Hepatitis B Surface Antigens ; blood ; immunology ; Hepatitis B, Chronic ; immunology ; Humans ; Immune System

Objective By using primary cultured mouse renal tubular epithelial cells (TECs) to develop an in vitro model of simulated ischemia-reperfusion (IR) injury.Methods The outer medulla of C57BL/6J mouse kidney was flushed and primary cultured after digestion in type Ⅰ collagenase,and then immunocytochemical staining was used to verify TECs.Primary cultured TECs were immersed in mineral oil to simulate the ischemic process,and 60 min later the whole culture medium was added to simulate reperfusion process.The cells were collected and RAN was extracted at indicated time points after medium replacement.The expression of TNF-α,IL-1β and IL-6 was detected by using real-time fluorescence quantitative RT-PCR. The culture supernatants were collected at 24 h after medium replacement for detection of the expression of cytokine protein by using ELISA.Results Primary cultured TECs were identified by cobblestone-shaped morphology and then verified by cytokeratin 18 (CK18) staining.In TECs of IR group after medium replacement the mRNA expression of TNF-α,IL-1β and IL-6 was higher than in control group.The expression of TNF-α after medium replacement was increased to a peak level at 0.5 h,about (24.45 ±6.51) times (P＜0.01 ) higher than the control group,and gradually declined thereafter.The mRNA expression of IL-1β after medium replacement kept an increasing tendency,about ( 15.27 ± 4.29) times (P＜0.05) higher than the control group at 6 h,and that of IL-6 after medium replacement was increased to a peak level at 3 h,about ( 11.19 ±4.55) times (P＜0.01) higher than the control group. In the IR group at 24 h after medium replacement,the protein expression of NF-α,IL-1β and IL-6 in the supernatants was significantly higher than in the control group.Conclusion High purity of primary cultured TECs was achieved from the outer medulla of mouse kidney by separation and digestion.The in vitro model of simulated IR in primary cultured mouse renal TECs was successfully created using paraffin oil.

ObjectiveTo investigate the effect and underling mechanism of water-soluble CO-releasing molecules (CORM-3)on the alleviation of allograft rejectionafter mouse kidney transplantation.Methods A mice kidney transplantation model was established using C.FVB-Tg (Itgax-DTR/GFP)57Lan/J or C57BL/6J (H-2Kb) mice as donors,and Balb/c (H-2Kd) mice as recipients.After donor nephrectomy,kidney was preserved in UW solution which contained CORM-3 or iCORM (inactive CO-releasing molecules) for 24 h in 4℃.Recipient survival after removal of both na? ve kidneys,serum creatinine as well as graft histology was observed.In the C.FVB-Tg(ItgaxDTR/GFP) 57Lan/J donors,rDCs were acquired in vitro and selected by magnetic cell sorting (MACS) after graft nephrectomy.The expression of activation markers,CD80 and CD86,on rDC was assessed by using flow cytometry.ResultsThe graft medium survival time was 40.5 days in the iCORM group and 70 days in the CORM-3 group respectively (P＜0.05).CORM-3 preserved the graft function as shown by significantly lower serum creatinine (P＜0.05; or P＜0.01) and alleviated graft pathology injury.Diffuse infiltration of mononuclear cells in the interstitial tissues,moderate tubulitis and partial glomerular sclerosis were found in the iCORM graft kidney,while the CORM-3 graft kidney displayed almost normal histology.Meanwhile,CORM-3 suppressed the expression of CD80 and CD86 in donor-derived rDC.ConclusionCORM-3 can alleviate allograft rejection,prolong the graft survival,and improve kidney function in mouse kidney transplantation,probably via inhibiting rDC activation.

The growth inhibition and pro-apoptosis effects of dracorhodin perchlorate on human prostate cancer PC-3 cell line were examined. After administration of 10-80 μmol/L dracorhodin perchlorate for 12-48 h, cell viability of PC-3 cells was measured by MTT colorimetry. Cell proliferation ability was detected by colony formation assay. Cellular apoptosis was inspected by acridine orange-ethidium bromide fluorescent staining, Hoechst 33258 fluorescent staining, and flow cytometry (FCM) with annexin V-FITC/propidium iodide dual staining. The results showed that dracorhodin perchlorate inhibited the growth of PC-3 in a dose- and time-dependent manner. IC50 of dracorhodin perchlorate on PC-3 cells at 24 h was 40.18 μmol/L. Cell clone formation rate was decreased by 86% after treatment with 20 μmol/L of dracorhodin perchlorate. Some cells presented the characteristic apoptotic changes. The cellular apoptotic rates induced by 10-40 μmol/L dracorhodin perchlorate for 24 h were 8.43% to 47.71% respectively. It was concluded that dracorhodin perchlorate significantly inhibited the growth of PC-3 cells by suppressing proliferation and inducing apoptosis of the cells.

Objective To evaluate the value of increased threshold of SUVmax and delayed imaging on diluted and filled bladder for improving the detection of bladder cancer with 18F-FDG PET/CT.Methods From July 2007 to October 2012,18 F-FDG PET/CT was performed on 63 suspected or treated (with bladder preserved) bladder cancer patients (55 males,8 females,average age 69.1 years).After routine imaging,all patients were given 1 500-2 000 ml of water orally three times and voided three times.Then they underwent delayed pelvic imaging at a full bladder status.The routine images were reanalyzed with increased SUVmax threshold (from 6-8 to 8-20).The final diagnosis was confirmed by pathology or follow-up (＞6 months).The differences of SUVmax in urine,18 F-FDG metabolism in lesions between routine and delayed imaging were compared.Paired t test was used to compare their differences.Results The SUVmax of urine on routine and delayed imaging was 15.11±11.11 and 4.73±2.00 respectively (t=4.15,P＜0.01).Among the 63 patients,there were 15 malignant and 3 benign cases confirmed by pathology,and 45 patients without obvious abnormality during follow-up.All 18 cases were detected by 18F-FDG PET/CT including the 3 benign false positive cases (2 were positive by CT though negative by PET,and 1 FDG-avid cystitis).All 15 true positive cases were confirmed as primary or recurrent bladder carcinoma and 1 false positive case as inflammation.The detection rates of early imaging with routine and increased display threshold of SUVmax were 18.8％(3/16) and 43.8％(7/16),respectively.Conclusion Increased SUVmax threshold for display and delayed imaging with diluted urine under full bladder status could effectively improve the detection rate of primary or recurrent bladder cancer with 18F-FDG PET/CT.

AIM: To explore the fundus fluorescein angiographic characteristics and relevant clinical significance of age-related macular degeneration(AMD).METHODS: Fundus fluorescein angiography (FFA) was performed on 149 eyes of 112 patients using Nikon NF-505 fundus camera.RESULTS: Out of 149 eyes, 90 eyes were atrophic AMD (60.4%), 59 eyes were exudative AMD (39.6%) which were further divided, according to the composition and location of lesion, into subfoveal choroidal neovasculari-zation (CNV)(7 eyes of classic type, 26 eyes of occult type, 9 eyes with disciform cicatrices, juxtafoveal CNV(2 eyes of classic type, 12 eyes of occult type), and extrafoveal CNV(3 eyes of occult type).CONCLUSION: FFA can show CNV of AMD patients and its quality and location, which is helpful to guide the treatment and evaluate the prognosis.

Objective To investgate the changes in the expression of hepatocyte growth factor (HGF)and c-met in the lungs in a rat model of pulmonary hypertension.Methods Eighty 7 week old male SD rats weighing 180-250 g were randomly divided into 2 groups ( n =40 each ):control group (group C) and pulmonary hypertension group (group PH).Pulmonary hypertension was induced by left pneumonectomy and subcutaneous monocrotaline (MCT) 60 mg/kg 2 weeks later.Pulmonary artery pressure and the ratio between the weight of right ventricle and left ventricle + interventricular septum ( RV/LV + S) were measured at 7,14,21 and 28 d after MCT administration.HGF and c-met protein and mRNA expression and TGF-β content in the lung tissue were determined.Results Pulmonary hypertension and right ventricular hypertrophy associated with hypertrophy of pulmonary artery tunica media and muscularization of small pulmonary arteries developed after MCT administration in PH group.In PH group HGF protein and mRNA expression in the lungs was significantly down-regulated as compared with group C.There were no significant differences in c-met protein and mRNA expression in the lungs between the 2 groups.The TGF-β content in the lungs was significantly increased in group PH as compared with group C.Conclusion Decrease in HGF production in the lungs plays an important role in the pulmonary hypertension.Increasing of pulmonary TGF-β may play an important role in the down-regulation of pulmonary HGF expression during pulmonary hypertension.

Objective To investigate the effect of human hepatocyte growth factor (hHGF) genetic modification on the ameliorating effects of mesenchymal stem cells (MSCs) implantation on pulmonary microvascular rarefaction in a rat model of pulmonary hypertension (PH).Methods MSCs were obtained from F344 rats and transduced with lentiviral vector modified with human HGF (hHGF-MSCs) or empty vector (EGFP-MSCs).Sixty-six 7 week old male F344 rats weighing 180-250 g were used in this study.PH was induced by left pneumonectomy and subcutaneous monocrotaline (MCT) 60 mg/kg injected at 2 weeks after operation.The animals with PH were randomly divided into 3 groups:control group (group C),EGFP-MSCs group (group E) and HGF-MSCs group (group H).Groups H and E received hHGF-MSCs or EGFP-MSCs 5 × 105 in DMEM 1 ml iv at 3 weeks after subcutaneous MCT injection,while group C received plain DMEM 1 ml.Mean pulmonary arterial pressure (mPAP) was measured and right ventricular hypertrophy and angiogenesis in the lung were assessed and the content of rat HGF (rHGF) and hHGF protein in lung tissue and pulmonary capillary density (by immuno-histochemistry) was measured at 2 weeks after MSCs implantation.The survival rates within 45 days after MCT administration were compared among the 3 groups.Results No hHGF was detected in groups C and E.Both hHGF-MSCs and EGFP-MSCs significantly reduced MPAP and right ventricular hypertrophy and increased pulmonary capillary density and survival rates in groups H and E as compared with group C and the efficacy of hHGF-MSCs was significantly greater than that of EGFP-MSCs.Barium angiography revealed that distal pulmonary vasculature was significantly increased in group H as compared with groups E and C.The survival of the rats receiving hHGF-MSCs was significantly longer in group H than that in groups E and C.Conclusion hHGF genetic modification can improve the ameliorating effects of MSCs implantation on PH-related microvascular rarefaction.

OBJECTIVETo explore the mechanism of miR-124 inhibiting the proliferative activity of prostate cancer PC3 cells.

METHODSLuciferase reporter gene assay was used to examine the specific binding ability of miR-124 to PKM2 mRNA 3'-UTR. After miR-124 was transfected mimic to PC3 cells, the expression levels of PKM2 mRNA and protein were detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot, respectively. The effects of miR-124 mimic and PKM2 siRNA on the proliferative activity of the PC3 cells were determined by MTT assay.

RESULTSThe expressions of PKM2 mRNA and protein were upregulated (5.12 +/- 0.35) times and (4.05 +/- 0.20) times respectively in the PC3 cells as compared with those in the RWPE-1 cells (P < 0.05). Luciferase reporter gene assay demonstrated that miR-124 targeted PKM2 3'-UTR. At 24 hours after transfection with miR-124 mimic, the PKM2 protein expression in the PC3 cells was downregulated (0.16 +/- 0.04) times (P < 0.05), while the PKM2 mRNA level was not changed significantly (P > 0.05), as compared with the control group. MTT assay showed that both miRNA-124 mimic and PKM2 siRNA could inhibit the proliferation of the PC3 cells, but the former exhibited a greater inhibitory effect than the latter. After transfection with miR-124 mimic and PKM2 siRNA, the cell growth rates were (66.20 +/- 5.10)% vs (82.10 +/- 6.35)% at 24 hours (P < 0.05) and (49.34 +/- 2.37)% vs (70.10 +/- 5.80)% at 48 hours (P < 0.05).

CONCLUSIONmiR-124 can suppress the proliferation of PC3 cells by regulating the PKM2 gene.

Carrier Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; genetics ; Humans ; Male ; Membrane Proteins ; genetics ; metabolism ; MicroRNAs ; genetics ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; Thyroid Hormones ; genetics ; metabolism ; Transfection