This study was aimed to clone the gene coding mouse fibroblast growth factor receptor-1 (fgfr1), to construct the recombinant lentiviral vector of truncated form fgfr-1 (Δfgfr1) carrying enhanced green fluorescence protein (EGFP) and to investigate its expression in eukaryotic cells (293FT cells). The full length fgfr1 gene was cloned by RT-PCR using brain tissue of BALB/c fetal mouse as template and inserted into PCR-Blunt vector, a truncated fgfr1 fragment was produced by site-directed mutagenesis for deleting intracellular phosphorylated domain, then was subcloned into a lentiviral vector and cotransfected into 293FT packaging cells together with envelope plasmid and packaging plasmid by lipofectamine 2000. Viruses were gathered and concentrated using ultracentrifuge, and then transfected into 293FT cells. Expression of EGFP was detected by fluorescent microscopy and flow cytometry (FCM), and the truncated FGFR1 protein was detected by Western blot. The results demonstrated that mouse fgfr1 gene was cloned and the lentiviral expression vector LV-IRES-EGFP-Δfgfr1 and control vector LV-IRES-EGFP were successfully constructed. The lentiviral particles were correctly packaged, and the virus titers were above 10(8) TU/ml in the supernatant after concentration. Expression of EGFP was detected by fluorescent microscopy in 293FT cells post transfection, and the transfection efficacy was > 95% determined by FCM. Expression of FGFR1 protein detected by Western blot was significantly higher than that in control group. It is concluded that the truncated gene fgfr1 along with the gene coding EGFP is successfully inserted into a lentiviral vector to construct a recombinant lentiviral vector, which can be expressed in eukaryotic cells.
Animals ; Cell Line ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Mice ; Mice, Inbred BALB C ; Plasmids ; Receptor, Fibroblast Growth Factor, Type 1 ; genetics ; Recombinant Fusion Proteins ; genetics ; Transfection

In order to construct a lentiviral vector carrying human VE-cadherin gene, and to express VE-cadherin in Sup-B15 cells, the VE-cadherin gene was amplified by RT-PCR from the human placenta, and then cloned into pCR-Blunt vector. The VE-cadherin DNA fragment was subcloned into pLB vector to generate a lentiviral vector pLB-VEC. Recombinant lentivirus was generated by co-transfection of three-plasmids into 293FT packing cells using lipofectamine 2000. The Sup-B15 cells were transfected by the lentivirus. The post-transfected Sup-B15 cells were observed by microscopy and flow cytometry. Western blot was used to determine the expression of VE-cadherin. The results showed that the VE-cadherin DNA fragment was amplified from human placenta and was cloned into pCR-Blunt vector, the recombinant lentiviral vector pLB-VEC was successfully constructed. High titer lentivirus was prepared by 3-plasmid packing system, and transfected into Sup-B15 cells in vitro effectively. The obviously morphological changes occurred in transfected cells, the expression of VE-cadherin protein could be detected in Sup-B15 cells via flow cytometry and Western blot. It is concluded that the lentiviral vector pLB-VEC carrying human VE-cadherin gene is successfully constructed; VE-cadherin gene is expressed in Sup-B15 cells via lentiviral vector transfection, which provides an optional tool for further study on the mechanism of VE-cadherin controlling leukemia development.
Antigens, CD ; genetics ; Cadherins ; genetics ; Cell Line, Tumor ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Plasmids ; Recombinant Fusion Proteins ; genetics ; Transfection

Standardized Manipulation of Acupuncture and Moxibustion Part 8: Intradermal Needle was compiled with the following principles. The compiling standard, technical features and clinic manipulations of intradermal needle were taken as the basic principle for compiling. Literature research, expert survey and clinic practice verification were applied as the drafting methods. The key issues were focused on the relationship between standardization and individualization, normalization and effectiveness, qualification and quantification. And the postural selection, reinforcing and reducing manipulations, fixing materials and embedding duration involved in intradermal needling were emphasized particularly. At the same time, details and the future way of thinking of intradermal needle were expounded in this article as well.
Acupuncture Therapy ; instrumentation ; standards ; China ; Humans ; Moxibustion ; standards ; Needles ; standards ; Reference Standards

OBJECTIVETo investigate the role of endothelial progenitor cell (EPC) injection in the restoration of vascular niche in bone marrow (BM) after allo-BMT in mice, and to observe its role on hematopoietic reconstitution.

METHODS6-8 weeks old female BALB/c (H-2(d)) were randomized to BMT (allo-BMT) group and combined EPC transplant (allo-BMT + EPC) group. For allo-BMT group, female BALB/c mice were lethally irradiated with 60Co source, and then were injected intravenously with 5×10(6) BM cells from donor mice. In allo-BMT + EPC group, recipient mice were injected intravenously with 5×10(6) BM cells and 5×10(5) EPC from donor mice. The recipients were monitored for histological changes of endothelial cells (EC) in BM. The recovery of hematopoiesis was determined by white blood cell counts and the proportion of reticulocytes in circulation and the proportion of hematopoietic stem cells (HSC) in BM. The histology of hematopoiesis in BM was also detected.

RESULTSThe in vitro induced EPC successfully homed to the bone marrow of recipients. The ECs of allo-BMT recipients were destructed severely, while the structures of ECs were restored in EPC treated recipients. 10 and 15 days after allo-BMT, the amount of Lin-c-kit(+)Sca-1(+) cells in the BM of the EPC treated group were (20.31 ± 2.65)×10(3) per mouse and (10.26 ± 2.19)×10(3) per mouse, while the allo-BMT group's were (9.61 ± 0.98)×10(3) per mouse and (4.09 ± 1.34)×10(3) per mouse; and 15 days after allo-BMT, the amount of white blood cell counts and proportion of reticulocytes of the EPC treated group were (1.20 ± 0.11)×10(9)/L and (2.35 ± 0.30)% comparing to the allo-BMT group which were (0.65 ± 0.10)×10(9)/L and (1.63 ± 0.20)%.

CONCLUSIONCo-transfer of donor EPC restores the ECs of bone marrow, which consequently promotes hematopoietic reconstitution in murine allo-BMT.

Animals ; Bone Marrow Cells ; cytology ; Bone Marrow Transplantation ; methods ; Endothelial Cells ; cytology ; Female ; Leukocyte Count ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Reticulocyte Count ; Stem Cell Niche ; Stem Cells ; cytology

This study was aimed to propagate and identify the prdm1 gene-knockout mice, so as to lay the foundation for studying Blimp-1 protein. Two kinds of transgenic homozygous mice with B6.prdm1(flox/flox) and B6.Lck-Cre were feed and propagated; after successful propagating, the first passage mice were obtained; after the first passage mice were copulated once again, the genotypes were obtained as follows: B6. prdm1(wild/wild). Lck-Cre, B6. prdm1(wild/wild), B6.prdm1(flox/flox). Lck-Cre, B6.prdm1(flox/wild). Lck-Cre, B6.prdm1(flox/flox), B6. prdm1(flox/wild). The genomic DNA of second passage mice was extracted, the Cre and loxp gene fragments were amplified by PCR, then the size of Cre and loxp genomic DNA were detected by agarose gel electrophoresis. The mice with B6.prdm1(flow/flox). Lek-Cre were used as conditionally prdm1-knockout mice, B6.prdm1(flox/wild). Lck-Cre mice, B6.prdm1(flox/flox) and B6 mice were used as controls. The spleen T lymphocytes and B lymphocytes were sorted by using magnetic beads, the blimp-1 target protein was identified by Western blot. The results showed that the two transgenic homozygous mice had the ability to reproduce, and the separation ratio of second passage mice generated from propagation of their offspring cach other meet Mendelian laws, and the prdm1 gene-knockout mice also could successfully obtained. It is concluded that the application of Cre-loxp system may successfully obtain plentiful prdm1 gene-knockout mice.
Animals ; Genotype ; Mice ; Mice, Inbred C57BL ; genetics ; Mice, Knockout ; genetics ; Reproduction ; Transcription Factors ; genetics

OBJECTIVETo prepare the genetic engineering regulatory T cells (Treg) via the self-inactivating (SIN) lentiviral vectors carrying Foxp3 gene, and assay the phenotype and abilities of its proliferation and immunosuppression.

METHODSThe bicistronic SIN lentiviral transfer plasmid containing Foxp3 gene and internal ribosomal entry site-green fluorescent protein gene (IRES-GFP) was constructed. Human embryonic kidney 293T cells were co-transfected using liposome by lentiviral packing system, which included the packaging plasmid Delta NRF, the transfer plasmid and the envelope plasmid VSVG. The efficiency of gene transduction and the expressions of Foxp3, CD25, GITR, CTLA-4 of CD4(+)CD25(-) T cells, which were isolated by magnetic beads from the spleen, and then co-cultured with 293T cells, were detected by flow cytometry (FCM). The proliferative and suppressive capacities of transduced T cells were estimated by Cell Count Kit-8 (CCK-8) and the cytokine production was performed by ELISA.

RESULTSThe lentiviral transfer plasmid pXZ208-Foxp3-IRES-GFP was successfully constructed, the virus titers were above 10(6) IU/ml in the supernatant. pXZ208-IRES-GFP was used as control group. After cocultured, the CD4(+)CD25(-) T cells expressed significantly higher Foxp3, CD25, GITR and CTLA-4 in experimental group than in control group. Upon stimulation with anti-CD3 epsilon and APCs, the proliferative capacity of Foxp3-transduced T cells and the production of IL-2, IL-4, IL-10, IFN-gamma were significantly lower than those in control group (P < 0.01); Foxp3-transduced T cells also significantly inhibited the proliferation of CD4(+)CD25(-) T cells.

CONCLUSIONSThe genetic engineering Treg mediated by SIN lentiviral vectors are successfully constructed and their phenotype and function are similar to natural CD4(+)CD25(+) Treg.

Animals ; Cell Proliferation ; Cells, Cultured ; Forkhead Transcription Factors ; genetics ; metabolism ; Genetic Engineering ; Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus ; genetics ; Mice ; Mice, Inbred BALB C ; Phenotype ; T-Lymphocytes, Regulatory ; cytology ; immunology ; metabolism ; Transfection

This study was purposed to investigate the effects of high-dose dexamethasone on structure and function of thymic epithelial cells (TEC). Male C57BL/6 mice aged 6 to 8 weeks were used as experimental animals. The mice were injected intraperitoneally with dexamethasone (20 mg/kg), and the other mice treated with saline were used as controls. Thymus was harvested at day 5 after treatment. The histological changes of the treated thymus were monitored by HE staining and in situ immunofluorescence staining. The ratio of each subset in the thymus were analyzed by using flow cytometry, and quantitative PCR was applied to detect the expression levels of IL-22 and Foxn1, which represent the regenerative function of thymus. The results showed that compared with control mice, the structure of TEC in mice treated with high-dose dexamethasone was damaged and the thymic cell number was declined dramatically (P < 0.05); the ratios of thymus cell subsets were changed, the number of double positive (DP) thymus cells among these subsets declined sharply (P < 0.05); the expression levels of Foxn1 and IL-22 increased by 34 and 8 folds respectively. It is concluded that the use of high-dose dexamethasone can lead to damage of the structure and function of TEC, and induce up-regulation of the expression of genes related to thymus repair.
Animals ; Dexamethasone ; administration & dosage ; adverse effects ; Epithelial Cells ; cytology ; drug effects ; Forkhead Transcription Factors ; metabolism ; Interleukins ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Thymus Gland ; cytology ; drug effects

This study was aimed to investigate the homing capacity of CXCR4 overexpressed mesenchymal stem cells (MSC) and their effect on hematopoietic recovery. The 293FT packaging cell line was transfected with the recombinant lentiviral vector LV-CXCR4-IRES-EGFP and LV-IRES-EGFP to produce lentivirus. Mouse MSC were then infected with viral supernatant. Male BALB/c mice were sublethally irradiated and then were injected intravenously with 5×10(5) MSC. General status and survival rate of mice were observed every day. On day 3, 7, 14, 21 and 28, peripheral blood samples were collected to calculate the number of white blood cells (WBC) and red blood cells (RBC), the ratio of reticulocyte to platelet, the number of platelet was detected by flow cytometry. The recovery of bone marrow and spleen was pathologically monitored. The proportion of MSC implantation was analysed by PCR. The results showed that the peripheral blood cells displayed the tendency of firstly increasing and then decreasing to their normal level. Generally, recovery of WBC level was earlier in mice infused with MSC (P < 0.05) . The histopathological examination of spleen and bone marrow showed a faster hematopoietic recovery in CXCR4-MSC group than the other two groups. And the donor MSC could be detected in the recipients on day 7, 14, 21 and 28. It is concluded that infusion of CXCR4-MSC enhances the implantation of hematopoietic stem cells and promotes hematopoietic recovery of the sublethally irradiated mice.
Animals ; Bone Marrow Cells ; cytology ; Genetic Vectors ; Hematopoiesis ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred BALB C ; Organisms, Genetically Modified ; Radiation Injuries, Experimental ; therapy ; Receptors, CXCR4 ; genetics ; Transfection ; Whole-Body Irradiation

OBJECTIVETo explore the silencing effect of short hairpin RNA (shRNA) on the CD28 of mice T lymphocytes by CD28-shRNA expressing plasmid evaluate the interfering effects (chronology and stability) mediated by shRNA and select out the most efficient CD28 shRNA sequence.

METHODSThree CD28 specific and one non-specific shRNA expressing plasmids were constructed and then transfected separately into mice spleen T lymphocytes. Non-transfected cells and non-specific shRNA were taken as controls. Inhibitory effect of CD28 shRNA was demonstrated by real-time quantitative PCR and Western blots. The sequence of the highest RNA interference (RNAi) efficacy was screened.

RESULTS(1) CD28 shRNA expressing plasmids were successfully constructed; (2) Three CD28 specific shRNAs effectively inhibited the expression of CD28 at the mRNA and protein levels, and there was a statistically significant difference comparing with the controls (P < 0.01): The copies of CD28 in mice spleen cells at the mRNA levels were persistently decreased by 99.62%, 99.89% and 99.80% respectively after 20 days, and so did at the protein level [(84.90 +/- 0.65)%, (96.49 +/- 0.03)%, (91.76 +/- 0.32)% respectively]. The highest inhibitory rate was in CD28 shRNA-2 group.

CONCLUSIONS(1) Specific shRNA can mediate long-term and stable silencing effects on CD28 gene; (2) shRNAs matching different sites of CD28 gene exert differential inhibitory effects.

Animals ; CD28 Antigens ; genetics ; Cells, Cultured ; Gene Expression Regulation ; Mice ; Mice, Inbred C57BL ; Plasmids ; genetics ; RNA Interference ; T-Lymphocytes ; metabolism ; Transfection

Recently, gene therapy has been become a promising approach to cure hemophilia A, a most common recessive bleeding disease. The aim of this study was to determine the perspective of lentiviral vector in hemophilia A gene therapy in vitro and in NOD/SCID mice. Lentivirus transfer vector pXZ9/BDDFVIII containing human B-domain-deleted Factor VIII-IRES-eGFP coding sequence and mock control pXZ9 were constructed. Lentivirus was prepared by co-transfecting 3 plasmids into 293FT cells. 293FT, HLF, human bone marrow mesenchymal stem cells and Chang-liver cells were transfected with the prepared virus. Coagulant activity of human FVIII, human FVIII antigen, human FVIII mRNA transcription and genomic integration were assayed by ELISA, one-step method, RT-PCR and PCR after infection. Lentiviral particles were concentrated by ultracentrifugation and NOD/SCID mice were transfected via portal vein injection. Human FVIII antigen in mouse blood plasma was analyzed by ELISA. eGFP expression was observed by fluorescent microscopy and human FVIII transcription in mouse liver was analyzed by RT-PCR at one month after transduction. The results showed that the high titer of recombinant virus was prepared and used to efficiently transduce the target cells in vitro. At 72 h after transfection, high levels of FVIII activity and FVIII antigen were detected. Human FVIII gene transcription could be detected in the liver of NOD/SCID mice received lentiviral particles carrying FVIII gene. Mouse hepatocytes were transfected with recombinant lentivirus efficiently in vivo. Human FVIII level in mouse blood plasma reached to (49 ± 6) mU, (54 ± 8) mU and (23 ± 4) mU at 72 h, one week and one month after transfection respectively. It is concluded that the lentiviral particles carrying BDDhFVIII gene can high efficiently transfect the target cells both in vitro and in vivo, and the transfected target cells can secrete hFVIII efficiently. The sustained expression of human FVIII in NOD/SCID mice is observed after lentivirus transfection via portal vein injection.
Animals ; Cell Line ; Factor VIII ; genetics ; Gene Expression ; Genetic Therapy ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Peptide Fragments ; genetics ; Plasmids