OBJECTIVETo culture rat prostate glandular epithelial cells and study their barrier functions in vitro.

METHODSRat prostate glandular epithelial cells were cultured in vitro. The expression of the tight junction protein claudin-1 was determined by immunohistochemistry, the structure and composition of the epithelial cells observed under the inverted microscope and transmission electron microscope. The transepithelial electrical resistances (TEERs) were monitored with the Millicell system. The permeability of the prostate glandular epithelial cells was assessed by the phenol red leakage test.

RESULTSCompact monolayer cell structures were formed in the prostate glandular epithelial cells cultured in vitro. Immunohistochemistry showed the expression of the tight junction protein claudin-1 and transmission electron microscopy confirmed the formation of tight junctions between the adjacent glandular epithelial cells. The TEERs in the cultured prostate glandular epithelial cells reached the peak of about (201.3 ± 3.5) Ω/cm2 on the 8th day. The phenol red leakage test manifested a decreased permeability of the cell layers with the increase of TEERs.

CONCLUSIONThe structure and function of rat prostate glandular epithelial cells are similar to those of brain capillary endothelial cells, retinal capillary endothelial cells, and intestinal epithelial cells. In vitro cultured prostate glandular epithelial cells have the barrier function and can be used as a model for the study of blood prostate barrier in vitro.

Animals ; Cell Membrane Permeability ; Cells, Cultured ; Claudin-1 ; metabolism ; Electric Impedance ; Epithelial Cells ; pathology ; physiology ; ultrastructure ; In Vitro Techniques ; Male ; Microscopy, Electron, Transmission ; Phenolsulfonphthalein ; pharmacokinetics ; Prostate ; metabolism ; pathology ; Rats ; Tight Junctions

Objective To explore the effect of macrolide antibiotics(erythromycin) on tumor necrosis factor(TNF)-α and interleukin(IL)-8 in hyperoxia-induced lung tissue of premature newborn rats,and to study the intervention effect of erythromycin on hyperoxia-induced lung injury.Methods One-day old preterm Sprague Dawley rats were randomly divided into four groups by random number table method:air + sodium chloride group,air + erythromycin group,hyperoxia + sodium chloride group,hyperoxia + erythromycin group.Hyperoxia groups were continuously exposed to oxygen (oxygen ＞ 0.85) and air group in room air.After 1,7,14 days of exposure,the preterm rats of four groups were sacrificed,whole lung of these rats were isolated,the lung histological changes were observed by hematoxylin-eosin staining,TNF-α and IL-8 in pulmonary tissue homogenate were detected by ELISA.Results The results showed that:(1) Compared with air + sodium chloride group,TNF-α and IL-8 expression in hyperoxia + sodium chloride group were significantly increased(P ＜ 0.05) after 1,7 days of exposure [1 d:TNF-α:(16.163 ± 0.574) ng/ml vs.(21.923 ±2.066) ng/ml,IL-8:(18.214 ±3.649) ng/ml vs.(23.546 ± 5.240) ng/ml ;7 d:TNF-α:(15.940 ±0.821) ng/ml vs.(19.688 ±0.764) ng/ml,IL-8:(18.541 ± 4.114) ng/ml vs.(24.255 ±4.692) ng/ml],in particular,TNF-α expression appeared to increase earlier,their expression became significantly weak in 14 days (P ＜ 0.05).(2) Compared with hyperoxia + sodium chloride group,TNF-α and IL-8 expression in hyperoxia +erythromycin group became significantly weak after 1,7,14 days of exposure(P ＜0.05) after the intervention of erythromycin [1 d:TNF-α:(21.923 ± 2.066) ng/ml vs.(18.903 ± 1.851) ng/ml,7 d:IL-8:(24.255 ±4.692) ng/ml vs.(23.508 ±3.543) ng/ml,14 d:TNF-α:(16.443 ±5.466) ng/ml vs.(14.453 ±0.963)ng/ml],but their expression became weaker in 14 days than that in 1,7 days.Conclusion The release of inflammatory mediators TNF-α and IL-8 induced by oxidation outbreak participates in the development of hyperoxia induced lung injury,erythromycin may regulate immune function,inhibits the levels of oxidant-mediated TNF-α and IL-8 induced by oxidation outbreak,and alleviate hyperoxia lung injury in premature rats.

Objectives To explore the effect of erythromycin on glutathione hormone (GSH) and γ-glutamyl cysteine synthetase (γ-GCS) in premature newborn rats exposed to hyperoxia, to study the intervention effect of erythromycin on hype-roxia-induced lung injury. Methods One-day old preterm SD rats were randomly divided into four groups:control group, eryth-romycin group, hyperoxia group, erythromycin+hyperoxia group. Hyperoxia group and hyperoxia+erythromycin group were continuously exposed to oxygen (oxygen concentration>0.85), control group and erythromycin group were in room air. Via cau-dal vein, the preterm rats was injected with erythromycin in erythromycin group and hyperoxia+erythromycin group, sodium chloride in control group and hyperoxia group daily. After 1,7,14 day(s) of hyperoxia (or air ) exposure, the preterm SD rats of four groups were killed, whole lung of these rats were isolated and histological changes were observed by hematoxylin-eosin (HE) staining, GSH andγ-GCS of pulmonary tissue homogenate were detected by double antibody sandwich enzyme linked im-munosorbent assay. Total lung RNA was extracted andγ-GCS mRNA was detected by reverse transcription polymerase chain re-action. Results The results showed that:After 1 and 7 day(s) of exposure, the expression of GSH、γ-GCS andγ-GCS mRNA in four groups showed significant differences(P<0.05). Among them, GSH expression in erythromycin + hyperoxia group was higher than that in the other three groups in 1,7,14 day(s) of exposure with significant differences (P<0.05);GSH expression in erythromycin+hyperoxia group and hyperoxia group reached the peak after 7 days of exposure. The expression ofγ-GCS andγ-GCS mRNA in erythromycin+hyperoxia group and hyperoxia group were higher than the other two groups after 1and 7 day(s) of exposure, the expression ofγ-GCS mRNA in erythromycin+hyperoxia group were higher than that of hyperoxia group with significant differences (P<0.05). Conclusions The expressions of GSH andγ-GCS in the lung of premature SD rats were abnor-mal by oxidation outbreak. Erythromycin may increase the activity ofγ-GCS, improve the anti-oxidation ability of GSH, and al-leviate hyperoxia mediated lung injury in premature rats.

Objective To study the influence factors of real-time shear wave elastography(SWE) on the elasticity of focal liver lesions (FLLs) measurement.Methods The mean Young's modulus of 160 FLLs were measured by SWE.The relation between elasticity with different aspects of FLLs were analyzed including postures (supine and left lateral position),respiratory phases (end of inspiratory and expiratory),the probe position (rib and intercostal space) and different operators.Results There were significant differences between different postures and respiratory phases in the elasticity measurement of FLLs by SWE (P ＜0.05).There was no significant difference between robe position and different operators in the elasticity measurement of FLLs by SWE(P ＞0.05).Conclusions The postures and respiratory phases can impact the elasticity measurement of FLLs by SWE.

Objective To investigate the expressions of Livin and vascular endothelial growth factor (VEGF)in pancreatic carcinoma and their cilinical significances.Methods The expressions of Livin and VEGF proteins were tested by immunohistochemistry in 68 cases of pancreatic carcinomas and 44 cases of adja-cent paracancerous tissues.Results The positive rates of Livin in pancreatic carcinomas and adjacent paracan-cerous tissues were 73.5% and 4.5% respectively,and the difference was statistically significant (χ2 =48.137,P<0.001).The positive rates of VEGF in pancreatic carcinomas and adjacent paracancerous tissues were 69.1% and 13.6% respectively,and the difference was statistically significant (χ2 =29.147,P <0.001).The expressions of Livin and VEGF were related with tumor differentiation (χ2 =6.061,P=0.014;χ2 =6.592,P=0.010),TNMstage (χ2 =4.175,P=0.041;χ2 =9.992,P=0.002),lymph node metasta-sis (χ2 =11.731,P=0.001;χ2 =12.002,P=0.001)and neural invasion (χ2 =9.950,P=0.002;χ2 =7.433,P=0.006).Significantly positive correlation was found between the expressions of Livin and VEGF by using Spearman correlation analysis (r=0.320,P=0.008).Survival analysis showed that the expressions of Livin and VEGF were independent prognostic factors in pancreatic carcinoma.Conclusion Livin and VEGF involve in the development,migration and metastasis of pancreatic carcinoma.Livin may upregulate the expres-sion of VEGF,which may lead to the angiogenesis and migration in pancreatic carcinoma.

Polymerase chain reaction(PCR) was employed to amplify the whole HBV X region from the serum of patients with chronic hepatitis B virus(HBV) infection, and then the PCR products were subcloned into pGEM Teasy vectors. Clones were randomly selected to be sequenced. Comparison of the cloned sequence was made to look for the differene. The sequencing results showed that each sequence of selected clones was differenct and there were HBV quasispecies groups in patients. There was hot deletion region near the 3' end of X gene. To address whether the mutations were responsible for the transactivating effect,the wild type(wt) and the mutants of HBV X genes were subcloned into the EcoRI site of pcDNA3 1( ) vectors, and the recombinant plasmids were cotransfected into HepG2 cells with reporter plasmid pSV lacZ,respectively. The activity of ? galactosidase controlled by SV40 early promoter/enhancer was detected, which reflected the transactivating function of HBx protein. The cotransfection results indicated that the wt HBV X gene acted as a transactivator on the SV40 early promoter/enhancer, and the mutations which caused premature termination of the X open reading frame reduced their transactivating effects to certain extent.

Objective To investigate the changes of hippocampal neurogenesis and cognitive dysfunction induced by cranial radiation therapy.Methods C57BL/6J mice aged 10 d were subjected to 10 Gy whole brain irradiation with 6 MV X-rays to develop irradiation-induced brain injury model.Morris water maze was designed to estimate spatial learning and memory.At different time post irradiation,brain tissue was removed to stain with hematoxylin-eosin for the pathological results.DCX and PCNA immunohistochemical staining was used to mark the level of neurogenesis in the hippocampus,and ED1immunohistochemical staining to mark the activation of microglia.The TUNEL assay was used to assess the apoptotic neuron death in situ in the hippocampus.Real-time PCR was supplied to inspect the expression of TNF-α and IL-1 β mRNA.Enzyme Linked Immunosorbent Assay (ELISA) was tested for the concentration of TNF-αt in the plasma.Results Pathological studies demonstrated that radiation could induce interstitial edema,inflammatory cell infiltration,cell degeneration,necrosis,apoptosis in the acute phase,edema subsiding,reduction of inflammatory cells,and cytothesis in the dentate gyrus of the hippocampus.IHC studies revealed that,at different time post irradiation,the number of DCX-positive cells and PCNA-positive cells decreased (F =4.9-12.5,5.2-15.7,P ＜ 0.05) but ED1-positive cells increased significantly (F =20.8,P ＜ 0.05).TUNEL-positive cells began to appear in the dentate gyrus of hippocampus 6 h post-irradiation,and its number reached to the highest level at 48 h post-irradiation (F =15.1,P ＜ 0.05).The formation of γ-H2AX foci got at the top 0.5 h post-irradiation (F =18.4,P ＜0.05) and then decreased.After irradiation,the expressions of TNF-α and IL-1β mRNA in the the irradiated group was higher than those of the control group (t =16.3,12.7,P ＜ 0.05).The concentration of TNF-α in the plasma of the irradiated group was higer than that in the control group 3 h post-irradiation,and maximized at 1 week post-irradiation (F =10.5,P ＜ 0.05).Morris water maze tests showed that the latency had no significant differences between the irradiated group and the control group at 1,2,3 d postirradiation,but the latency in the irradiated group was longer than that in the control group with a significant differences at 4,5,6 d post-irradiation (F =7.01,8.17,4.22,P ＜ 0.05).Conclusions Irradiation-induced cognitive dysfunction may be caused by microglial activation and suppression in hippocampal neurogenesis following cranial radiation therapy.

Objective To observe the immunostaining of endothelin-1(ET-1) and von Willebrand factor(vWF) in pulmonary vascular of ventricular septal defect(VSD) with pulmonary hypertension(PH),and the possible roles of ET-1 and vWF in the development of PH and remodeling in pulmonary artery.Methods VSD with non-PH were choosed as control group,20 cases with VSD with PH were divided into Ⅰgroup and Ⅱgroup according to mean pulmonary artery pressure. The specimens from middle lobe of right lung was obtained and stained by immunohistochemical technique.The degree of immunoreaction was measured with light density.Results The light density of ET-1 and vWF in small pulmonary arteries had significant statistical difference between every group,and Ⅱgroup was higher than those of Ⅰgroup,but no difference in small pulmonary veins.In every group,the light density of ET-1 and vWF had significant difference between small pulmonary arteries and pulmonary veins.There were statistical correlation among mean pulmonary pressure,VSD/A,ET-1 and vWF.Conclusions ET-1 and vWF are concerned with the development of PH and mediate pulmonary vascular remodeling.The data of small pulmonary veins suggest that the main origin of ET-1 in pulmonary tissue is small pulmonary artery.

This study aims to clarify out the anti-inflammatory mechanism of Qingfei Xiaoyan Wan. Chemical constituents of Qingfei Xiaoyan Wan identified by UPLC Q-TOF, were submit to Molinspiration, PharmMapper and KEGG bioinformatics softwares for predicting their absorption parameters, target proteins and related pathways respectively; and the gene chip and real time-PCR were carried out to investigate the expression of inflammatory genes on lung tissue of guinea pigs or human bronchial epithelial cell lines. The predicted results showed that 19 of the 24 absorbable constituents affected at 9 inflammation-related pathways through 11 protein targets; Qingfei Xiaoyan Wan treatment can significantly reduce the infiltration of cytokines through ERK1 gene and 5 inflammatory pathways (Focal adhesion, Fc epsilon RI, Toll-like receptors, NK cell-mediated cytotoxic, and ERK/MAPK). The results of real time-PCR further confirmed that the anti-inflammatory effects of Qingfei Xiaoyan Wan were due to active ingredients such as arctigenin, cholic acid and sinapic acid intervened focal adhesion, Fc epsilon RI signaling and ERK/MAPK pathways. The novel approach of 'drug-target-pathway' will present an effective strategy for the study of traditional Chinese medicines.

Objective To explore the efficacy and safety of mild hypothermia (MH) in treating the infants with moderate-to-severe neonatal hypoxic-ischemic encephalopathy(HIE),and to make a follow-up of the nerve motor development of the infants at 18 months old after discharge.Methods Totally 61 neonates with moderate-to-severe HIE in Neonatal Intensive Care Unit (NICU) from Jan.2007 to Dec.2013 were retrospectively analyzed.According to before and after MH therapeutic apparatus was used by NICU of Shanghai Children's Hospital,61 neonates of HIE were divided into 2 groups,the conventional treatment group(25 cases) and MH treatment group(36 cases).The patients in both groups were measured respectively by using the amplitude integrated electroencephalography (aEEG) before MH treatment and at 72 hours after M H treatment,by neonatal behavioral neurological assessment(NBNA) on the 28th day after birth,and by adopting Bayley Scales of Infant Development at 18 months old.The adverse reactions,serious disability cases and deaths of MH treatment were recorded.Results Compared with the conventional treatment group,aEEG recording before treatment showed no statistically significant differences in MH treatment group [maximum voltage:(22.4 ±3.1) μV vs(18.6 ±2.5) μV,maximum voltage:(8.2 ±2.6)μV vs(6.5 ±1.9) μV,t =1.264,0.852,all P ＞ 0.05].However,aEEG recording at 72 h after treatment showed statistically significant differences in MH treatment group [maximum voltage:(24.1 ± 3.2) μV vs (30.6 ± 2.8) μV,maximum voltage:(9.7 ± 3.4) μV vs (13.3 ± 2.2) μV,t =6.376,4.257,all P ＜ 0.05].Severe disability cases [24.0％ (6/25 cases) vs 5.6％ (2/36 cases),x2 =4.405,P ＜ 0.05] and deaths [16.0％ (4/25 cases) vs 0 (0/36 case),x2 =6.1 64,P ＜ 0.05] in MH treatment group were significantly decreased,and there was significantly difference in NBNA on the 28th day after birth[(35.9 ± 2.1) vs(39.1-± 1.6),t =3.361,P ＜ 0.05],and scales of neurobehavioral evaluation through follow-up of 18 months old [mental development index (MDI):(85.2 ± 10.7) vs (96.5-± 13.1),t =7.839,P ＜ 0.05].Very few neonates had apnea,coagulation dysfunction,arrhythmia and other adverse reactions in MH treatment course.Conclusions MH treating moderate-to-severe HIE is safe and effective.MH is effective in reducing death and major disabilities in neonates with moderate-to-severe HIE and without significant side effects.MH can obviously improve the development of nervous system disorders in 0-18 months infants,and can significantly improve these infants' Bayley developmental scale neurobehavioral scores.