Induction of apoptosis with Salvia miltiorrhiza monomer IH764-3 via downregulating focal adhesion kinase in H2O2-stimulated hepatic stellate cells

AIM: To investigate the effect of IH764-3 on the apoptosis of H₂O₂-stimulated hepatic stellate cells(HSCs) and the alteration of focal adhesion kinase (FAK). METHODS: The proliferation of HSCs was examined by direct cell count and the apoptosis was determined by Annexin-V/PI labeling, while the morphological change was observed by light microscopy and transmission electron microscopy. In addition, FAK mRNA was detected by RT-PCR. RESULTS: H₂O₂ promoted the proliferation of HSCs. IH764-3 induced the apoptosis of HSCs in a dose-dependent manner. The HSC apoptotic rates of different groups were 6. 35%,9. 28%,15. 10%,19. 69%,respectively, after treated with different concentrations of IH764-3 for 48 h while H₂O₂ group showed 2. 30%. In 30 mg/L group, the apoptosis rates were 6. 73%、10. 34%、15. 10% for the indicated time periods(12 h, 24 h, 48 h). In the presence of IH764-3, FAK mRNA decreased. The FAK mRNA reduction began at 2 h after adding IH764-3. CONCLUSION: IH764-3 induced the apoptosis of HSCs. Down-regulating the expression of FAK mRNA may be one of its mechanisms.     

Bcl-2 antisense oligodeoxynucleotide increases the sensitivity of HL60 and K562 cells to daunorubicin

AIM:To investigate whether the bcl-2 antisense oligonucleotide increases the sensitivity of HL60 and K562 cell lines to daunorubicin.METHODS:IC50 for HL60 and K562 was determined with MTT method, the expression levels of Bcl-2 protein were assayed by immunofluorescence using fluoresce isothiocyanate labeling. In addition, apoptosis was detected by morphological observation and flow cytometric analysis of DNA fragmentation.RESULTS:It was found that the two oligonucleotides directed against the coding region and the translation initiation of bcl-2 mRNA, combined respectively with daunorubicin, inhibited expression of bcl-2 protein, increased apoptosis in HL60 and K562 cells, and decreased IC50 of daunorubicin significantly (P＜0.05). Compared to the antisense oligonucleotide directed against the translation initiation of bcl-2 mRNA, the antisense oligonucleotide directed against the coding region showed stronger effects in the aspects of increasing the sensitivity of HL60 cells to daunorubicin (P＜0.05).CONCLUSIONS:These two antisense sequences in the translation initiation and the coding region of bcl-2 mRNA increased the sensitivity of HL60 and K562 cell lines to daunorubicin in a sequence-specific manner.     

Effect of Liucha extract on tumor K562 cell growth

AIM:To investigate the effects of Liucha extract on the growth of tumor cells in vitro and its possible mechanism.METHODS:The capability of colony forming of human leukemia K562 cell in vitro and the cells metabolism were studied by semi-solid agar culture and MTT staining. Then , the changes in morphology in the tumor cells were examined using electronic microscope.RESULTS:Semi-solid agar culture and MTT colorimetric analysis showed that Liucha extrats could significantly inhibit the growth of the tumor cells and their capability of colony forming. Also,under the electronic microscope,it was found that the tumor K562 cell had a narrower perinuclear space,condensation of chromatin and an enlarged mitochondria , in which the cristase disappeared.CONCLUSION:The extract from Liucha possesses an inhibitory effect on K562 cell growth in vitro through affecting the metabolism of the tumor cells.     

Bone marrow-derived stromal cells and cartilage tissue engineering

Articular cartilage repair is one of the most challenging issues which remains to be resolved in clinic work. Discovering of bone marrow stromal cells (BMSC) and its application in tissue engineering provide new methods for the treatment of cartilage defects. High seeding density, appropriate cytokines and three-dimensional culture play important roles in the process of inducing BMSC differentiating into chondrocytes, suitable scaffold is also essential in reconstructing cartilage in vitro by methods of tissue engineering.     

Selection and amplification of the liver stem cell subset from rat bone marrow cells with a medium containing cholestatic serum in vitro

AIM: To explore the feasibility of direct separat and selective enlargement of the bone marrow-derived liver stem cells (BDLSC) from bone marrow cells with a culture system containing cholestatic serum in vitro. METHODS: Bone marrow cells of rats were cultured with selective media containing 2%, 5%, 7% and 10% cholestatic rat serum, respectively. The BDLSC were then induced to proliferate with the addition of hepatocyte growth factor (HGF) on the firth day. BDLSC were characterized using immunocytochemistry and RT-PCR for lineage markers, glycogen staining and urea synthetic assay for functions 2 weeks later. RESULTS: Bone marrow cells were unble to form colony in the presence of 2% cholestatic serum and apopotosis appeared gradually in 7% or 10% cholestatic serum. The BDLSC survived in the medium containing 5% cholestatic serum while the other types of cells did not. The survival cells proliferated with a high speed during the second week and then formed hepatocyte-like colony-forming units (H-CFU). Cells in the H-CFU expressed the characteristic proteins of fetal hepatocytes. Furthermore, they had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes. CONCLUSION: The selective micro-environment effectively selected BDLSC from the bone marrow cell, and will be a new way to provide an abundant source of donor hepatocytes for clinical cell therapy.     

Sca-1+ cells from mouse fetal liver can differentiate into neurons in vitro

AIM: To study whether Sca-1⁺ cells from fetal liver can be induced to differentiate into neuronal cells in vitro. METHODS:Sca-1⁺cells from 14 5-days-old murine fetal liver were isolated with a magnetic cell sorting kit, and were cultured in Dulbecco s modif ied Eagle s medium(DMEM)/F12 supplemented with 10%fetal bovine serum(FBS), and passaged at a rat io of 1 3 when cells reached more than 80%confluence.The 5 passage cells were induced by 10^-3mol/Lβ-mercaptoethanol(β-ME)and 5×10^-7 mol/L all-trans-retinoic acid(RA)for 24 hours, and then incubated in serum-free medium for 5 hours to 5 days.The characteristics of treated cel s were assayed by immunocytochemistry staining analysis at 5 hours, or 5 days.RESULTS: Cells treated with β-ME and RA exhibited neuronal phenotype and expressed neuron-specific protein such as neuron-specific nuclear protein (NeuN), neuronfilament-M, and neuron-specific tubulin-1 (TuJ-1) but not tau, MAP-2, or the astrocyte-specific marker glial fibrillary acidic protein (GFAP).CONCLUSION: Sca-1⁺ cells from fetal liver, of which most are regarded as hematopoietic stem cells, could differentiate into early immature neuronal cells in vitro. These findings suggest that Sca-1⁺ cells from fetal liver may be an alternative source in cell therapy and gene therapy of neural dysfunction.     

Study on adherent precursors from human cord blood

AIM: To confirm the existence of the endothelial progenitor cells in human cord blood and to study its differentiation and development process. METHODS: The mononuclear cells in human cord blood were isolated using lymphocyte separation solution. Then the mononuclear cells were cltured in MCDB131 containing 20% fetal bovine serum. The effects of 5 μmol/L dexamethasone, the extract from bovine brain, insulin and hypoxanine on the proliferation and differentiation of the adherent cells were observed. The morphology of the adherent cells were examined twice daily by inverted phase contrast microscope. CD34 and CD14 expression were determined by FACS. Immunohistochemistry was used to confirm the expression of factor Ⅷ. RESULTS: The proliferative endothelial progenitor cells existed within the CD34^-adherent mononuclear cells of human cord blood. Dexamethasone and hypoxanine decreased the number of spindle-shaped cells and caudated cells. Bovine brain extract, insulin and FCS enhanced the number of spindle-shaped cells and caudated cells. CONCLUSION: The existence of endothelial progenitor cells within the CD34 - adherent monouclear cells of the human cord blood was observed and these cells were able to differentiate into endothelial-like cells in vitro.     

Biological characteristics of long passaging rat mesenchymal stem cells

AIM:To investigate multi-potential of rat bone marrow mesenchymal stem cells (rBMMSC) and mutation inclination, the rBMMSC were long passaged in vitro. METHODS:Cellular cycles of different passages were assayed by FACSan 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 strong proliferation; passage 6, passage 25 and passage 45 all showed normal karyotype. CONCLUSION:Long-term culture and passage of rBMMSC still remains strong proliferation. With this capability, the mutation inclination is not enhanced.     

Effect of mutant β-catenin gene on the proliferation of hepatocytes

AIM:The β-catenin is a key molecule in the Wnt signal pathway, which plays a critical role in normal development and tumorigenesis. However, the mechanisms of the β-catenin on the cell growth control are still not completely defined. The aim of this study was to test the hypothesis that the mutant β-catenin may regulate the hepatocyte proliferation. METHODS: The immortalized murine hepatocyte cell line, AML12, was used for this study. A plasmid that contain mutant β-catenin S33Y was transfected into the AML12 cells and a stable cell line AML12S33Y was established. The cell growth property of this cell line and the parental cell were compared by flow cytometry analysis and direct cell count. The cells were also tested for the ability to form soft agar colonies, and the ability to form tumors in the severe immune deficient mice (SCID). RESULTS:1. The mutant β-catenin containing cell line AML12S33Y has higher proliferating index compared with the parental AML12 cells (P<0.01), suggesting that mutant β-catenin promotes cell growth. 2. The mutant β-catenin cells formed small colonies in soft agar after 4 weeks of culture, but did not generate tumor in SCID mice. CONCLUSION:The mutant β-catenin promotes liver cell growth.     

Effects of TGF-β1 and TNF-α antisense PS-ODNS on ex vivo expansion of hematopoietic stem/progenitor cells(HSPC)

AIM:To study the effect of TGF-β₁ and TNF-α antisense PS-ODNS on ex vivo expansion of hematopoietic stem/progenitor cells (HSPC). METHODS:CD₃₄⁺cells were purified from fresh umbilical cord blood by immunomagnetic beads, and mononuclear cells were purified from bone marrow by Ficoll-hypaque. The effects of TGF-β₁ and /or TNF-α antisense PS-ODNS on ex vivo expansion of CD₃₄⁺ cells、CFU-GEMM、CFU-GM、CFU-E and BFU-E were detected by using liquid and semi-solid culture systems.RESULTS:TGF-β₁ antisense PS-ODNS cooperated with cytokines increased the number of CD₃₄⁺ cells, CFU-GEMM, CFU-GM, CFU-E and BFU-E, which was as 4, 2.6, 2.7, 1.8, 2.1 times as that of the control (the cytokines combination), respectively. TNF-α antisense PS-ODNS cooperated with cytokines respectively increased the number of CD₃₄⁺ cells, CFU-GEMM, CFU-GM, CFU-E and BFU-E by 4, 2.9, 2.6, 1.7, 1.8 times as that of the control. The above two antisense PS-ODNS cooperated with cytokines could respectively increased the number of CD₃₄⁺ cells, CFU-GEMM, CFU-GM, CFU-E and BFU-E by 5.3, 2.1, 2.7, 1.9, 1.8 times as that of the control.CONCLUSION:Inhibition of endogenous TGF-β₁ and TNF-α by antisense PS-ODNS will be one of the effective methods to expand HSPC ex vivo.