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Objective: To investigate the effects of conditioning regimen on mice liver injury during hematopoietic stem cell transplantation., Methods: Forty healthy 8-10 weeks old female BALB/c mice were randomly divided into control group, non-myeloablative total body irradiation group, myeloablative total body irradiation group, busulfan group and cyclophosphamide group with 8 mice each. The general condition of each mouse was continuously observed. Peripheral white blood cells were counted and pathological changes of liver were examined by hematoxylin and eosin stain under microscope. The ultrastructure changes of hepatocyte and liver vascular endothelium were detected under transmission electron microscope., Results: Compared to control group, each experimental group showed that the count of white blood cells was decreased significantly after conditioning pretreatments (P < 0.05). The conditioning regimen had injury effects on the mice liver in each experimental group. There were various degrees of damage to the hepatocyte and liver vascular endothelium., Conclusion: Total body irradiation, Busulfan and Cyclophosphamide all led to the damage of liver vascular endothelium in hematopoietic stem cell transplantation, and may be the important triggers of hepatic veno-occlusive disease.

Objective: To construct red fluorescent protein (DsRed) and mouse vascular endothelial growth factor (mVEGF) lentiviral expression vector in order to obtain the DsRed and VEGF expression in 293T cells., Methods: The mVEGF was amplified from the lungs of fetal mouse by RT-PCR. The IRES-DsRed was obtained from plasmid pLV-tTRKRAB-red which contains IRES-DsRed sequence by PCR. Then they were cloned into lentiviral expression vector pTK-208. Human embryonic kidney 293T cells were co-transfected with the three plasmids by lipofectamine, and the expression of DsRed was examined under fluorescent microscope 24 h and 48 h after transfection. 48 h and 72 h later the viral supernatant was used to infect 293T cells, the expression of DsRed was examined under fluorescent microscope and Western blot assays was used to examine the expression of mVEGF in cells and the culture medium., Results: The recombinant lentiviral vector pTK208-mVEGF-IRES-DsRed was constructed, the virus titres were above 10(6) PFU/mL in the supernatant. After infection, DsRed and mVEGF proteins were successfully expressed in 293T cells., Conclusion: The lentiviral vector pTK208-mVEGF-IRES-DsRed contains DsRed and mVEGF were successfully constructed and expressed in 293T cells, it could provide a basis for further researches on the pathophysiological mechanism of VEGF and lay the foundation of gene therapy.

Objective: To investigate the effect of allo-HSCT irradiation preconditioning on the homing of donor endothelial progenitor cells (EPCs)., Methods: The mononuclear cells were collected form mice bone morrows, then cultured in EGM-2 medium. The cells were identified after 5-7 days and were labeled with CFSE. The mice were divided randomly into four groups, control, EPCs transplantation, irradiation, and irradiation EPCs transplantation. The circulating ECs, circulating EPCs and CFSE positive cells in the peripheral blood, spleen and bone morrows of the mice were measured by FCS. The histological examination, electron microscope and immunofluorescence analyses of those tissues were also performed. The distribution of labeled EPCs was observed., Results: The cells were identified as CD45low, CD133+ CD31+, Dil-ac-LDL+. The number of those cells in the irradiation group increased gradually after preconditioning and reached peak at day 3 or 5 and sustained the peak level for 2 days, which was significantly different from the control (P < 0.05). Under the light microscopy and TEM, damaged endothelium was detected. In the irradiation and EPCs transplantation groups, CFSE labeled cells increased and reached peak at 18 hours, and then decreased till 72 hours. Green fluorescent cells were observed in different tissues at different times, and the strongest fluorescence intensity occurred at 36 hours., Conclusions: The EPCs can be induced from mice bone morrow mononuclear cells, with expressed phenotypic characterization. Irradiation in transplantation preconditioning can cause severe endothelium injury, which can start the mobilization of EPCs. The endothelium injury can induce extrinsic EPCs homing to the tissues which are badly injured. Endothelial progenitor cells

Objective: To construct the self-inactivating lentiviral vector containing murine Foxp3 gene and detect its expression in the CD4+ CD25- T cells., Methods: The bicistronic 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 by lentiviral packing system, which including the packaging plasmid deltaNRF, the transfer plasmid and the envelope plasmid VSVG using liposome. Then manifested the efficiency of gene transduction with Flow Cytometry (FCM) and the expressions of Foxp3 mRNA and protein were assayed by RT-PCR and Western blotting., Results: The lentiviral transfer plasmid pXZ208-Foxp3-IRES-GFP was constructed, the virus titres were above 10(6) IU/mL in the supernatant. After coculture with thus cells, the CD4+ CD25- T cells in experimental group expressed significantly higher amounts of Foxp3 mRNA and protein than control group, and the efficiency of gene transduction was about 55.95%., Conclusions: The self-inactivating lentiviral vector containing murine Foxp3 gene are successfully constructed. This gene delivery system can transduce Foxp3 gene into CD4+ CD25- T cells with highly efficient expression.

Objective: To develop a method for quantitative assay of alpha-Gal on pig cell surface., Methods: After centrifugation of the anticoagulated blood samples, the white blood cell layer was collected for addition of FITC-BSIB4 and analysis using FCM as well as Laser Scan Confocal Microscope (Bio-Rad)., Results: With human blood group B as negative control, and mouse myeloma SP2/0 cells as positive control. the peripheral blood cells of two Chinese pigs were all positive, there were about 9.16 x 10(5) alpha-Galactosyl epitopes expressed on the cell-membrane of granulocytes 1.37 x 10(5) on the monocytes of Banna Minipig Inbred Line (BMI), and 1.16 x 10(6) on the granulocyte and 2.45 x 10(5) on the monocytes of Sichuan White Pig (SWP). The levels of alpha-Galactosyl expression on the surface of blood cells in BMI and SWP differed in various cells, the sequence from high to lower expression levels being on the platelets, granulocytes, monocytes, erthrocytes in each species of pig., Conclusion: The method is useful for quantitative assay of alpha-Gal on pig cell surface, and the assay may be used for alpha-Gal for on-site large-scaled screening of living pigs.