Objective To prepare a targeted ultrasound micro bubble,which carried the HSV-TK gene,and investigate the in vitro target searching ability of the micro bubbles and inhibitory effect on the proliferation of HepG2 cells.Methods Ultrasonic micro bubbles were prepared by mechanical vibration method,construction of targeted HSV-TK ultrasound micro bubbles by biotin affinity bridge construction.To detect the general characteristics of ultrasound micro bubbles,and to test its effect on the proliferation of HepG2 cells in vitro.Results HSV-TK targeted ultrasound microbubbles more gathered on the surface of HepG2 cells,through detection of PCNA and MTT,it was found that the proliferation of gene targeting microbubble group was obviously decreased,cell apoptosis increased significantly,Cells invade experiments showed that the number of cells in genetic microbubble group (22.18 ± 2.01) decreased significantly compared with the control group and the nontargeted group,can effectively inhibit the proliferation and invasive ability of HepG2 cells.Conclusion Targeted ultrasound microbubble carrying target gene have better inhibitory effect on HepG2 cells in vitro.

Objective:To identify the signature of tumor-infiltrating lymphocyte (TIL) subtypes that may affect cytokine expres-sion between different outcomes of hepatocellular carcinoma (HCC) patients by analyzing the CD molecular expression profiles of non-cancerous hepatic tissues. Methods:Surface markers of TIL in noncancerous hepatic tissues from 146 HCC patients were determined by using immunohistochemical method and flow cytometry. Univariate and multivariate Cox proportional hazards models and Kaplan-Meier method were used to analyze the association of their expression levels with tumor recurrence and survival. Results:More than 86.4%of TILs in patients were quiescent, as measured via CD4+or Foxp3 expression. Meanwhile, more than 90%of CD3+T cells ex-pressed CD8+. The proportion of T cells was low compared with CD8+T cells. The proportion of CD19 and CD20 in distant nontumor tissues almost was zero. The proportion of T cell subgroups isolated from HCC circulating whole blood did not show a significant shift compared with the normal control, as follows:CD4+T/CD8+T=1.167 ± 1.04, CD8+T/CD3+T=0.288 ± 0.116, and CD4+T/CD3+T=0.429 ± 0.178. The proportion of CD8+T cells in noncancerous hepatic tissues was higher than that in blood (P<0.001).Conclusion:TILs in HCC noncancerous hepatic tissues are increased and contain a subpopulation of CD3+CD8+T cells. CD8+T cells in cancerous tissues, rather than noncancerous tissues, show significant differences between different prognostic groups.

0.05 by ? 2 test (? 2=0.487). CT angiography of SSD, MIP, and CT virtual vascular endoscopy could show the location, extent and degree of occlusion or stenosis of internal carotid arteries clearly. CT angiography could also detect calcific plaque in 21 internal carotid arteries and soft plaque in 15 internal carotid arteries. Conclusion Two slices CT perfusion imaging could be made with regular helical CT scanner. CTA could also evaluate the status of plaque. The combination of CT perfusion imaging and CTA are useful not only in observing the morphology of internal carotid arteries, but also in observing the hemodynamic information of the brain, which are important for further general individual analysis.

Objective To construct the eukaryotic expression vector for mouse microRNA miR-21 and identification its expression activ-ity in 293 cells. Methods The genomic sequence containing pre-miR-21 was amplified from mouse genomic DNA by PCR and cloned into the pRC/CMV plasmid. The constructed recombinant plasmid pRC/CMV-mmu-miR-21 was transfected to 293 cells by lipofectamine 2000, and the stably transfected cells were screened with G418,from which total RNA was extracted for detecting the expression of mature miR-21 by northern blot. In the meantime,a luciferase report plasmid examing the activity of miR-21 named pmiR-21-Luc reporter was also construc-ted,and luciferase activity analysis indicated the product of pRC/CMV-mmu-miR-21 indeed had biological activity. Results Both restriction enzyme digestion analysis and sequencing proved the recombinant plasmids were constructed correctly. The miR-21 was highly expressed in the screened clones of 293 cells and it had good biological activity. Conclusion The eukaryotic expression plasmid of mouse miR-21 was successfully constructed,which laid the foundation of further investigation of the role of miR-21 during skin wound healing.

OBJECTIVETo establish a long-term proliferation culture system for mouse spermatogonial stem cells.

METHODSTestis tissues were obtained from 30 newborn male ICR mice on postnatal day 2-6. Testis cell suspension was collected by two-step enzymatic digestion prior to culture. The dissociated cells were aliquoted into tissue culture plates and cultivated with a modified system composed of serum-free defined medium on mouse embryonic fibroblasts (MEF) feeders. Their proliferation was determined by the BrdU incorporation test and the cultured cells identified by alkaline phosphatase (AP) activity, immunofluorescence staining and RT-PCR assay.

RESULTSThe cultures remained in a steady state and continued to generate germ cell colonies. The undifferentiated state was confirmed by strong positivity for AP activity, immunofluorescent staining of GFRalpha-1+ /Oct-4+ /VASA+ /SCP3- and GFRalpha-1+ /Oct-4+/SCP3- at the gene expression levels.

CONCLUSIONMouse spermatogonial stem cells could be expanded in our defined culture system and passaged steadily in vitro. The harvested cells remained in an undifferentiated state, which has provided a good platform for the study of spermatogenesis in vitro.

Alkaline Phosphatase ; genetics ; metabolism ; Animals ; Cell Culture Techniques ; Cell Proliferation ; Cells, Cultured ; Fluorescent Antibody Technique ; Male ; Mice ; Mice, Inbred ICR ; Reverse Transcriptase Polymerase Chain Reaction ; Spermatogonia ; cytology ; metabolism ; Stem Cells ; cytology ; metabolism ; Time Factors

0.05).Conclusion Coverage of medical insurance and effective antiviral therapy for the patients with CHB could affect their QOL.

Purpose　The aim is to invest the mechanism and activity of kringle1～3 gene of angiostatin. Methods　Kringle1～3 gene of angiostatin was abtained from hepatocyte of normal human by RT-PCR and inserted into expression vector pPIC9K in Pichia expression system. The recombinant protein was induced secrete , purified and test the activity by MTT.Results　　Krigle1～3 protein could inhibit vascular endothelial cell proliferative activity.Conclusion　The reaction similar apoptosis induced by kringle1～3 recombinant protein of angiostatin inhibit cell proliferation.

[Objective] To investigate the clinical characteristics and risk factors of lower-extremity arterial disease in the patients with newly diagnosed type 2 diabetes mellitus combined with nonalcoholic fatty liver disease (NAFLD). [Methods] One hundred fifty-one patients were investigated respectively. The patients were divided into two groups (NAFLD-Group and non-NAFLD group) by liver ultrasonography and disease history, then their clinical data were collected and compared in order to find the differences of biochemical indicators and the morbidity of lower-extremity arterial disease between two groups. [Results] Ninety-two cases (60.93%) were complicated with NAFLD. NAFLD group had higher levels of fast insulin and C peptide level, postprandial insulin and C peptide level, uric acid, body mass index (BMI), homeostasis model assessment (HOMA-IR) and lower level of high-density lipoprotein cholesterol and insulin sensitive index than those of without NAFLD (P<0.05). One hundred and one cases(66.89%) were complicated with lower-extremity arterial disease. The morbidity of lower-extremity arterial diseases was higher in NAFLD group than that of without NAFLD group (75% vs. 54.24%, P<0.01). [Conclusion] Both lower-extremity arterial disease and NAFLD are common complicated with type 2 diabetes. The morbidity of lower-extremity arterial diseases was higher in NAFLD group than that of without NAFLD group.

Objective To evaluate the efficacy of preoperative biliary drainage (PBD) in the pancreaticoduodenectomy for malignant obstructive jaundice.Methods Database including PubMed,EMBASE,Cochrane Central Register of Controlled Trials,Academic Degree Dissertation Database and Conference Database were searched with malignant obstructive jaundice,pancreaticoduodenectomy,preoperative biliary drainage,comparative study.Literatures about the randomized controlled trials of PBD (PBD group) and efficacy of early surgery (ES group) in the pancreaticoduodenectomy were retrieved from January 2001 to December 2013,and then a Meta analysis was carried out based on the data.The count data were analyzed using the odds ratio (OR),relative risk (RR) and 95％ confidence interval (95％ CI),and the measurement data were analyzed using mean difference (MD) and 95％ CI.The heterogeneity of the data was analyzed using the I2 test.Data were integrated by fixed or random effect model.Results Twelve literatures including 1 982 patients were selected.There were 1 029 patients in the PBD group and 953 in the ES group.The results of Meta analysis showed that the operation time,volume of blood loss and rate of postoperative wound infection in the PBD group were significantly different from those in the ES group (MD =10.50,107.92,95％ CI:6.34-14.66,16.43-199.42;RR =1.62,95％CI:1.19-2.21,P ＜0.05).There were no significant differences in the postoperative mortality,incidence of pancreatic fistula,incidence of bile leakage,incidence of delayed gastric emptying and duration of hospital stay between the 2 groups (RR=0.69,95％CI:0.52-0.92;OR =0.68,1.35,95％CI:0.38-1.21,0.93-1.95;MD =0.69,95％CI:-0.67-2.05;RR =0.00,95％ CI:-0.02-0.01,P ＞0.05).Conclusion PBD in the pancreaticoduodenectomy for malignant obstructive jaundice cannot reduce postoperative mortality and incidence of complications in patients,and should not be used as the conventional management in the perioperative period.

Background and purpose: TFPI-2 is a new serine proteinase inhibitor,it is related to many tumors.In this study,the effect of TFPI-2 gene on cell proliferation and apoptosis of human pancreatic cancer cell line Panc-1 were investigated.Methods:The growth curve was drawn for the 3 groups,Panc-1-TFPI-2、Panc-1-V and Panc-1.DNA strand breaks were used to detect the apoptosis of the 3 groups,and the change of cell cycle and apoptosis index was evaluated by flow cytometry.Results:Compared with nontransfected cells,the growth of Panc-1 cell transfected with TFPI-2 was inhibited significantly.The transfected cells showed a significant increase in G1/G0 phase.The apoptosis of Panc-1-TFPI-2 cell could be identified by DNA strand breaks and flow cytometry in comparison with the control group.The apoptosis index of the Panc-1-TFPI-2 cell(6.9?0.5)% was higher than the group Panc-1-V and group Panc-1[(3.0?0.4)% and(3.5?0.4)%]P