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.

Objectives To investigate the effects of lipoxin receptor agonist BML-111 on IFN-βand IE86 mRNA expression of macrophages infected by human cytomegalovirus (HCMV). Methods Macrophages were infected with HCMV (MOI=0.5), and the cultured cells were randomly divided into control group, HCMV group, HCMV+BML-111 group, and HCMV+MP group. The cells were collected at 0,1,2,4,8 and 12 h after infection, and the levels of IFN-βand IE86 mRNA were tested by real-time PCR. Results Compared with HCMV group, the levels of IFN-βmRNA in HCMV+BML-111 group increased significantly (P < 0.05), while the levels of IFN-βmRNA in HCMV+MP group decreased significantly (P < 0.05); Compared with HCMV group, there were no significant differences of the levels of IE86 mRNA in HCMV+BML-111 group (P>0.05), while the levels of IE86 mRNA in HCMV+MP group increased significantly (P < 0.05). Conclusion BML-111 exerts antiviral activity by promoting the expression of IFN-βmRNA at the early stage of HCMV infection.

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 explore the correlation between the expression of IL-17A and the degree of spleen damage in acute mouse cytomegalovirus(MCMV) disseminated infection in vivo and to understand the mechanism about how IL-17A involved in the pathological damage of the spleen in MCMV infection.Methods An acute disseminated MCMV infection model was established in mice.BALB/c mice were randomly divided into two groups.Mice in group one were infected with MCMV Smith to establish disseminated infection.Mice in another group were sham-infected control.Three mice from each group were randomly chosen to be sacrificed on days 3,7,14 and 28 after the infection.Viral titers in spleen tissues were determined using a standard plaque assay.The expression of IL-17A mRNA and MCMV mRNA in the splenocytes were measured by RT-PCR.The expression of IL-17A in spleen tissues was observed by immunohistochemical staining.The pathology of the infected mice was assessed by histological examination of H&E stained spleen sections.Results Viral titers and MCMV mRNA in the spleen peaked on day 3,but quickly diminished on day 7.Virus was no longer detectable in the spleen on day 14 after the infection.The expression of IL-17A mRNA was significantly increased during the acute infection and reached the highest level on day 14,then decreased on day 28.It is significantly higher than that of the mock infection group.Immunohistochemistry assay also indicated that the expression of IL-17A in spleen tissue gradually increased to climax on day 14,then decreased on day 28.Accordingly,the pathological damages of spleen tissue in the infected mice deteriorated until day 14,then showed signs of recovery on day 28.The most severe pathological injury of spleen tissue and the highest expression of IL-17A appeared in the same period of time.Conclusion Our results showed a close correlation between IL-17A and the pathological damage in spleen.Thus,IL-17A may contribute to the spleen pathological damage during the acute disseminated MCMV infection.

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 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.

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