[Establishment of Humanized Mouse Model by Using Transplantation of Mobilized Peripheral Blood Stem Cells].

OBJECTIVE: To investigate the hematopoietic reconstitution in immunodeficiency NPG(TM) mice after transplantation of G-CSF-mobilized peripheral blood CD34(+) hemopoietic stem cells. METHODS: CD34(+) cells were isolated from peripheral blood stem cells (PBSC) by magnetic activated cell sorting (MACS), and then were transplanted into NPG(TM) mice irradiated with sublethal dose of X ray by marrow cavity transplantation. The hemogram of mice after transplantation for 2, 4 weeks was observed; human cell populations (CD45(+), CD19(+)) in the peripheral blood of mice were dynamically analyzed by flow cytometry (FCM) at 4, 6, 8, 10 and 12 weeks after transplantation. Until the planned harvest at the 12 week after transplantation, the CD45(+), CD19(+) level in bone marrow, liver, spleen from each mouse were detected by flow cytometry; the expression of human Alu gene in the bone marrow cell of mouse was detected by PCR. RESULTS: The purity of CD34(+) cells accounted for 96.3%; after irradiation, the nucleated cells and megalokaryocytes in the marrow cavity of NPG mice were reduced significantly or were lost, and reached the myeloablative effect. At week 4 after transplantation, components of blood cells in peripheral blood of transplanted mice were recovered to the level before irradiation; all the mice survived, human CD45(+), CD19(+) cells were found by FCM in the peripheral blood of all the surviving mice in transplantation group at week 4, 6, 8, 10, 12 after the transplantation; at the 12th week, the human Alu gene could be detected in the bone marrow of all the mice in transplantation group. CONCLUSION: The human-mouse chimeric model is successfully established in irradiation-induced NPG mouse by transplantation of CD34(+) HSC from G-CSF-mobilized peripheral blood via marrow cavity.

Induction of neutralizing antibodies against four serotypes of dengue viruses by MixBiEDIII, a tetravalent dengue vaccine.

The worldwide expansion of four serotypes of dengue virus (DENV) poses great risk to global public health. Several vaccine candidates are under development. However, none is yet available for humans. In the present study, a novel strategy to produce tetravalent DENV vaccine based on envelope protein domain III (EDIII) was proposed. Tandem EDIIIs of two serotypes (type 1-2 and type 3-4) of DENV connected by a Gly-Ser linker ((Gly4Ser)3) were expressed in E. coli, respectively. Then, the two bivalent recombinant EDIIIs were equally mixed to form the tetravalent vaccine candidate MixBiEDIII, and used to immunize BALB/c mice. The results showed that specific IgG and neutralizing antibodies against all four serotypes of DENV were successfully induced in the MixBiEDIII employing Freund adjuvant immunized mice. Furthermore, in the suckling mouse model, sera from mice immunized with MixBiEDIII provided significant protection against four serotypes of DENV challenge. Our data demonstrated that MixBiEDIII, as a novel form of subunit vaccine candidates, might have the potential to be further developed as a tetravalent dengue vaccine in the near future.

[Morphology and microRNA expression profiles of drug-resistant cells in hepatocellular carcinoma].

OBJECTIVE: To compare morphological differences of three drug-resistant hepatocellular carcinoma (HCC) cell subclones (Huh-7/ADM, Huh-7/CBP, Huh-7/MMC) and their parental Huh-7 cell line, to analyze differential microRNA (miRNA) expression profiles in these cells and, finally to screen for the abnormal expressed miRNAs in drug-resistant HCC cells. METHODS: Cellular morphology was observed by histology and transmission electron microscopy. MiRNA microarray was used to analyze the differential miRNA expression profiles in these cells (Huh-7, Huh-7/ADM, Huh-7/CBP, Huh-7/MMC) followed by real time quantitative PCR validation. RESULTS: The drug-resistant cells had more intracytoplasmic organelles and were larger in size along with increased cytological pleomorphism than the parental Huh-7 cells. Compared with the parental Huh-7 cells, 32 simultaneously up-regulated and 22 down-regulated miRNAs were found in three drug-resistant cells. Up-regulation of miR-15a, miR-16, miR-27b, miR-30b, miR-146a, miR-146b-5p, miR-181a, miR-181d and miR-194 was verified by RT-qPCR. CONCLUSION: Drug-resistant HCC cells have abnormal expressed miRNAs, which may be explored to further investigate the association of miRNA expressions with multidrugs resistance in HCC.

[Removal nitrogen of integrated vertical-flow constructed wetland under aeration condition].

Oxygen is an important limit factor of nitrogen removal in constructed wetlands, so it is the key point for improving nitrogen removal efficiency of constructed wetlands that the optimization of oxygen distribution within wetlands. Therefore, oxygen status, nitrogen removal and purification mechanism of integrated vertical-flow constructed wetland (IVCW) under aeration condition in summer and winter have been studied. The results showed that both oxygen levels and aerobic zones were increased in the wetland substrates. The area of oxic zone I (expressing with depth) extended from 22 cm, 17 cm to 53 cm, 44 cm, in summer and winter, respectively. The electric potential (Eh) profiling demonstrated that artificial aeration maintained the pattern of sequential oxic-anoxic-oxic (O-A-O) redox zones within the aerated IVCW in winter, while only two oxic-anoxic (O-A) zones were present inside the non-aerated IVCW in the cold season. The decomposition of organic matter and nitrification were obviously enhanced by artificial aeration since the removal efficiency of COD, TN and NH4(+) -N were increased by 12.2%, 6.9% and 15.1% in winter, respectively. There was no significant accumulation of NO3(-) -N in the effluent with an aeration cycle of 8 h on and 16 h off in this experiment. Moreover, we found that oxic zone I was the main region of pollutants removal in IVCW system, and artificial aeration mainly acted to enhance the purification capacity of this oxic zone in the aerated IVCW. These results suggest that aeration is important for optimization and application of IVCW system.

[Expression of survivin, p27 and PTEN in hepatocellular carcinoma and their clinical significances].

OBJECTIVE: To detect the expression of survivin protein, survivin mRNA, p27 protein, p27 mRNA and PTEN protein in hepatocellular carcinomas (HCC) and their clinical significances. METHODS: Tissue microarrays were constructed. The expression of survivin protein, p27 protein and PTEN protein were evaluated by immunohistochemical methods and in expression of survivin mRNA and p27 mRNA were evaluated by in stiu hybridization respectively in tumor tissues from 141 HCC patients, 128 samples of para-carcinoma liver tissues, 97 liver tissues far from the carcinomas and normal liver tissues from non HCC patients. The relationship of survivin, p27 and PTEN were investigated and a prediction model of HCC was constructed. RESULTS: The expressions of survivin protein (Ridit 95% CI = 0.689+/-0.048, P < 0.01), survivin mRNA (Ridit 95% CI = 0.690+/-0.049, P < 0.01) and p27 protein (Ridit 95% CI = 0.556+/-0.053, P < 0.05) in HCC tissues were significantly increased, while the expression of PTEN protein (Ridit 95% CI = 0.282+/-0.048) in HCC tissues was significantly reduced (P < 0.01). CONCLUSION: Overexpressions of survivin mRNA and p27 protein and reduced expression of PTEN protein might be a valuable marker to predict the presence of HCC.

[Construction of genomic full-length cDNA of SARS coronavirus BJ01 strain and identification of its biological characteristics].

Severe acute respiratory syndrome (SARS) is an important emerging infectious disease which caused by SARS coronavirus (SARS-CoV), and the study of its pathogenesis is needed for the treatment and prevention of this disease. To study the pathogenesis of SARS-CoV using reverse genetics technology, by in vitro ligation using 7 contiguous cDNAs that span the entire genome of the SARS-CoV BJ01 strain, a genomic full-length cDNA was assembled, then using T7 RiboMAX Large Scale RNA Production Systems with the genomic full-length cDNA as template, the RNA transcript was attained. The typical SARS-CoV-resulted cell pathogenic effects were observed when RNA transcript was electroporated into Vero E6 cells. The results of RT-PCR and sequencing of the rescued virus showed that it originated from transcript which derived from the full-length cDNA construct. Rescued virus-infected cells were detected by indirect fluorescent antibody staining demonstrated that it can specifically reaction with SARS-CoV. By CPE method and plaque assay, the titers of the rescued virus and wild-type virus were assessed, which demonstrated there are no significant difference between the viruses and they have similar biological characteristics. Construction of the genomic full-length cDNA of SARS-CoV BJ01 stain successfully and study of the biological characteristics of the rescued virus will provide a useful tool serving for the discovery of molecular pathogenesis and development of candidate vaccines against SARS-CoV.

[Expression and nuclease activity analysis of staphylococcus nuclease in E.coli].

AIM: To express staphylococcus nuclease (SN) in E.coli and prepare rabbit antisera against SN. METHODS: The SN gene was amplified by high-fidelity PCR from plasmid pPLC-SN and then subcloned into expression vector pLEX to obtain the recombinant plasmid pLEX-SN. The expression of recombinant protein was induced by tryptophan. The expressed SN was used to immunize a rabbit to prepare specific antibody. RESULTS: SDS-PAGE analysis showed that the relative molecular mass (M(r)) of the expressed SN was about 17,000 and the expressed SN accounted for about 37% of total bacterial proteins. The prepared antisera were specific to react with recombinant SN. CONCLUSION: Expression vector of SN has been successfully constructed and rabbit antibody against SN was prepared. These results lay the foundation for developing SN as antiviral protein.

[Capsid-targeted viral inactivation for dengue virus infection].

To explore the feasibility of capsid-targeted viral inactivation for dengue virus infection, a newly-discovered antiviral strategy, a mammalian cell line stably expressing staphylococcal nuclease fused to the capsid protein of dengue 2 virus was established and the effects on the production of infectious virus particles were examined. The results presented evidence that the enzymatically active staphylococcal nuclease fused to capsid protein could be incorporated into the nascent virions during wild virus assembly, resulting in degradation of viral genomic RNA and decrease in infectivity. Comparing the effects of incorporated SN and SN*, an enzymatically inactive missense mutant form of SN, on the infectivity of progeny virions, nucleolytic activity of incorporated SN was responsible for the major antiviral effects. These results paved the road of developing capsid-targeted viral inactivation as a new antiviral strategy against dengue.