Objective: To assess the incidence and independent risk factors for clinical anastomotic leakage (AL) in patients undergoing anterior resection (AR) or low anterior resection, (LAR) for rectal cancer. Methods: This was a retrospective case-control study of 550 patients with rectal cancer who underwent AR or LAR from April 2007 to March 2017 in Beijing Friendship Hospital, Capital Medical University. The relationship between the incidence of AL and clinicopathological manifestations was analyzed by Chi-squared test and Fisher exact test, and the independent risk factors of AL were analyzed using logistic regression analysis. AL is defined as a defect (including necrosis or abscess formation) of the intestinal wall at the anastomotic site, leading to a communication between the intra- and extra-luminal compartments. AL can be divided into three grades. Grade A anastomotic leakage results in no change in the management of patients, whereas grade B leakage requires active therapeutic intervention but is manageable without re-laparotomy. Grade C anastomotic leakage requires re-laparotomy. Results: AL was noted in 32 (5.8%) of 550 patients with rectal cancer who underwent AR or LAR, including 15 (46.9%) , 4 (12.5%) , and 13 patients (40.6%) with Grades A, B, and C, respectively. Five patients (0.9%, 5/550) died peri-operatively. AL- and non-AL-related deaths occurred in 3 (9.4%, 3/32, all cases were Grade C) and 2 patients (0.4%, 2/518) , respectively, with the two mortality rates being significant difference (P = 0.002) . Chi-squared test or Fisher exact test showed that the incidence of AL was associated with neoadjuvant chemoradiotherapy (P = 0.011) , intraoperative bleeding (≥100 ml) (χ² = 11.980, P = 0.001) , and tension-reducing suture of anastomosis (P = 0.015) . The results of logistic regression analysis showed that the independent risk factors of AL were neoadjuvant chemoradiotherapy (OR = 2.402, 95%CI: 1.004 - 5.749, P = 0.049) , intraoperative bleeding (≥100 ml) (OR = 2.971, 95%CI: 1.269 - 6.957, P = 0.012) and tension-reducing suture of anastomosis (OR = 2.304, 95%CI: 1.008 - 5.263, P = 0.048) . Conclusion The incidence of AL in patients undergoing AR for rectal cancer is 5.8%. The high-risk factors for AL are neoadjuvant chemoradiotherapy, intraoperative bleeding (≥100 ml) , and tension-reducing suture of anastomosis. Patients with these three risk factors have a high risk of AL rate, and a defunctioning stoma should be performed.

Objective To detect the mRNA and protein expressions of desmoglein 1 (DSG1) and DSG3 in different types of keratinocytes (KCs).Methods Two keratinocyte cell lines HaCaT and A431,as well as primary keratinocytes from human abdomenal skin served as the object of this study.Direct immunofluorescence assay was performed to observe and quantify the expressions of DSG1 and DSG3,and quantitative PCR (qPCR) to determine the mRNA expressions of DSG1 and DSG3,in these cells.Results Both DSG1 and DSG3 were expressed in all the three types of keratinocytes,and the fluorescence intensity of DSG1 and DSG3 in HaCaT cells was higher than that in primary keratinocytes but lower than that in A431 cells.Similarly,all the keratinocytes expressed DSG1 and DSG3 mRNA,with the relative expression levels of DSG1 and DSG3 mRNA in primary keratinocytes being 291.7％ and 237.4％ of those in HaCaT cells respectively (both P ＜ 0.01),and those in A431 cells being 0.1％ and 18.8％ of those in HaCaT cells respectively (both P ＜ 0.05).Conclusions HaCaT cells,A431 cells and primary keratinocytes all can be used for the study of DSG1 and DSG3,of which,A431 cells show the strongest expressions of DSG1 and DSG3,and primary keratinocytes display the highest expressions of DSG1 and DSG3 mRNAs.

Objective To investigate the relationship between disease severity and enzyme-linked immunosorbent assay (ELISA) index values of anti-desmoglein (Dsg) 1 and-Dsg3 antibodies in 56 patients with pemphigus,and to characterize fluctuations in anti-Dsg1 and-Dsg3 antibodies in different forms of pemphigus.Methods Fifty-six patients with pemphigus (including 36 cases of pemphigus vulgaris (PV) and 20 cases of pemphigus foliaceus (PF)) were enrolled in this study.Blood samples were obtained from these patients before treatment and at the following four time points:when the condition was relieved and the taper of glucocorticoids began,the dose of glucocorticoids was tapered to half of their initial dose,the maintenance treatment started,and when the duration of maintenance treatment reached two years.ELISA was performed to determine the levels of anti-Dsg1 and-Dsg3 antibodies in these serum samples.Spearman correlation analysis was carried out to assess the relationship between disease severity and ELISA index values,and independent sample's t test to compare the levels of anti-Dsg antibodies among these time points.Results The severity of pemphigus was correlated with anti-Dsg antibody ELISA index values.Both anti-Dsg1 and anti-Dsg3 ELISA index values were significantly reduced at the remission of pemphigus compared with those before treatment (all P ＜ 0.01).At the end of the two-year maintenance treatment,10 (50％) patients with PF and 7 (19.4％) patients with PV became negative for anti-Dsg1 ELISA,whereas only 1 (2.7％) patient with PV became negative for anti-Dsg3 ELISA.Conclusions Anti-Dsg antibody ELISA index value is correlated with disease severity in patients with pemphigus,which may serve as a useful marker for assessing disease severity and activity as well as evaluating therapeutic efficacy.

Objective To study the effect of paeonol on the proliferation and apoptosis of A375 human melanoma cells and its mechanism.Methods Cell counting kit-8 (CCK8) was used to evaluate the proliferative activity of A375 cells treated with paeonol of 0.5,1,2,4,8 mmol/L for 24,48 and 72 hours respectively.Subsequently,A375 cells were treated with paeonol of 1.25,2.5 and 5 mmol/L for 24 hours followed by double staining with annexin V and propidium iodide for the detection of cell apoptosis,fluorometric assay for the estimation of caspase 3,caspase 8 and caspase 9 activity,and Western blot for the determination of the levels of p53,nuclear factor-κB proteins and some of their target proteins.The A375 cells receiving no treatment served as the blank control group.Statistical analysis was carried out by t test.Results Within the investigated concentration and time ranges,paeonol significantly inhibited the proliferative activity of A375 cells in a concentration-and timedependent manner.Compared with the blank control group,a significant increase was observed in the early apoptosis rate in A375 cells treated with paeonol of 1.25,2.5 and 5 mmol/L for 24 hours (13.74％-± 1.73％,25.95％ ± 0.57％ and 46.44％ ± 0.81％ vs.3.11％ ± 0.53％,P ＜ 0.05 or 0.01),as well as in the activity of caspase 3,8 and 9 in A375 cells treated with paeonol of 2.5 and 5 mmol/L for 24 hours (P ＜ 0.05 or 0.01).After 24-hour treatment,the protein levels of p53 and Bax were elevated,but those of nuclear factor-κB,Bcl-2 and Bcl-XL were decreased in A375 cells with the increase of paeonol concentration.Conclusions Paeonol can inhibit the proliferation but induce the apoptosis of A375 cells,and the apoptosis-inducing effect may be realized through intrinsic and extrinsic pathways by modulating nuclear factor-κB and p53 genes.

Objective To optimize the concentration of a microRNA-21 (miR-21) inhibitor and a miR-494 mimic for the transfection of A375 human melanoma cells,and to estimate the effect of the miR-21 inbihitor and miR-494 mimic on the proliferation of A375 cells.Methods A miR-21 inbihitor and a miR-494 mimic were designed and constructed.To optimize the concentration of the miR-21 inbihitor and miR-494 mimic for transfection,six concentrations (70-250 nmol/L) of the inbihitor and mimic were transfected into A375 cells separately by using LipofectamineTM2000.Then,quantitative fluorescence-based PCR was performed to determine the expression of miR-21 and miR-494 in A375 cells.Some A375 cells were classified into five groups:Mock blank control group remaining untransfected,miR-21 inhibitor group transfected with the miR-21 inhibitor,miR-21 control group transfected with the miR-21 inhibitor negative control,miR-494 mimic group transfected with the miR-494 mimic,and miR-494 control group transfected with the miR-494 mimic negative control.Mter another 48-hour culture,the cells were collected for the analysis of cell apoptosis and cycle by using flow cytometry.Meanwhile,Cy5-labelled miR-494 mimic negative control was transfected into A375 cells for the evaluation of the transfection efficiency by using an inverted fluorescence microscope.Results miRNAs were successfully extracted from A375 cells.As quantitative PCR revealed,the A375 cells transfected with the miR-21 inhibitor at 120 nmol/L showed the lowest expression level (2-△△Ct) of miR-21 (average:0.80; range:0.65-0.92),and those transfected with the miR494 mimic at 250 nmol/L displayed the highest expression level of miR-494 (average:126.82; range:111.52-144.22).The transfection efficiency in A375 cells was higher than 90％.Compared with the corresponding negative control groups,the miR-21 inhibitor group and miR-494 mimic group showed increased apoptosis rate ((27.74 ± 1.39)％ vs.(12.93 ± 0.65)％,(34.30 ± 2.35)％ vs.(15.54 ± 1.02)％,both P ＜ 0.01),percentage of G1-phase cells ((61.61 ± 3.25)％ vs.(50.34 ± 5.62)％,(61.05 ± 3.17)％ vs.(49.95 ± 2.58)％,both P＜ 0.05),but decreased proliferation index ((38.39 ± 3.25)％ vs.(49.66 ± 5.62) ％,(38.95 ± 3.17)％ vs.(50.05 ± 2.58)％,both P ＜ 0.05).Conclusions Both the miR-21 inhibitor and miR-494 mimic can promote the G1-phase arrest and apoptosis in A375 cells,and miR-21 may act as a protooncogene accelerating the proliferation of A375 cells,while miR-494 may founction as a tumor suppressor inhibiting the proliferation of A375 cells.

Objective To investigate the expression of CD200 in peripheral lymphocytes and hair follicles from patients with alopecia areata (AA).Methods Flow cytometry was used to detect the expression of CD200 in peripheral lymphocytes from 43 patients with AA and 43 healthy controls.Immunohistochemistry was carried out to quantify the expression of CD200 and cytokeratin 15(CK15,a marker for basal cells of the outer root sheath) in resected scalp specimens from 8 patients with AA and 8 healthy controls.Differences in the expression of CD200 and CK15 between the patients and controls were assessed by independent-samples t test and rank sum test.Data were processed by the software SPSS17.0.Results The percentage of CD200-expressing cells in peripheral blood lymphocytes and T lymphocytes was significantly lower in the patients with AA than in the healthy controls (5.73％ ± 3.46％ vs.12.01％ ± 4.90％,8.85％ ± 4.80％ vs.12.31％ ± 3.12％,t =6.865,3.964,respectively,both P ＜ 0.05).However,no significant difference was observed in the percentage of CD200-expressing cells in peripheral blood B lymphocytes between the patients and controls (74.68％ ± 8.12％ vs.75.75％ ± 9.45％,t =0.570,P ＞ 0.05).Further more,the patients showed a lower expression of CD200 (P ＜ 0.05),but a similar expression of CK15 (P ＞ 0.05) in hair follicles compared with the controls.Conclusion The decrease in CD200 expression in peripheral lymphocytes and hair follicles may be involved in the pathogenesis of AA.

Objective To study the effects of triptolide on the apoptosis in and proliferation of a human melanoma cell line M14.Methods M14 cells were cultured with the presence of 5 concentrations (12.5,25,50,100,200 nmol/L) of triptolide for 24,48 and 72 hours respectively,and cell counting kit-8 (CCK-8) was used for the detection of cell proliferation.Some M14 cells were treated with triptolide at 10 nmol/L,20 nmol/L and 30 nmol/L for 48 hours followed by the analysis of cell cycle by flow cytometry and detection of cell apoptosis by flow cytometry following annexin V-fluorescein isothiocyanate (FITC)/propidium iodide double staining.The morphological changes of M14 cells treated by triptolide at 30 nmol/L for 48 hours were observed by Hoechest 33258 staining.Results Compared with untreated M14 cells,an increase of cell population in S phase was observed in triptolide-treated cells,along with a decline in cell population in G2/M phase.The apoptosis rate was (2.92 ± 0.17)％,(20.99 ± 0.40)％,(34.28 ± 2.04)％ and (63.38 ± 0.71) ％ respectively in M14 cells treated with triptolide at 0,10,20 and 30 nmol/L for 48 hours,suggesting that triptolide enhanced the proliferation of M14 cells in a dose-dependent manner.After treatment with triptolide of 30 nmol/L,M14 cells showed morphological changes characteristic of apoptosis.Conclusion Triptolide could inhibit the proliferation of and induce the apoptosis in M14 human melanoma cells.

Objective To assess the relationship of methylation status of CpG islands in the promoter region of insulin-like growth factor binding protein 7 (IGFBP7) gene with the expression of IGFBP7 gene in human melanoma cell lines and primary melanocytes.Methods Primary melanocytes from human forcskin tissue as well as 4 human melanoma cell lines,including A375,M14,SK-MEL-1 and MV3,were used in this study.Bisulfite sequencing PCR (BSP) was applied to detect the methylation status of 54 CpG sites in the 5'-flanking promoter region of IGFBP7 gene in all of the melanoma cell lines and primary melanocytes.Results As hierarchical cluster analysis showed,IGFBP7-positive cells (including A375,M14 and SK-MEL-1 ) differed significantly from IGFBP7-negative cells (including MV3 cells and primary melanocytes) in the methylation pattern of IGFBP7 gene promoter region.Conclusion The methylation status of CpG island in the promoter region of IGFBP7 gene may be associated with its expression in melanoma cell lines.

ObjectiveTo investigate the correlation between the methylation status of HLA class Ⅰ genes(HLA-A, -B and -C) in psoriatic epidermis and disease severity in patients with psoriasis vulgaris. MethodsDNA specimens were obtained from the lesional and nonlesional epidermis of 46 patients with psoriasis vulgaris and from the normal skin of 28 human controls. Methylation specific PCR (MSP) was conducted to detect the methylation status of CpG islands in the promoter region of HLA-A, -B and -C genes. The severity of psoriasis was evaluated by psoriasis area and severity index(PASI) scores. ResultsThe percentage of promoter methylation of HLA-B and HLA-C genes was 4.35％(2/46) and 21.74％(10/46), respectively in nonlesional epidermis, 4.35％ (2/46) and 4.35％ (2/46), respectively in lesional epidermis from these patients. No methylation was observed for the promoter of HLA-A, -B or -C gene in the normal control epidermis or for that of HLA-A gene in the nonlesional or lesional epidermis from the patients. The frequency of HLA-C gene promoter methylation in the nonlesional epidermis was significantly higher than that in the lesional epidermis and control epidermis, but was uncorrelated to the disease severity. No significant difference was observed for the methylation frequency of HLA-A or -B gene promoter among the three groups of specimens. Conclusion Abnormal methylation of HLA-C gene promoter is observed in patients with psoriasis vulgaris.

Objective To investigate the expression of CXCR7 in several cutaneous malignant tumors including cutaneous squamous cell carcinoma (SCC),basal cell carcinoma (BCC) and invasive cutaneous malignant melanoma and their cell lines,as well as its significance.Methods Tissue specimens were obtained from the lesions of 30 patients with cutaneous squamous cell carcinoma,25 patients with basal cell carcinoma and 30 patients with cutaneous malignant melanoma.Immunohistochemistry was performed to detect the expression of CXCR7 protein in these tissue specimens and several cell lines (A375 human melanoma cells,M14 human melanoma cells,A431 human epidermoid carcinoma cells,HaCaT human keratinocytes).The mRNA expression of CXCR7 in these cell lines was measured by reverse transcription PCR.Results CXCR7 protein was apparently expressed in invasive cutaneous malignant melanoma.The high expression rate of CXCR7 protein was significantly elevated in cutaneous malignant melanoma tissue specimens compared with SCC and BCC tissue specimens [80％ (24/30) vs.26.67％ (8/30) and 8％ (2/25),x2 =17.16,28.36,both P ＜ 0.05].CXCR7 mRNA was expressed in A375,M14 and A431 cells,but not in HaCaT cells,with the strongest expression observed in A375 cells.Immunohistochemistry revealed the expression of CXCR7 protein only in A375 cells.Conclusions CXCR7 is highly expressed in cutaneous malignant melanoma and A375 cells,which may be involved in the malignant invasion and metastasis of melanoma.