Objective To establish and identify transgenic mice model that specifically express delta drosophila homolog-like 1 (DLK1) in liver for the functional study of DLK1 on liver differentiation,hepatopathy,and hepatocellular carcinoma.Methods The coding sequence of DLK1 cDNA was inserted downstream of mouse albumin gene enhancer/promoter to construct a liver-specific DLK1 expression recombinant vector.The DNA fragment of transgene digested from the recombinant vector by Pine I was transfected to Hepl-6 cells to verify the expression of DLK1 in vitro.Then the transgene fragment was microinjeeted into fertilized eggs of C57×CBA F1 mice and 20 transgenic founder mice were generated.The F1 mice of DLK1 transgenie founder mice were used to identify the expression of the transgene in liver and other tissues.Results RT-PCR and cellular immunofluorescence showed DLK1 expression when the transgene fragment was transfeeted into Hep1-6.For transgenic mice,RT-PCR and immunohistochemistry showed that DLK1 was specifically expressed in adult F1 mice liver.Conclusion Successfully established the liver-specific DLK1 expression transgenic mice model.

0.05). It is decrease greatly, than those in anti-VEGF control (P0.05).The toxicity of CRM9 control are markedly higher than those in blank control groups (P

Blood Gas Analysis ; Hepatopulmonary Syndrome ; therapy ; Humans ; Male ; Middle Aged ; Portasystemic Shunt, Transjugular Intrahepatic ; Treatment Outcome

Objective: To establish primary immune thrombocytopenia (ITP) animal model induced by anti-platelet membrane glycoprotein GPⅠbα antibodies AN51 and R300. Methods: Twenty guinea pigs (6-8 week) were divided into 4 groups. Five guinea pigs in each group were intravenously injected with different doses of AN51 (0.05, 0.1, 0.2 μg/g) and 0.2 μg/g IgG as control. The whole blood was collected from inner angular venous plexus. Platelets number was determined by an automated cell counter and Swiss-Jim method. Then, the similar protocol was used to establish ITP nude mice model by intraperitoneal injection of different concentrations of anti-platelet GPⅠbα antibody R300, respectively. Results: ①Five minutes after intravenous injection of AN51 at 0.05, 0.1 and 0.2 μg/g, the platelet counts of guinea pigs reduced about 0-5%, 50%-60% and 70%-80% compared to the control group, respectively. The difference was statistically significant (P<0.01) . ②Six hours after intraperitoneal injection of R300 at 0.05, 0.1, 0.2 μg/g, the platelet counts of nude mice decreased about 20%-30%, 60%-70% and 80%-90% compared to the control group, respectively. The difference was statistically significant (P<0.01) . The nude mice, injected 0.2 μg/g R300 once a day for 2 weeks, showed typical ITP clinical manifestations including large number of petechiaes or ecchymoses on limbs, head and abdomen. Conclusion AN51 at 0.2 μg/g and R300 at 0.2 μg/g could establish stable ITP model in guinea pigs and nude mice respectively.

Objective:To investigate the role of HIV-1 negative regulator (Nef) in HIV-1 neuropathogenicity.Methods:Five different HIV-1 nef genes were obtained from the central nervous system (CNS) and peripheral regions (basal ganglia, frontal cortex, meninges, temporal cortex and spleen) of a patient with HIV-1-associated dementia (HAD). The recombinant pcDNA3.1- nef eukaryotic expression vectors were constructed by connecting them with pcDNA3.1 vector and transfected into human glioma cell line U87 respectively. The expression of Nef protein was detected by immunohistochemistry and Western blotting at 22ndhour, 27 th hour, 32nd hour, 37th and 42nd hour after transfection. The result were analyzed quantitatively by JEDA801D and JD-801 software. The supernatant of U87 cells was collected every 5 hours from 27th hour to 62nd hour after transfection. The levels of TNF-α and IL-1 β in the supernatant were determined by ELISA, and the dynamic expression of the two cytokines was analyzed. Results:Five recombinant pcDNA3.1- nef eukaryotic expression vectors of nef genes from different tissues of an HAD patient were successfully constructed and transfected into U87 cells. The result of immunohistochemistry showed that Nef protein began to express at 42nd hour after transfection, which was further confirmed by Western blot. The result of ELISA showed that the levels of cytokines in the supernatant of each group increased gradually with time from 22ed hour to 37th hour after transfection, but there was no significant difference among the groups (TNF-α: F=0.445, P=0.837; F=0.579, P=0.742; F=0.617, P=0.714; F=2.728, P=0.057. IL-1β: F=2.656, P=0.062; F=0.485, P=0.809; F=0.165, P=0.982; F=2.463, P=0.093); The levels of TNF-α and IL-1 β in the experimental group were significantly higher than those in the control group from the 42nd hour ( P<0.05); after 42nd hour, the levels of cytokines in each group gradually decreased, and the levels of TNF-α and IL-1 β remained stable from the 57th hour to the 62nd hour, while the levels of TNF-α and IL-1 β in the experimental group were higher than those in the control group from the 42nd hour to the 62nd hour, the difference was still statistically significant (TNF-α: F=241.310, P<0.001; F=242.638, P<0.001; F=250.114, P<0.001; F=143.877, P<0.001; F=146.172, P<0.001. IL-1β: F=251.578, P<0.001; F=188.816, P<0.001; F=276.240, P<0.001; F=238.136, P<0.001; F=163.361, P<0.001), and there was no significant difference among the experimental groups ( P>0.05). Conclusions:HIV-1 Nef protein can induce and enhance the secretion of TNF-α and IL-1 β in U87 cells. However, the amino acid variation of HIV-1 Nef protein from different sources in an HAD patient had no effect on the secretion of TNF-α and IL-1 β.

OBJECTIVETo study the effect of reactive oxygen species (ROS) on the regulation of platelet apoptosis.

METHODSWashed healthy human platelets were pre-incubated with N-caetyl-Lcysteine (NAC), and then stimulated with dibucaine or thrombin. The production of ROS and depolarization of mitochondrial membrane potential (∆ ψm) were detected by flow cytometry. The activation of caspase-3 and expression of Bcl-xL were analyzed by Western blot.

RESULTS(1)The average ROS fluorescence value of NAC+dibucaine group was lower than that of dibucaine group(0.66 ± 0.11 vs 1.06 ± 0.08, P<0.01), while that of NAC+thrombin group was also lower than that of thrombin group(0.45 ± 0.05 vs 0.71 ± 0.11, P=0.001). (2)The percentage of platelets with normal ∆ψm in NAC+Dibucaine group was higher than that of dibucaine group[(86.30 ± 9.37)% vs (13.52 ± 3.01)%, P=0.000], while that of NAC+thrombin group was also higher than that of thrombin group[(93.00 ± 3.03)% vs (76.58 ± 5.28)%, P=0.000]. (3)Fragmentation generated by caspase-3 activation in dibucaine group was much more than that in DMSO control group, while the fragmentation in NAC+dibucaine group was significantly decreased. (4)The expression of anti-apoptosis protein Bcl-xL of NAC+dibucaine group was significantly higher than that of the dibucaine group, while that of NAC+thrombin group was also higher than that of thrombin group.

CONCLUSIONThrough the regulation of ROS, NAC could inhibit the platelet apoptosis induced by dibucaine or thrombin.

Acetylcysteine ; pharmacology ; Apoptosis ; drug effects ; physiology ; Blood Platelets ; drug effects ; metabolism ; physiology ; Caspase 3 ; metabolism ; Dibucaine ; pharmacology ; Humans ; Membrane Potential, Mitochondrial ; physiology ; Reactive Oxygen Species ; metabolism ; Thrombin ; pharmacology ; bcl-X Protein ; metabolism

OBJECTIVES: To investigate the reliability and accuracy of deep learning technology in automatic sex estimation using the 3D reconstructed images of the computed tomography (CT) from the Chinese Han population. METHODS: The pelvic CT images of 700 individuals (350 males and 350 females) of the Chinese Han population aged 20 to 85 years were collected and reconstructed into 3D virtual skeletal models. The feature region images of the medial aspect of the ischiopubic ramus (MIPR) were intercepted. The Inception v4 was adopted as the image recognition model, and two methods of initial learning and transfer learning were used for training. Eighty percent of the individuals' images were randomly selected as the training and validation dataset, and the remaining were used as the test dataset. The left and right sides of the MIPR images were trained separately and combinedly. Subsequently, the models' performance was evaluated by overall accuracy, female accuracy, male accuracy, etc. RESULTS: When both sides of the MIPR images were trained separately with initial learning, the overall accuracy of the right model was 95.7%, the female accuracy and male accuracy were both 95.7%; the overall accuracy of the left model was 92.1%, the female accuracy was 88.6% and the male accuracy was 95.7%. When the left and right MIPR images were combined to train with initial learning, the overall accuracy of the model was 94.6%, the female accuracy was 92.1% and the male accuracy was 97.1%. When the left and right MIPR images were combined to train with transfer learning, the model achieved an overall accuracy of 95.7%, and the female and male accuracies were both 95.7%. CONCLUSIONS The use of deep learning model of Inception v4 and transfer learning algorithm to construct a sex estimation model for pelvic MIPR images of Chinese Han population has high accuracy and well generalizability in human remains, which can effectively estimate the sex in adults.
Adult ; Female ; Humans ; Male ; Deep Learning ; Imaging, Three-Dimensional ; Pelvis ; Reproducibility of Results ; Tomography, X-Ray Computed ; Young Adult ; Middle Aged ; Aged ; Aged, 80 and over

To prepare polyclonal antibodies (PcAb) against UspA1 of Moraxella catarrhalis (Mc), we used bioinformatic analysis to determine the surface exposed region in this protein that holds the antigen epitopes. Then the corresponding coding sequences for this fragment was artificially synthesized according to the codon usage of Escherichia coli. The gene fragment was then subcloned into the prokaryotic expression vector pET-28a(+) and expressed in E. coli rosseta (DE3), and then the recombinant UspA1-His proteins were purified. Two New Zealand white rabbits were immunized with this protein to prepare antiserum. The resulting PcAb was then purified from the antiserum with Protein A affinity column. The results of fluorescence antibody assay, enzyme linked immunosorbent assay and Western blotting analysis showed that the PcAb could specifically recognize the surface exposed region of UspA1 on Mc. The preparation of the PcAb laid a foundation of further development of rapid detection technique for M. catarrhalis.