Objective To establish an immortalized tree shrew corneal stromal cells(CSCs)line and to study its response to virus infection.Methods Primary tree shrew CSCs were isolated and cultured by the tissue block adhesion method.CSCs were then transfected with a lentivirus carrying the SV40T gene and monoclonal cells were selected for passage culture.The characteristics of the CSCs were investigated by morphological observation and compared with 40 generations until the 50 generations or more,immunofluorescence identification of vimentin and SV40T genes,karyotype examination,and cell proliferation curve.The CSCs were infected with herpes simplex virus-1(HSV-1)(McKrae strain),Zika virus(ZIKV,GZ01 strain),Dengue virus typeⅡ,and H1N1(PR8).Results The immortalized tree shrew CSCs after＞50 passages appeared spindle-shaped with good cell morphology and structure compared with 40 generations.Positive immunofluorescence expression of vimentin and SV40T genes.The cell growth curve showed that the cells were in logarithmic-phase growth on days 4～5 and grew vigorously.The number of chromosomes in the primary cells was stable at 62,while immortalized CSCs had 64 chromosomes at P21 and P56.The virus titer results showed that the immortalized tree shrew CSCs were sensitive to HSV-1(McKrae strain),ZIKV(GZ01 strain),Dengue virus typeⅡ,and H1N1(PR8),with virus titers of 1.32×105,5.62×106,2.69×107,and 7.76×104 CCID50/mL,respectively.Conclusions The immortalized tree shrew CSCs were established successfully,suggesting that this cell line is suitable for studies of the mechanisms of HSV,ZIKV,Dengue virus,and influenza A virus infection in relation to corneal diseases and antiviral drugs.

Global Burden of Disease ; Risk Factors ; Quality-Adjusted Life Years

Influenza is a highly contagious disease that mainly affects the respiratory system and often leads to lung complications. Also it can cause a variety of very rare and serious neurological complications, including Guillain-Barre syndrome, transverse myelitis, meningoencephalitis and others. In recent years, neurological complications caused by influenza A virus have been reported in many countries and regions, and gradually attracted international attention. However, the pathogenesis of this complication remains unclear, and there are few related cases and animal experimental studies,and no specific treatment. Therefore, the authors summarized the study of neurological complications caused by influenza A virus in human and laboratory animals, in order to have a comprehensive understanding of the neurological diseases caused by influenza A virus.

OBJECTIVES: Urinary tract infection (UTI) is the most common infection complication after kidney transplantation, and the reports of the incidence vary greatly among different centers. This study aims to explore the risk factors for UTI after kidney transplantation with the donation from brain death (DBD) and the impact on graft function, thus to provide theoretical basis for comprehensive prevention and treatment of UTI after kidney transplantation. METHODS: The clinical and laboratory data of DBD kidney transplantation from January 2017 to December 2018 in Xiangya Hospital, Central South University were collected and retrospectively analyzed. Patients were assigned into an UTI group and a non-UTI group. The base line characteristics, post-transplant complications, and graft function were compared between the 2 groups. Multivariate logistic regression was used to analyze the risk factors for UTI. RESULTS: A total of 212 DBD kidney transplant recipients were enrolled in this study. UTI occurred in 44 (20.75%) patients after transplantation. The female, the time of indwelling catheter, and postoperative urinary fistula were independent risk factors for UTI after DBD kidney transplantation. A total of 19 strains of gram-positive bacteria, 12 strains of gram-negative bacteria , and 10 strains of fungi were isolated from the urine of 44 UTI patients. The UTI after kidney transplantation significantly increased time of hospital stay ( CONCLUSIONS UTI after DBD kidney transplantation transplantation affects the renal function at 3 months and increases the patient's economic burden.
Brain Death ; Female ; Humans ; Kidney Transplantation/adverse effects* ; Retrospective Studies ; Risk Factors ; Urinary Tract Infections/etiology*

OBJECTIVETo investigate the effects of Panax notoginseng saponins (PNS) on the proliferation, apoptosis and cell cycle of K562 cells and explore the molecular mechanisms underlying these effects.

METHODSPNS-induced growth inhibition of K562 cells was detected by MTT assay; the cell apoptosis was evaluated by AO/EB staining and Annexin V-FITC/ PI staining; flow cytometry was used to detect cell cycle changes in the treated cells. The mRNA expressions of the molecules in mTOR signaling pathway were examined by RT-PCR, and the cellular expressions of cleaved caspeas-3, cyclin D1 and major proteins in mTOR signaling pathway were detected using Western blotting.

RESULTSMTT assay showed that treatment with 100-800 µg/mL PNS significantly inhibited the proliferation, promoted the cell apoptosis, and caused cell cycle arrest in G0/G1 phase in K562 cells. Western blotting revealed increased protein expression of cleaved caspase-3 and decreased expression of cyclin D1 in PNS-treated cells, in which the proteins expressions of mTOR, p-mTOR, p-p70S6K and p-4E-BP 1 and the mRNA expression of mTOR were all decreased.

CONCLUSIONPNS can inhibit the proliferation, induce apoptosis and cause cell cycle arrest in K562 cells possibly by up-regulating cleaved caspase 3 and down-regulating cyclin D1 and mTOR signaling pathway.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Cycle ; drug effects ; Cell Cycle Checkpoints ; Cell Proliferation ; drug effects ; Cyclin D1 ; metabolism ; Humans ; K562 Cells ; drug effects ; Panax notoginseng ; chemistry ; Saponins ; chemistry ; Signal Transduction ; TOR Serine-Threonine Kinases ; metabolism ; Up-Regulation

OBJECTIVE: To determine the effect of transient withdrawal of immunosuppressive agents during the treatment of pulmonary infection on long-term survival of patients and graft s. METHODS: A total of 104 patients with post-transplant pulmonary infection were enrolled in this study. These patients received renal transplantation in Center for Organ Transplantation, Xiangya Hospital, Central South University, during December 2005 and August 2014. Among them, 50 patients stopped immunosuppressive agents during the treatment of infection. These patients served as stopping drug (SD) group, whereas the remaining patients who served as a control group did not stop immunosuppressive drugs. The five-year cumulative patient survival, graft survival, and laboratory results were compared between the 2 groups. RESULTS: The five-year cumulative patient survival rates in the SD group were significantly lower than those in the control group [(69.8 ± 7.0)% vs (94.2 ± 3.2)%, P=0.001]. There was no significant difference in the allograft survival rates between the 2 groups [(81.7 ± 6.6)% vs (90.9 ± 4.3)%, P=0.113]. In patients who survived from pulmonary infection, there was no significant difference in long-term survival rates between the 2 groups (P=0.979). CONCLUSION Pulmonary infection impacts allograft survival after patients underwent renal transplantation. Transient stopping immunosuppressive agents during the treatment of infection is a safe and necessary treatment strategy for patients with serious post-transplant pulmonary infection.
Graft Rejection ; Graft Survival ; Humans ; Immunosuppressive Agents ; administration & dosage ; Kidney Transplantation ; Lung Diseases ; therapy ; Postoperative Complications ; Survival Rate ; Transplantation, Homologous

Objective To examine the effect of fibronectin connecting segment-1 (CS1) peptidefacilitated blockade of inflammatory cells-fibronectin adhesion on a rat liver transplantation model of prolonged ex vivo cold ischemia.Methods A model of liver transplantation in Wistar→Wistar rat was established.The donors of the CS1 treatment group received CS1 peptides through the tail vein for 3 days before operation.Another two doses of CS1 peptides were administered into the liver intraportally during procurement and before transplantion.Recipients received an additional 3-day course of CS1 peptides after transplantation.Rats in control group received scrambled peptides.Rats were sacrificed at 6,24 and 72 h after transplantion,and plasma transaminase activity and hepatic pathological changes were studied.The inflammatory cells and liver sinusoidal endothelial cells were visualized histochemically.Real-time PCR was used to detect tumor necrosis factor-α (TNF-α),interleukin-1β (IL-1β) and vascular endothelial growth factor (VEGF) mRNA expression in the liver.Results The plasma transaminase activity and hepatic necrosis areas in CS1 treatment group were significantly lower than in control group (P＜0.05).CS1 peptides treatment significantly decreased the number of Kupffer cells after transplantation and greatly inhibited the recruitment of neutrophils to the graft liver as compared with control group (P＜0.05).After prolonged cold ischemia,only a few hepatic endothelial cells exhibited positive staining of hepatic sinusoidal endothelial cell biomarker SE-1.Lots of hepatic sinusoidal endothelial cells positive for SE-1 staining could be detected in CS1 group at 72 h after transplantation,while much less SE-1 positive cells presented in the control goup.Prolonged cold ischemia caused a significant increase of TNF-α,IL-1β and VEGF mRNA expression in the graft liver of control group after transplantation.The expression of TNF-α mRNA at 6 and 24 h and VEGF mRNA expression at 24 h were significantly lower in CS1 group than in control group (P＜0.05).Conclusion Peptide-mediated blockade of inflammatory cells-fibronectin interaction decreased the mRNA expression of inflammatory cytokines,prevented hepatic sinusoidal endothelial cells from injury and subsequently protected against severe ischemia/reperfusion injury of the graft liver after transplantation.

OBJECTIVE: To design short hairpin RNA (shRNA) interference sequence to silence glutathione S-transferase P1 (GSTP1) gene of androgen independent prostate cancer cell line DU145, and to explore its effect on proliferation and sensitivity to chemotherapeutics. METHODS: The target sequence was picked up to form the shRNA, and the 3 shRNA expression vectors were shRNA255, shRNA554 and shRNA593. The DNA template was cloned to plasmid pGPU6/GFP/Neo. The shRNA was identified by enzyme digesting and gene sequencing. The screening experiment was done to pick up the shRNA expression vector with the highest transfection ratio and best gene silencing results. DU145 cells were divided into a blank plasmid group and a shRNA transfected group. According to the chemotherapeutics the DU145 cells were divided into a fluorouracil (FU) group and a paclitaxel (PA) group, and the 2 groups were subdivided into 4 subsets according to the chemotherapeutic concentrations (FU: 30, 60, 120, and 240 μg/mL; PA: 0.2, 2, 10, and 20 μg/mL), meanwhile a blank control group was included respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate the proliferation after the transfection. MTT and terminal de-oxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were used to detect the inhibition effect of different concentrations of 5-FU or PA on the proliferation and induction of apoptosis of DU145. RESULTS: The transfection ratio of the 3 shRNA expression vectors (shRNA255, shRNA554, and shRNA593) was (63.30±1.04)%, (76.20±0.68)%, and (72.70±0.33)%, and the transfection ratio of shRNA554 was the highest. there was significant difference among the above 3 shRNA expression vectors (P<0.01). After the transfection, the mRNA was 128.31±2.50, 43.24±4.30 and 85.62±6.30, the GSTP1 protein was 163.92±12.40, 65.38±9.30 and 114.25±16.70. After the transfection of shRNA554, the mRNA and protein of GSTP1 were the lowest level. there was significant difference among the above 3 shRNA expression vector (P<0.01). MTT analysis showed that before the transfection, the survival ratio of cells under different concentrations of FU (30, 60, 120, and 240 μg/mL) was (95.60±2.11)%, (90.20±0.86)%, (83.10±3.12)% and (74.60±1.32)%; however after the transfection, the survival ratio of cells was (91.30±1.43)%, (84.60±2.13)%, (73.20±1.52)%, and (65.5±0.942)%. TUNEL assay showed that before the transfection, the apoptosis ratio of cells under different concentrations of FU (30, 60, 120, and 240 μg/mL) was (5.50±0.88)%, (10.20±1.64)%, (15.20±2.39)%, and (25.10±2.59)%; however after the transfection, the apoptosis ratio of cells was (10.8±0.62)%, (15.7±1.32)%, (20.4±1.89)%, and (34.9±2.54)%. After the transfection, the cell survival ratio decreased under the same concentration of FU, and the apoptosis ratio increased, with statistical significance (both P<0.01). MTT analysis showed that before the transfection, the survival ratio of cells under different concentrations of PA (0.2, 2, 10, and 20 μg/mL) was (98.50±2.34)%, (95.20±1.32)%, (89.40±0.68)%, and (82.70±1.73)%; after the transfection the survival ratio of cells was (94.20±0.78)%, (86.50±2.13)%, (78.70±1.34)%, and (70.10±0.76)%. TUNEL assay showed that before the transfection, the apoptosis ratio of cells under different concentrations of PA (0.2, 2, 10, and 20 μg/mL) were (2.40±1.07)%, (5.20±1.33)%, (10.50±2.41)%, (20.70±1.92)%; after the transfection the apoptosis ratio of cells was (5.46±2.13)%, (13.80±1.24)%, (21.20±2.39)%, and (29.20±2.21)%. After the transfection, the cell survival ratio decreased under the same PA concentration, and the apoptosis ratio increased, with statistical significance (both P<0.01). CONCLUSION gene GSTP1 silence via shRNA transfection to androgen independent prostate cancer cell line DU145 can inhibit its proliferation in time dependent manner, and induce apoptosis and raise its sensitivity to chemotherapeutics.
Androgens ; metabolism ; Antineoplastic Agents ; pharmacology ; Apoptosis ; genetics ; Cell Line, Tumor ; Cell Proliferation ; Gene Silencing ; Glutathione S-Transferase pi ; genetics ; Humans ; Male ; Prostatic Neoplasms ; genetics ; pathology ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

An introduction to the practice of introducing the“ surgeon qualification admittance system”in the hospital and a description of its management framework,basic principles,rules of classification for surgical operations,application and approval procedure,and supportive management regulations.The authors hold that this system has normalized behavior of medical activities,clarified operation authorities of medical doctors at various levels,and intensified the awareness of risk exposure.Therefore this system can safeguard the quality and safety of surgical operations and its efficiency to a great extent,in addition to better building the team and backup forces.

Introduction to the specific practice and policies of the hospital for academic promotions. The emphasis is placed on "quality priority" and "work performance", making an overall assessment of the applicants on their professional virtue, academic skills, research outcome and social services. Among all these indicators, appraisal of the clinical skills and teaching skills is prioritized,encouraging the creativity and incentives of clinicians for clinical and teaching work.