Background Hypoxia is the main factor of retinal neovascularization and is closely associated with retinal ganglial cells (RGCs) degeneration.However, the study of retinal neural tissue lesions is rare.Objective This study was to investigate the influence of hypoxia environment on the expression of annexin A2 (ANXA2) in mouse RGC-5 cells and explore the mechanism of RGCs damage induced by hypoxia.Methods Immortalized mouse RGC-5 cells were cultured in high glucose DMEM with 10％ fetal bovine serum.The cells were identified by detecting the expression of Thy-1 ,a specific biomarker of RGCs.CoCl2 was added into the medium at the final concentrations of 50,100,200 and 300 μmol/L, and the cells without CoCl2 served as the control group.Cell viability (absorbance) was assayed by cell counting kit-8 (CCK-8) method in 12,24 and 48 hours after addition of CoCl2.The hypoxic cell models were established in DMEM with 100 μmol/L CoCl2 and divided into the hypoxic 3-hour group, hypoxic 6-hour group, hypoxic 12-hour group and hypoxic 24-hour group,with the normal cultured cells as the normal control group.Apoptotic cells were determined by using hoechst 33342 stain.The expression levels of ANXA2 mRNA and protein in the cells were detected by real-time quantification PCR and Western blot,respectively.The expression and location of ANXA2 in the cells were examined by using immunofluorescence technique.Results The cultured cells grew well and showed the fusiform and polygonal shape,with positive expression of Thy-1 protein.Compared with the normal control group, the viabilities of the cells were insignificantly changed in the 50 μ mol/L CoCl2 group and 100 μmol/L CoCl2 group (all at P＞0.05) ,but the cell viabilities were significantly reduced in the 200 μμmol/L CoCl2 group and 300 μmol/L CoCl2 group in various time points (all at P＜0.05).Hoechst 33342 staining showed that the apoptotic cells with nuclear condensation and high green fluorescence intensity were obtained in the hypoxia groups.The relative expression levels of ANXA2 mRNA were significantly lower in the hypoxic groups than those in the normal control group (all at P ＜ 0.05).The relative expression levels of ANXA2 protein were significantly lower in the hypoxia 3-,6-, 12-and 24-hour group than those in the normal control group (all at P＜ 0.05).Apoptotic cells were seen in the hypoxic 3-hour group, hypoxic 6-hour group, hypoxic 12-hour group and hypoxic 24-hour group compared with the normal control group, showing the bright blue fluorescence in cellular nucleus for hoechst 33342.The relative expressing levels of ANXA2 mRNA in the cells were 0.80±0.14,0.67±0.33, 0.49±0.17 and 0.39±0.02 in the hypoxic 3-hour group,hypoxic 6-hour group, hypoxic 12-hour group and hypoxic 24-hour group, which were significantly declined in comparison with the normal control group, with a statistically difference among the groups (F=434.354, P =0.000).The relative expression values of ANXA2 protein were 0.552 6±0.012 3,0.425 9± 0.033 4,0.344 9 ± 0.017 8 and 0.382 7 ± 0.022 1 in the hypoxic 3-hour group, hypoxic 6-hour group, hypoxic 12-hour group and hypoxic 24-hour group,which were remarkably lower than 0.602 1 ±0.001 4 in the normal control group, showing considerably difference among the groups (F =3.057, P =0.000).ANXA2 proteins were highly expressed in the cellular nucleus and less expressed in the cell membrane and cytoplasm in the normal cells.Compared with the normal control group, the ANXA2 protein showed weak expression in the hypoxia group and primarily in the cytoplasm.Conclusions The expression of ANXA2 down-regulates in hypoxic mouse RGC-5 cells,which may participate in the apoptosis process of RGCs in high glucose environment.

Objective To select omeprazole content changes smaller with dispensing method and to seek for rationality of off-label uses.Methods To measure content change of omeprazole sodium for injection mixed by different subscriptions at different time through HPLC, and compared effect of different dispensing methods on content of omeprazole sodium for injection.Results 10 mL 0.9%sodium chloride injection was chosed as dissolvent,the change of omeprazole sodium for injection content would be minor, and stability of drug solution was superior.Conclusion Dispensing methods of drug impact on its'security and validity, which is part of discuss category about medicine rational use as well.Off-label uses could not vest in unreasonable use, which should contingent on specific document,data and actual environment of medical treatment.

Background Retinal neovascularization is pathological basis of a variety of fundus diseases,but its pathogenesis is unclear.Studies showed that the expression level of radixin in retina is remarkably increased in retinal neovascularization-related diseases.It is presumed that silencing or down-regulating the abnormal expression of radixin is helpful for curing retinal neovascularization-related diseases.Objective This study was to investigate the inhibitory effect of radixin short hairpin RNA (shRNA) plasmid on expression of radixin gene in retina of oxygen-induced retinopathy (OIR) mice.Methods Sixty-four 7-day-old C57BL/6J mice were randomly divided into normal control group, model control group, radixin shRNA plasmid group and shRNA plasmid group by random number table.There were 16 mice in every group.OIR models were established by exposing the mice in an environment of (75±2) ％ oxygen for 5 days and then returned to the normal air in the model control group,radixin shRNA plasmid group and shRNA plasmid group,while the mice of the normal control group were fed in the normal air environment.Radixin shRNA plasmid or control shRNA plasmid at the dose of 1 μg was intravitreally injected in 12-day-old mice of the radixin shRNA plasmid group or shRNA plasmid group, respectively.Five days later, FD-2000S angiography was performed on the mice of each group and then retinal flatmounts were prepared for the observation of retinal vessels.The mice from various groups were sacrificed and retinal sections were prepared.The vascular endothelial nucleus and new blood vessels extending inner limiting membrane (ILM) were examined by hematoxylin and eosin staining;the expression of radixin in the retinas was detected using immunochemistry;the relative expression levels of radixin mRNA and protein were quantitative assayed by real-time quantitative RCR and Western blot, respectively.The use and care of the animals adhered to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.Results The distribution of retinal vessels was normal in the normal control group.Non-perfusion zone at the posterior pole of retina, circuity of blood vessels,leakage of vessel wall and new blood vessels were found in the mice of the model control group.Non-perfusion zone and microaneurysms were also exhibited in the shRNA plasmid group.However,these findings were slight in the radixin shRNA plasmid group.The surface of ILM was in discontinuity in the model mice and shRNA-injected mice with more vascular endothelial cell nucleus and more tubes extending ILM than that in the radixin shRNA plasmid group.The immunochemistry results showed that the expressions of radixin in the normal control group and radixin shRNA plasmid group were weaker than those in the model control group and control shRNA plasmid group.The relative expression levels of radixin mRNA were 1.002±0.043,2.236-±0.093,0.556±0.015 and 2.272±0.096 in the normal control group, model control group,radixin shRNA plasmid group and control shRNA plasmid group,and those in the radixin shRNA plasmid group were significantly reduced in comparison with the normal control group, model control group and the shRNA plasmid group (all at P＜0.01).The relative expression levels were 1.000±0.082,1.193±0.021,0.263± 0.016 and 1.235±0.005 in the normal control group,model control group,radixin shRNA plasmid group and shRNA plasmid,with the lowest expression level in the radixin shRNA plasmid group (all at P＜0.01).Conclusions Radixin shRNA can downregulate the expression of radixin gene in the retinas of OIR mice and further inhibit pathological retinal neovascularization.

Objective:To study the expression and regulation of TLR2/4 in mycobacterium tuberculosis heat shock proteins 16. 3 (mycobacterium tuberculosis heat shock proteins 16. 3,MTB Hsp16. 3) effect on mouse bone marrow-derived macrophages in vitro. Methods:Bone marrow cells were isolated from tibia and femurs of BALB/c mice and incubated with GM-CSF,then detected the expression of CD11b and F4/80 with flow cytometry and observed morphology. The M0 macrophages were stimulated with MTB Hsp16. 3 for 0 h,12 h,24 h,36 h,48 h and 72 h. Real-time PCR detected the expression of TLR2/4 in intracellular at different time point. Silencing macrophages cell surface TLR2/4 molecules by siRNA technology which stimulated with MTB Hsp16. 3 for 0 h,12 h,24 h,36 h,48 h and 72 h. Real-time PCR detected the expression of TLR2/4,Ym-1,Fizz1,IL-10,TNF-α,iNOS and TGF-βin intracellular at different time point. Results:Morphology analysis showed that MTB Hsp16. 3 stimulated macrophages were round cells stretching out pseudopodia,whereas MTB Hsp16. 3 stimulated silencing TLR2/4 macrophages had elongated fibroblastoid. Real time PCR detected the expression of TLR2/4 were upregulated after MTB Hsp16. 3 stimulated M0 macrophages. MTB Hsp16. 3 stimulated silencing TLR2/4 macrophages the expression of IL-6, TNF-α, iNOS were upregulated, whereas IL-10, TGF-β, Ym-1 and Fizz1 were downregulated. Conclusion:MTB Hsp16. 3 may stimulated M0 macrophages to M2 macrophages and suppress M1 macrophages through binding with TLR2/4 receptor,which may be involved the progresss of MTB evaded macrophage phagocytosis.

Objective:To evaluate the fetal adrenal gland volume (AGV) and corrected adrenal gland volume (cAGV) in intrauterine growth restriction (IUGR) fetuses and observe their associations with the adverse perinatal outcomes.Methods:From February 2021 to August 2022, 32 IUGR fetuses who underwent fetal ultrasound examination in the Second Xiangya Hospital of Central South University were prospectively selected as the IUGR group, and 32 normal fetuses matched for gestational age during the same period were selected as the control group. Three-dimensional ultrasound was used to obtain fetal adrenal volume images, and the virtual organ computer-aided analysis (VOCAL) was used to measure AGV, then the cAGV was calculated. The values of AGV and cAGV were appropriately compared between the IUGR and the control groups. The pregnancy outcomes were noted. Multiple logistic regression analysis was employed to evaluate the relationship between the cAGV and adverse perinatal outcomes in IUGR fetus, with maternal age and the CPR included as covariates to control for confounding factors.Results:A total of 32 fetuses with IUGR and 32 controls were involved in this prospective study. There was no significant difference in the AGV between these two groups ( P=0.417). The cAGV of the IUGR fetus was substantially larger than that of the normal fetus ( P=0.034). In the multivariate logistic regression analysis, after adjusting for maternal age and fetal CPR, the fetal cAGV was noticeably associated with the fetal distress (adjusted OR=0.005, 95% CI=0.000-0.587, P=0.029) and the total adverse perinatal outcomes (adjusted OR=0.014, 95% CI=0.000-0.475, P=0.018). Conclusions:The value of cAGV is increased in the IUGR fetuses and associated with adverse perinatal outcomes. The evaluation of fetal AGV could be beneficial to monitoring and managing IUGR fetuses.

ObjectiveConstruction of a negative control line for the Drosophila transgenic system based on ΦC31 integrase and vector plasmid pUASTattB to provide a more scientific negative control for transgenic Drosophila research experiments. MethodsThe vector plasmid pUASTattB was microinjected into four different genetic backgrounds Drosophila lines attP-25C6, attP-68A4, attP-75B1 and attP-86F8 embryos carrying ΦC31 integrase. All of the injected embryos were incubuated to get G0 adults, and each of them was crossed with balancer stock ywR13S separately in a single vial (1 adult of the G0 generation and 3 of the ywR13S in each vial). The probability of successful insertion was calculated by observing the colour of the compound eyes of the G1 generation of Drosophila to determine whether there was a mini-White insertion. The G1 generation Drosophila adults successfully inserted into mini-White were then selected to make single-vial crosses (one G1 generation male Drosophila crossed with three virgins of balancer Drosophila line) with each of the three balancer Drosophila strains DB, ywR13S and yw122, respectively, for balanced seed preservation. The genomic DNA of the conserved Drosophila lines was extracted and the vector plasmid pUASTattB was identified for transfer by PCR. Results12 Drosophila strains were obtained, all of which were red-eyedDrosophila melanogaster carrying the mini-White marker, and were identified by PCR as having the pUASTattB sequence insertion. ConclusionThe 12 transgenic Drosophila strains can meet the negative control requirements for the transgenic fly research experiments that constructed with pUASTattB as the vector basically, enriching the Drosophila resources in the National Drosophila Resource Center of China.