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.

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.

OBJECTIVETo explore the effect and mechanism of hirudin on atherosclerotic plaques in apolipoprotein E knockout (ApoE(-/-)) mice.

METHODSTotally 24 ApoE(-/-) mice, 7-8 weeks old were fed with high fat diets. They were randomly divided into the recombinant hirudin treatment group (drug group) and the model group according to body weight and different dens, 12 in each group. Twelve C57BL/6J mice, 7-8 weeks old fed with high fat diet were recruited as the normal control group. Recombinant hirudin (0.25 mg/kg) was intraperitoneally injected to mice in the drug group from the 10th week old once every other day for five successive weeks. Equal volume of normal saline was injected to mice in the model group. Mice in the normal control group received no treatment. All mice were sacrificed after fed with high fat diet until they were 20 weeks old. Serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), high-sensitive C-reactive protein (hs-CRP), E-selectin, interleukin-6 (IL-6), and stromal metalloproteinase-2 (MMP-2) were detected. The plaque/lumen area and extracellular lipid composition/ plaque area were analyzed by HE staining and morphometry. Changes of signaling molecules in store-operated calcium channels, including stromal interacting molecule 1 (STIM1), Orail protein, and transient receptor potential channel 1 (TRPC1) were determined by Western blot. Results Lipid plaque formed in the aorta vessel wall of 20-week old mice in the model group. Compared with the normal control group, serum levels of TC, TG and LDL increased (P<0.01), hs-CRP, E-selction, IL-6, and MMP-2 obviously increased (P<0.01, P<0.05) in the model group; expression levels of STIM1, TRPC1, and Orail significantly increased (P<0.01). Compared with the model group, the plaque/lumen area and the extracellular lipid composition/plaque area significantly decreased in the drug group (P<0.05, P<0.01); serum levels of TC and LDL, hs-CRP, E-selction, IL-6, and MMP-2 obviously decreased (P<0.05, P<0.01); expression levels of STIM1, TRPC1, and Orail were significantly down-regulated (P<0.05, P<0.01).

CONCLUSIONHirudin could significantly improve lipids and endothelial functions of ApoE(-/-) mice, down-regulate expression levels of STIM1, Orai1, and TRPC1, and thus delaying the occurrence and development of atherosclerosis.

Animals ; Aorta ; Apolipoproteins E ; metabolism ; Atherosclerosis ; C-Reactive Protein ; Cholesterol ; Diet, High-Fat ; Drugs, Chinese Herbal ; E-Selectin ; Hirudins ; metabolism ; Interleukin-6 ; Lipids ; Lipoproteins, HDL ; Lipoproteins, LDL ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Plaque, Atherosclerotic ; metabolism ; Recombinant Proteins ; metabolism ; Triglycerides

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.

OBJECTIVETo compare the effect of the extracts from Decoction for resuscitation (DRE) and its component herbs on prostacyclin (PGI2), thromboxane A2 (TXA2) and nitric oxide (NO) release from rat vascular endothelial cells under hypoxia.

METHODAfter treatment with the extracts from DRE and its component herbs, the contents of 6-keto-prostaglandin F1alpha(6-keto-PGF1alpha), thromboxane B2 (TXB2) as well as nitrite (NO), which were degradation products of PGI2, TXA2 and NO respectively, in culture medium of rat vascular endothelial cells under hypoxia were measured with radioimmunoassay and Griess Reaction.

RESULTCompared with the control group, the results indicated that DRE, prepared licorice root extract (LE), dried ginger extract (GE), aconite root extract (AE), extracts of aconite root and prepared licorice root (ALE), extracts of aconite root and dried ginger (AGE) increased significantly the content of 6-keto-PGF1alpha and the ratio of 6-keto-PGF1alpha/TXB2, but had no effect on the content of TXB2 in culture medium of rat vascular endothelial cells under hypoxia. The content of 6-keto-PGF1alpha in the DRE group was higher than that in the groups of LE, GE, AE, ALE, AGE. The ratio of 6-keto-PGF1alpha/TXB2 in the DRE group was higher than that of the groups of GE, AE, ALE. Compared with the control group, DRE, LE, GE, AE, ALE, AGE increased significantly the content of NO2- in culture medium of rat vascular endothelial cells under hypoxia. Moreover, the content of NO2- in the DRE group was higher than that of the groups of GE, AE, ALE.

CONCLUSIONThe results suggested that DRE increased significantly the content of PGI2 and the ratio of PGI2/TXA2 as well as the content of NO. The effect of DRE on the parameters in culture medium of rat vascular endothelial cells under hypoxia was better than that of the extracts from its component herbs.

6-Ketoprostaglandin F1 alpha ; metabolism ; Aconitum ; chemistry ; Animals ; Aorta, Abdominal ; cytology ; Cell Hypoxia ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Endothelial Cells ; metabolism ; Ginger ; chemistry ; Glycyrrhiza uralensis ; chemistry ; Nitric Oxide ; metabolism ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar ; Thromboxane B2 ; metabolism

OBJECTIVETo investigate the relationship between plasma levels of fibrinogen, the-beta455 G/A fibrinogen gene polymorphism and the severity of periodontal inflammation and to explore the possible role of fibrinogen in the association of periodontitis with coronary heart disease (CHD).

METHODSA total of 121 patients with moderate to severe periodontitis and periodontally healthy and gingivitis controls were enrolled in the study. Peripheral blood samples were collected and the plasma fibrinogen levels were determined by the clotting method of Clauss. Polymerase chain reaction and restriction fragment length polymorphism analysis with Hae III were used to examine the -beta455 G/A fibrinogen gene polymorphism.

RESULTSFibrinogen levels were significantly higher in moderately or severely chronic periodontitis patients [(3.45 +/- 0.68) g/L] than periodontally healthy and gingivitis controls [(2.47 +/- 0.42) g/L, P < 0.001]. The carrier status of the A allele at position -455 in the beta fibrinogen gene was associated with elevated fibrinogen levels and the frequency of the-A455 allele in the beta fibrinogen gene in the patient group was significantly higher than in the control group (P = 0.032). Carriers of the -A455 allele were about 3-fold more likely to have moderate or severe periodontitis as compare to individuals without the -A455 allele( OR = 3. =135, P= 0.008).

CONCLUSIONSFg-beta455 G/A polymorphism may contribute to the elevated plasma fibrinogen levels and put individuals at higher risk of having severe periodontitis. As the independent risk factor of CHD, fibrinogen levels and Fg-beta455 G/A polymorphism may play a role in the pathogenesis of periodontitis.

Adult ; Alleles ; Case-Control Studies ; Chronic Periodontitis ; genetics ; Coronary Disease ; genetics ; Female ; Fibrinogen ; analysis ; genetics ; Genotype ; Humans ; Male ; Middle Aged ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length

OBJECTIVETo investigate the correlation between moderately and severely chronic periodontitis and coronary heart disease, as well as the role of fibrinogen in the mechanisms responsible for the correlation between periodontitis and coronary heart disease.

METHODS95 subjects who were systemic health or patients of coronary heart disease with or without periodontitis were enrolled. All the subjects were placed into 4 groups based on their periodontal status and cardiovascular health. The 4 groups were healthy control group (HC), moderately and severely chronic periodontitis group (MSP), coronary heart disease group(CHD), and MSP coexisted with CHD group (MSP+CHD). Clinical periodontal index were examined, at the same time, plasma fibrinogen levels and serological changes used in diagnosing of cardiovascular disease routinely were determined. ANOVA and ANCOVA were used in the statistical analysis.

RESULTSFibrinogen levels of HC, MSP, CHD, and MSP+CHD group were (2.36+/-0.37), (3.63+/-0.73), (4.08+/-0.84), and (4.14+/-0.96) g/L, respectively. Fibrinogen levels of MSP and MSP+CHD group were significantly higher than that of healthy controls (P<0.01). The patients with moderately to severely chronic periodontitis were more likely to have coronary heart disease as compared to periodontally healthy controls (OR=2.527, P=0.047) after adjusted for blood pressure and body mass index.

CONCLUSIONModerately and severely chronic periodontitis maybe a risk factor of coronary heart disease and fibrinogen could be one of the biological basis which links periodontitis with coronary heart disease.

Chronic Periodontitis ; Coronary Disease ; Female ; Humans ; Male ; Middle Aged ; Periodontal Index ; Periodontitis ; Risk Factors

OBJECTIVETo study the clinic application of compound flap pedicled with arterial arch of palpebral margin in repairing severe full defect of eyelid.

METHODSAccording to eyelid structure and the defect size, the two compound flaps were designed beside the defect based on the arterial arch of the palpebral margin. If the defective area was too large, the lateral compound flap may be extended to lower or upper eyelid 0.5 cm away from the outer canthus, then cut and propelled the two compound flaps to repair the full eyelid defect.

RESULTS20 cases had been cured with this method since 1998. In this cases, 4 cases were basal cell carcinoma of eyelid, 2 cases were squamous carcinoma, 3 angiomas, 6 chromatophore nexuses, 3 traumatic defects, 2 congenital defects. The largest length of eyelid full defect was 1.7 cm and the smallest was 0.8 cm. 6 cases were upper eyelid defect and 14 cases were lower eyelid defect. All the compound flaps survived completely without any complications. All cases obtained satisfactory results functionally and esthetically.

CONCLUSIONSRepairing full eyelid defect with the compound eyelid flap is the same kind tissue repairing. It can not only provide enough tissues to primary repair large full defect of the upper or lower eyelid to restore normal anatomical structure and appearance of the eyelid, but also is easy to be operated without severe secondary deformities. The arterial arch of the palpebral margin is constant and the blood supply of the compound flap is reliable. It is an ideal method of repairing the eyelid defect.

Adult ; Aged ; Child ; Child, Preschool ; Eyelids ; blood supply ; transplantation ; Female ; Humans ; Infant ; Male ; Middle Aged ; Ophthalmic Artery ; transplantation ; Reconstructive Surgical Procedures ; methods ; Surgical Flaps

OBJECTIVETo investigate the expression of human epididymal secretary protein 2 isoform human epididymal protein 2beta1(HE2beta1) in the testis and epididymis of adolescent male rats along with its significance.

METHODSImmunohistochemical staining was used to detect the expression and localization of HE2beta1 in the testis and epididymis of 15 adolescent SD rats.

RESULTSHE2beta1 immunoreactive staining was detected in the testis and epididymis. In the epithelia of the epididymal duct, HE2beta1 expressed mainly in the supranuclear region of the principle cells and the basement membrane of some epithelial cells; there were no immunostaining in the n clear cells, halo cells and basal cells. The immunopositive reaction was detected, weak in the distal caput, strong in the proximal, middle corpus and the cauda, but negative in the initial segment. Immunopositive results of HE2beta1 were also observed in some of the nuclei of spermatogonia and Sertoli cells with negatively-stained cytoplasm.

CONCLUSIONImmunohistochemical staining is a fairly sensitive method for detecting HE2beta1 expression. The localization and expression level of HE2beta1 in the genital duct of adolescent male rats exhibited a region- and cell-specific expression pattern, which suggests that HE2beta1 may play an important role in spermatogenesis, maturation and epididymal epithelial innate defense mechanisms.

Animals ; Antigens, Surface ; biosynthesis ; Epididymis ; metabolism ; Glycopeptides ; biosynthesis ; Humans ; Immunohistochemistry ; Leydig Cells ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Testis ; metabolism

Objective To investigate the general pattern and character-istics of adverse drug reaction ( ADR) induced by ribonucleic acid Ⅱ. Methods Pareto optimal analysis was adopted to analyze 39 cases of ADR induced by ribonucleic acid Ⅱoccurred in our hospital from Octo-ber 2010 to January 2014.Results Among 39 cases of ADR reports , the majority were people aged 40-59 years old (66.7%) , and all were caused by intravenous route of administration .24 patients ( 61.6%) were treated with more than three kinds of drugs .Most of cases occurred after 16 minutes ( 64.1%) .The clinical manifestations of ADR were mainly lesions of skin and its appendages ( 46.2%) , followed by ana-phylactoid reaction (38.4%) .Conclusion Clinical doctors and phar-macists should understand the pattern and characteristics of ADR induced by ribonucleic acid Ⅱ, and strengthen monitoring of its use in order to reduce occurrence of ADRs.