ABSTRACT
Objective:To preliminarily evaluate the effect of tension stimulation on the biological activity of and expression of fibrosis marker genes in keloid fibroblasts (KD-Fbs) .Methods:Three patients who were diagnosed with keloids and received surgical treatment were collected from the Department of Dermatology, Tangdu Hospital, the Fourth Military Medical University from January to March 2017. Human KD-Fbs were isolated from resected keloid tissues, and subjected to primary culture. The third- to sixth-passage KD-Fbs were divided into tension group and control group to be cultured in the tension-based chamber and control chamber respectively, and subjected to tension stimulation and normal culture respectively. Cell counting kit-8 (CCK8) assay was performed to assess the proliferative activity of KD-Fbs after 1-, 2-, 3- and 4-day culture, and the scratch assay to evaluate the migratory ability of KD-Fbs after 1- and 2-day culture. After 48-hour treatment, real-time quantitative PCR and Western blot analysis were performed to determine the mRNA and protein expression of fibrosis markers type Ⅰ collagen, fibronectin and α-smooth muscle actin (α-SMA) in KD-Fbs respectively. Two-independent-sample t test was used for comparisons between 2 groups. Results:CCK8 assay showed that the proliferative activity of KD-Fbs was significantly higher in the tension group than in the control group after 1-, 2-, 3- and 4-day culture ( t=3.05, 7.00, 16.65, 15.19, respectively, all P< 0.05) . After 1- and 2-day culture, the scratch assay showed that the migration rate of KD-Fbs was significantly higher in the tension group (48.65%±3.96%, 100.00%, respectively) than in the control group (9.36%±1.14%, 50.35%±4.23%, t=16.53, 20.35, respectively, both P< 0.01) . Real-time quantitative PCR showed that the mRNA expression of type Ⅰ collagen, fibronectin and α-SMA was significantly higher in the tension group (3.04±0.20, 2.16±0.10, 3.76±0.24, respectively) than in the control group (1.00; t=17.57, 21.01, 20.25, respectively, all P< 0.01) . As Western blot analysis revealed, changes in the protein expression of the 3 fibrosis markers were consistent with their mRNA expression changes (all P< 0.05) . Conclusion:Tension may participate in the fibrosis in keloids by promoting the expression of fibrosis marker genes, and enhancing the proliferative and migratory ability of KD-Fbs.
ABSTRACT
[Objective] To study the methylation status of CD40L gene regulatory regions in peripheral blood CD4+ T cells from patients with systemic sclerosis (SSc).[Methods] Peripheral blood mononuclear cells were isolated from the venous blood of 21 SSc patients and 20 healthy controls by density gradient centrifugation.CD4+ T cells were separated by using magnetic beads.Genomic DNA was isolated from the CD4+ T cells and treated with sodium bisulfite.Nested PCR was perfonned to amplify the desired regulatory sequences (including the promotor and enhancer) of CD40L,and the amplicons were transformed into the Escberichia coli DH5α.Subsequently,8 independent clones were selected and sequenced for each of the amplified fragments.[Results] In healthy female controls,half of the cloned fragments of CD40L regulatory sequences were unmethylated,and the other half were methylated.The mean methylation levels of CD40L promoter and enhancer from female SSc patients were significantly lower than those from healthy female controls (both P ＜ 0.01 ).Almost all of the cloned fragments of CD40L promoter and enhancer were unmethylated in healthy male controls and male SSc patients,with no significant difference in the methylation level between male SSc patients and healthy controls (both P ＞ 0.05,respectively).[Conclusion]s There is a low methylation level of CD40L regulatory elements on the inactive X chromosome in female SSc patients,which may contribute to the CD40L overexpression in CD4+ T cells.
ABSTRACT
Objective To investigate the expression levels of CD40L in CD4 + T cells from systemic sclerosis (SSc) patients.Methods PBMC ( peripheral blood mononuclear cells) cells were isolated from the peripheral venous blood of SSc patients (16females,10males) and healthy donors (15females,10males) by density gradient centrifugation.CD4 + T cells were isolated using magnetic beads.mRNA levels of CD40L in CD4 + T cells were measured by real-time quantitative polymerase chain reaction (RTPCR).Flow cytometric analysis was used to detect the CD40L protein on the surface of CD4 + T cells.Results Both CD40L mRNA and protein expression in CD4 +T cells was significantly elevated in female SSc patients compared with female healthy controls [5.61 ± 1.86 vs 2.80 ±0.94,P ＜0.01 ; (6.70 ±3.55)％vs (2.37 ± 1.39)％,P ＜ 0.05,respectively].No significant increase in CD40L mRNA and protein expression was observed in male SSc patients compared with male controls [2.59 ± 0.89 vs 1.92 ± 0.56,P ＞0.05; (2.06±1.09)％ vs (2.13±0.87)％,P ＞0.05,respectively].Conclusions CD40L was overexpressed in CD4 + T cell of female patients but not males,which maybe one of the important reasons for female susceptibility to SSc.