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 By comparing the organ system based medical integration teaching mode and traditional teaching mode to evaluate the effect of the integration of curriculum implemen-tation. Methods Through a questionnaire survey of 63 students who implement integrated curriculum and 183 students who are not implementing integrated curriculum as well as 76 teachers, we evaluate students' emotional, cognitive and motor skills, using SPSS statistical analysis, chi-square test. Results In the affective domain, most of the teachers thought the experimental class was superior to the com-mon one in learning interest, lifelong learning and independent learning, critical thinking, the contrast of the two classes were P=0.000, P=0.031 and P=0.001 respectively, all of significant difference. In the cognitive domain, the contrast of the two classes in memory, understanding and application were P=0.000, all of significant difference. The experimental class has high degree of recognition. In the psychomotor domain, more than 55.3%(42/76) of the teachers thought that the experimental class was superior to the common class. According to the students' questionnaire survey, the contrast of the two classes in clinical operation skills, Interpersonal communication skills were P=0.077, P=0.031 respec-tively. There was no statistically significant difference. Conclusions The integrated curriculum teach-ing model can greatly promote students' abilities in most areas such as interest in learning, lifelong learning awareness, clinical thinking ability; interpersonal communication skills etc. In some respects the differences remains to be further studied such as memory of knowledge, systematic knowledge, and clinical operation skills, etc.

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.

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.

70 bpm during scan),the proportion of segments that could not be assessed because of motion artifact were 0.1%(1/759),1.1%(7/649),2.5% (10/407),42.6%(103/242),and 75.5%(108/143),respectively.With conventional selective coronary angiography as the golden standard,the sensitivity,specificity,positive and negative prediction values to detect≥50% stenotic lesions in the assessable segments were 79.2%,96.0%,83.8%,and 94.6%,respectively.There was a significant correlation between the number of segments per patient not assessable because of motion artifact and heart rate during the scan(r=0.655,P=0.000).Conclusion MSCT is capable of achieving high accuracy for detection of coronary artery stenosis,and is a reliable technique to diagnose coronary artery disease.

Objective To evaluate the effectiveness of arthroscopically assisted percutaneous reduction and internal fixation with eannulated screws.Methods The fracture was reduced by closed manipulation or percutaneous leverage force by using the Kirschner wire.Then the patella was temporarily fixed by using a large size towel clamp or Kirschner wires.Under the guidance of knee arthroscopy,a micro-incision was made at the size of cannulated screw placement,the pilot holes were drilled at a proper depth,and the thread was configured.Two titanium screws were inserted parallelly.Results Following-up chekups for 4～24 months in 18 cases showed a satisfactory recovery of knee functions.According to the Bostman' standard,excellent effects were obtained in 16 cases and good effects in 2 cases.Conclusion Treatment of patellar fractures by percutaneous cannulated screw fixation under arrhroscope of- fered advantages of minimal invasion,accurate reduction,reliable fixation,and excellent recovery of joint functions.