ObjectiveTo investigate the role of caspase 3 in HMME-induced apoptosis in hypertrophic scar fibroblasts (HSFs).MethodsFibroblasts were obtained from 10 patients with untreated hypertrophic scar,and subjected to a primary culture.After 4 to 6 passages of culture,the HSFs were divided into 3 groups to remain untreated(control group),be treated with HMME followed by photodynamic therapy (HMME-PDT group),or the combination of HMME and Z-DEVD-FMK followed by photodynamic therapy (caspase 3 inhibitor group).At 12 hours after the therapy,HSFs were collected and immunofluorescence microscopy was used to observe the fluorescence intensity of caspase 3 after staining with fluorescein isocyanate (FITC) and popodium iodide (PI),flow cytometry was performed to determine the percentage of caspase 3-positive HSFs and apoptosis rate in HSFs after single staining with FITC and PI respectively.Results The fluorescence intensity of caspase 3 was weak in the control group and caspase 3 inhibitor group,but was strong in the HMME-PDT group.An increased percentage of caspase 3-positive HSFs was noted in the HMMEPDT group compared with the control group and caspase 3 inhibitor group(30.86％ ± 1.21％ vs.3.12％ ±0.28％ and 2.46％ ± 0.18％,t =19.92,21.76,both P ＜ 0.05).The apoptosis rate in HSFs was significantly higher in the HMME-PDT group and caspase 3 inhibitor group than in the control group(30.54％ ± 3.78％ and 10.46％ ± 2.15％ vs.2.45％ ± 0.22％,t =35.90,27.97,both P＜ 0.05),and higher in the HMME-PDT group than in the caspase 3 inhibitor group.ConclusionsThe apoptosis in HSFs induced by HMME-PDT is closely related to the activation of caspase 3,while caspase 3 seems to be dispensable for the apoptosis.

Objective To investigate the apoptotic effects of hypertrophic scar fibroblast (HSF) induced by HMME-PDT.Methods Fibroblasts were cultured from nontreated hypertrophic scars,and cells at passages 4-6 were used for the experiments (photosensitizer dose 4 μg/ml,λ630 nm,pow er density 10 mw/cm2,energy fluence 2.5 J/cm2).Morphological and biochemical changes in fibroblasts were assessed by Hoechst 33258 staining and fluorescence microscopy.The rate of apoptotic or necrotic cells was detected by flow cytometry (FCM) through double staining of Annexin V -FITC and popodium iodide (PI),respectively.Results Marked morphological features of cell apoptosis were viewed under the fluorescent microscope through Hoechst 33258 staining.The analysis of FCM indica ted that the apoptotic rate was significantly increased after HMME PDT [(34.82 ± I.42) ％ vs (3.12±0.28) ％,P＜0.05],and apoptotic rate was higher than necrosis rate [(14.65±1.02) ％ vs (34.82±1.42) ％,P＜0.05].Conclusions Low level exposure to 630 nm PDT mediated by HMME appears to induce fibroblast apoptosis.

Adult ; Asthma, Occupational ; therapy ; Bronchoalveolar Lavage ; Humans ; Male ; Textiles ; adverse effects

OBJECTIVETo observe the effect of umbilical sticking therapy (UST) with Qitou Xiaugu Plaster (QXP) on hemodynamics of portal system in patients with liver cirrhosis.

METHODSOne hundred and twenty patients of liver cirrhosis with portal hypertension were assigned to two groups. On the basis of conventional therapy, UST was applied in the 66 patients in treated group, which was exchanged once every 3 days with an interval of 1-day rest. The 54 patients in the control group were orally administered with propanolol. The therapeutic course for both groups was 1 month. Before and after treatment, the hemodynamic changes in portal or splenic veins were observed by color Doppler ultrasonograph, and the changes of liver function, blood coagulation and patients' subjective symptoms were observed as well.

RESULTSAfter treatment, portal vein diameter and splenic vein diameter significantly decreased (P < 0.05, portal venous flow velocity and splenic venous flow velocity apparently increased (P < 0.05), and portal venous flow apparently decreased in both groups (P < 0.05), while no significant change was found in the splenic venous flow (P > 0.05). The liver function and blood coagulation indexes in both groups were improved. The improvement of clinical symptoms in the treated group was superior to that in the control group.

CONCLUSIONUST with QXP could decrease the portal vein pressure in a short time, with the therapeutic effect comparable to propanolol, and with no adverse reaction.

Adult ; Aged ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Hemodynamics ; drug effects ; Humans ; Hypertension, Portal ; drug therapy ; physiopathology ; Liver Cirrhosis ; drug therapy ; physiopathology ; Male ; Middle Aged ; Portal Pressure ; drug effects ; Portal Vein ; drug effects ; physiopathology ; Splenic Vein ; drug effects ; physiopathology ; Umbilicus ; blood supply ; Young Adult

Cell Transformation, Neoplastic ; drug effects ; genetics ; Dimethyl Sulfoxide ; pharmacology ; Electroporation ; methods ; HL-60 Cells ; Humans ; Membrane Proteins ; genetics ; Neoplasm Proteins ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Stromal Interaction Molecule 1 ; Transfection

Background To study diabetic retinopathy (DR) related risk factors is very important in the prevention of DR.Platelet endothelial cell adhesion molecule-1 (PECAM-1) is an important mediator that mediates high blood glucose-induced vascular diseases in diabetic patients.However,its mechanism is still below understood.Objective This clinical study was to investigate the effect of serum level changes of PECAM-1 on DR in type 2 diabetic patients.Methods Fifty-four patients with type 2 diabetes were enrolled from the endocrinology department of the Third People' s Hospital of Nantong City.Fundus examination was performed using the ophthalmoscope and fundus fluorescence angiography (FFA) on all the patients,and these patients were grouped as the non-DR (NDR)group (18 cases),non-proliferative DR(NPDR) group (20 cases) and proliferative DR group (PDR) (16 cases) based on the DR staging criterion of the Chinese Medical Association (1987 version).Eighteen age-and gender-matched normal subjects served as the normal control group.Peripheral blood was collected,and serum PECAM-1 levels were assayed using ELISA.Serum HbA1c levels were detected using the high performance liquid colorimetric(HPLC) method.The correlation of serum PECAM-1 level with serum HbA1c level was analyzed.All results were compared among the groups.Results The serum PECAM-1 levels were (10.907 ± 2.792),(7.024 ±2.377),(5.231 ± 1.816) and (3.817 ± 1.045) μg/L,respectively,in the PDR group,NPDR group,NDR group and normal control group,showing a significant difference among the 4 groups (F =12.630,P =0.02).Serum PECAM-1 content was significantly higher in the PDR group when compared with the NPDR group,NDR group and normal control group (P＜0.05).The serum HbA1c levels were (12.596±3.148)％,(9.118±3.356)％,(5.491±1.017)％ and (4.992 ± 0.725)％ in the PDR group,NPDR group,NDR group and normal control group,respectively,with a significant difference among these 4 groups (F =7.130,P =0.015),and those in the PDR group and NPDR group were significantly elevated in comparison with the NDR group and normal control group (P＜0.05).Significantly positive correlations were seen between serum PECAM-1 level and HbA1 c level in the PDR group,NPDR group and NDR group (r=0.799,P＜0.01 ;r =0.647,P＜0.01 ;r =0.685,P＜0.01).Significantly more patients with a disease course of ≥ 10 years were in the NPDR group in comparison with the PDR group (P =0.023).Conclusions Increase of serum PECAM-1 level is closely associated with blood glucose level,and it is an important factor in the pathogenesis and development of DR.These results imply that control of blood glucose is crucial for the prevention of DR in patient with type 2 diabetes.

Objective:To construct lentiviral vector specific for mouse B7-1 RNA interference and study lentivirus-mediated B7-1 gene silencing effects in L929 fibroblast cells.Methods:Three candidate sequences for B7-1 RNAi selected from coding sequence of mouse B7-1 transcription were used to design short hairpin RNA ( shRNA ) templates and then cloned into lentiviral expression plasmid followed with correctness identification of inserted sequence by DNA sequencing.Recombinant lentivirus were prepared by co-transfecting lentiviral expression vector and packaging plasmids into 293T cells.Then the resulting culture supernatant containing infectious lentiviral particles was pooled and centrifuged via ultra-centrifugation.Infectious titer of the preparations was determined by detecting the expression of GFP in 293T cells after transfected by lentivirus.Cultured L929 cells were transfected with lentivirus to deter-mine transduction efficiency and silencing efficacy of B7-1 expression by flow cytometry.Transducted L929 cells were then screened using puromycin to generate stable cell clones followed by flow cytometry analysis of GFP and B7-1 expression.A mixed reaction system consisting of stable B7-1 silencing L929 cells and mouse splenic T cells was used to analyze ability of the established cell line to trigger T cells proliferation.Results: Lentiviral expression vector for mouse B7-1 RNAi was correctly constructed with inserted sequences as designed.Recombinant RNAi lentivirus were prepared with titers ranging (3-5) ×108 TU/ml and efficacy to mediate GFP transgene expression and B7-1 silencing.B7-1 expression and the ability to trigger T cells proliferation of stable L929 cells were suppressed significantly ( P<0.05 ).Conclusion: We generated lentiviral vector specific for mouse B7-1 RNAi with high performance of transduction efficiency as well as B7-1 silencing efficacy and the recombinant RNAi lentivirus can mediated stable B7-1 gene silencing in L929 cells and inhibition of T cells proliferation induced by B7-1/CD28 co-stimulatory signal.

OBJECTIVE To evaluate whether the IDO1 inhibitor 1- methyl- L- tryptophan (1- MT) combine calcium influx inhibitor carboxyamidotriazole (CAI) could further enhance the suppression of programmed death 1 (PD-1) in CD8 + T cells and investigate the curative effect of the combined use. METHODS CD8 +T cells were isolated from normal mice spleen by negative selection using magnetic cell separation. The isolated CD8 +T cells were cultured in RPMI 1640 medium containing 10% FBS and 100 U·mL- 1 IL-2 and activated by the addition of anti-CD3 and anti-CD28 (1 g·L- 1 each mabs). CD8 + T cells were pretreated for 48 h with drug and the fluo- 3 as a marker of intracellular calcium concentration was detected by flow cytometry. The calcineurin (CaN) levels were assayed with ELISA in CD8+T cells after 48 h incubation with 10 μm CAI. The nuclear translocations of NFAT and AHR were detected by immunofluorescent staining after 48 h of drug treatment. The expression of PD-1 in CD8+T cells was analyzed by flow cytometry. RESULTS Intracellular fluorescent intensity was markedly debase due to CAI treatment(P<0.01). Meanwhile, the changes of CaN content had a resembled correlation (P<0.01). Immunofluorescence experiment showed that after combination therapy the transfer of NFAT and AHR in nuclear substantially reduced. Flow cytometry revealed that after the combination caused a significant decrease in PD-1 expression in CD8+T cells. CONCLUSION CAI and 1-MT could inhibit markedly the expression of PD-1 in CD8 +T cells by inhibiting the nuclear translocation of NFAT and AHR, respectively and the combination of them has synergetic effect.

Objective To observe the phosphorylation of Smad3 in hyperplastic scar fibroblasts (HSFs) induced by hematoporphyrin monomerthyl ether (HMME) followed by photodynamic therapy (PDT).Methods Fibroblasts were isolated from the hypertrophic scar tissues of 10 patients and subjected to culture in vitro.After 3-5 passages,the HSFs were divided into 4 groups:control group receiving no treatment,PDT group pretreated with HMME of 4 μg/ml followed by PDT,HMME group induced by HMME alone,and laser group irradiated with laser alone.Fluorescence microscopy was used to observe the expression of Smad3 after immunofluorescent staining with anti-Smad3 antibody,and Western blot to detect the expression of Smad3 and phosphorylated Smad3 in these HSFs.Paired t test was conducted to compare the difference in Smad3 and phosphorylated Smad3 expression between these groups.Results The total fluorescence intensity of Smad3 was similar between these groups,but the intranuclear fluorescence signal was significantly weaker in the PDT group than in the control group.The level of phosphorylated Smad3 was statistically decreased in the PDT group compared with the control group (0.20 ± 0.02 vs.0.92 ± 0.15,P ＜ 0.05),but no significant difference was observed between the HMME group and laser group (P ＞ 0.05).Conclusion PDT may inhibit the proliferation of HSFs via attenuating the phosphorylation of Smad3.

Insulin sensitivity,β-cell function and serum high-sensitivity C-reactive protein (hs-CRP)levels were observed in obese and non-obese normoglycemic first-degree relatives of type 2 diabetic patients (FDR). The results showed that there existed insulin resistance,β-cell dysfunction and increased serum hs-CRP level in obese FDR of type 2 diabetic patients. Moreover, insulin resistance and increased CRP level were positively related to waist circumference.