Objective To construct recombinant lentivirus silence vector aiming at rictor gene in mTORC2 specific protein, and to investigate its regulation on mTORC2/SGK1 signal pathway and the effect on pulmonary alveolar epithelial sodium ion chan-nel,as well as the role in acute respiratory distress syndrome(ARDS)and acute lung injury.Methods The interfering vector plas-mid and empty vector plasmid of target gene rictor were constructed,which and the lentivirus packaging system were co-transfected to 293T cells.The viral supernatant was collected,centrifuged,concentrated and purified for obtaining recombinant lentivirus.The virus titer was detected and the virus was infected to A549 cells.Stable cell lines were screened.RT-PCR was used to confirm the silencing situation of target gene rictor.The expression situation of various signal indexes in this pathway was detected by PCR and Western blot.Results The recombinant lentivirus of silence gene rictor was successfully constructed and transfected to A549 cell for obtaining stable cell lines.Compared with blank and control groups,the mRNA levels of rictor,downstream SGK1 andα-,β-andγ-ENaC in the shRNA-rictor group were significantly decreased (P <0.05 ).Meanwhile,the protein levels of rictor,downstream SGK1,P-SGK andα-,β-andγ-ENaC in the shRNA-rictor group were significantly decreased compared with the other two groups(P<0.05).Conclusion Silence rictor gene has the obvious regulation effect on mTORC2/SGK1 signal pathway,meanwhile affects the expression of pulmonary alveolar epithelial cellular α-,β-and γ-ENaC at gene and protein level.It is speculated that mTORC2/SGK1 may be an important signal pathway for regulating the clearance capacity of pulmonary alveolar epithelial cells on pulmonary alveolar fluid and simultaneously affecting the pulmonary edema formation.

As one of the first resident standardized training bases, department of ophthalmology of Shanghai Ruijin Hospital participated in this reform process from 2010. Relevant rules and regulations (training management system , training scheme implementation system and evaluation system ) were strictly obeyed. When new problems emerged, under the guidance of department in charge, a series of regimens were formulated and improved gradually by Ophthalmology Professional Committee of Shanghai Resident Standardized Training Department. Based on reviewing and summarizing the work in the last 3 years, some thoughts and suggestions on the resident standardized training in future were put forward ,in-cluding how to better solve the“heavily used, lightly cultured” problem, the“disregarding medical ethics establishment”problem, the“disregarding assessment of teachers”problem and the“disregard-ing obtaining employment”problem.

Objective To investigate the effect of pigment epithelium-derived factor (PEDF) on rat retinal Müller cells under high glucose conditions. Methods Müller cells cultured in 25 mmoL/L glncose were incubated with 100 ng/ml PEDF or 10 ng/ml interleukin-1β(IL-1β) for 24 h. The expression of IL-1β or PEDF in Müller cells was measured by indirect immunofluorescence, Western blot or realtime RT-PCR. The survival of Müller cells was detected by MTT assay. Results Under high glucose conditions, expression of IL-1β or PEDF was decreased after treated with 100 ng/ml PEDF or 10 ng/ml IL-1β for 24 hours by the methods of immunocytochemistry, Western blot or realtime PCR (P ＜ 0.05). Activity of Müller cells was increased significantly by PEDF (0.48±0.09 vs 0.64±0.17, P＜0.05). Conclusion In mimic diabetic conditions, PEDF decreases expression of IL-1β in rat retinal Müller cells and enhances the cell activity. To some degree, PEDF may block the process of inflammation in diabetic retinopathy.

OBJECTIVETo explore the association of serum HBV DNA level with HBV-specific and nonspecific cytotoxic T lymphocytes (CTL) in patients with HBV-induced hepatic cirrhosis.

METHODS120 patients with HBV-induced hepatic cirrhosis who were positive for HBV DNA, HBeAg and human leucocyte antigen (HLA)-A2 were enrolled in this study. The level of HBV DNA was determined by real time fluorescence quantitative polymerase chain reaction (PCR). HBV-specific and nonspecific CTL were detected by flow cytometry. Liver function tests were done in the 120 patients. The 120 patients were divided into group A and B based on their HBV DNA levels. In group A, there were 68 patients with HBV DNA level of 3-4 log10 copy/ml, and in group B, 52 with 5-6 log10 copy/ml. HBV-specific and nonspecific CTL and liver function were compared between the two groups.

RESULTSHBV DNA levels were (3.68 +/- 0.19) and (5.97 +/- 0.32) log10 copy/ml in Group A and B respectively with P < 0.001. HBV-specific CTL was higher in group A (0.33% +/- 0.04%) than in group B (0.11% +/- 0.01%) with P < 0.001. HBV-nonspecific CTL were (11.99% +/- 1.51% ) and (11.91% +/- 1.61%) in group A and B respectively with P > 0. 05.

CONCLUSIONThe level of serum HBV DNA is related to the levels of HBV-specific CTL in patients with HBV-induced hepatic cirrhosis. Patients with higher HBV DNA had lower levels of HBV-specific CTL, and the damage to liver function was severe because of higher levels of HBV DNA. Patients with lower HBV DNA had higher levels of HBV-specific CTL which predict good anti-viral effect.

Adult ; Case-Control Studies ; DNA, Viral ; blood ; Female ; HLA-A2 Antigen ; genetics ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; genetics ; immunology ; physiology ; Humans ; Liver Cirrhosis ; blood ; immunology ; virology ; Liver Function Tests ; Male ; Middle Aged ; T-Lymphocytes, Cytotoxic ; immunology ; Young Adult

OBJECTIVETo explore relationship between HBV DNA level and peripheral blood follicular helper T lymphocyte (Tfh) in patients with chronic hepatitis B (CHB) and its significance.

METHODSHBV DNA levels of 179 cases of CHB patients with positive HBV DNA, positive HBeAg and positive human leukocyte antigen(HLA)-A2 were tested with real time fluorescent quantitative PCR. Tfh and HBV specific CTL were tested with flow cytometry. IL-21 was also tested. 179 cases of CHB patients were divided into group A and group B based on HBV DNA levels, 86 cases in group A, HBV DNA levels were 10(4)-10(5) copies/ml, 93 cases in group B, HBV DNA levels were 10(6)-10(7) copies/ml. Above testing indexes of the two groups were compared.

RESULTSHBV DNA levels of group A were (4.85 +/- 0.37) log10 copies/ml, HBV DNA levels of group B were (6.83 +/- 0.31 ) log10 copies/ml, t = 27.31, P < 0. 001; Tfh of group A was (5.96 +/- 1.59)%, higher than that of group B (3.71 +/- 2.15)%, t = 4.92, P < 0.01; IL-21 of group A was (42.61 +/- 15.11)ng/L, higher than that of group B (14.91 +/- 3.15) ng/L, t = 8.62, P < 0.01; HBV specific CTL of group A was (0.36 +/- 0.08)%, higher than that of group B (0.18 +/- 0.06)%, t = 19.99, P < 0.001.

CONCLUSIONSerum HBV DNA level of CHB patients is related to the level of peripheral blood Tfh level: patients with low HBV DNA level have high Tfh level, high IL-21 level and high HBV specific CTL level. Patients with high HBV DNA level have low Tfh level, low IL-21 level and low HBV specific CTL level. The mechanism of baseline HBV DNA level affecting anti-viral therapy may be related to Tfh level.

Adult ; CD4 Lymphocyte Count ; DNA, Viral ; blood ; genetics ; Female ; HLA-A2 Antigen ; immunology ; Hepatitis B virus ; genetics ; isolation & purification ; Hepatitis B, Chronic ; blood ; immunology ; virology ; Humans ; Interleukins ; immunology ; Male ; T-Lymphocytes, Helper-Inducer ; cytology

OBJECTIVETo explore relations between ALT level and hepatitis B virus (HBV) specific CTL and non-specific CTL in patients with chronic hepatitis B (CHB).

METHODS148 cases of CHB were divided into three groups according to ALT level. 35 cases in group A, ALT > or =2 x upper limit of normal value (ULN)--5 x ULN (100-250 IU/L); 53 cases in group B, ALT > 5 x ULN-- < or =10 x ULN (251-500 IU/L); 60 cases in group C, ALT > 10 x ULN ( > 500 IU/L). Flow cytometry is used to determine non-specific CTV. HBV specific CTL was tested on 74 cases of CHB (17 in group A, 27 in group B and 30 in group C) with positive (HLA)-A2. Compare HBV specific CTL, non-specific CTL, HBV DNA levels and positive rate of HBeAg.

RESULTSHBV specific CTL: Group A (0.42 +/- 0.10)% is higher than group B (0.25 +/- 0.08)%, t = 6.37, P < 0.01, group B is higher than group C (0.17 +/- 0.004)%, t = 5.14, P < 0.01; Non-specific CTL: Group A (15.01 +/- 3.01)% is lower than group B (18.1 +/- 5.02)%, t = 2.81, P < 0.01, group B is lower than group C (21.5 +/- 6.11)%, t = 3.07, P < 0.01; HBV DNA level: Group A [(4.97 +/- 0.86) log10 copies/ml] is lower than group B [(5.92 +/- 0.92) log10 copies/ml], t = 4.87, P < 0.01. Group B is lower than group C [(6.37 +/- 0.71) log10 copies/ml], t = 2.92, P < 0.01; Positive HBeAg: Group A (15 cases, 42.86%) is lower than group B (32 cases, 60.38%), chi2 = 2.59, P > 0.05. Group B is lower than group C (41 cases, 68.33%), chi2 = 0.78, P > 0.05. Group A is lower than group C, chi2 = 5.929, P < 0.05.

CONCLUSIONThe higher the non-specific CTL of patients with CHB is, the higher the ALT level would be, whereas the lower the HBV specific CTL is, the stronger the HBV replication would be.

Adult ; Alanine Transaminase ; metabolism ; Female ; Hepatitis B virus ; genetics ; immunology ; physiology ; Hepatitis B, Chronic ; enzymology ; immunology ; virology ; Humans ; Lymphocyte Count ; Male ; T-Lymphocytes, Cytotoxic ; immunology ; Virus Replication ; Young Adult

OBJECTIVETo study the role of autophagy in the death of dopaminergic neurons induced by 6-hydroxydopamine (6-OHDA).

METHODSRat models of Parkinson disease (PD) were established by stereotaxic administration of 6-OHDA (8 μg) into the unilateral substantia nigra par compact (SNpc). Autophagosomes in the SNpc were observed with transmission electron microscopy (TEM), and the expression of autophagy-related protein LC3 was determined with immunofluorescence (IF) assay.

RESULTSUnder TEM, the autophagosomes were found in the ipsilateral SNpc 6-24 h after 6-OHDA injection, which suggested the activation of autophagy. IF assay showed significantly increased LC3 expression in 6-OHDA-damaged TH-positive neurons as compared to the control group.

CONCLUSIONSThe increase of autophagosomes and activation of autophagy may play a role in dopaminergic neuron death induced by 6-OHDA.

Animals ; Autophagy ; drug effects ; Cell Death ; drug effects ; Disease Models, Animal ; Dopaminergic Neurons ; cytology ; drug effects ; Male ; Microtubule-Associated Proteins ; metabolism ; Oxidopamine ; pharmacology ; Parkinson Disease, Secondary ; chemically induced ; metabolism ; Phagosomes ; metabolism ; Rats ; Rats, Sprague-Dawley ; Substantia Nigra ; drug effects

Traditional treatment of chronic idiopathic thrombocytopenic purpura (CITP) is usually adopted as hormonal therapy. If necessary, it also can be given immunosuppressive therapy. In order to investigate the curative effect of rhIL-11 on patients with ITP, 26 patients were divided into control group and rhIL-11 group, each group with 13 patients. Control group was given traditional hormonal therapy and immunosuppressive therapy, while rhIL-11 group was given rhIL-11 on base of traditional therapy. rhIL-11 25 microg/(kg.d) was injected s.c. for 28 days. The results showed that 23 out of 26 patients obtained obviously curative effect in platelet count, especially in rhIL-11 group, but another 3 patients had no response on therapy. The platelet counts of control and rhIL-11 groups increased from 26.15 x 10(9)/L and 27.84 x 10(9)/L before treatment to 66.62 x 10(9)/L and 105.62 x 10(9)/L after treatment. The platelet count of rhIL-11 group after treatment was remarkably higher than that of the control group (p < 0.05). Platelet count of 8 patients in rhIL-11 group recovered to normal. It is concluded that rhIL-11 combined with traditional hormone-immuno-suppressive therapy is effective to CITP.
Female ; Humans ; Immunosuppressive Agents ; therapeutic use ; Interleukin-11 ; therapeutic use ; Male ; Middle Aged ; Platelet Count ; Purpura, Thrombocytopenic, Idiopathic ; therapy ; Recombinant Proteins ; therapeutic use ; Treatment Outcome

Objective To explore the protective effect of neuregulin-1 (NRG-1) on heart function and myocardium in rats with sepsis and its mechanism . Methods Healthy male Sprague-Dawly (SD) rats were divided into three groups according to random number table method, with 6 rats in each group. Sepsis model was established by cecal ligation and puncture (CLP group); rats in sham operation group (sham group) underwent the same procedure except ligation. Rats in NRG-1 pre-treatment group (NRG group) were intravenously injected with recombinant human NGR-1 (rhNRG-1) at the dose of 10 μg/kg through tail vein; rats in CLP group and sham group were treated with the same amount of saline. At 24 hours after CLP, hemodynamic method was used to evaluate the cardiac function, and myocardial morphology was observed with hematoxylin and eosin (HE) staining, enzyme linked immunosorbent assay (ELISA) was used to detect the levels of cardiac troponin T (cTnT), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) in serum and macrophage migration inhibitor factor (MIF) in myocardial tissue. Results ① heart function: compared with the sham group, the mean arterial pressure (MAP), left ventricular systolic pressure (LVSP) and left ventricular pressure maximal rate of rise and fall (±dp/dt max) were significantly decreased in CLP group and NRG group, while the MAP, LVSP and ±dp/dt max in NRG group were significantly higher than those in CLP group [MAP (mmHg, 1 mmHg = 0.133 kPa): 125.78±8.15 vs. 113.05±5.85, LVSP (mmHg): 151.27±6.79 vs. 139.39±8.05, +dp/dt max (kPa/s): 4 389.59±332.38 vs. 3 706.85±451.31, -dp/dt max (kPa/s): 4 291.42±323.72 vs. 3 691.17±515.44, all 1 <0.05]. ②Myocardial injury: compared with the sham group, the levels of serum cTnT in CLP group and NRG group were significantly increased, while the levels of serum cTnT in NRG group were significantly lower than those in CLP group (ng/L: 206.37±67.28 vs. 344.13±80.95, 1 < 0.05), and the HE staining showed that myocardial pathological changes in NRG group were improved compared with the CLP group. ③Inflammatory mediators level: compared with the sham group, the levels of serum TNF-α, IL-1β and myocardial MIF were significantly increased in CLP group and NRG group, while the indicators in NRG group were lower than those in CLP group [TNF-α(ng/L): 52.77±3.43 vs. 97.19±13.98, IL-1β (ng/L): 40.25±5.48 vs. 56.05±6.88, MIF (μg/L): 1.92±0.16 vs. 2.87±0.10, all 1 <0.05]. Conclusion NRG-1 can reduce circulating levels of inflammatory factors in rats with sepsis, adjust myocardial MIF level, and alleviate myocardial cell injury, thereby improving cardiac function, and play a role in myocardial protection.

Objective To explore the effect of nuclear transcription factor kappaB(NF-κB) pathway on proliferation of rat pulmonary artery smooth muscle cells (PASMC) induced by platelet-derived growth factor (PDGF).Methods PASMC isolated from rats and cultured in vitro were divided into 3 groups according to the randomization principle:control group(cultured by M199),PDGF treatment group(cultured by M199 and stimulated by PDGF),PDGF + parthenolide treatment group (cultured by M199 and stimulated by PDGF,and intervented by the NF-κB inhibitors parthenolide).MTT colorimetric assay and flow cytometry were performed to detect cell proliferation and cell cycle distribution.Immunohistochemistry was performed to detect the expressions of NF-κB and COX-2 protein.Fluorescence quantitative RT-PCR was performed to detect NF-κB and COX-2 mRNA expressions.One-way ANOVA was used for statistical analysis,multiple comparisons were analyzed by LSD.Results Compared with the control group,MTT value of PASMC was increased significantly when induced by PDGF at each time points(all P ＜ 0.05).MTT value decreased dramatically after the intervention of NF-κB inhibitor parthenolide(P ＜0.05).Data from flow cytometry detection showed that cell proportion of S phase and G2 + M phase increased significantly in PDGF treatment group,which had statistical difference compared with control group (all P ＜ 0.05).Compared with PDGF induced group,after the intervention of parthenolide,cell proportion of S phase and G2 + M phase ratio decreased dramatically (P ＜ 0.05).The expressions of NF-κB and COX-2 protein and mRNA were promoted in the PDGF induced group compared with the control group (all P ＜ 0.05).Compared with PDGF induced group,after the intervention of parthenolide,the expressions of NF-κB and COX-2 protein and mRNA decreased dramatically(all P ＜ 0.05).Conclusions PDGF can induce proliferation of PASMC,promote cell cycle process and enhance the expressions of NF-κB and COX-2 protein and mRNA.NF-κB pathway involves in the proliferation of PASMC induced by PDGF.