The article reviews the biologica l characteristics of stem cells and discusses the possibility of utilizing stem ce lls from various origins for diabetes therapy.

BACKGROUND: Dendritic cells play an important role in antigen present in vivo, and the mechanism of tumor cells in escaping the antigen presentation of dendritic cells existed in the patients with tumor.OBJECTIVE: To sensitize dendritic cells from human peripheral blood with apoptotic hepatoma cells induced by mitomycin.DESIGN: A randomized control trial by taking apoptotic hepatoma cell sensitized dendritic cells as the observed subjects.SETTINGS: Institute of Field Surgery, Daping Hospital, the Third Military Medical University of Chinese PLA; Institute of Radiation Medicine,Chinese PLA Academy of Military Medical Sciences.MATERIALS: The experiments were carried out in the Institute of Radiation Medicine, Chinese PLA Academy of Military Medical Sciences from April 1998 to May 1999. The cell strain was the QBC939 bile duct cancer cell strain, and mitomycin was used as the antitumor drug.METHODS: Mononuclear cells were isolated from the peripheral blood of normal people, 50μg/L granulocyte-macrophage colony stimulating factor(GM-CSF) and 1 000 U/mL interleukin-4 (IL-4) were added, once every other day for 4 times. On the 3rd day of culture, the apoptotic bile duct cancer cells induced by mitomycin was added, and then cultured in vitro for 4 days, finally the dendritic cells were collected.MAIN OUTCOME MEASURES: ① The identification of the cultured dendritic cells was observed; ② The dendritic cells were co-cultured with necrotic and normally cultured bile duct cancer cells respectively, and the phagocytized apoptotic body loaded antigens were observed; ③ The immunostimulatory activity of dendritic cells (1×103, 5×103 and 1×104/well)and that after loaded by antigen were detected, and the mononuclear cells were taken as controls.RESULTS: ① The cultured and amplified dendritic cells expressed high levels of costimulatory molecules of CD1a and B7, and there were typical irregular processes on the surface. ② The tumor cells formed apoptotic bodies when they were induced by mitomycin, which were arrested and phagocytized by dendritic cells. ③ The ability of the antigen loaded dendritic cells in stimulating the proliferation of allogenic lymphocyte T was further enhanced.CONCLUSION: The apoptotic tumor cells induced by mitomycin can induce the mononuclear cells from human peripheral blood differentiating into the dendritic cells with the concomitance of GM-CSF and recombinant IL-4 and amplify dendritic cells. Meanwhile, the dendritic cells can effectively present the antigens of apoptotic bile duct cancer cells, and it probably becomes a new effective approach for tumor antigen to sensitize dendritic cells.

Objective To explore the chemotherapeutic agents-induced modulating effects of Egr-1 promoter sequence on GM-CSF expression and its protective effect against injury to haematopoiesis due to chemotherapy.Methods Human GM-CSF cDNA and enhanced green fluorescent protein(EGFP) cDNA were linked to internal ribosome entry site(IRES) respectively,and then recombined to pCIneo vector containing Egr-1 promoter(Egr-EG).The vector was transferred into human bone marrow stromal cell line HFCL by lipofection,and the HFCL/EG cells were then finally constructed.MTT assay was performed to determine the effects of cisplatin(DDP),5-fluorouracil(5-FU),gemcitabine(GEM) and paclitaxel(PTX) on the survival rates of HFCL/EG and HFCL cells,and the IC50 of such agents against HFCL/EG and HFCL cells were calculated.The percentage of HFCL/EG cells which positively expressed EGFP was assessed by both flow cytometry and inverted fluorescence microscope.The effects of the active oxygen inhibitor N-acetylcysteine(NAC) on the expression of GM-CSF,which was modulated by promotor Egr-1,were detected by ELISA.The effects of HFCL/EG supernatant on CFU-GM after being exposed to chemotherapeutic agents were examined.Results With different sensitivity to DDP,5-FU,GEM and PTX,HFCL/EG cells were successfully constructed.The drugs showed higher IC50 value against HFCL/EG cells than HFCL cells(P

To elucidate the relationship between WT1 expression and differentiation of leukemia cells and the role of WT1 gene in differentiation of leukemia cells, HL 60 and K562 cell lines were induced for 5 days by all trans retinoic acid(ATRA).Then the degree of differentiation and WT1 expression of cell lines were determined by NBT reduction assay and RT PCR respectively. The resultsshowed that only differentiation of HL 60 cells but no K562 cells could be induced by ATRA. When HL 60 cells were induced to differentiate to granulocytes by ATRA, the expression of WT1 decreased markedly during differentiation. However, WT1 transcripts were not significantly altered in K562 cells in which differentiation wasn't found. It suggested that WT1 gene expression may relate to the differentiation of leukemia cells.

The human GM CSF cDNAandEGFP cDNA were linked together with IRES and then inserted into the expression vector pCI Egr 1, which was constructed by substituting CMV promoter in pCIneo with the Egr 1 promoter(Egr EG).The vector was transfered into human bone marrow stromal cell line HFCL by Lipofectin TM . The results indicated that the activity of EGFP in transfected cells increased at 18h after exposure to 2.5 Gy. The amounts of secreted GM CSF and CFU GM in serum free supernatants of Egr EG was significantly higher than the control group. EGFP and GM CSF cDNA were successfully integrated and expressed in the cells, which were confirmed by FACS and RT PCR analysis respectively. These in vitro data provide an experimental basis for the in vivo use of gene therapy of GM CSF gene regulated by Egr 1 promoter to protect hematopoiesis from total body irradiation.

Objective:To eastablish the efficient presentation of antigen from apoptotic cells by human DC from peripheral blood. Methods: using recombinant human granulocyte/macrophage colonystimulating factor(GM- CSF) and interleukin 4 (IL- 4 ) we have established dendritic cells (DC)from peripheral blood monocyte that maintain the antigen capturing and processing capacity characteristic of immature dendritic cells in vivo. GM - CSF 50ng/ml , IL- 41 000ng/ml once two days(total four). on the 3 rd day of culture, immature DC and apoptotic hepatochlangioma cells were in coculture lasting 7 days. Results:these cells had typical dendritic morphology, express high levels of CD1a ,B7 and acquired antigen from apoptotic cells and induced an increased T cell stimulatory capacity in MLR. Conclusions:we have established DC from blood mononuclear tells using GM- CSF and IL- 4 and DC can be efficiently drived from apoptotic cells and can induce the increase of T cells obviously. It probably becomes an effective approach of antigen transduced with DC.

Objective To investigate the survival, differentiation and action of neural stem cell transplanted into striatum of Parkinson disease rat model. Methods Neural stem cells were isolated and cultured. The character of self-renewing was confirmed by technique of single cell clone culture. The ability of multi-directional differentiation and specific antigen-nestin was showed by immunocytochemistry(ICC). PD rat models were established, and the DAPI-labelled neural stem cells were transplanted into striatum. The 6-HHDA induced rotary behavior was measured, distribution and migration of the cell were checked by fluoresce activating. ICC and HPLC are used to see its differentiation and action of secrete neurotransmitter dopamine(DA) and it’s metabolites. Results The cultured stem cell express nestin and have the ability of self-renewing and multi-directional differentiation. 6-HHDA induced rotation decreased after transplantation of NSC into the PD rat striatum(P

Embryonic stem cells(ESCs) are pluripotent cells derived from the inner cell mass of blastocyst-stage embryos, possessing permanent self-renewal and indefinite proliferative capacity in vitro. And ESCs could differentiate into the hematopoietic cell fate. Therefore ESCs may provide an alternative for hematopoietic stem cell transplantation and blood cells transfusion. Furthermore, ESCs differentiation could also provide a powerful model system to better understand the hematopoietic development and the mechanism involved. The current status for efforts to differentiate ESCs into hematopoietic lineages were reviewed.

Current therapies for myocardial infarction and congestive heart failure are limited in efficacy or in applicability. The plasticity of adult stem cells and cellular transplantation offer a novel therapeutic approach to improve cardiac function. This review describes the latest progress in research, summarizes recent studies of adult stem cells and their application in myocardial regenerative medicine in China and abroad, and discusses the future directions of cell transplantation as a new therapy to repair injured hearts. (J Geriatr Cardiol 2004;1(2) :77-82. )

AIM: To facilitate the suicide gene delivery into neoplasm, a chimeric gene of HSV-tk and green fluorescent protein (gfp) was constructed. METHODS: Molecular cloning technique was used to construct this kind of eukaryotic vector. The internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV), which could coordinate expression of two genes in a single vector, was optioned. By using liposome-mediated transfection, eukaryotic expression vector tgCMV/hytk-IRES-gfp was transfected into human bladder carcinoma cells EJ. RESULTS: A bicistronic eukaryotic vector carrying gfp and hygromycin phosphotransferase-thymidine kinase fusion (hytk) gene was constructed. The results of PCR and microscopy detection show that the hytk-IRES-gfp gene was successfully transferred into EJ cells. There were no differences in the growth pattern or the morphology between EJ and EJ/hytk-GFP cells. In vitro experiments demonstrated dose- and time-dependent cell killing by transduction of the hytk-IRES-gfp gene followed by GCV treatment. The IC50 (the concentration required to elicit 50% growth inhibition) was 2.16 mg/L in treatment with GCV for 72 hours. CONCLUSION: These results suggest that this new kind of eukaryotic vector could serves as a new tool and method for neoplasm gene therapy.