Objective To establish and optimize the method of isolation and cultivation of rat bone marrow mesenchymal stem cells(MSCs),and to investigate the condition and capability of hepatic differentiation of MSCs.(Methods) Rat bone marrow cells were collected from rats of 5～6 week and mononeuclear cells were isolated by Percoll density gradient centrifugation.MSCs were cultivated and purified by adherence method.Morphology,RT-PCR and immunocytochemistry were used to identify the role of substrates,the kinds of the cytokines and concentrations of cytokines on the hepatic differentiation potential.Results The quality and quantity of the isolated bone marrow mononuclear cells reached the peak by using 57% Percoll density gradient centrifugation.Discarding the suspending cells after 24h incubation and subculturing the cells with low seeding density were helpful for acquiring MSCs with improved reproductive capability and active function.MSCs could be successfully induced under HGF/FGF-4 induction on the substrate of(10 ?g/mL) fibronectin.MSCs exhibited round in shape after differentiation,instead of fibroblast-like morphology before.Albumin mRNA and protein were positively expressed in MSCs,withoutdetection of alpha-fetoprotein(AFP).Conclusion The optimization of rats' age,the density of Percoll,the methods of cell cultivation are conducing to obtaining a pure population of MSCs with good differential activity.MSCs are inclined to differentiate into mature hepatocyte-like cells under the induction of HGF/FGF-4.

Fecal microbiota transplantation(FMT)is a method to recover the gut microbiota and treat the intestinal or non-intestinal diseases through transplanting the fecal liquid from healthy population into patients' gastrointestinal tract.There are limited data about the FMT in children.The clinical practice needs professional workgroups,strict indications,normalized and precise procedures.

Objective To establish the murine congenital infection model by MCMV and observe the pathological changes and infection status of brain tissue.Methods After anesthesia,mice who were pregnant 11-13.5 days (E11-13.5 d) were intra-amniotic injected one uterus by one with virus (MCMV K181 suspension,1 μl,1×103 PFU).The control group of the same period was intra-anmiotic injected with culture medium DMEM (1 μl).Carefully reset the uteruses and close the abdomen.After 5 days of separated feeding,kill the pregnant mice,take the fetus out of the uterus,anesthetize and kill them.Make frozen sections of these fetal brains.Some sections were stained using conventional HE method,to observe the pathological changes under the light microscope.Detect MCMV early antigen in the brain tissue by immunohistochemistry staining and immunofluorescence assay.Results The survival rates of the infected group were 71.9％.Compared with the control group,intra-amniotic inoculation of MCMV does not affect the rate of fetal survival,fetal absorption,fetal death and the average weight of the heads,but decrease their average weight of the bodies.The pathological changes are found in the brain tissue of the mouse in the infection group.Through enzyme immunohistochemistry assay,there are many MCMV infected cells in brain-ventricular zone,brain subependymal zone,cerebral cortex and hippocampus area in the infection group.Similar findings were observed by immunofluorescence method.Conclusion By intra-amniotic injection of MCMV suspension,murine model of MCMV congenital infection can be successfully established.This model could be used to study the mechanisms of encephalodysplasia caused by congenital CMV infection in vivo.

Objectives To investigate the value of hepatobiliary scintigraphy in evaluation of the hepatic uptake and excre-tion function in neonatal intrahepatic cholestasis caused by citrin deifciency (NICCD). Methods Hepatobiliary scintigraphy with SPECT was used to detect the hepatic uptake and excretion function of 12 patients with NICCD conifrmed by SLC25A13 gene analysis, and the results were compared with the results of blood biochemical tests. Results The hepatic uptake and excretion function were obviously impaired in all of 12 NICCD patients in the initial scintigraphy. The scintigraphy were performed again in 5 patients in the follow-up after treatment, and showed that the hepatic uptake and excretion function was recovered. It was sug-gested that the hepatic uptake and excretion function was consistent with the level of liver enzymes and the degree of cholestasis. Conclusions Hepatobiliary scintigraphy is of value in evaluation of the hepatic uptake and excretion function in NICCD patients.

Amphotericin B ; administration & dosage ; therapeutic use ; Antifungal Agents ; administration & dosage ; therapeutic use ; Biomarkers ; blood ; Child ; Cough ; diagnosis ; drug therapy ; etiology ; Cryptococcosis ; Fever ; diagnosis ; drug therapy ; etiology ; Fluconazole ; administration & dosage ; therapeutic use ; Humans ; Lung ; diagnostic imaging ; pathology ; Lung Diseases, Fungal ; complications ; diagnosis ; drug therapy ; Male ; Radiography, Thoracic ; Tomography, X-Ray Computed

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.

Murine cytomegalovirus (MCMV) IE3 protein is a multifunctional viral protein that interacts with several target proteins of both viral and host cellular origin. To investigate the biological function of IE3 in the pathogenesis of the brain disorders caused by CMV, a screening for host cellular proteins that could interact with IE3 was performed. By yeast two-hybrid screening, ankyrin repeats domain 17 (Ankrd17, also known as Gtar) was identified as a host factor that could interact with IE3. This interaction was verified by yeast two-hybrid assay and chemiluminescent co-immunoprecipitaion. Mapping analysis suggested that the 1-148 residues of IE3 were responsible for the interaction. These results suggested that the interaction between Ankrd17 and IE3 may play a key role in the pathogenesis of MCMV-associated disease.

Objective To investigate the nature of Th17 cells in murine cytomegalovirus(MCMV)infection during the acute stage,we characterized the frequency of IL-17A-producing CD4 T cells and the level of Th17 cytokines,IL-17A,in MCMV-infected mice.Methods BALB/c mice were randomly divided into two groups.One was infected with MCMV Smith for establishing disseminative infection; the other was sham-inoculated control.On day 3,7,14 and 28 of the experiment,three mice of each group were randomly chosen to be killed separately.Real-time PCR was used to detect MCMV loads in organs of MCMV-infected mice,the pathology of spleen was observed by HE staining.The frequency of CD4+IL-17A+ T cells in total splenocytes of mice was detected by flow cytometry.The level of IL-17A in culture supernatants of splenocytes was measured by double antibody sandwich ELISA.Results MCMV loads in salivary gland reached the peak on day 14 after MCMV infection,the most severe spleen injury was also shown on day 14,the frequencies of CD4+IL-17A+ T cells in total splenocytes increased significantly( all P＜0.01 ) in MCMV-infected mice than those in controls,and reached the peak on day 14 ( 1.14％ ±0.09％ vs 0.19％ ±0.04％,t =17.551,P=0.000).The levels of MCMV-specific IL-17A in culture supernatants were increased dramatically in MCMV-infected mice than those in controls on day 14 [ (81.98± 12.37) pg/ml vs (44.96±8.44)pg/ml,t=4.281,P=0.006].In MCMV-infected mice,correlation was positive between the levels of MCMV-specific IL-17A in culture supernatants and MCMV loads in salivary gland tissues (r=0.54,P＜0.05 ),the levels of IL-17 A in culture supernatants were higher in more severe spleen injury.Conclusion Thl7 cells and IL-17A were involved in the immunity response during acute MCMV infection.They may correlate with the persistence of MCMV and the pathology of spleen in infected mice.

ObjectiveTo observe the changes of AIM2 ( absent in melanoma 2) inflammasome during early murine cytomegalovirus (MCMV) infection.MethodsBALB/c mice were randomly divided into two groups.One was infected with MCMV Smith for establishing disseminated infection,the other was sham-inoculated control.On days 1,3,5 and 7 of the experiment,three mice of each group were randomly chosen to be killed separately.The expression of AIM2,ASC and caspase-1 in splenic macrophages was detected by Western blot,the levels of IL-1β and IL-18 in sera were measured by double antibody sandwich ELISA,and the viral titers in salivary gland tissues were quantified by a standard plaque assay.Results The MCMV titers in salivary gland tissues were gradually increased in MCMV-infected mice on days 3,5 and 7,while the expressions of AIM2 in macrophages were began to increase on day 1 and significantly increased and reached the highest level on day 3 but gradually decreased afterwards.The relative intensity of AIM2 on day 3 differed significantly between the MCMV-infected mice and the controls (1.121±0.243 vs 0.240±0.046,P＜0.01,t test),as did ASC ( 1.318±0.333 vs 0.248±0.090,P＜0.01 ) and caspase-1 ( 1.085±0.243 vs 0.247±0.064,P＜0.01 ).Meanwhile,the levels of IL-1β and IL-18 in MCMV-infected mice were (112.72±5.20) pg/ml and (42.74±4.23) pg/ml,and the levels were significantly higher (P＜0.01 ) than those in controls [ (47.86±4.35) pg/ml and (22.60±2.82) pg/ml].ConclusionThese results demonstrate that AIM2 inflammasome is activated in macrophages during early MCMV infection and could be as a therapeutic target for CMV-induced diseases.

Objective To explore the role of CD4+CD25+regulatory T cells(Treg)in the co-culture svstem consisting of T cells and mouse embryo fibroblasts(MEF)infected with murine cytomegalovirus (MCMV)in vitro.Methods A co-culture system of T cells and MCMV infected MEF(MEFMCMV)was established.The viral load in the supernatants was determined by plaque assay.TH1/TH2 differentiation-specific transcription factors T-bet/GATA-3 were assayed by Western blot.The levels of cytokines IL-4,IL-10and IFN-γ in the co-culture supenatants were measured by double-antibody sandwich ELISA.Results After 3 d incubation,the viral load in the supernatants of TdepTreg-MEFMCMC group,in which the T cells depleted Treg(TdepTreg)were co-cultured together with MEFMVMV,was significantly lower than that in MEFMCMV group.And in the co-cultivation of MEFMCMV and T cells without Treg the expressions of T-bet/GATA-3,IL-4,IL-10 and IFN-γ incteased significantly.Compared with the virus content in the TdepTreg-MEFMCMV group,the MCMV load in the"add-on Treg group"increased and the levels of T-bet/GATA-3 and IFN-γ were lower,and the expression of IL-4 didn't show any significant difference. Compared with the level of IL-10 in the TdepTreg-MEFMCMV group,the IL-10 level in the"add-on TREG group"didn't show any significant differece when the Treg ratio among total T cells was 1%～2%,and increased significantly when the Treg ratio was more than 5%.These eflfects were all correlated with Treg ratios in a dose-dependent manner.Conclusion MCMV infected MEF can induce the proliferation and activation of effector T cells,but Treg can inhibit the T cell-mediated protective effect on MCMV infection.