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Objective To build the lentiviral vectors of pigment epithelial derived factor (PEDF) gene,and investigate their expression in human umbilical cord mesenchymal stem cells (hUCMSCs).Methods The PEDF lentiviral vectors (LV-PEDF) were built by DNA recombination and confirmed by DNA sequencing.hUCMSCs were transfected by LV-PEDF with MOI 10,30,50,respectively.The transfection efficiency was observed under fluorescence microscope.Cell immunofluorescence,immunocytochemistry and real-time PCR methods were used for detecting the expression of PEDF and VEGF.Results The PEDF cDNA was sub-cloned into pCDH-CMV-MCS-EF 1-copGFP vector successfully.DNA sequencing analysis confirmed that PEDF gene sequence was exactly the same with that reported in GenBank.pCDH-PEDF infected cells could show green fluorescence under fluorescence microscope.The transfection efficiency was 72.1％ in PEDF-MSCs.Immunofluorescence and immunochemical staining confirmed that PEDF protein was overexpressed in hUCMSCs.The relative expression of PEDF mRNA in experimental group and control group was (0.170±0.028) and (0.015 ± 0.007) respectively by RT-PCR,the difference was statistically significant (P＜0.00 1).The relative expression levels of VEGF mRNA in the two groups were (0.265 ± 0.022) and (0.285 ± 0.049),respectively,with no significant difference (P＞0.05).Conclusions We successfully built a lentivius vector carrying PEDF gene and obtained hUCMSCs with overexpressed PEDF.
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Background Retina fixed flat-mount perfused by Evans blue (EB) is a common method for the evaluation of blood-retinal barrier (BRB).However,previous method is inconvenient for some laboratories because the retinal specimen can not be observed by gereral microscope rather than confocal laser scanning microscope after the fixation.Objective This study was to modify the preparing way of flat-mounted retina in order to obtain transparent specimen for the observation of rat retinal vessels and the evaluation of leakage under the ordinary fluorescence microscope.Methods Forty male SD rats were divided into the control group,diabetes mellitus (DM) 1-month group,DM 3-month group and DM 6-month group according to the random number table.Streptozotocinum (STZ) of 2％ dissolved in 0.05 mmol/L sodium citrate-hydrochloric acid buffer was intraperitoneally injected in SD rats to establish DM models,and the equal volume of solvent was injected in the same way in the control rats.One month,three months and six months after injection,EB of 30 g/L was injected via rat femoral vein in the dose of 45 mg/kg.Fifteen minutes after injection of EB,the rats were sacrificed and the retinas were isolated and cut radially to prepare the flat-mounted retinas in PBS immediately and then were dried till the specimens were transparent.The specimens were examined under the fluorescence microscope.The percentage of EB leakage was quantitatively calculated by IPP 6.0 software.All procedures were performed following approval of the institutional animal care and use committee of Tianjin Medical University.Results The retina morphology was normal in the control group,and EB filled the vessels,exhibiting the red fluorescence under the fluorescence microscope.Compared with the control group,retinal background fluorescence was enhanced slightly in the DM 1-month group,and focal leakage of the EB from capillaries and focal dilated vessels were found in the DM 3-month group,further,vascular caliber inequality,retinal hypoperfusion area and a larger number of hyperfluorescence areas were seen in the DM 6-month group.The percentage of leakage area was (0.05 ±0.02) ％,(0.27 ±0.06) ％,(1.17 ±0.18)％ and (4.77 ±0.66)％ in the control group,DM 1-month group,DM 3-month group and DM 6-month group,respectively,showing a significant difference among the four groups (F =795.800,P＜0.001),and the leakage area was obviously larger in the DM 3-month group and DM 6-month group than that in thecontrol group (q'=10.338,q'=43.475,both at P＜0.001).Conclusions Modified EB-perfused retinal wholemount method is easy and helpful for clear visualization of retinal vessel leakage induced by BRB breakdown in the diabetic rats under the common fluorescence microscope.
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Objective To investigate the interactional roles corticosterone and stress played during the PTSD-Like memory impairment.Methods First we established the model of PTSD-like memory using three levels of electric shock:0 mA,0.8 mA and 1.4 mA.The freezing time percent of each group were calculated 24 hours later in order to find which group of rats obtained the PTSD-like memory,and generalizing tests was used to verify it.After the model was established,rats received adrenalectomy (ADX) were operated bilaterally in order to investigate the effect of corticosterone and fed with corticosterone in the drinking water 7 days before fear conditioning.These rats were divided into four groups:control group (no corticosterone supplement,0 mA shock),Cort group (corticosterone supplement,0 mA shock),shock group (no corticosterone supplement,1.4 mA shock),Cort-shock group (corticosterone supplement,1.4 mA shock).A two-way factorial analysis of variance was used to determine whether there was a significant interaction between corticosterone and shock.Results The freezing time percent of group 0.8 mA raised compared with the group 0 mA,whereas the freezing time percent of group 1.4 mA showed reversely and the generalizing effects appeared,compared with group 0.8 mA.In two-way factorial analysis design,the freezing time percent of Cort-shock group significantly increased (P＜0.05) compared with other groups,and there was no significant variance among the other groups.Corticosterone (F=6.464,P＜0.05) and stress (F=53.419,P ＜0.01) played parts in the formation of PTSD-like memory impairment,and they interacted with each other (F=11.580,P＜0.05).Conclusion PTSD-like memory impairment can be induced on rats with high (1.4 mA) electrical shock,and they have interactional effect with each other in the process.
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OBJECTIVE:To improve the writing quality prescriptions of traditional Chinese medicines and to facilitate the standardization of traditional Chinese medicine prescriptions.METHODS:A total of 15 000 prescriptions were sampled in our hospital in 2006 for an analysis of the problems in accordance with the related standards specified in Chinese Pharmacopoeia(CP,2005 edition)and the new "Prescription management method".RESULTS:The problems manifested as nonstandard in drug name and footnotes,or overdosage and so on.CONCLUSION:We should strengthen the management of the traditional Chinese drugs and improve our pharmaceutical care.