in eukaryotes. In order to research genome-wide methylation levels and patterns in foxtail millet (Setaria italica), the Methylation Sensitive Amplified Polymorphism (MSAP) analysis (employing double digestion with EcoR I and Hpa II/Msp I) was established and applied in two foxtail millet cultivars (Chaogu 58 and Yugu 1). The results showed that 32 pairs of MSAP primers were selected from 100 MSAP primers, and 1 615 and 1 482 clearly distinguishable and reproducible bands were amplified from Chaogu 58 and Yugu 1 respectively, including 3 types of methylation patterns. Cytosine methylation levels of CCGG context in Chaogu 58 and Yugu 1 were characterized as 6.93% and 8.77% respectively. Such different genomic DNA methylation levels between two foxtail millet varieties may provide a preliminary reference for the cultivation of this crop from a novel epigenetic viewpoint.
DNA Methylation
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Genome, Plant
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Genomics
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Polymorphism, Genetic
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Setaria Plant
9. Observation on clinical effects of recombinant human growth hormone on the treatment of children with severe burn
Zhigang CHU ; Ze LI ; Aihua WANG ; Qiongfang RUAN ; Hong WU ; Jingjing RUAN ; Weiguo XIE
Chinese Journal of Burns 2018;34(8):522-525
Objective:
To observe the clinical effects of recombinant human growth hormone (rhGH) on children with severe burn.
Methods:
Clinical data of 94 children with severe burn, hospitalized in our burn unit from April 2012 to December 2016, conforming to the study criteria, were retrospectively analyzed. According to the use of rhGH, children were divided into rhGH group (
10. Clinical effects of sequential laser treatments on early stage hypertrophic burn scars
Weiguo XIE ; Fang LEI ; Juan WANG ; Juan XU ; Jingjing RUAN ; Ze LI
Chinese Journal of Burns 2018;34(9):615-623
Objective:
To observe the clinical effects of sequential treatments of pulsed dye laser (PDL) and ablative fractional carbon dioxide laser on early stage hypertrophic burn scars.
Methods:
From January 2016 to December 2017, 221 patients with 228 hypertrophic scars in all parts of body within 6 months post healing, conforming to the study criteria and treated in our department, were included in this prospective study. They were first treated by PDL, repeated at an interval of one month until the vascularity score of scar fell below 2 points, and then treated by ablative fractional carbon dioxide laser, repeated at an interval of 3 months. Their start time and numbers of treatment and follow-up time were recorded. Before the first treatment (hereinafter referred to as before treatment) and at the last follow-up (hereinafter referred to as after treatment), the vascularity, thickness, and pliability of scars were scored by a self-made scar rating scale. The scores of patients with the observation time between 6 to18 months post healing were compared among scars of patients grouped by age, body site of scar, starting time of treatment and numbers of treatment. The laser speckle contrast imaging technique was used to measure the blood flow value of scars. The itching symptom of the scars was evaluated by the Verbal Rating Scale. The satisfaction to the final effects of the doctors and patients was investigated and scored separately by Likert scale after treatment. The therapeutic or adverse reactions were recorded during the treatment. Data were processed with paired

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