Objective To construct the pseudovirus containing nucleic acid(NA)fragments of Marburg virus,Zaire Ebola virus,Sudan Ebola virus,Lassa fever virus and Yellow fever virus by using a lentiviral vector system in order to provide a reference standard for the detection of the five viruses.Methods The gene fragments of the above five viruses were synthesized in vitro,connected into a single gene by fusion PCR technique,and cloned into lentiviral vectors with its auxiliary vector.After co-transfecting into 293T cells,the supernatants were collected on 48 h and 72 h post transfection. The naked NA was cleaned from the supernatants using DNase and RNase digestion before pseudotype virus was purified and concentrated.After the NA of the pseudotype virus,were extracted normal PCR and real-time PCR were conducted. Results Sequence analysis showed that the five target genes in vitro synthesis were properly connected and inserted into lentivirus vectors.Using the NA of the pseudotype virus as the template,both normal PCR and real-time PCR could sensitively amplify the target gene with the primers and probes of the above five,viruses respectively.The result indicated that the pseudovirus particles containing the five kinds of hemorrhagic fever virus target genes were successfully packaged. Conclusion The pseudovirus particles containing gene fragments of five viruses are constructed,which can be used as a common reference standard for NA detection.

AIM:To observed the protective effect of diminazene aceturate(DIZE),an angiotensin-converting enzyme 2(ACE2)activator,on rats with diabetic cardiomyopathy(DCM).METHODS:Male Wistar rats(n=30)were randomly divided into normal control group ,DCM group and DIZE treatment group(DIZE group).The rats in DCM group and DIZE group were intraperitoneally injected with streptozotocin(65 mg/kg )to establish diabetic model.After 12 weeks,the diabetic rats were infused with DIZE at 15 mg· kg-1 · d-1 or the same volume of saline for 4 weeks using os-motic minipump.The cardiac function was measured at the end of the 16th week.The methods of Mason staining and HE staining were used to observe the morphological changes of the myocardial tissue.Western blot ,ELISA and immunohisto-chemistry were used to observe the changes of ACE2,angiotensin(Ang)Ⅱ,Ang-(1-7),interleukin(IL)-1,IL-6 and connective tissue growth factor(CTGF).RESULTS:DIZE significantly improved the expression of ACE 2 in diabetic rats(P<0.05).Compared with DCM group,the levels of IL-1 and IL-6 in DIZE group were significantly decreased ,and the cardiac function in DIZE group was significantly improved(P<0.05).CONCLUSION:ACE2 endogenous agonist DIZE significantly increases the ACE 2 level and reduces the level of inflammation ,thus protecting the heart function of DCM rats.

OBJECTIVETo identify factors associated with YMDD mutation in patients with chronic hepatitis B before and after lamivudine treatment in Zunyi region.

METHODS53 patients with chronic hepatitis B were enrolled in this study, HBV DNA,HBV markers, ALT, AST, TBil, albumin in the serum were examined at 0, 3, 6, 12, 18 and 24 months after lamivudine treatment. HBV genotype and YMDD mutation were determined by sequencing before lamivudine treatment. YMDD mutation was checked again if serum HBV DNA rebound to more than 1 x 10(4) copies/ml after the initial decrease.

RESULTSHBV genotype in Zunyi region is constitute of B, C and B+C genotype. YMDD mutation occurred in 18 cases after lamivudine treatment, the rate of YMDD mutation was 15.1%, and 34.0% after 1 year and 2 years treatment. There are four types of mutation: rtL180M/M204V, rtL180M/M204I, rtM204I, rtL180M. rtM204V mutation in C gene was always accompanied by rtL180M mutation (100%). The rate of rtL180M/M204V mutation in genotype C group was significantly higher than that in genotype B group (77.8% to 25.0%), the same was true for the rtL180M/ M204I mutation (22.2% to 12.5%). There was no point mutation in genotype C group. The point mutation of rtM204I and rtL180M appeared only in genotype B group. Gender, nation, family history of hepatitis B and HBeAg were not associated with YMDD mutation (P more than 0.05), while the mutation rate was associated with the disease course and severity of disease. YMDD mutation did not occur in patients with low HBV DNA level (less than 10(5) copies/ml).

CONCLUSIONYMDD mutation after lamivudine therapy is associated with HBV genotype and P gene mutation type, and prolonged treatment increases the the mutation rate. In order to reduce the incidence of YMDD mutation, patients with shorter disease course, lower HBV DNA level, more serious liver damage should be treated with lamivudine.

Adult ; Alanine Transaminase ; blood ; Antiviral Agents ; pharmacology ; therapeutic use ; Aspartate Aminotransferases ; blood ; China ; epidemiology ; DNA Mutational Analysis ; DNA Primers ; DNA, Viral ; blood ; genetics ; Drug Resistance, Viral ; Female ; Genotype ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; blood ; drug therapy ; virology ; Humans ; Lamivudine ; pharmacology ; therapeutic use ; Male ; Mutation ; Polymerase Chain Reaction

SUMMARY Thehumanembryonicstemcells(hESCs)serveasaself-renewable,genetically-healthy, pluripotent and single source of all body cells,tissues and organs.Therefore,it is considered as the good standard for all human stem cells by US,Europe and international authorities.In this study,the standard and healthy human mesenchymal progenitors,ligament tissues,cardiomyocytes,keratinocytes,primary neurons,fibroblasts,and salivary serous cells were differentiated from hESCs.The human cellular health-safety of NaF,retinoic acid,5-fluorouracil,dexamethasone,penicillin G,adriamycin,lead ace-tate PbAc,bisphenol A-biglycidyl methacrylate (Bis-GMA)were evaluated selectively on the standar-dized platforms of hESCs,hESCs-derived cardiomyocytes,keratinocytes,primary neurons,and fibro-blasts.The evaluations were compared with those on the currently most adopted cellular platforms.Parti-cularly,the sensitivity difference of PM2.5 toxicity on standardized and healthy hESCs derived fibroblasts, currently adopted immortalized human bronchial epithelial cells Beas-2B and human umbilical vein endo-thelial cells (HUVECs)were evaluated.The results showed that the standardized hESCs cellular plat-forms provided more sensitivity and accuracy for human cellular health-safety evaluation.

Objective:To observe the effect of combining biofeedback therapy (BFT) based on virtual reality technology with repeated transcranial magnetic stimulation (rTMS) on dysphagia among stroke survivors.Methods:Eighty patients were randomly divided into a control group, an rTMS group, a BFT group and a combined treatment group, each of 20. In addition to routine dysphagia rehabilitation, the rTMS and BFT groups were given those treatments, while the combined treatment group was given both for 4 weeks. Swallowing function was evaluated before and after the treatment using the standardized swallowing assessment (SSA) and the functional oral intake scale (FOIS). Videofluoroscopy was used to quantify the subjects′ oral and pharyngeal phases and their aspiration status.Results:Significant improvement was observed in the average FOIS and SSA scores, as well as in the average oral and pharyngeal phases and in aspiration. The combined treatment group′s results were significantly better in all those aspects than those of the other 3 groups.Conclusion:The combined application of biofeedback therapy based on virtual reality technology and repeated transcranial magnetic stimulation can improve the swallowing function of stroke survivors with dysphagia. It is worthy of clinical promotion.

Objective To evaluate the relationship between the changes of diffusion tensor imaging(DTI)and the clinical stages in patients with cerebral infarction. Methods One hundred and seven patients with cerebral infarction were chosen to perform conventional MRI and DTI,respectively.With double-blind method,the signal intensity of fractional anisotropy(FA)and isotropic apparent diffusion coefficient(ADCiso)in responsible to infraction regions was observed and based on their results,the relationships between the changes of DTI and the clinical stages were analyzed. Results In 29 cases of cerebral infarction in acute stage,10 were manifested as type I(hypointensity on ADCiso map,hyperintensity on FA map)and type Ⅱ(hypointensity on ADCiso map and FA map with hyper intensive circumference)on DTI;3 were manifested as type Ⅲ on DTI(iso-/Paypo-intensity on ADCiso map and FA map with normal circumference);6 were manifested as type Ⅳ on DTI(hyperintensity on ADCiso map,hypointensity on FA map).In 34 cases of cerebral infarction in sub-acute stage,2 were manifested astype Ⅰ on DTI;20 were manifested astype Ⅱ;8 were manifested as type Ⅲ and 4 were manifested as type IV.In 44 cases of cerebral infarction chronic stage.1 Was manifested as type Ⅱ on DTI;2 were manifested as type Ⅲ and 41 were manifested as type IV.Positive correlation was found between changes of DTI and clinical stages(r=0.693,P=0.000). Conclusion The intensity of infraction on DTI can directly demonstrate the clinical stages,providing precise evidence for classifying the stages of cerebral infarction.

Objective To investigate the effect of growth differentiation factor 15 (GDF15) on H9c2 cardiomyocytes against ischemia/reperfusion injury(I/R) by constructing GDF15 stable over expression of myocardial cell lines.Methods To establish the model of hypoxia/reoxygenation,transfect targeted GDF15 over expression lentivirus and negative empty vector lentivirus into H9c2 cells,and select the H9c2 cells of GDF15 gene over expression stably.Randomly assigned to control group,transfected with empty vector(H9c2/Sham) and GDF15 over expression group (H9c2/GDF15).Cell viability was detected by MTT.And lactate dehydrogenase (LDH) in supernatant of cell culture was measured by LDH kit.The expression levels of apoptosis-related protein Caspase-3,Bcl-2,Bax and autophagy-related protein Beclin-1,LC3-B protein were observed with Western-blotting.Results Compared with control group (60.47 ± 5.03) ％ and H9c2/Sham group (63.21 ± 17.13) ％,the H9c2/GDF15 group (74.93 ± 10.03) ％ increased significantly,the difference was statistically significant after I/R 2 h (P ＜ 0.01).Compared with control group 108.72 ± 7.20,the activity of LDH in H9c2/GDF15 group 50.29 ± 10.05,the difference was statistically significant (P＜0.01).To apoptosis-related protein Caspase-3,the expression in H9c2/GDF15 group (gray value 67.68 ±0.47) was lower than control group (gray value 99.73 ±0.61),the difference was statistically significant after I/R2 h(P ＜0.01).The Bcl-2/Bax in H9c2/GDF15 group 82.52 ± 1.39 was higher than control group 72.52 ±2.05 and H9c2/Sham group 39.55 ± 0.46,the difference was statistically significant after I/R 2 h (P＜0.01).To autophagy-related protein Beclin-1,the expression in H9c2/GDF15 group (gray value 88.39 ±0.72) was higher than control group (gray value 58.93 ± 0.64) and H9c2/Sham group (gray value 36.71 ±0.35),the difference was statistically significant(P ＜0.01).The LC3-Ⅱ/LC3-Ⅰ in H9c2/GDF15 group (3.32 ±0.04) was higher than control group(2.11 ± 0.02),the difference was statistically significant (P ＜0.01).Conclusion Up-regulation of GDF15 in H9c2 cells protected myocardial cells H9c2 and its protective mechanism may be achieved by promoting the autophagy of cells.

Objective:To clarify the role of polypyrimidine tract binding protein (PTB) in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and explore its mechanism.Methods:The bone marrow blood of 4 patients with alveolar cleft treated in Plastic Surgery Hospital of Chinese Academy of Medical Science was collected. Human BMSCs were cultured and induced to osteoblasts, RT-PCR and Western blotting were applied to detect the expression of PTB at different time points. PTB expression in BMSCs was knocked down using shRNA, alizarin red S staining and real-time PCR were applied to evaluate its effect on osteogenic differentiation. Human BMSCs were also treated in vitro with TNF-α, an inflammatory factor closely related to osteoporosis, and PTB expression and osteogenic differentiation were detected by Real-time PCR and alizarin red S staining. The rat osteoporosis model was established and the expression of PTB in the BMSCs was detected by Real-time PCR. Flow cytometry was used to detect intracellular ROS in human BMSCs after PTB knockdown. All quantitative data were displayed as mean ± standard deviation, student t test was applied for the comparison between the two groups and the difference was statistically significant when P<0.05. Results:PTB expression increased during osteogenic differentiation of BMSCs in vitro. Compared with the control, the fold changes of relative expressions of osteogenic gene RUNX2 and ALP in BMSCs with PTB knockdown were 0.74±0.15 and 0.29±0.18, respectively, and the decreases were statistically significant( t=3.490, P=0.039; t=7.983, P=0.004). Consist with that, the formation of mineralized nodules decreased after PTB was knocked down. TNF-α at both 10 ng/ml ( t=3.528, P=0.039) and 40 ng/ml ( t=4.306, P=0.023) inhibited PTB expression and osteogenic differentiation of human BMSCs. The relative expression of PTB in BMSCs from ovariectomized rat osteoporosis models (0.001 25±0.000 12) was significantly lower than the sham-operated group(0.001 66±0.000 18)( t=3.217, P=0.032). Compared with the control group (1204.0±300.1), ROS content in BMSCs with PTB knockdown (720.7±129.7) significantly decreased ( t=4.872, P=0.040). Conclusions:PTB promotes the osteogenic differentiation of BMSCs, and PTB knockdown mediated down-regulation of ROS content is an important mechanism for its regulation of osteogenic differentiation of BMSCs.

Inflammation plays an important role in the development of acute lung injury (ALI). Severe pulmonary inflammation can cause acute respiratory distress syndrome (ARDS) or even death. Expression of proinflammatory interleukin-1β(IL-1β) and inducible nitric oxide synthase (iNOS) in the process of pulmonary inflammation will further exacerbate the severity of ALI. The purpose of this study was to explore the effect of Palrnatine (Pa) on lipopolysaccharide (LPS)-induced mouse ALI and its underlying mechanism. Pa, a natural product, has a wide range of pharmacological activities with the potential to protect against lung injury. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed to detect the expression and translation of inflammatory genes and proteins in vitro and in vivo. Immunoprecipitation was used to detect the degree of P65 translocation into the nucleus. We also used molecular modeling to further clarify the mechanism of action. The results showed that Pa pretreatment could significantly inhibit the expression and secretion of the inflammatory cytokine IL-1β, and significantly reduce the protein level of the proinflammatory protease iNOS, in both in vivo and in vitro models induced by LPS. Further mechanism studies showed that Pa could significantly inhibit the activation of the protein kinase B (Akt)/nuclear factor-κB (NF-κB) signaling pathway in the LPS-induced ALI mode and in LPS-induced RAW264.7 cells. Through molecular dynamics simulation, we observed that Pa was bound to the catalytic pocket of Akt and effectively inhibited the biological activity of Akt. These results indicated that Pa significantly relieves LPS-induced ALI by activating the Akt/NF-κB signaling pathway.

Objective:To clarify the role of polypyrimidine tract binding protein (PTB) in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and explore its mechanism.Methods:The bone marrow blood of 4 patients with alveolar cleft treated in Plastic Surgery Hospital of Chinese Academy of Medical Science was collected. Human BMSCs were cultured and induced to osteoblasts, RT-PCR and Western blotting were applied to detect the expression of PTB at different time points. PTB expression in BMSCs was knocked down using shRNA, alizarin red S staining and real-time PCR were applied to evaluate its effect on osteogenic differentiation. Human BMSCs were also treated in vitro with TNF-α, an inflammatory factor closely related to osteoporosis, and PTB expression and osteogenic differentiation were detected by Real-time PCR and alizarin red S staining. The rat osteoporosis model was established and the expression of PTB in the BMSCs was detected by Real-time PCR. Flow cytometry was used to detect intracellular ROS in human BMSCs after PTB knockdown. All quantitative data were displayed as mean ± standard deviation, student t test was applied for the comparison between the two groups and the difference was statistically significant when P<0.05. Results:PTB expression increased during osteogenic differentiation of BMSCs in vitro. Compared with the control, the fold changes of relative expressions of osteogenic gene RUNX2 and ALP in BMSCs with PTB knockdown were 0.74±0.15 and 0.29±0.18, respectively, and the decreases were statistically significant( t=3.490, P=0.039; t=7.983, P=0.004). Consist with that, the formation of mineralized nodules decreased after PTB was knocked down. TNF-α at both 10 ng/ml ( t=3.528, P=0.039) and 40 ng/ml ( t=4.306, P=0.023) inhibited PTB expression and osteogenic differentiation of human BMSCs. The relative expression of PTB in BMSCs from ovariectomized rat osteoporosis models (0.001 25±0.000 12) was significantly lower than the sham-operated group(0.001 66±0.000 18)( t=3.217, P=0.032). Compared with the control group (1204.0±300.1), ROS content in BMSCs with PTB knockdown (720.7±129.7) significantly decreased ( t=4.872, P=0.040). Conclusions:PTB promotes the osteogenic differentiation of BMSCs, and PTB knockdown mediated down-regulation of ROS content is an important mechanism for its regulation of osteogenic differentiation of BMSCs.