Differentiation Character of Adult Mesenchymal Stem Cells andTransfection of MSCs with Lentiviral Vectors

This study examined the differentiation character and pluripotency of mesenchymal stem cells (MSCs) under different conditions. Adult MSCs were initially isolated from the bone marrow of rats, cultured in vitro and identified by flow cytometry. After MSCs were transferred to osteogenic and adipogenic medium respectively, the morphological characterization of induced cells was observed. The expression of marker genes was detected by RT-PCR analysis. Then MSCs were transfected with lenti- viral vectors pGC-FU-Sox9-EGFP. Enhanced green fluorescence protein (EGFP) expression and trans- fection efficiency were determined by fluorescence microscopy. The results demonstrated that EGFP caused no effect on the multilineage potential of adult MSCs. Sox9 gene expression of high level was maintained stable in the transfected MSCs and induced MSCs to differentiate into chondrocytes. Ag- gracan was positive in chondrogenic lineages and the expression of aggracan and type Ⅱ collagenwas significantly increased during MSCs chondrogenic differentiation. It was concluded that Sox9 gene-modified adult MSCs may be promising candidate cells for further studies on tissue engineering. EGFP facilitates the research on MSCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.

Recombinant Vaccinia Virus is an Effective and Non-perturbing Vector for Human Dendritic Cells Transfected with Epstein-Barr Virus Latent Membrane Protein 2A

ObjectiveTo study the effects of dendritic cells (DC) transfected with recombinant vaccinia virus encoding Epstein Barr virus (EBV) latent membrane protein 2A(LMP2A) gene,and to provide evidence for further investigation on the therapeutic vaccines against EBV associated malignancies. MethodsMature DC were transfected with EBV LMP2A recombinant vaccinia virus (rVV LMP2A). Before and after the transfection,the expression of surface antigens on mature DC including CD1a,CD83,CD40,CD80,HLA DR was measured by fluorescence activated cell sorter (FACS) and the function of DC to stimulate allogeneic T cells proliferation was measured by mixed leukocyte reactions (MLR). ResultsLMP2A protein was highly expressed (66.1 %) in DC after the transfection of rVV LMP2A. No significant changes in the primary surface antigens expression and in the MLR were detected during the transfection. Transfected DC still had strong potential in stimulating the proliferation of allogeneic T cells. ConclusionRecombinant vaccinia virus was an effective and non perturbing vector to mediate the transfection of LMP2A into DC. The functions of mature DC were not affected significantly by the transfection of Vac LMP2A. This study could provide evidence for the further immunotherapy of EBV associated malignancies,e.g. nasopharyngeal carcinoma (NPC).

Construction and expression of retroviral vector pLEGFP-N1-TERT in preparation of seed cells for skin tissue engineering

Objective:To construct the retroviral vector pLEGFP-N1-telomerase reverse transcriptase(TERT)and to investigate the expression of TERT in neonatal mouse bypodermal cells.Methods:The polymerase chain reaction(PCR)-amplified TERT gene was inserted into plasmid pLEGFPN1.The positive clone was identified by restriction enzyme digestion and sequencing,then was transfected into packaging cells to produce retrovirus particles.Neonatal mouse hypodermal cells were infected with the virus to generate a stable cell line.The TERT mRNA expression level,telomerase activity,and enhanced green fluorescent protein(EGFP)expression level were analyzed.Results:Retroviral vector pLEGFP-N1-TERT was constructed successfully,and a stable cell line of neonatal mouse hypodermal cells expressing EGFP was established.Western blot and immunohistochemical assay showed that the expression level of TERT was significantly elevated in the neonatal mouse hypodermal cells.Conclusions:A high titer of retrovirus pLEGFP-N1-TERT mediates high-level expression of the exogenous TERT gene in the neonatal mouse hypodermal cells.This protocol has potential applications for skin tissue engineering and cell transplantation therapy.

Interleukin-2 expression and glioma cell proliferation following Vaccinia vector gene transfection in vivo

BACKGROUND： The effectiveness of gene therapy is closely related to the efficiency of vector transfection and expression. OBJECTIVE： This study was designed to transfect a human brain glioma cell line with recombinant Vaccinia virus expressing the interleukin-2 （rVV-IL-2） gene, and to observe IL-2 expression and glioma cell proliferation potential after transfection. DESIGN： Experimental observation. SETTING： Department of Neurosurgery, Shenyang Military Area Command of Chinese PLA. MATERIALS： The rVV-IL-2 vectors were obtained through homologous recombination and screening in the Second Military Medical University of Chinese PLA. The human brain glioma cell line and IL-2-dependent cells were produced by the Second Military Medical University of Chinese PLA. Human IL-2 was produced by Genzyme Corporation. METHODS： At passage day l, Veto cells were amplified l ; 1 for virus and cells. A human brain glioma cell line was transfected using amplified Vaccinia viral vectors at varying multiplicities of infection （MOI）. At 2, 4, 6, 8, 12, and 24 hours post-transfection, superuatant was collected to determine by MTT assay IL-2 expression levels in IL-2 dependent cells. The transfected and non-transfected cells were divided into 4 groups, namely MOI1 ： 1, MOI 5 ： 1, MOI 10 ： 1, and control groups. MAIN OUTCOME MEASURES： IL-2 expression at different time points after transfection of human brain glioma cells with varying MOI of Vaccinia viral vectors; in vitro proliferation capacity of human brain glioma cells among the 4 groups. RESULTS： IL-2 expression was detectable 4 hours after Vaccinia viral vector transfection and reached 300 kU/L by 8 hours. There was no significant difference in the proliferating rate of human brain glioma cells among the 4 groups （P 〉 0.05）. CONCLUSION： Vaccinia viral vectors can transfect human brain glioma cells in vitro and express high levels of IL-2. Vaccinia virus and high IL-2 expression do not influence the proliferation rate of human brain glioma cells in vitro.

Effect of MSTN Propeptide and shRNA Co-expression Vector on Proliferation of Skeletal Muscle Satellite Cells

Myostatin （MSTN） is a negative regulator of skeletal muscle growth, in order to study the effect of inhibition MSTN expression on the proliferation of bovine skeletal muscle satellite cells, we constructed co-expression vector pcDNA3.1-Pro- MSTNshRNA, transfected it into muscle satellite cells by Liposome 2000, and detected cell proliferation changes by CCK-8 method and flow cytometry after 48 h. The expressions of P21 and CDK2 were detected by Western blot and real-time PCR. The results showed that the cell vitality of experimental groups significantly increased than that of the negative control, and cells in S phase also increased significantly （P〈0.05）. After knocked down MSTN gene, P21 expression decreased （P〈0.05）, but CDK2 gene expression increased （P〈0.05）. These results indicated that MSTN gene expression was associated with P21 and CDK2, the proliferation of skeletal muscle satellite cells could be promoted while MSTN was inhibited, which provided a theoretical basis for the study on transgenic cattle.

Gene Cloning of Murine α-Fetoprotein Gene and Construction of Its Eukaryotic Expression Vector and Expression in CHO Cells

To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was amplified by RT PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.

Expression of Green Fluorescent Protein Gene with Baculovirus Vectorin Insect Cells

The green fluorescence of bioluminescent jellyfish Aequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10 3 dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus insect cell expression system.

THE ROLE OF RECOMBINANT Rb GENE ADENOVIRUS VECTOR IN THE GROWTH OF LUNG ADENOCARCINOMA CELLS

Objective: To study the role of the most extensively studied tumor suppressor gene, retinoblastoma (Rb) gene, on the growth of lung adenocarcinoma cell line GLC 82 and explore a gene therapy approach for lung adenocarcinoma Methods: The recombinant Rb gene adenovirus vector was constructed, the control virus which carries LacZ gene was producted by the same method Infection effects were detected by biochemical staining of β gal and immunohistochemical analysis of Rb protein The Rb cDNA of infected cells were determined by PCR The cell growth rate and cell cycle were observed by cell counting and flow cytometry Results: The constructed recombinant adenovirus vector could infect effectively the cells with high level expression of Rb cDNA and Rb protein The transfection of wild type Rb gene could suppress GLC 82 cell proliferation and decrease the cellular DNA synthesis Conclusions: These results showed the possibility of using recombinant Rb gene adenovirus vector in the gene therapy of cancer to inhibit the growth of cancer

The Flux of Stem Cells to the Area of the Retina

Stem cell transplantation for the blind is a promising area of research, but it is still in the early stages of development. Our aim in this article is to think about geometric-mathematical tools so that by the flux of stem cells into open and curved spaces in the retina of recently blind people and macular degeneration patients, (AMD) patients, we will enable the growth of visual cells in their retinas.

Construction of a replication-competent hepatitis B virus vector carrying secreted luciferase transgene and establishment of new hepatitis B virus replication and expression cell lines

BACKGROUND Previously,we have successfully constructed replication-competent hepatitis B virus(HBV)vectors by uncoupling the P open reading frame(ORF)from the preC/C ORF to carefully design the transgene insertion site to overcome the compact organization of the HBV genome and maintain HBV replication competence.Consequently,the replication-competent HBV vectors carrying foreign genes,including pCH-BsdR,carrying blasticidin resistance gene(399 bp),and pCH-hrGFP,carrying humanized renilla green fluorescent protein gene(720 bp),were successfully obtained.However,the replication efficiency of the former is higher but it is tedious to use,while that of the latter is poor and cannot be quantified.Hence,we need to search for a new reporter gene that is convenient and quantifiable for further research.AIM To establish a helpful tool for intracellular HBV replication and anti-viral drugs screening studies.METHODS We utilized the replication-competent HBV viral vectors constructed by our laboratory,combined with the secreted luciferase reporter gene,to construct replication-competent HBV vectors expressing the reporter gene secretory Nanoluc Luciferase(SecNluc).HepG2.TA2-7 cells were transfected with this vector to obtain cell lines with stably secreted HBV particles carrying secNluc reporter gene.RESULTS The replication-competent HBV vector carrying the SecNluc reporter gene pCHsNLuc could produce all major viral RNAs and a full set of envelope proteins and achieve high-level secreted luciferase expression.HBV replication intermediates could be produced from this vector.Via transfection with pTRE-sNLuc and selection by hygromycin,we obtained isolated cell clones,named HBV-NLuc-35 cells,which could secrete secNLuc recombinant viruses,and were sensitive to existing anti-HBV drugs.Using differentiated HepaRG cells,it was verified that recombinant HBV possessed infectivity.CONCLUSION Our research demonstrated that a replication-competent HBV vector carrying a secreted luciferase transgene possesses replication and expression ability,and the established HBV replication and expression cell lines could stably secrete viral particles carrying secNluc reporter gene.More importantly,the cell line and the secreted recombinant viral particles could be used to trace HBV replication or infection.

用TEM CELL测量辐射发射的新研究

首次从天势和洛仑互易定理出发，通过关量和张量运算得出横电磁波空中包括电四极子的辐射模型的被测物的辐射发射的更一般的结论，通过实验验证，结果较好。

用TEM CELL测量辐射发射的理论与实验

本文首次从矢势和洛仑兹互易定理出发，通过矢量和张量运算得出横电磁波室中包括电四极子辐射模型被测物的辐射发射的更一般结论。通过实验验证，结果较好。

Expression of the Human Growth Hormone Genein Insect Cells

The insect cellvirus (AcNPV) system was used to express heterologous protein. The recombinant transfer vector pVL1393/hGH was reconstructed in which the human Growth Hormone (hGH) gene was inserted under the control of the polyhedron gene promoter. The Spodoptera frugiperda (Sf9) cells was cotransfected with the plasmid DNA containing hGH gene and wildtype AcNPV DNA. The hGH gene was transferred to the AcNPV genome DNA through homologous recombination, and the recombinant virus rAcVhGH was obtained by multiple plaque purification. The high level of production of hGH (40 μg/mL) in supernatant of the infected monolayer culture was determined by immunochemiluminescent assay.

Hepatocyte nuclear factor 4α induces a tendency of differentiation and activation of rat hepatic stellate cells

AIM: To investigate the effect of hepatocyte nuclear factor 4α(HNF4α) on the differentiation and transformation of hepatic stellate cells(HSCs).METHODS: By constructing the recombinant adenovirus vector expressing HNF4α and HNF4αshRNA vector, and manipulating HNF4α expression in HSC-T6 cells, we explored the influence of HNF4α and its induction capacity in the differentiation of rat HSCs into hepatocytes.RESULTS: With increased expression of HNF4αmediated by AdHNF4α, the relative expression of Nanog was downregulated in HSC-T6 cells(98.33 ±12.33 vs 41.33 ± 5.67, P < 0.001). Consequently, the expression of G-P-6 and PEPCK was upregulated(G-P-6:14.34 ± 3.33 vs 42.53 ± 5.87, P < 0.01; PEPCK: 10.10± 4.67 vs 56.56 ± 5.25, P < 0.001), the expression of AFP and ALB was positive, and the expression of Nanog, Type Ⅰ collagen, α-SMA, and TIMP-1 was significantly decreased. HNF4α also downregulated vimentin expression and enhanced E-cadherin expression. The ultrastructure of HNF4α-induced cells had more mitochondria and ribosomes compared with the parental cells. After silencing HNF4α expression,EPCK, E-cadherin, AFP, and ALB were downregulated and α-SMA and vimentin were upregulated.CONCLUSION: HNF4α can induce a tendency of differentiation of HSCs into hepatocyte-like cells. These findings may provide an effective way for the treatmentof liver diseases.

An efficient strategy for generation of transgenic mice by lentiviral transduction of male germline stem cells in vivo

Background： Male germline stem cells（MGSCs） are a subpopulation of germ cells in the testis tissue. MGSCs are capable of differentiation into spermatozoa and thus are perfect targets for genomic manipulation to generate transgenic animals.Method： The present study was to optimize a protocol of production of transgenic mice through transduction of MGSCs in vivo using lentiviral-based vectors. The recombinant lentiviral vectors with either EF-1 or CMV promoter to drive the expression of enhanced green fluorescent protein（e GFP） transgene were injected into seminiferous tubules or inter-tubular space of 7-day-old and 28-day-old mouse testes. At 5 or 6 wk post-surgery, these pre-founders were mated with wild-type C57BL/6J female mice（1.5 to 2.0-month-old）.Results： Sixty-seven percent of F1 generation and 55.56 % of F2 offspring were positive for eG FP transgene under the control of EF-1 promoter via PCR analysis. The transgenic pups were generated in an injection site-and age-independent manner. The expression of transgene was displayed in the progeny derived from lentiviral vector containing CMV promoter to drive transgene, but it was silenced or undetectable in the offspring derived from lentiviral vector with transgene under EF-1 promoter. The methylation level of g DNA in the promoter region of transgene was much higher in the samples derived lentiviral vectors with EF-1 promoter than that with CMV promoter,suggesting e GFP transgene was suppressed by DNA methylation in vivo.Conclusion： This research reported here an effective strategy for generation of transgenic mice through transduction of MGSCs in vivo using lentivirus vectors with specific promoters, and the transgenic offspring were obtained in an injection site-and age-independent manner. This protocol could be applied to other animal species, leading to advancement of animal transgenesis in agricultural and biomedical fields.

CELL体系的结构、性能及其应用前景

最初为PS3设计的CELL处理器是一个高性能分布式体系结构处理器,CELL包括硬件CELL和软件CELL。硬件CELL采用多核技术,内核采用RISC指令集结构,指令的动态调度,寄存器换名等都由软件实现,从而大大提高了硬件速度。

Effects of Over-expression of ANXA10 Gene on Proliferation and Apoptosis of Hepatocellular Carcinoma Cell Line HepG2

The effects of over-expression of ANXA10 gene on proliferation and apoptosis of hepatocellular carcinoma cell line HepG2 were elucidated.The human ANXA10 gene was subcloned into the lentiviral vector,PGC-FU,to generate the lentiviral expression vector,PGC-FU-ANXA10.The corrected ANXA10 was confirmed by endoenzyme digestion,and sequencing.Recombinant lentiviruses were produced by 293T cells following the co-transfection of PGC-FU-ANXA10 with the packaging plasmids pHelper1.0 and pHelper2.0.The resulting recombinant lentiviruses carrying ANXA10 were then used to infect human embryonic kidney epithelial cells,and lentiviral particles were produced.The ANXA10 expression in 293T cells was detected by using fluorescent microscope and Western blotting.HepG2 cells were infected,and divided into PGC-Fu-ANXA10 group,PGC-Fu group and HepG2 cell group.The changes of ANXA10 mRNA and protein expression were detected by using RT-PCR and Western blotting respectively.Flow cytometry and MTT assay were performed to examine the changes in cell apoptosis and proliferation respectively.The recombinant PGC-FU-ANXA10 vector was successfully constructed,the ANXA10 protein was detected by using Western blotting,and virus titer was 2×108 TU/mL.The recombinant lentiviruses were effectively infected into HepG2 cells in vitro and the infection efficiency was 70%.At 72 h after infection,the ANXA10 mRNA and protein expression levels in PGC-Fu-ANXA10 group were significantly higher than in PGC-Fu group and HepG2 cell group (P<0.05);the in vitro growth inhibition rate of HepG2 cells in PGC-Fu-ANXA10 group was 24.65%,significantly higher than that in PGC-Fu group and HepG2 cell group (P<0.05),but there was no significant difference between PGC-Fu group and HepG2 cell group;the apoptosis rate in PGC-Fu-ANXA10 group,PGC-Fu group and HepG2 cell group was (51.92±1.41)%,(19.00±1.12)% and (3.59±0.89)% respectively.The apoptosis rate in PGC-Fu-ANXA10 group was significantly higher than in PGC-Fu group and HepG2 cell group (P<0.05).The recombinant lentiviruses PGC-FU-ANXA10 were constructed successfully and infected into HepG2 cells.The overexpression of ANXA10 gene can significantly inhibit proliferation and promote apoptosis of HepG2 cells in vitro.

Overexpression of heme oxygenase-1 protects smooth muscle cells against oxidative injury and inhibits cell proliferation

To investigate whether the expression of exogenous heme oxygenase-1 (HO-1) gene within vascular smooth muscle cells (VSMC) could protect the cells from free radical attack and inhibit cell proliferation, we established an in vitro transfection of human HO-1 gene into rat VSMC mediated by a retroviral vector. The results showed that the profound expression of HO-1 protein as well as HO activity was 1.8- and 2.0-fold increased respectively in the transfected cells compared to the non-transfected ones. The treatment of VSMC with different concentrations of H2O2 led to the remarkable cell damage as indicated by survival rate and LDH leakage. However, the resistance of the HO-1 transfected VSMC against H2O2 was significantly raised. This protective effect was dramatically diminished when the transfected VSMC were pretreated with ZnPP-IX, a specific inhibitor of HO, for 24 h. In addition, we found that the growth potential of the transfected cells was significantly inhibited directly by increased activity of HO-1, and this effect might be related to decreased phosphorylation of MAPK. These results suggest that the overexpression of introduced hHO-1 is potentially able to reduce the risk factors of atherosclerosis, partially due to its cellular protection against oxidative injury and to its inhibitory effect on cellular proliferation.

miRNA-155 Modulates the Malignant Biological Characteristics of NK/T-Cell Lymphoma Cells by Targeting FOXO3a Gene

This study investigated the effects of miRNA-155 on malignant biological characteristics of NK/T-cell lymphoma cell lines and the possible mechanism. The expression of miRNA-155 was detected in lymphoma cell lines from different sources （SNK-6, YTS, Jurkat and DOHH2） by real-time PCR. Lentiviral vectors （pLL3.7） that could overexpress or downexpress miRNA-155 were constructed. Recombinant lentiviral particles were prepared and purified, and their titers determined. The expression of miRNA-155 in the infected SNK-6 cells and the cell proliferation were detected by PCR and CCK-8, respectively. Flow cytometry was used to determine the apoptosis of infected SNK-6 cells. The target of miRNA155 was predicted from Targetscan website. The effect of miRNA155 on FOXO3a expression was examined by Western blotting. The results showed that among the human NK/T-cell lymphoma cell lines SNK-6, YTS, Jurkat and DOHH2, the expression of miRNA-155 was highest in SNK-6. The infection efficiency of the recombinant lentivirns in SNK-6 was more than 70% at multiplicity of infection （MOI） of 100. The expression of miRNA-155 was significantly increased in SNK-6 cells infected by lentivirus vectors with high expression of miRNA-155 （4 times higher than the control group）, and profoundly decreased in those infected with lentivirnses with low expression of miRNA-155. The proliferation of letivirns-infected SNK-6 cells was decreased as the expression of miRNA-155 reduced. The apoptosis rate was increased with the reduction in the expression of miRNA-155. FOXO3a was found to be a possible target of miRNA155, as suggested by Targetscan website. Western blotting showed that the expression of FOXO3a was significantly elevated in SNK-6 cells with miRNA-155 inhibition. It was concluded that reduction in miRNA-155 expression can inhibit the proliferation of SNK-6 lymphoma cells and promote their apoptosis, which may be associated with regulation of FOXO3a gene.