The human canstatin cDNA was amplified by RT-PCR and then directionally cloned into pU? expression vector. The recombinant pU?-Can vector was connected with the screening marker (bar box), to construct a eukaryotic expression vector called pU?-Can-Bar. This expression vector was introduced into the D.salina by glass beads method. The screening culture of transformants of D.salina was performed in solid media containing 5 ?g/ml PPT, and the analyses of transformants were carried out through PCR and Southern blot. PCR results revealed that specific 700 bp products were detected in the different transformants of D.salina but not in negative control. Southern blot analysis further demonstrated that human canstatin gene was integrated into the D.salina genome. Moreover, the results of genetic stability analyses of transformants demonstrated that canstatin gene was stably inherited in the D.salina transformants. The successful preparation of the D.salina transformants will provide the experimentation evidence for producing canstatin protein cosmically by using the D.salina bioreactor and give a better prophase work basis for clinic application of canstatin protein early.

Objective:To investigate the effect of metformin and arsenic trioxide on KG1a cells proliferation of acute myeloid leukemia and its possible mechanism.Methods:CCK-8 method was used to detect the killing effect of metformin,arsenic trioxide and combined application on KG1a cells.Annexin V-FITC/P1 Dual Stain Flow Cytometry was used to detect the effect of combined application on apoptosis of KG1 a cells.Western blot was used to detect the expression of intracellular apoptosis-,autophagy-related protein.Results:Metformin and arsenic trioxide alone or in combination could inhibit the proliferation of KG1 a cells and induce apoptosis of KG1 a cells,and the proliferation inhibition rate and apoptosis rate in the combined drug group were higher than those in the drug group alone(P＜0.05).The combination of drugs induced upregulation of Caspase 8 protein and P62 protein expression and was higher than that in the drug group alone(P＜0.05).Conclusion:Metformin can synergize with arsenic trioxide to kill KG1a cells,and its mechanism of action may be related to inducing apoptosis and enhancing autophagy.

In this study, the effect of heparin-derived oligosaccharide (HDO) on platelet-derived growth factor (PDGF) induced vascular smooth muscle cells (VSMCs) proliferation and the related signal transduction mechanisms were investigated. MTT assays were used to measure VSMCs proliferation. Cell cycle distribution was analyzed by flow cytometry. The level of key regulatory proteins in PKC, MAPK and Akt/PI3K pathways were determined by RT-PCR, Western blot and immunocytochemical methods. Meanwhile, mRNA expressions of some proto-oncogenes were assayed by RT-PCR method. Our data showed that HDO (0.01, 0.1 and 1 μmol · L(-1)) inhibited 30 ng · mL(-1) PDGF-induced VSMCs proliferation in a dose-dependent manner, blocked the G1/S transition and inhibited the level of key regulatory proteins and some proto-oncogenes (P < 0.05). The results showed that HDO may decrease the key regulatory proteins expression, hence suppress the transcription of proto-oncogene and G1/S transition, finally inhibiting VSMCs proliferation.
Cell Cycle ; Cell Proliferation ; drug effects ; Cells, Cultured ; Flow Cytometry ; Heparin ; pharmacology ; Humans ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Oligosaccharides ; pharmacology ; Platelet-Derived Growth Factor ; pharmacology ; Signal Transduction

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.

Objective To investigate the function of soluble stem cell factor receptor(s-kit)and stem cell factor(SCF) in chronic aplastic anemia(CAA).Methods ELISA assay was employed to determine the levels of s-kit and SCF in peripheral blood of CAA patients and umbilical cord blood.Results The levels of s-kit and SCF in peripheral blood of CAA patients are lower than those of normal group and umbilical cord blood group.Conclusions The decreased levels of s-kit and SCF show that s-kit and SCF may play a role in CAA mechanism.The raised levels of s-kit and SCF show that s-kit and SCF may be applied in the field of cord blood transplantation.

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 effects of exercise training(ET)on the expression of glucogen synthase kinase 3 bate(GSK 3?)in the adipose tissues of insulin resistant(IR)rats on a high fat diet(HFD). Methods Thirty male Wistar rats were randomly divided into a control group(n=10)and a model group(M group,n=20).Insulin resistance was established by feeding a HFD to the M group for 4 w,while rats in the control group were fed a normal diet.The IR rats were then randomly divided into two subgroups:an IR group and an ET group.All rats in the IR and ET groups were fed a HFD,but ET was administrated to the ET group for 6w.The expres- sion of GSK 3?protein in the rats'epididymis adipose tissue was detected using Western blotting,and body weight (BW),the concentrations of serum triglyceride and cholesterol(TG and TC),fasting plasma glucose(FPG)and serum insulin(FINS),as well as insulin sensitivity index(ISI)were regularly detected.Results Compared with the con- trol group,BW and the concentrations of serum TG and TC,FPG and FINS in the model group were significantly in- creased(P＜0.05),while ISI was decreased(P＜0.01).Compared with the control group,there was no difference in GSK 3 protein expression in the ET group,but the expression of GSK 3?protein in the ET group was obviously de- creased in comparison with that in the IR group(P＜0.05).Conclusion ET can ameliorate IR by decreasing GSK 3?protein expression in adipose tissues and enhancing the ingestion of glucose and the synthesis of glycogen.

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.

BACKGROUND:The anti-inflammation and protective effects of lipoxin have been verified in several immunity-related disease models. Preliminary studies of our research group have shown that, lipoxin receptor agonist BML-111 has negative regulation effects on the human cytomegalovirus (HCMV)-induced immunological injury. However, the effect of BML-111 on the HCMV replication remains unclear.
OBJECTIVE:To observe the influence of lipoxin receptor agonist BML-111 on HCMV replication and proliferation in THP-1 macrophages and human embryonic lung fibroblasts.
METHODS:THP-1 macrophages were infected by HCMV AD169 strain, and were divided into three groups:mock infection, HCMV infection, HCMV+BML-111. The final concentration of BML-111 was 100 nmol/L. cells in each group were col ected at 0, 1, 2, 4, 12, 36, 48 hours, the mRNA levels of IE86 and pp65 in the THP-1 macrophages were tested by RT-PCR method. Human embryonic lung fibroblasts were infected with HCMV (MOI=0.1), and were divided into two groups:HCMV infection and HCMV+BML-111. The patho-morphous changes of human embryonic lung fibroblasts were observed under light microscope, and the cellnumber was measured. The infective virus titer changes in human embryonic lung fibroblasts were examined by plaque assay.
RESULTS AND CONCLUSION:After the macrophages were infected by HCMV, compared with the mock infection group, the mRNA levels of IE86 and pp65 in the HCMV group and HCMV+BML-111 group were increased significantly;compared with the HCMV infection group, the mRNA levels of IE86 and pp65 in the HCMV+BML-111 group were increased significantly in the early stage (within 4 hours) after infection, but the pp65 mRNA levels were decreased significantly in the medium and late stages (24-72 hours) after infection. After human embryonic lung fibroblasts were infected by HCMV, the degree of the patho-morphous in the HCMV+BML-111 group reached 100%2 days earlier than the of HCMV infection group. The infective virus titer reached the peak 2 days earlier than the HCMV infection group, but no significant difference was found between the two groups. BML-111 accelerates the replication of HCMV in the early stage of infection, but inhibits the expression of pp65 gene in the late stage. BML-111 has no impact on the proliferation of the infective HCMV titer in vitro.