AIM: The expression of the mouse ? 2 microglobulin (? 2 m) in NIH3T3 cells transfected with the mouse ? 2m sense and antisense RNA was detected to clarify the effect of mouse ? 2m sense and antisense RNA on the expression of MHC classⅠgene. METHODS: The mouse sense and antisense RNA, pcDNA3-? 2mSN and pcDNA3-? 2mAN, were constructed and were transfected into NIH3T3 cells by lipofectamine. RT-PCR and Western blot were used to detect the expression of ? 2m in those cells. RESULTS: The expression of the mouse ? 2m in the cells transfected with pcDNA3-? 2mSN was increased, while it was decreased in those cells transfected with pcDNA3-? 2mAN. CONCLUSION: pcDNA3 -? 2mAN can downregulate the expression of the ? 2m in NIH3T3 cells.

AIM: To detect CD23 mRNA expression in B-cell lymphoma. METHODS: 20 pathological diagnostic conformed B-cell lymphoma samples were selected. Surface CD23 protein expression was examined by SP-immunohistochemistry and CD23 mRNA was detected by in situ hybridization with digotoxin labeled CD23 DNA probe.RESULTS: The positive rates of both surface CD23 protein and CD23 mRNA expression in B-cell lymphoma samples were more than 90%. CONCLUSIONS: The high expression of CD23 was showed in B-cell lymphoma both in mRNA and in protein levels. The results of this study was useful for understanding the molecular mechanism of B-cell lymphoma and for clinical diagnosis and treatment of the disease.

AIM: To construct a recombinant adenovirus expression vector containing CTLA4Ig gene.METHODS: The CTLA4Ig gene derived from the plasmid PCDNA3.0/CTLA4Ig by using polymerase chain reaction (PCR) was inserted into the backward position of cytomegalovirus (CMV) immediate early promoter of the shuttle plasmid (pAdTrack-CMV). After being identified by endonuclease, PCR and sequencing, the recombinant shuttle plasmid pAdTrack-CTLA4Ig was co-transformed into E.coli. BJ5183 cells with the adeoviral backbone plasmid pAdEasyl-1 to obtain the homologous recombination. The adenovirus was generated in 293 cells. A series methods such as PCR and fluorescence microscope was employed to identify the generated recombinant adenovirus. RESULTS: Recombinant CTLA4Ig adenoviruses were constructed and the titer of virus was generally up to 1.65?10 12 phaque forming units per liter (PFU/L). CONCLUSION: Success in constructing recombinant pAdTrack-CTLA4Ig will be the base of the further research on its expression in the mammalian cells, and be potenially used in the prevention of transplant rejection and autoimmunity diseases.

AIM: To investigate the differentiation potential of human bone marrow mesenchymal stem cells (hBMMSCs) into hematopoietic cells in vivo. METHODS: hBMMSCs prepared from the bone marrow-aspirate sample obtained from healthy human donors were culture-expanded in vitro with 5-8 passages. hBMMSCs(P5-8, 4.8?10 5 cells/mouse) were injected into the severe combined immuodeficiency (SCID) mice treated by cyclophosphamide(CPA) and various tissues were analyzed at 35 days post-transplant for the presence of differentiated human cells. RESULTS: hBMMSCs(P5-8) viability, which was determined by typan blue staining at the end of the harvest and before infusion, was greater than 95% in every infusate at both time points. Cells characterized by flow cytometry using human MSC-specific monoclonal antibodies were uniformly positive for CD29, CD44, CD90, CD105, CD106, CD166 and negative for CD11a, CD14, CD34, CD38, CD45, CD80, CD86 which are common on cells of the hematopoietic lineages. Analysis of PB demonstrated that 5 of 6 hBMMSCs transplanted SCID mice had low level of circulating human CD45 +/ H-2D d- cells(range from 0.17% to 0.36%)and CD34 +/ H-2D d- cells(range from 0.10% to 0.50%). Analysis of BM for the presence of hematopoietic chimerism demonstrated human CD45 +/ H-2D d- cells and CD34 +/ H-2D d- cells in the marrow of 4 out of 6 hBMMSCs transplanted SCID mice (0.10%-0.19% and 0.03%-0.52%, respectively). Human hematopoietic cells with these same phenotype were also detected in the spleen 4 of the hBMMSCs transplanted SCID mice (range from 0.19% to 1.65% ,from 0.20% to 0.26%, respectively). No human hematopoietic cell was seen either in the PB, BM or spleen of all control animals. CONCLUSION: hBMMSCs have the ability to differentiate into blood cells of multiple lineages, including CD34 + hematopoietic stem cells/progenitor cells (HSC/HPC).

AIM: To construct recombinant adenovirus vector containing brain derived neurotrophic factor, (BDNF) gene using bacterial homogenous recombination, and investigate the expression in expanded rat mesenchymal stem cells (rMSC) in vitro. METHODS: BDNF gene and proBDNF gene were subcloned into adenovirus shuttle plasmid pAdTrack-CMV containing enhanced green fluorescent protein gene (EGFP) expression cassette, forming shuttle vector of pAdTrack-BDNF, and pAdTrack-proBDNF, and co-transformed into BJ5183 bacterial cells with adenovirus backbone vector pAdEasy-1 using chemical transformation. After the recombinant adenovirus vector was obtained, the identified recombinant adenovirus plasmid DNA was digested with Pac I and transfected to 293 cells to package recombinant adenovirus particles. rMSC were infected by recombinant adenovirus and EGFP expression was detected using fluorescent microscope. Infection efficiency was assessed by flow cytometrics. Western blotting identified expression of Ad -proBDNF and Ad-BDNF in rMSC. rMSC infected with Ad -proBDNF and Ad-BDNF were induced to differentiate into neuron-like cells. rMSC infected with Ad -proBDNF and Ad-BDNF were injected into nude mice and assessd in vivo. RESULTS: We successfully constructed the recombinant adenovirus Ad -proBDNF and Ad-BDNF that expressed in expanded rMSC in vitro.CONCLUSION: Recombinant adenovirus high-effectively mediates Ad -proBDNF and Ad-BDNF expression in expanded rMSC in vitro and in vivo.

AIM: To study the effects of cotransplantation of donor-derived bone marrow mesenchymal stem cells on graft versus host disease in a rat allogeneic bone marrow transplantation model. METHODS: Fisher 344 rat bone marrow MSCs were isolated and cultured to the fifth passage (P5) in vitro . The recipient Wistar rats were conditioned with lethal total body irradiation and transplanted with F344 rat bone marrow cells and spleen cells in the presence or absence of (P5) MSCs. The onset time of graft versus host disease (GVHD), incidence of GVHD and survival time were monitored. RESULTS: Cotransplantation of MSCs deferred the onset time of GVHD[(19.1?1.7) d vs (15.6?1.5) d, P

AIM: To investigate multi-potential of rat bo ne marrow mesenchymal stem cells (rBMMSC) and mutation inclination, the rBMMSC w ere long passaged in vitro. METHODS: Cellular cycles of diff erent passages were assayed by FA CSan flow cytometry and karyotypes of passage 6, passage 25 and passage 45 were compared by G-binding analysis. RESULTS: The early passages and long-term passages all showed st rong proliferation; passage 6, passage 25 and passage 45 all showed normal karyo type. CONCLUSION: Long-term culture and passage of rBMMSC still remain s strong proliferation. With this capability, the mutation inclination is not enhanced.

AIM: To investigate the differentiation from human mesenchymal stem cells (hMSC) to adipocytes.METHODS: hMSC were separated from rib marrow and expanded in culture medium. To detect the surface antigens, the labeled cells were analysed on a FACScan flow cytometer. hMSC were induced with dexamethasone, insulin, 1-methy1-3-isobutylxanthine and indomethacin which acted as adipocyte differentiation inducer. The cells were stained with Oil Red O. The number of adipocytes were counted on a phase-contrast microscope.RESULTS: hMSC were expanded as undifferentiated cells in culture for more than 5 passages. The isolated cultured MSC comprised a single phenotypic population and displayed a fibroblast-like morphology. These expanded, attached MSC were uniformly positive for CD29, CD44, CD90, CD105, CD166 and didn't express CD14, CD34, CD45, CD11a. After induced with induction medium, lipid vacuoles were first detectable within the cells at 48 hours. Two weeks later, more than 85% MSC differentiated into adipocytes which displayed a perinuclear accumlation of lipid vacuoles, as detected by Oil Red O. CONCLUSION: hMSC can be induced to differentiate into adipocytes. [

AIM: To observe the relationship between the immune deviation of Th1 and Th2 cell clones and class Ⅱ major histocompatibility complex (MHC) antigen expression in different stages of acute rejection in transplanted hearts. METHODS: Heart transplantation were performed in rats.Isografts and non-transplanted animals were used as control group. Donor class II MHC antigen expression were detected with monoclonal antibodies and immunostaining technique and the amount of type Ⅰ and Ⅱ cytokines mRNA expression were detected by semiquantitative RT-PCR in cardiac allografts. RESULTS: Myocardial IL-2 mRNA and donor class Ⅱ MHC antigen expression were significantly in creased, accompanied with development of acute rejection( P

AIM: To investigate the differentiation from human mesenchymal stem cells (hMSC) into osteoblasts. METHODS: MSC were separated from human marrow with Ficoll-Paque reagent and expanded in cuture medium. To detect the surface antigens, The labeled cells were analysed on a FACScan flow cytometer. hMSC were induced to differentiate from mesenchymal stem cells into osteoblasts with dexamethasone, vitamin C, ?-GP. Cell morphology?AP activity?calcium deposition and osteopontin were detected. P10 MSC were compared to P3 MSC in the tendency of osteoblastic differentiation. RESULTS: The cultured MSC comprised a single phenotypic population and displayed a fibroblast-like morphology. hMSC showed a strong self-renewal capacity. After primary culture, approximately (5-6)?10 5 cells were obtained. These expanded attached MSC were uniformaly positive for CD29,CD44,CD59,CD105,CD166 and didn't express CD11a, CD14, CD33, CD34, CD45, CD38, CD80, CD86, CD117. After osteoblasts induction, the cells changed from spindle-shape to cuboidal and polygonal in cell morphology. The AP activity increased gradually and many scattered calcium nodes were observed. The expression of osteopontin was positive. CONCLUSION: hMSC can be induced to differentiate into osteoblasts.