Background T helper cell 17 (Th17),a newly discovered subset of CD4+ T cells,have been found to play an important role in dry eye disease in animal model.Further investigation should be done on the immunopathogenesis of Th17 cells in dry eye patients.Objective This study was to analyze the expression status of interleukin-17 (IL-17) and IL-22 in tear and supernatant of peripheral blood mononuclear cells (PBMCs) of dry eye patients and their correlation with clinical symptom and sign.Methods Twenty Sj(o)gren syndrome (SS)patients,twenty non-Sj(o)gren syndrome (NSS) patients were included in Wuxi Second Hospital from 2010 to 2011,and twenty healthy volunteers were recruited at the same period.All of subjects understood the purpose and procedure of research and written informed consent was obtained form each subject initial of this study.Dry eye symptom questionnairs were self-answered and multiple dry eye disease-related clinical tests,including the breakup time of tear film (BUT),Schirmer Ⅰ test (S Ⅰ t) and corneal fluorescein staining were performed.The periphery blood of 3 ml and tear were collected in all the subjects,and IL-17 and IL-22 levels in supernatant of PBMCs and tear were detected by enzyme-linked immunosorbent assay (ELISA).The correlations between levels of IL-17 and IL-22 with BUT,S Ⅰ t,corneal fluorescein staining and dry eye scores were analyzed.Results The dry eye scores reduced,BUT prolonged,S Ⅰ t increased and corneal fluorescein dye decreased from SS group,NSS group to normal control group,with significant differences among the three groups (dry eye scores:H =40.81,P＜0.01 ; BUT:H =40.15,P＜0.01 ;S Ⅰ t..H=50.07,P＜0.01 ;corneal dye scores:H=40.52,P＜0.01).The concentration of IL-17 in the supernatant of PBMCs in the SS patients,NSS patients and normal controls were (964.92±124.83)ng/L,(718.85± 115.89)ng/L and (341.95±85.08) ng/L,showing a statistically significant difference among them (F=162.95,P＜0.01).The levels of IL-17 in the tear were (440.69±126.09) ng/L,(364.33±126.85) ng/L and (61.16±11.60) ng/L in the SS group,NSS group and normal control group respectively,exhibiting an elevated level in the SS group and NSS group compared with the control group (F=75.27,P＜0.01).In addition,the levels of IL-22 in the supernatant of PBMCs in the SS patients,NSS patients and normal controls were (98.77± 11.27) ng/L,(79.65 ± 11.01) ng/L and (32.78±9.34) ng/L,and those in the tear were (22.22 ± 8.96) ng/L,(14.92 ±4.35) ng/L and (10.47 ± 2.67) ng/L,with significant differences among the three groups (F =206.27,P＜0.01 ;F =19.87,P＜0.01).The significant correlations were found between the IL-17 and IL-22 concentration in the supernatant of PBMCs and tear with corneal fluorescein staining scores and S Ⅰ t.Conclusions The contents of IL-17 and IL-22 in PBMCs and tear upregulate in the SS and NSS patients,indicating that Th17 plays a key role in the immunity mechanism of dry eye.

Background Mechanism of dry eye disease has not been established clearly.Some increasing evidences showed that inflammation plays an important role in pathogenesis and development of dry eye disease.However,the association of interleukin-6 (IL-6) with dry eye has not been clarified.Objective This study was to assess the correlation of IL-6 levels in tear and serum of patient with dry eye disease and clinical symptom.Methods Twenty patients with Sj（o）gren syndrome (SS) and 20 cases with non-Sj（o）gren syndrome (NSS) were enrolled from Second Affiliated Hospital of Nanjing Medical University,and 20 healthy volunteers were recruited as the normal controls.Symptom questionnaires were self-answered and multiple dry eye-related clinical tests were performed,including tear film breakup time (BUT),Schirmer Ⅰ test (S Ⅰ t) and corneal fluorescence staining.IL-6 concentrations in serum and tear were detected by enzyme-linked immunosorbent assay (ELASA).The correlation between IL-6 level and tear film and ocular surface parameters was analyzed by Spearman rank correlation.Written informed consent was obtained from each subject initial of this trial.Results The scores of symptom questionnaires were 18.50 (11.75,23.00),23.00 (19.00,32.00) and 2.00 (0.25,4.00) in the SS group,NSS group and the normal control group,and significantly increasing values were seen in the SS group and NSS group compared with the normal control group (P=0.00,0.00).BUT values in the SS group and NSS group were 2.50 (2.00,3.88) seconds and 3.50 (2.13,4.00) seconds and were lower than 15.00 (13.13,17.00) seconds in the normal control group (P=0.00,0.00).S Ⅰ t values in the SS group,NSS group and the normal control group were 2.00 (1.50,2.88),4.00 (3.00,5.75) and 19.00 (18.00,25.00)mm,showing significant differences between the SS group and the normal control group (P =0.00),the SS group and the NSS group (P =0.00) or the NSS group and the normal control group (P=0.00).The marked elevation of keratoepithelioplasty score was seen between the SS group and the normal control group (5.75 (4.00,7.75) vs.0.00 (0.00,0.75) (P =0.00) or the NSS group and the normal control group (4.50(3.63,6.00) vs.0.00(0.00,0.75)) (P=0.00).Concentrations of IL-6 in serum and tear in the SS group,NSS group were (131.47±21.04) μg/L and (77.81 ± 15.68) μg/L and were significantly higher than (31.62±8.57) μg/L of the normal control group,and those in the tear were (33.44±8.01) μg/L,(18.78 ±5.73) μg/L and (8.77 ± 3.43)μg/L,respectively in the three groups,with a significant reduce in the normal control group (all P=0.00).tL-6 levels in serum and tear were positively correlated with the score of symptom questionnaires or keratoepithelioplasty and negatively correlated with BUT and S Ⅰ t (all P =0.00).Conclusions IL-6 levels in the tear and serum of the dry eye patient are associated with the severity of the disease.IL-6 levels in serum and tear may be objective,sensitive and reliable indicators of dry eye.

Objective To investigate the mechanism of the inhibitory effect of ?-ray irradiation on rat vascular smooth muscle cells(VSMC). Methods The ef fect of ?-ray irradiation on proliferation of VSMC was observed by 3?H-TdR incor poration. After ?-ray irradiation, the VSMC cell cycle change was detected b y flo w cytometry. The expression of p53, cyclin D and PCNA was investigated by Wester n Blot. Results The inhibitory effect of ?-ray irradiation on VSMC proliferati on was dose-dependent. After ?-ray irradiation, VSMC was arrested in G 1 st age, w ith the expression of p53 increased but the expression of cyclin D and PCNA decr eased. Conclusions ?-ray irradiation can inhibit the proliferation of VSMC. T he main mechanism is probably due to the induction of cell cycle arrest and inhi bition of the VSMC mitosis ,during which process, p53,cyclin D and PCNA all pla y an important role .

OBJECTIVETo investigate the effects of pregnancy malnutrition on the occurrence of insulin resistance (IR) in rat offspring during adult stage and to find out the relationship between TNF-alpha and IR; and to find out a reasonable early nutritional intervention measure for the prevention of IR, through giving different diets to offspring.

METHODSAn IUGR model was built by maternal nutrition restriction. 80 newborn IUGR female pups were randomly divided into 4 groups, the mother rats were given the following diet respectively for 3 weeks after delivery, pups were fed by mother milk: (1) The IUGR (intrauterine growth retardation) rat model was used and the animals were divided into: IUGR control group (group S/N) fed with normal diet, (2) IUGR high-caloric diet group (group A), (3) IUGR high-protein and high-caloric diet group (group B) and (4) IUGR high-protein isocaloric diet group (group C). Each group had 20 pups and another 20 normal female pups were fed with normal diet as the normal control group (group C/N). All pups were weaned at the 4th week of age and fed with normal diet till the end of the experiment. At the 12th week (adulthood) and 48th week (senility) of life, body weight and length, the fasting blood glucose, insulin concentration, TNF-alpha of adipose tissue and body weight were measured. Body mass index (BMI), ISI (insulin sensitive index), IRI (insulin resistant index) and HBCI (beta cell insulin excretion index) and their correlation to TNF-alpha were calculated.

RESULTSAt 12th week and 48th week of life, the insulin sensitivity of IUGR model group was significantly lower than group C/N, although there was no significant difference of body weight between these two groups. TNF-alpha was negatively correlated with ISI, positively correlated with IRI and no relation to HBCI. Group A and B was fatter and developed more severe IR. There were no significant differences in ISI, IRI, HBCI and TNF-alpha between group C and group C/N.

CONCLUSIONSIUGR offspring of pregnancy malnutrition mother rats showed IR at the age of 12th week. TNF-alpha was closely related to the occurrence of IR in IUGR pups. IUGR pups fed with high caloric diet or high protein and caloric diet at the early postnatal period amplified the metabolic abnormality. The high protein isocaloric diet is effective early nutritional intervention measure for the prevention of occurrence of IR at adulthood.

Animals ; Animals, Newborn ; growth & development ; Body Weight ; drug effects ; Dietary Proteins ; pharmacology ; Female ; Fetal Growth Retardation ; blood ; etiology ; Insulin Resistance ; physiology ; Malnutrition ; physiopathology ; Pregnancy ; Pregnancy Complications ; Prenatal Exposure Delayed Effects ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate effects of paraquat on the mRNA expression of key elements of Notch signaling (Notch1, Jagged1 and DTX1) during differentiation process of human neural stem cells (hNSCs).

METHODShNSCs exposed to PQ at the concentrations 0.10, 1.00, 10.00 M. Cell proliferation ability was assessed using MTT assay and mRNA expressions of Notch1, Jagged1 and DTX1 were detected by Real-time RT-PCR at 2, 4, 8, 12 d of differentiation.

RESULTSCompared with control group, NOTCH1, JAG1 mRNA expression levels exposed to PQ at the concentration of 0.10 M significantly reduced at 2, 4, 8 d and significantly went up at 12d (P < 0.01). Compared with control group, NOTCH1, JAG1 and DTX1 mRNA expression levels exposed to PQ at the concentration of 10.00 M significantly reduced at 2, 8, 12 d (P < 0.01). PQ could down-regulate Notch1, Jagged1 and DTX1 mRNA expressions at the early stage of differentiation, then up-regulate Notch1 mRNA expression, and down-regulate Notch1, Jagged1 and DTX1 mRNA expressions at the end of differentiation.

CONCLUSIONNotch signaling pathway may be involved in differentiation of neural stem cell exposed to PQ.

Calcium-Binding Proteins ; metabolism ; Cell Differentiation ; drug effects ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; drug effects ; metabolism ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Jagged-1 Protein ; Membrane Proteins ; metabolism ; Neural Stem Cells ; cytology ; drug effects ; metabolism ; Paraquat ; pharmacology ; Receptor, Notch1 ; metabolism ; Serrate-Jagged Proteins ; Signal Transduction ; drug effects ; Ubiquitin-Protein Ligases ; metabolism

Adolescent ; Adult ; Ambulatory Surgical Procedures ; instrumentation ; methods ; Child ; Child, Preschool ; Female ; Humans ; Male ; Middle Aged ; Radio Waves ; Tonsillectomy ; instrumentation ; methods ; Young Adult

The aim of the present study was to investigate the role of caveolin-1 in the inhibition of endothelin-1 induced proliferation of vascular smooth muscle cells (VSMCs) by 17beta-estradiol. In the cultured rat thoracic aortic VSMCs, proliferation of VSMCs was determined by using [(3)H]-thymidine incorporation and the expression of caveolin-1 protein was measured by immunofluorescence assays and Western blotting. The measurement demonstate VSMCs exposed to various concentrations of endothelin-1 (1-100 nmol/L) for 24 h induced an increase in [(3)H]-thymidine incorporation. Pretreament with various concentrations of 17beta-estradiol (0.1-10 nmol/L) for 24 h inhibited the proliferation effect of endothelin-1. Immunofluorescence assays showed that after 24 h treatment of VSMCs with endothelin-1 (100 nmol/L), the expression of caveolin-1 in VSMCs was significantly increased, whereas pretreament with 17beta-estradiol (10 nmol/L) for 24 h inhibited the effect. Western blotting results further proved that endothelin-1 inhibited and 17beta-estradiol increased the expression of caveolin-1 in VSMCs. These results demonstrate that 17beta-estradiol inhibits the VSMCs proliferation induced by endothelin-1, and that the effect of estradiol is probably mediated by caveolin-1.
Animals ; Aorta, Thoracic ; cytology ; Caveolin 1 ; physiology ; Cell Division ; Cells, Cultured ; Endothelin-1 ; antagonists & inhibitors ; physiology ; Estradiol ; pharmacology ; Muscle, Smooth, Vascular ; cytology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo evaluate the safety and efficiency of local paclitaxel delivery using the double-balloon perfusion catheter to prevent restenosis in the canine coronary artery.

METHODSTwenty domestic canines underwent bare-mental stent implantation after balloon injure of the left coronary artery. A novel double-balloon perfusion catheter was used to deliver the drug locally in the canine coronary artery. In the treatment group (n = 15), paclitaxel (10 ml, 20 micromol/L) was delivered using the double-balloon perfusion catheter before stent implantation. In the control group (n = 5), 10 ml saline was delivered using the double-balloon perfusion catheter before stent implantation. The perfusion time in both groups was (26.45 +/- 5.18) s. Animals underwent coronary angiography and optical coherence tomography (OCT) 90 days after stent implantation and were sacrificed. Vessels were perfusion-fixed and morphometric analysis was performed using conventional techniques.

RESULTSCoronary angiography results showed restenosis rate in control group was significantly higher than that in treatment group (60% vs. 33.33%, P < 0.05). The parameters of OCT showed in treatment group and control group: the neointimal thickness was (0.19 +/- 0.08) mm and (0.38 +/- 0.03) mm, the neointimal area was (1.52 +/- 0.49) mm2 and (2.51 +/- 0.47) mm2, the lumen area was (3.50 +/- 0.66) mm2 and (2.78 +/- 0.57) mm2, the extent of stenosis was (30.13 +/- 8.56)% and (47.40 +/- 4.50)%, and all the variances above were significantly different between the two groups (P < 0.05). The histologic parameters showed in treatment group and control group: the neointimal thickness was (0.22 +/- 0.10) mm and (0.47 +/- 0.05) mm, the neointimal area was (1.85 +/- 0.78) mm2 and (3.43 +/- 0.25) mm2, the lumen area was (3.15 +/- 0.43) mm2 and (1.85 +/- 0.55) mm2, the extent of stenosis was (36.00 +/- 10.97)% and (65.40 +/- 8.23)%, and all the variances above were also significantly different between the two groups (P < 0.05). The stents of both the groups were fully endothelialized. No thrombus or aneurysm was found in stents.

CONCLUSIONLocal delivery of paclitaxel with the double-balloon perfusion catheter to prevent restenosis in coronary stents is safe and efficient.

Angioplasty, Balloon, Coronary ; Animals ; Catheters ; Coronary Restenosis ; prevention & control ; Disease Models, Animal ; Dogs ; Injections ; Paclitaxel ; administration & dosage ; therapeutic use ; Stents

OBJECTIVETo establish an effective method for isolating and culturing outer root sheath (ORS) bulge cells, dermal sheath cells (DSCs) and dermal papilla cells (DPCs) derived from human hair follicle.

METHODSSmall scalp specimens were incubated in the presence of dispase at 37 degrees celsius; for 2 h, the hair shafts with ORS embedded in the dermal sheath (DS) were extracted under dissecting microscope, and the ORS tissue were inoculated onto Petri dish. The specimens were transected at the interface between the dermis and subcutaneous tissue. The portions of DS and DP (linked with and enclosed by DS) embedded in the adipose tissue were pulled out and incubated with collagenase at 37 degrees celsius; for 6-8 h, and the DP and DSCs were isolated by repeated low-speed centrifugation and cultured respectively on Petri dishes. The cultured ORS bulge cells were identified by immunohistochemistry with K19 antibody and DPCs and DSCs by immunohistochemistry with alpha-actin antibody.

RESULTSPurified ORS bulge cells, DSCs and DPCs could be harvested from the same human hair follicle.

CONCLUSIONThis new method allows efficient, rapid, and simultaneous isolation and culture of ORS bulge cells, DSCs and DPCs.

Cell Culture Techniques ; methods ; Cell Separation ; Cells, Cultured ; Dermis ; cytology ; Hair Follicle ; cytology ; Humans ; Immunohistochemistry ; Scalp ; cytology

In the present study, confluent bovine aortic endothelial cells (BAECs) were used to study the rapid nongenomic effects of 17beta-estradiol and the membrane impermeable conjugated 17beta-estradiol (E(2)BSA) on the activation of endothelial nitric oxide synthase (eNOS) and mitogen activated protein kinase (MAPK). eNOS activation was assessed in whole cells by measuring [(3)H]L-arginine conversion to [(3)H]L-citrulline. MAPK activity was determined by Western blotting. The results obtained show that the addition of various concentrations of E(2) (0.001-1 micromol/L) resulted in 122+/-29, 186+/-17, 83+/-20 and 157+/-29% increases in eNOS activity, respectively, in BAECs within 15 min of exposure to the hormone. E(2) (0.01 mol/L)-stimulated eNOS activity was detectable during 5-, 15- and 30- min incubation which yielded increases of 37+/-6, 56+/-9 and 38+/-8%, respectively. The increase reached a plateau from 15 through 30 min and rapidly declined thereafter. E(2)BSA 17.5 ng/ml also enhanced eNOS activity by an increase of 35+/-9% above the basal activity. The effect of E(2) and E(2)BSA on eNOS activation was unaffected by actinomycin D 25 microg/ml but was obviously inhibited by tamoxifen (0.1 micromol/L) and PD98059 (50 micromol/L). Compared with control E(2) and E(2)BSA stimulation of BAECs for 15 min caused an increase in MAPK activity by 428+/-17 and 360+/-14% respectively. This effect was blocked by tamoxifen. These results suggest that there might be the membrane estrogen receptor localized on BAECs, which mediates the rapid nongenomic effect of estrogen on eNOS activation through MAPK pathways.
Animals ; Aorta ; cytology ; Cattle ; Cell Membrane ; metabolism ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Estradiol ; pharmacology ; Mitogen-Activated Protein Kinases ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Receptors, Estrogen ; physiology