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.

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.

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.

Objective To investigate the influence of murine cytomegalovirus on the expansion of CD+CD25+ Foxp3+ regulatory T cell (Treg) and the activation of CD4+ CD25+Foxp3 - effector T cell (TE) in vivo. Methods Forty-two BALB/c mice were intraperitoneally inoculated with appropriate amount ofMCMV Smith strain for establishing the model of infection, another 42 mice served as mocked-infected con-trois. Day 28 post MCMV infection was determined to be the demarcation point of the acute and chronic in- fection based on the viral load of major visceral organs. On day 1,3, 7, 14, 28, 45 and 60 post infection, splenocytes were prepared by means of routine method. The proportions of CD4+CD25+ Foxp3+Treg and CD4+CD25+Foxp3- activated TE in T lymphocyte were measured by flow cytometry. Results The propor- tion of CD+CD25+ Foxp3+ T cells in T lymphocytes was persistently suppressed since day 7 post infection, and fell to the lowest level on day 28 post infection (P <0.01), then zoomed and reached the peak value on day 60 post infection (P < 0.05). CD4+CD25+ Foxp3 - TE proportion was up to the highest on day 3 post infection(P < 0. 01), then suppressed and in significantly lower level since day 45 post infection (P < 0.05). Treg/CD+TE ratio was in lower level on day 3 to 14 post infection(P <0.05) ; but on day 45 and day 60 post infection Treg/CD+ TE ratio was markedly increased (P < 0.05). Conclusion MCMV infec- tion can increase the CD+CD25+Foxp3+ Treg proportion, and inhibit CD4+T cells activation in chronic in- fection phase, which is likely to suppress the function of antiviral immunity in the infected host to cause a persistent latent infection.

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.

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.

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 investigate the biological function of M122 in pathogenesis of MCMV in developmental brain disorders and brain damage, screening for mouse brain cDNA library interacting with M122 was performed by a yeast two-hybrid system. Methods The reconstructed bait plasmid pGBKT7-M122 was transformed into yeast cells AH109 and screened on the nutrient deficiency medium SD/-Trp. After express of the bait protein in AH109 yeast strains was detected by Western blot analysis, yeast-two hybrid screening was performed by mating AH109 with Y187 containing mouse brain cDNA library plasmid. The diploid yeast cells were plated on the nutrient deficiency medium SD/-Trp/-Leu/-His/-Ade. The second screening was performed with SD/-Trp/-Leu/-His/-Ade containing X-α-gal. The plasmids in positive colonies were extracted and transformed into E. coli JM109 cells. After plasmid DNA in JM109 cells were extracted form positive colonies and sequenced, the results were analyzed by bioinformatic methods. The interactions between M122 protein and the protein obtained from positive colonies were further confirmed by repeating yeast-two hybrid. Then, autoactivations of the proteins obtained from positive colonies were detected.Results The reconstructed bait plasmid was transformed into yeast cells AH109 successfully. The bait protein expressed in the yeast cells AH109 stably. 24 proteins interacting with MCMV M122 were screened, including syntaxin 8 ( Stx8 ), phosphoglucomutase 2 ( Pgm2 ), potassium voltage-gated channel, shaker-related subfamily, beta member 1 ( Kcnab1 ), collagen, type ⅪⅩ, alpha 1 ( Col19a1 ), archain 1 ( Arcn1 ), cytidylate kinase( Cmpk), DnaJ(Hsp40) homolog, subfamily A, member 1 (Dnaja1), ATPase, Na+/K + transporting, beta 3 polypeptide( Atp1b3 ), SH3-domain GRB2-like ( endophilin ) interacting protein 1 ( Sgip1 ),ankyrin repeat domain 17 (Ankrd17), Smg-7 homolog, nonsense mediated mRNA decay factor(Smg7),sperm associated antigen 9 ( Spag9 ), FK506 binding protein 1a ( Fkbp1a), MYST histone acetyltransferase monocytic leukemia 4 ( Myst4), hyaluronan and proteoglycan link protein 1 ( Hapln1), autophagy-related 3 (Atg3), splicing factor, arginine/serine-rich 5 ( Sfrs5 ), zinc finger, C3HC-type containing 1 ( Zc3hc1 ),thioredoxin-related transmembrane protein 1 ( Txndc1 ), adaptor protein complex AP-1, gamma 1 subunit (Ap1g1), Cullin 1 ( Cul1 ), and so on. Three of them were formerly unknown proteins. M122 protein could interact with the proteins obtained from positive colonies in the yeast cells AH109. Ap1g1 and Cul1 were proved to have autoactivation. Conclusion A class of proteins in brain interacting with M122 has been obtained. It is presumed that these proteins are correlated with neuropathogenesis of the brain disorders caused by CMV, but the candidates still need further confirmation for the interaction.

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.