Objective To investigate the influence of murine cytomegalovirus ( MCMV) infection on the expression of downstream differentiation related target genes of Wnt signaling pathway in neural stem cells (NSCs) in vitro and explore the molecular mechanism of fetal encephalodysplasia caused by CMV infection. Methods NSCs were separated from fetal BALB/c mouse and cultured in vitro. The NSCs infected by MCMV at a MOI (multiplicity of infection) of 5, 1 and 0.1, respectively, were cultured in differentiation medium. The dynamic expression of the downstream differentiation related target genes ( c-myc, cyclinD1, ngn-1 and ngn-2) of Wnt signal pathway in NSCs were measured by Western blot. Real-time RT-PCR was employed to measure the expression levels of the key differentiation genes ngn-1 in Wnt signal pathway of NSCs post infection. Results The protein levels of c-myc in the infected groups were significantly lower than that in the normal control at 0.5-5 d (P＜0.05) ; At 0. 5 d and 1 d post-infection (p. i. ) , the protein levels of cyclinDl in the infected groups were lower than that in the normal control (P＜0.05). At 2 d and 3 d p. i. , the cyclinD1 expression in the infected groups was higher than that in the control group (P ＜ 0. 05). However, at 4 d and 5 d p. i. , the cyclinD1 levels in the group of the MOI of 5 were lower than in other three groups (F＜0.05). The expression of ngn-1 protein in the infected groups was reduced importantly compared with normal control at 1 -5 d p. i. ( P ＜ 0.05 ). The expression of ngn-1 mRNA in the infected groups was lower than that in the control group at all time points (P ＜ 0. 05 ). The expression of ngn-2 protein decreased at first and then increased, which was opposite to the normal control. The peak of ngn-2 expression in groups of the MOT of 0.1 and 1 occurred later and were significantly lower than that in the normal control (P ＜0. 05). No distinct peak was seen in the group of the MOI of 5. At 1 d p. i. , the expression of ngn-2 of all infected groups was significantly lower than that in the normal control ( P ＜ 0. 05 ). At 2 d p. i. , the expression of in the group of the MOI of 5 was still lower (P ＜ 0.05). While at 3 d, 4 d and 5 d p. i. , the protein levels in all infected groups were higher than that in the normal control (P ＜ 0. 05). The protein expression of these genes increased following the increase of MOI. Conclusion MCMV inhibited the protein expression of c-myc and ngn-1 in differentiated NSCs, repressed the mRNA expression of ngn-1 and caused the perturbed expression of cyclinDl and ngn-2 in a MOI-dependent manner. These data suggest that inhibition of or interference with the protein expression of downstream differentiation related target genes of Wnt signaling pathway in NSCs by MCMV may be one of the important mechanisms, by which proliferation and differentiation of NSCs are inhibited and thus fetal brain is impaired after MCMV infection.

BackgroundEndostatin (ES) is currently the strongest endogenous angiognesis inhibitor,and it can inhibit the occurrence of neovascularization.Various studies demonstrated that the poly RGD sequence can enhance the function of the ES gene.ObjectiveThis study was to evaluate the use of gene therapy of modified ES for alkaline burn-induced corneal neovascularization (CNV).MethodsOne hundred and two clean SD rats were randomly divided into the normal control group,the pCI empty vector group,the pCI-ES group,and the pCI-RGDRGDES group.Corneal neovascularization models were established by placing a piece of 3 mm filter paper with 1 mol/L NaOH at the central cornea for 40 seconds.3 μg of the pCI blank vector,ES-tranfected pCI blank vector,or RGDRGD-ES-transfected pCI vector was injected into the superior bulbar conjunctiva after the alkali burn twice at 1-week intervals.Area of CNV and edema of the cornea in the various groups of rats were examined daily under the slit lamp biomicroscope.1,4,7 and 14 days after operation,the rats were sacrificed by the excessive anesthesia method and corneal tissues were obtained to evaluate pathological changes.The expression of CD34 in vascular endothelial cells was detected by immunochemistry to calculate the corneal neovascular density.The expressions of VEGF mRNA and Flk-1 protein in the corneas were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis.The use and maintenance of animals followed the Statement of ARVO.Results Seven to fourteen days after corneal alkali-burning,the corneal neovascular area was smaller in the pCI-ES group and pCI-RGDRGD-ES group compared with the normal control group and pCI blank vector group (P＜0.05,P＜0.01 ),and nevascular area in the pCI-RGDRGD-ES group was smaller than that in the pCI-ES group (P＜0.05).The expression level of CD34 was significantly lower in the pCI-ES group and pCI-RGDRGD-ES group than that in the normal control group and pCI blank vector group (P＜0.05,P＜0.01 ),and the expression level of CD34 was further declined in the pCI-RGDRGD-ES group compared with the pCI-ES group (P＜0.05 ).Compared with the normal control group and pCI vector group,the expressions of the Flk-1 protein and VEGF mRNA were decreased in the pCI-ES group and pCI-RGDRGD-ES group on the fourth day after corneal alkali-burning (P＜0.01,P＜0.05 ),and those in the pCI-RGDRGD-ES group were less than the pCI-ES group (P＜ 0.05,P＜ 0.05 ).Conclusions Subconjunctival injection of both ES and modified RGDRGD-ES genes result in significant suppression of CNV in vivo,and modified RGDRGD-ES appears to be more effective than native ES.The main mechanism of ES in inhibiting neovascularization is to downregulate the expression of VEGF and Flk-1.

Objective To study the feature that murine cytomegalovirus(MCMV)infect macrophage strain RAW264.7 and the influence of virus infection on proliferation and apoptosis of RAW264.7 in vitro.Methods RAW was infected by MCMV Smith with multiplicity of infection(MOI)1,0.1 and 0.01,respectivelv.The cells and culture supernatant were collected at 6,12,24,36,48,72,96 and 120 h post-infection(P.i.).Cytopathic effect(CPE)was found with microscope.Virus particles and uhrastructural changes of RAW were observed by transmission electron microscope(TEM). Early antigen(EA)expression was assaved bv immunohistochemical method.The proliferation of MCMV was studied by plaque formation assay.The influence of virus infection on proliferation and apoptosis of RAW were measured by MTT method and flow cytometry.The mouse embryo fibroblast(MEF)susceptible to MCMV infection was positive contro1.Results RAW was swollen and desquamated on 24-48 h P.i..The full-grown virus particles and swollen organelles in RAW were displayed with TEM.Preliminary positive expression of EA was demonstra ted from 6 h(MOI=1 and 0.1)to 12 h(MOI=0.01)P.i..Virus titer in RAw supernatant increased obviouslv on 24 h p.i.and reached the peak on 96-120 h P.i..The proliferation of RAW could be obviously inhibited by MCMV on 72-120 h p.i..When infected by virus with MOI=0.1,necrotic cells of RAW increased on 72-120 h D.i.and the influence of MCMV infection on apoptosis of RAW was not obvious.Conclusion Macrophage strain RAW264.7 is susceptible to MCMV,and it emerges faster cytolytic and productive infection than MEF.MCMV can inhibit the proliferation of RAW but not influence the apoptosis of it.These results can provide a practical experimental model for studying immunological pathogenic mechanism of cytomegalovirus in vitro.

Objective To investigate the influence of murine cytomegalovirus(MCMV) infection on differentiation and differentiation gene expression of neural stem cells (NSCs) in vitro for studying the mechanisms of brain abnormalities calmed by congenital cytomegalovirns infection. Methods NSCs were separated from fetal BALB/c mouse and cultured and identified in vitro. The differentiation potency of NSCs was observed by immunnfluorescence. The NSCs infected by MCMV at dosage of multiplicity of infection (MOI) equaled to 5, I and 0. 1, respectively, were cultured in differentiation medium. The morphological changes of the cells were observed by inverted microscope. The ratios of NSCs and its differentiated cells were detected by flow cytometry. The expression changes of nestin, GFAP and NSE, markers of NSCs and its differentiated cells, were studied by immunofluorescence ( MOI = 1 ). The expression of early antigen (EA) of MCMV was detected to observe the infection process. Real-time RT-PCR method was employed to measure the expression levels of the key differentiation genes Wnt-3 and Wnt-7a in Wnt signal pathway of NSCs at early phage of differentiation culture. Results NSCs isolated from embryonic mouse brains could proliferate to form neurnspheres and strongly express Nestin and differentiate into NF-200 positive neurons or GFAP positive astrocytes. The NSCs of the infected groups couldn't adhere to the wall and appear differentia-tion growth, but showed swollen gradually after differentiation culture. The nostin expression of the infected groups downregulated slowly and was higher than that of the control groups ( P < 0.05 ). The GFAP and NSE expression of the infected groups were lower than that of the control groups (P <0.05). The EA of MCMV could be always detected in the cells of the infected groups. The ratios of nestin positive cells of the infected groups were higher than that of the control groups, but the ratios of GFAP and NSE positive cells of the for-mer were lower than that of the latter from 3rd to 9th day after differentiation culture ( P < 0.05 ). The levels of Wnt-3 mRNA and Wnt-7a mRNA of the infected groups were markedly lower than that of the control groups from 1st to 2nd clay and from 12th hour to 2nd day after differentiation culture respectively ( P < 0.05 ) . These changes of the infected groups became more obvious as MCMV MOI increased . Conclusion MCMV could inhibit significantly NSCs differentiate to neurons and astrocytes and lead to the decrease of dif-ferentiated cells. MCMV could inhibit or interfere with the gene expression of Wnt-3 and Wnt-7a in Wnt sig-nal pathway of NSCs. The effect that MCMV inhibited the differentiation and the differentiation gene expres-sion of NSCs showed dose-dependent with MCMV MOI. The inhibitory effect of MCMV on the differentiation of NSCs might be induced by interfering the differentiation gene expression of NSCs, which is possibly the one of primary causes of brain development disorders caused by congenital CMV infection.

AIM: The influence of MCMV infection on differentiation and differentiation gene expression in neural stem cells ( NSCs) in vitro were investigated for studying the mechanisms of brain abnormalities caused by congenital cytomegalovirus infection. METHODS: NSCs were separated from fetal BALB/C mouse, and cultured and identified in vitro. The differentiation potency of NSCs was observed by immunofluorescence. The NSCs infected by MCMV at dosage of MOI( multiplicity of infection) equaled to 5, 1 and 0.1 .respectively, were cultured in differentiation medium. The morphological changes of infected cells were observed under inverted microscope. The ratios of NSCs and its differentiated cells were detected by flow cytometry. The expressions of nestin, GFAP and NSE, markers of NSCs and its differentiated cells, were studied by immunofluorescence( MOI = 1). The expression of early antigen ( EA ) of MCMV was detected to observe the infection process. Real - time RT - PCR method was employed to measure the expression levels of the key genes Neurog2, Myc and Ccnd1 in Wnt signal pathway of NSCs at early stage of differentiation culture. RESULTS: NSCs isolated from embryonic mouse brains proliferated to form neurospheres, strongly expressed nestin and differentiated into NF - 200 positive neurons or GFAP positive astrocytes. The infected NSCs did not adhere to the wall and appeared differentiation growths, but showed swollen gradually after differentiation culture. The nestin expression in the infected cells downregulated slowly and was higher than that in control groups ( P < 0.05). The GFAP and NSE expressions of the infected cells were lower than those in control groups ( P <0.05). The early antigen ( EA) of MCMV was always detected in the cells in infected groups. The ratios of nestin positive cells in infected groups were higher than those in control groups, but the ratios of GFAP and NSE positive cells of former were lower than that of the latter from 3rd to 9th d after differentiation culture(P < 0.05 ). The levels of Neurog2 mRNA and Myc mRNA in infected groups were markedly lower than those in normal control groups on 1st d and from 1st to 4th d after differentiation culture, respectively( P <0.05). The levels of Ccnd1 mRNA of infected groups were obviously lower than those in normal control groups from 12th h to 1st d( P <0.05 ). These changes in infected groups became more obvious as MCMV MOI increased. CONCLUSION: MCMV significantly inhibits differentiation of NSCs to neurons and astrocytes, and leads to the decrease in differentiated cells. MCMV inhibits or interferes with the gene expression of Newog2, Myc and Ccnd1 in Wnt signal pathway of NSCs. The effect that MCMV inhibits the expressions of differentiation and the differentiation genes in NSCs shows dose - dependent with MCMV MOI. The inhibitory effect of MCMV on the differentiation of NSCs might be induced by interfering with the expression of differentiation gene in NSCs, which is possibly the one of primary causes of brain development disorders induced by congenital CMV infection.

Background Corneal epithelial abrasion results in corneal ulcer and stroma cloudy evenb irreversible visual impairment.Previous drugs for corneal epithelial injury can only alleviate the inflammatory irritation.So it is very important to seek a drug which regulate the growth of corneal epithelium.Objective This study was to investigate the effects of recombinant human BIGH3 protein eye drops on corneal epithelial abrasion.Methods Fifty right eyes of 50 clean adult New Zealand white rabbits were collected.Two rabbits were sacrificed right away following establishment of corneal epithelial abrasion models (0 hour group).The other 48 rabbits were randomly divided into recombinant human epidermal growth factor (EGF) derivative group (positive control group),normal saline solution group (negative control group),0.25％ or 0.5％ recombinant human BIGH3 protein eye drops group.Corneal abrasion models were created with alcohol corrosion method with a defect area of 7 mm2.The corresponding eye drops were used separately in 4 groups for four times per day after operation.Experimental eyes were examined by the slit lamp microscope,and fluorescein vital staining were performed 12,24,36,48,72 hours after operation.Planimetry was performed and the corneal photographs were analyzed with computer software.The rabbits were sacrificed 12,24,36,48 and 72 hours after operation,respectively,and the histopathological examination of corneal tissue was carried out.Results No obvious irritation response was seen after administered of eye drops in the recombinant human EGF derivative group,normal saline solution group,0.25％ and 0.5％ recombinant human BIGH3 protein eye drops groups.Histopathological examination revealed a full-thickness defect of corneal epithelium after modeling.The defect area was gradually smaller with time lapse,and corneal epithelium migrated from periphery toward the center zone.Corneal epithelial cells increased with time lapse.Compared with normal saline solution group,the defect area of corneal epithelium lessened 12,24,36,48 hours after operation in the 0.25％,0.5％ recombinant human BIGH3 protein eye drops groups and recombinant human EGF derivative group (all at P =0.000),but at 12and 24,36 hours after operation,no significant differences were found between the recombinant human EGF derivative group and normal saline solution group (P =0.321,0.057,0.126).The defect area was smaller in the 0.5％recombinant human BIGH3 protein eye drops group than that of the recombinant human EGF derivative group at various time points (P=0.042,0.039,0.025,0.008).However,significant smaller defect area was exhibited only at 12 hours and 24 hours after operation in the 0.25％ recombinant human BIGH3 protein eye drops group (P=0.047,0.042).No significant differences were seen in corneal defect area at various time points between 0.25％ and 0.5％recombinant human BIGH3 protein eye drops groups (P =0.358,0.259,0.108,0.062).In addition,the corneal defect area was (0.51 ±0.42)mm2 72 hours after operation in the normal saline group;while that in the recombinant human EGF derivative group and recombinant human BIGH3 protein eye drops groups was disappeared.The repairing curves in the recombinant human BIGH3 protein eye drops groups were superior to those of the recombinant human EGF derivative group and normal saline solution group.Conclusions 0.25％ and 0.5％ recombinant human BIGH3 protein eye drops have facilitation effect on the growth of corneal epithelial cells and the healing of corneal injury.

Objective:Using an area in east of China as a case,the paper exploit the methodology to define and visualize the scope of the medical insurance pharmacy service through using ArcGIS and its function modules to analy-zing the basic data including this area's population distribution,address of drugstores,administrative districts,road network and soon.Plan A based on the 15-minute walk distance norm for defining the scopes, shows that this area need to increase 548 medical insurances designated drugstore,the effect of which was service area can be increased by 12.36%,service population can be increased by 10.82%, designated drugstore healthy competition rate can be increased by 8.36%;Plan B based on the 10-minute walk distance norm for defining the scopes, displays that this area need to increase 1197 medical insurance designated drugstore, the effect of which was service area can be in-creased by 15.23%,service population can be increased by 20.49%,designated drugstore healthy competition rate can be increased by 19%.

AIMTo investigate the correlation between the gastric adaptive cytoprotection and the low concentration alcohol intake in a chronic drinking rat model and the effect of chronic ethanol exposures on the cell turnover of the gastric mucosa and its possible role in adaptive cytoprotection.

METHODSSprague-Dawley rats received the drinking water containing 6% (v/v) ethanol as their only water intake for 28 days. In the different stages of the 28 days (1st, 3rd, 7th, 14th and 28th days), the stomachs of the rats were cannulated and perfused with pure ethanol, and the severity of mucosal lesions was measured in 2 hours at the end of perfusion respectively. The cell proliferation and apoptosis in gastric mucosa of rats in different groups were analyzed by flow cytometer, immunohistochemistry and computer image analysis.

RESULTSPure ethanol caused ulcer and haemorrhagic damage in the corpus and antral mucosa of the control rats. These lesions were prevented by pretreatment of the animals with ethanol exposure in the 3 rd to 14 th days. The damage index was decreased by 80%, as compared with those in control rats. There was no significant difference in the rats exposed to the ethanol in the 1st and 28th days. Compared with control, the cell apoptosis in gastric mucosa of the rats was enhanced during they exposure to the ethanol in the 3rd to 28th days. Otherwise the cell proliferation was increased in the 3rd to 28th days, and decreased in the 28th days, respectively.

CONCLUSIONChronic adequate alcohol intake may enhance the cell turnover of gastric mucosa and lead to an adaptive cytoprotection. Long-term stimulus with the low concentration ethanol may cause the atrophy of gastric mucosa and reduce the gastric mucosal cytoprotective effect.

Alcoholism ; Animals ; Apoptosis ; Cell Proliferation ; Cytoprotection ; Ethanol ; adverse effects ; Gastric Mucosa ; cytology ; pathology ; Male ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect of ischemic preconditioning on chaperone hsp70 expression and protein aggregation in the CA1 neurons of rats, and to further explore its potential neuroprotective mechanism.

METHODSTwo-vesseloccluded transient global ischemia rat model was used. The rats were divided into sublethal 3-min ischemia group, lethal 10-min ischemia group and ischemic preconditioning group. Neuronal death in the CA1 region was observed by hematoxylineosin staining, and number of live neurons was assessed by cell counting under a light microscope. Immunochemistry and laser scanning confocal microscopy were used to observe the distribution of chaperone hsp70 in the CA1 neurons. Differential centrifuge was used to isolate cytosol, nucleus and protein aggregates fractions. Western blot was used to analyze the quantitative alterations of protein aggregates and inducible chaperone hsp70 in cellular fractions and in protein aggregates under different ischemic conditions.

RESULTSHistological examination showed that ischemic preconditioning significantly reduced delayed neuronal death in the hippocampus CA1 region (P < 0.01 vs 10-min ischemia group). Sublethal ischemic preconditioning induced chaperone hsp70 expression in the CA1 neurons after 24 h reperfusion following 10-min ischemia. Induced-hsp70 combined with the abnormal proteins produced during the secondary lethal 10-min ischemia and inhibited the formation of cytotoxic protein aggregates (P < 0.01 vs 10-min ischemia group).

CONCLUSIONIschemic preconditioning induced chaperone hsp70 expression and inhibited protein aggregates formation in the CA1 neurons when suffered secondary lethal ischemia, which may protect neurons from death.

Animals ; Brain Ischemia ; pathology ; Cell Count ; methods ; Cell Death ; Disease Models, Animal ; Gene Expression Regulation ; physiology ; HSP70 Heat-Shock Proteins ; metabolism ; Hippocampus ; blood supply ; metabolism ; pathology ; Ischemic Preconditioning ; Male ; Neurons ; metabolism ; Proteins ; metabolism ; Rats ; Rats, Wistar ; Time Factors

Ischemic stroke leads to high potentiality of mortality and disability.The current treatment for ischemic stroke is mainly focused on intravenous thrombolytic therapy.However,ischemia/reperfusion induces neuronal damage,which significantly influences the outcome of patients with ischemic stroke,and the exact mechanism implicated in ischemia/reperfusion injury remains unclear,although evidence shows that oxidative stress is likely to be involved.Betulinic acid is mainly known for its anti-tumor and anti-inflammatory activities.Our previous study showed that betulinic acid could decrease the reactive oxygen species (ROS) production by regulating the expression of NADPH oxidase.Thus,we hypothesized that betulinic acid may protect against brain ischemic injury in the animal model of stroke.Focal cerebral ischemia was achieved by using the standard intraluminal occlusion method and reperfusion enabled after 2 h ischemia.Neurological deficits were scored.Infarct size was determined with 2,3,5-triphenyltetrazolium chloride monohydrate (TTC) staining and the mRNA expression of NADPH oxidase 4 (NOX4) was determined by RT-PCR in infarct tissue.ROS generation and apoptosis in ischemic tissue were analyzed by measuring the oxidative conversion of cell permeable 2',7'-dichloro-fluorescein diacetate (DCF-DA) to fluorescent dichlorofluorescein (DCF) in fluorescence microplate reader and TUNEL assay,respectively.In Kunming mice,2 h of middle cerebral artery (MCA) occlusion followed by 24 or 72 h of reperfusion led to an enhanced NOX4 expression in the ischemic hemisphere.This was associated with elevated levels of ROS generation and neuronal apoptosis.Pre-treatment with betulinic acid (50 mg/kg/day for 7 days via gavage) prior to MCA occlusion prevented the ischemia/reperfusion-induced up-regulation of NOX4 and ROS production.In addition,treatment with betulinic acid could markedly blunt the ischemia/reperfusion-induced neuronal apoptosis.Finally,betulinic acid reduced infarct volume and ameliorated the neurological deficit in this stroke mouse model.Our results suggest that betulinic acid protects against cerebral ischemia/reperfusion injury in mice and the down-regulation of NOX4 may represent a mechanism contributing to this effect.