Establishment of Humanized Mouse Model by Using Transplantation of Mobilized Peripheral Blood Stem Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the hematopoietic reconstitution in immunodeficiency NPG(TM) mice after transplantation of G-CSF-mobilized peripheral blood CD34(+) hemopoietic stem cells.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Construction and expression of recombinant adenovirus vector Ad5-hTRX-EGFP / 中国实验血液学杂志

This study was purposed to construct and prepare the recombinant adenovirus vector carrying human thioredoxin (hTRX) and enhanced green fluorescence protein (EGFP), and transfect it into HEK293 cells, so as to lay a foundation for further gene therapy. The PCR-amplified products of hTRX with a pair of primers containing Not I and EcoR V restriction sites were subcloned into shuttle plasmid pDC316-mCMV. HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid pDC316-hTRX-EGFP and large adenovirus-helper plasmid pBHGlox (delta) E1, 3Cre in mediation of liposome. The obtained replication-defective recombinant adenovirus pAd-hTRX-EGFP was co-transfected in HEK293 cells, purified by CsCl gradient centrifugation, counted for virus particles and determined for titer. The recombinant adenovirus was identified by PCR. The HEK293 cells were then transfected with adenoviruses and assayed by flow cytometry. The expression of hTRX was confirmed by Western blot. The results showed that according to PCR and restriction endonuclease assay, the target gene was inserted into recombinant adenovirus vector successfully. The sequence of fusion gene was the same as that of designed fragments. The titer of the purified recombinant adenovirus pAd-hTRX-EGFP was 5.558×10(10) pfu/ml. A transfection efficiency of 92.25% could be achieved at MOI = 100. Western blot further confirmed that hTRX was efficiently expressed in HEK293 cells. It is concluded that recombinant adenovirus vector containing hTRX has been constructed successfully and obtained highly efficient virus that can express efficiently in HEK293 cells, which laid a foundation for further investigation.

Reconstruction and preparation of lentiviral vector system expressing dual-reporter genes / 中国实验血液学杂志

This study was aimed to construct, package and purify the recombinant lentivirus vector carrying the firefly luciferase gene (FLUC) and red fluorescent protein gene (RFP) and to transfect the recombinant lentivirus into HeLa cells, so as to observe the expression levels of these two genes. The FLUC and RFP genes were amplified by RT-PCR and inserted in the lentiviral expression vector (pLenti-Bi-cistronic) to construct the lentiviral vector pLenti-FLUC-RFP. The viral particles were generated by cotransfection of 293T cells with pLenti-FLUC-RFP and three packaging vectors, and the virus titer was determined by calculating the percentage of RFP positive cells. After transfection of pLenti-FLUC-RFP into HeLa cells, the expression of RFP was observed by fluorescent microscopy, and the activity of FLUC was determined by luciferase reporter gene assay kit. The results showed that the inserting orientation of the RFP and FLUC genes in the lentiviral vector pLenti-FLUC-RFP were verified by restriction analysis. Targeted RFP and FLUC sequences were confirmed by DNA sequencing. The final titer obtained was 1×10(7)TU/ml. The expressions of RFP and FLUC were observed in the transfected HeLa cells. It is concluded that the pLenti-III-FLUC-RFP recombinant lentivirus vector carrying RFP gene and FLUC gene with high viral titer is constructed and packaged successfully, and provides experimental basis for studying dynamic distribution of mesenchymal stem cells in vivo.

Involvement of MAPK pathway in the osteoblastic differentiation of mouse mesenchymal stem cells / 中国实验血液学杂志

This study was purposed to investigate the effect of mitogen-activated protein kinase (MAPK) pathway on the osteoblast differentiation of mouse mesenchymal stem cells (MSCs), MSCs were isolated from mouse compact bone and serially passaged. After being cultured in osteogenic induction medium, the phosphorylation levels of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 were detected by Western blot. The effects of corresponding pathway inhibitors including PD98059, JNK II and SB203580 on alkaline phosphatase (ALP) and calcium accumulation in the osteoblastic differentiation of MSCs were determined by ALP staining and von kossa staining respectively. The results showed that MAPK pathway including ERK, JNK and p38 was activated in differentiation of MSCs into osteoblasts. ALP activity of MSCs increased in the early phase by addition of PD98059 treatment, whereas ALP activity and calcium accumulation were not observed via JNK II treatment. However, SB203580 strongly inhibited the ALP expression and the calcium accumulation. It is concluded that p38 plays a positive role in the osteogenic differentiation of MSCs, and ERK is probably a negative factor at the early phase of differentiation, but the effect of JNK is not essential.

Immunoregulatory function of mesenchymal stem cells and application of mesenchymal stem cells in therapy of autoimmune disease / 中国实验血液学杂志

Mesenchymal stem cells (MSCs) are multipotent cells derived from many adult tissues, which can differentiate into cells of the mesodermal lineage, such as adipocyte, osteocyte and chondrocyte, as well as cells of other embryonic lineages. They are a promising tool for tissue engineering. In addition, MSC interacts with immune system, suppressing T cell, B cell and NK cell function and dendritic cell activities. MSC migrates to injured tissue to promote the survival of damaged cells and induces peripheral immune tolerance. The role of MSC in reducing the incidence and severity of graft versus host disease (GVHD) clinically has recently been reported. The immunoregulatory function of MSCs also shows a growing promise in the therapeutic application in autoimmune diseases. This review discusses the mechanism of MSC immunomodulatory ability and its therapeutic potential in autoimmune diseases.