Objective: To explore the effects of silencing Smad ubiquitination regulatory factor 2 (Smurf2) on the secretion of transforming growth factor beta 1 (TGF-β₁), alpha-smooth muscle actin (α-SMA), and collagen type Ⅰ by human hypertrophic scar-derived fibroblasts. Methods: The human normal skin-derived fibroblasts and hypertrophic scar-derived fibroblasts were cultured with explant culture technique from the normal skin and hypertrophic scar tissue, which was obtained from 9 patients with hypertrophic scars after burn. Two kinds of fibroblasts of the third passage were both divided into 6 groups according to the random number table, with 9 wells in each group. Fibroblasts in blank control group were cultured for 72 h without transfection of any small interfering RNA (siRNA), fibroblasts in negative control group were for cultured for 72 h after transfected with non-target siRNA, fibroblasts in Smurf2 siRNA group were cultured for 72 h after transfected with 100 nmol/L Smurf2 siRNA, fibroblasts in blank control+ TGF-β₁ group were cultured for 72 h without transfection of any siRNA and then treated with 10 ng/mL TGF-β₁ for 6 h, fibroblasts in negative control+ TGF-β₁ group were cultured for 72 h after transfected with non-target siRNA and then treated with 10 ng/mL TGF-β₁ for 6 h, fibroblasts in Smurf2 siRNA+ TGF-β₁ group were cultured for 72 h after transfected with Smurf2 siRNA and then treated with 10 ng/mL TGF-β₁ for 6 h. (1) The protein and mRNA expression levels of Smurf2 of the two kinds of cells in blank control group, negative control group, and Smurf2 siRNA group were assessed by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR), respectively. (2) The content of TGF-β₁ in the cell culture supernatant of the two kinds of cells in blank control group and Smurf2 siRNA group was determined by enzyme-linked immunosorbent assay (ELISA). (3) The protein expression levels of α-SMA of the two kinds of cells in the 6 groups were assessed by Western blotting. The content of collagen type Ⅰ in the cell culture supernatant of the two kinds of cells in the 6 groups was determined by ELISA. (4) The mRNA expression levels of α-SMA and collagen type Ⅰ of the two kinds of cells in the 6 groups were assessed by RT-PCR. The sample numbers of each group in the above experiments were all 9. Data were processed with analysis of variance of factorial design and Bonferroni test. Results: (1) The protein and mRNA expression levels of Smurf2 of the two kinds of cells in Smurf2 siRNA group were significantly lower than those in blank control group and negative control group (with P values below 0.05). The protein and mRNA expression levels of Smurf2 of the two kinds of cells in blank control group and negative control group were close (with P values above 0.05). (2) The content of TGF-β₁ in the cell culture supernatant of hypertrophic scar-derived fibroblasts in blank control group and Smurf2 siRNA group was respectively (4.34±0.56) and (2.14±0.28) pg/mL, which was significantly higher than (1.52±0.20) and (1.41±0.18) pg/mL of normal skin-derived fibroblasts respectively (with P values below 0.05). In hypertrophic scar-derived fibroblasts, the content of TGF-β₁ in the cell culture supernatant in Smurf2 siRNA group was significantly lower than that in blank control group (P<0.05). In normal skin-derived fibroblasts, the content of TGF-β₁ in the cell culture supernatant in Smurf2 siRNA group was close to that in blank control group (P>0.05). (3) The protein expression levels of α-SMA and content of collagen type Ⅰ in the cell culture supernatant of the two kinds of cells in blank control+ TGF-β₁ group were significantly higher than those in blank control group (with P values below 0.05). The protein expression levels of α-SMA and content of collagen type Ⅰ in the cell culture supernatant of the two kinds of cells in negative control+ TGF-β₁ group were significantly higher than those in negative control group (with P values below 0.05). The protein expression levels of α-SMA and content of collagen type Ⅰ in the cell culture supernatant of the two kinds of cells in Smurf2 siRNA group were close to those in blank control group and negative control group (with P values above 0.05). The protein expression levels of α-SMA and content of collagen type Ⅰ in the cell culture supernatant of the two kinds of cells in Smurf2 siRNA+ TGF-β₁ group were significantly lower than those in blank control+ TGF-β₁ group and negative control+ TGF-β₁ group (with P values below 0.05). (4) The mRNA expression levels of α-SMA and collagen type Ⅰ of the two kinds of cells in blank control+ TGF-β₁ group were significantly higher than those in blank control group (with P values below 0.05). The mRNA expression levels of α-SMA and collagen type Ⅰ of the two kinds of cells in negative control+ TGF-β₁ group were significantly higher than those in negative control group (with P values below 0.05). The mRNA expression levels of α-SMA and collagen type Ⅰ of the two kinds of cells in Smurf2 siRNA group were close to those in blank control group and negative control group (with P values above 0.05). The mRNA expression levels of α-SMA and collagen type Ⅰ of the two kinds of cells in Smurf2 siRNA+ TGF-β₁ group were significantly lower than those in blank control+ TGF-β₁ group and negative control+ TGF-β₁ group (with P values below 0.05). Conclusions Silencing Smurf2 in human hypertrophic scar-derived fibroblasts can reduce the autocrine of TGF-β₁ and inhibit the TGF-β₁-induced α-SMA expression and collagen type Ⅰ synthesis.

Adult ; Aged ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Mucin-2 ; Mucin-6 ; Mucins ; analysis ; Neoplasms, Cystic, Mucinous, and Serous ; chemistry ; Pancreatic Neoplasms ; chemistry

Objective: To investigate the expression of SnoN in human hypertrophic scar fibroblasts and the mechanism of its participation in hypertrophic scar formation. Methods: Eight patients with hypertrophic scar after burn in need of surgery were admitted in our unit from January to October 2013, and then hypertrophic scar tissue and normal skin tissue of full-thickness skin donor site resected by surgery of the patients were collected. Hypertrophic scar fibroblasts and normal skin fibroblasts of patients were isolated with method of explant culture and then sub-cultured. Cells of the third to fifth passage were used in the following experiments. (1) The protein expressions of SnoN of hypertrophic scar fibroblasts and normal skin fibroblasts were assessed with Western blotting. (2) The mRNA expressions of SnoN of another batch of hypertrophic scar fibroblasts and normal skin fibroblasts were determined with reverse transcription polymerase chain reaction. (3) Another batch of hypertrophic scar fibroblasts and normal skin fibroblasts were treated with 10 ng/mL transforming growth factor beta1 (TGF-β₁) for 30 min, 1 h, 2 h, and 6 h, respectively, and then the protein expressions and mRNA expressions of SnoN of untreated cells and treated cells were detected as above. Data were processed with one way analysis of variance and independent sample t test. Results: (1) The protein expression of SnoN of hypertrophic scar fibroblasts was 0.020±0.003, significantly lower than that of normal skin fibroblasts (0.032±0.005, t=7.19, P<0.05). (2) The mRNA expression of SnoN of hypertrophic scar fibroblasts was 0.407±0.157, with no significant difference from that of normal skin fibroblasts (0.339±0.095, t=-1.29, P>0.05). (3) The protein expression of SnoN of normal skin fibroblasts was increased in a time-dependent fashion with the TGF-β₁ stimulation, and the protein expressions of SnoN of cells treated with TGF-β₁ for 30 min, 1 h, 2 h, and 6 h were significantly higher than those of untreated cells (with t values from 2.27 to 27.89, P values below 0.05). The protein expression of SnoN of hypertrophic scar fibroblasts was decreased in a time-dependent fashion with the TGF-β₁ stimulation, and the protein expressions of SnoN of cells treated with TGF-β₁ for 30 min, 1 h, 2 h, and 6 h were obviously lower than those of untreated cells (with t values from 10.80 to 13.85, P values below 0.05). (4) The mRNA expressions of SnoN of normal skin fibroblasts and hypertrophic scar fibroblasts were both increased in a time-dependent fashion with the TGF-β₁ stimulation, and the mRNA expressions of SnoN of the two types of cells treated with TGF-β₁ for 30 min, 1 h, 2 h, and 6 h were both significantly higher than those of untreated cells (with t values from 18.16 to 58.22, P values below 0.05). Conclusions The protein expression of SnoN in hypertrophic scar fibroblasts is reduced, which weakens its inhibitory effect on TGF-β₁ signal, thus amplifying the TGF-β₁ signal, and it may participate in the formation of hypertrophic scar.

OBJECTIVETo establish a predictive scoring system which may serve for the prediction of sustained response to conventional interferon-alpha (IFN-alpha) treatment on chronic hepatitis B.

METHODSA total of 474 IFN-alpha treated hepatitis B virus e antigen (HBeAg)-positive patients were enrolled in the present study. The patients' baseline characteristics, such as age, gender, aminotransferases, activity grading (G) of intrahepatic inflammation, score (S) of liver fibrosis, hepatitis B virus (HBV) DNA and genotype were evaluated; therapy duration and response of each patient at the 24th wk after cessation of IFN-alpha treatment were also recorded. A predictive scoring system for a sustained complete response (CR) to IFN-alpha therapy was established based on genetic algorithm. About 10% of the patients were randomly drawn out as the test set. Responses to IFN-alpha therapy were divided into CR, partial response (PR) and non-response (NR). The mixed set of PR and NR was recorded as PR + NR.

RESULTSFor the scoring system, the sensitivity and specificity were 78.8% and 80.6%, respectively.

CONCLUSIONThis SCR scoring system has satisfying prediction efficiency and is easily employed in clinical practice. With this scoring system, practitioners can propose individualized decisions that have an integrated foundation on both evidence-based medicine and personal characteristics.

Adolescent ; Adult ; Antiviral Agents ; therapeutic use ; Child ; Dose-Response Relationship, Drug ; Female ; Hepatitis B, Chronic ; drug therapy ; Humans ; Interferon-alpha ; therapeutic use ; Male ; Middle Aged ; Predictive Value of Tests ; Prognosis ; Sensitivity and Specificity ; Treatment Outcome ; Young Adult

Child ; Hernia, Inguinal ; surgery ; Herniorrhaphy ; methods ; Humans ; Laparoscopy ; methods