Radix Astragali-induced differentiation of rat bone marrow-derived mesenchymal stem cells

BACKGROUND： Chemical induction has been shown to be effective at promoting the differentiation of bone marrow-derived mesenchymal stem cells （MSCs）. However, these inductors have cytotoxicity side effects that may damage cells over time. Traditional Chinese medicines avoid this disadvantage while still producing effective induction. OBJECTIVE： To investigate the influence of RadixAstragafi （Huangql） on the differentiation of MSCs. DESIGN, TIME AND SETTING： In vitro study of traditional Chinese medicine in neural stem cell differentiation. The experiment was performed at the Central Laboratory of Hebei North University between April and June 2007. MATERIALS： Radix Astragafi solution （lot No. 060105; license No. Z53021585） was purchased from Dali Pharmaceutical Co., Ltd., China; rabbit anti-rat nestin, rabbit anti-rat neuron-specific enolase （NSE）, mouse anti-rat microtubule-associated protein 2, and rabbit anti-rat glial fibrillary acidic protein were purchased from Wuhan Boster, China. METHODS： Whole bone marrow was isolated from the femur and tibia of 6-week-old male Wistar rats and subcultured. The fourth passage of MSCs were harvested and induced by different concentrations （50, 100, 200, 400 g/L） of Radix Astragali. MAIN OUTCOME MEASURES： Hematoxylin-eosin staining was used to observe MSC morphology after 24 hours of induction. Immunocytochemistry was employed to observe the expression of NSE （specific neuronal marker）, nestin （marker of neural stem cell）, glial fibrillary acidic protein and microtubule-associated protein 2 （markers of astrocytes）. RESULTS： Following Radix Astragali treatment, changes occurred in cell morphology including： cell body pyknosis; thin and long processes formed in some cells, with growth corresponding to drug concentration and induction time; and the formation of network-like connections between some cells. With increasing drug concentration and induction time, nestin expression was upregulated, and the number of positive cells increased; cells produced NSE, glial fibrillary acidic protein and microtubule-associated protein 2; nestin was expressed earlier than glial fibrillary acidic protein and microtubule-associated protein 2 expression. In addition, the number of NSE-positive cells was increased significantly more than glial fibrillary acidic protein-positive cells. CONCLUSION： Radix Astragafi promoted process formation in stem cells. It may induce the differentiation of MSCs into neural stem cells, and subsequently into neuronal- and glial-like cells. Radix Astragafi exhibits stronger inductive effect on neuronal differentiation than glial differentiation of MSCs.

Lethal effect of mononuclear cells derived from human umbilical cord blood differentiating into dendritic cells after in vitro induction of cytokines on neuroblastoma cells

BACKGROUND： Dendritic cell is the most major antigen presenting cell of organism. It is proved in recent studies that human umbilical cord blood mononuclear cells induced and cultured in vitro by recombinant human granulocyte-macrophage colony stimulating factor （rhG-MCSF） and recombinant human interleukin-4 （rhlL-4） can generate a great many dendritic cells and promote the lethal effect of T cells on human neuroblastoma, but it is unclear that whether the lethal effect is associated with the most proper concentration of dendritic cells. OBJEETIVE： To investigate the lethal effect of human umbilical cord blood mononuclear cells induced in vitro by cytokines differentiating into dendritic cells on human neuroblastoma, and its best concentration range. DESIGN ： Open experiment SEI-FING： Department of Pediatrics, the Medical School Hospital of Qingdao University MATERIALS ： The study was carried out in the Shandong Provincial Key Laboratory （Laboratory for the Department of Pediatrics of the Medical School Hospital of Qingdao University） during September 2005 to May 2006. Human umbilical cord blood samples were taken from the healthy newborn infants of full-term normal delivery during October to November 2005 in the Medical School Hospital of Qingdao University, and were voluntarily donated by the puerperas. Main instruments： type 3111 CO2 incubator （Forma Scientific, USA）, type 550 ELISA Reader （Bio-Rad, USA）. Main reagents： neuroblastoma cell line SK-N-SH （Shanghai Institute of Life Science, Chinese Academy of Sciences）, RPMI-1640 culture fluid and fetal bovine serum （Hyclone）, rhlL-4 （Promega, USA）, rhG-MCSF （Harbin Pharmaceutic Group Bioengineering Co.Ltd）, rat anti-human CDla monoclonal antibody and FITC-labeled rabbit anti-rat IgG （Xiehe Stem cell Gene Engineering Co.Ltd）. METHODS： ① Human umbilical cord blood mononuclear cells obtained with attachment methods differentiated into human umbilical cord blood dendritic cells, presenting typical morphology of dendritic cells after in vitro induction by rhG-MCSF and rhlL-4. ② Different concentrations of dendritic cells[ dendritic cells： neuroblastoma cells=20：1,50：1,100：1 （2×10^8 L^-1,5×10^8 L^-1,1×10^9 L^-1）], 1×10^9 L^-1 T cells and 1×10^7 L^-1 neuroblastoma cells were added in the experimental group. 1 ×10^9 L^-1 T cells and 1 ×10^7 L^-1 neuroblastoma cells were added in the control group. ③ Main surface marker CDla molecules of dendritic cells were detected with indirect immunofluorescence, and the percent rate of dendritic cells was counted with ultraviolet light and expressed as the expression rate of CD1a^＋ cells. ④Single effector cells and target cells were respectively set in the experimental group and control group to obtain the lethal effect. The lethal effect of dendritic cells on neuroblastoma cells was indirectly evaluated by detecting cellular survival with MTT assay. The lethal effect（%）= （1-A experimentat well-A effector cell /A target cell well）×100%.⑤The expenmental data were presented as Mean ±SD, and paired t test was used. MAIN OUTCOME MEASURES： ① Morphological characters of dendritic cells in the process of induction and differentiation. ②CD1a^＋ cellular expression rate. ③Lethal effect of dendntic cells on neuroblastoma cells. RESULTS： ①Morphological characters of dendritic cells in the process of induction and differentiation： On the 15^th day after human umbilical cord blood mononuclear cells were induced by rhG-MCSF and rhlL-4, typical morphology of dendritic cells could be seen under an inverted microscope. ②Expression rate of CD1a^＋ cells was （43.12±5.83）%. ③Lethal effect of dendritic cells on neuroblastoma cells： Lethal effect of dendritic cells stimulated T cells in each experimental group （ dendritic cells： neuroblastoma cells=100：1,50：1, 20：1 respectively） on neuroblastoma cells was significantly higher than that in control group[（31.00 ±4.41 ）%, （30.92±5.27）%,（33.57±5.35）%,（26.23±5.20）%, t=3.51,2.98,4.24, P〈 0.01 ）; But the lethal effect of dendntic cells on neuroblastoma was significantly lower when their ratio was 100：1 and 50：1 in comparison with 20：1 （t=2.01,2.36, P 〈 0.05）, and no significant difference in lethal effect existed between the ratio at 100：1 and 50：1 （t=0.06,P 〉 0.05）. CONCLUSION： Dendritic cells differentiated from human umbilical cord blood mononuclear cells after in vitro induction of cytokines can promote the lethal effect of T cells on neuroblastoma cells. The lethal effect is associated with the concentration of dendritic cells within some range.

Effects of arotinoid acid on induction of apoptosis and differentiation and telomerase activity and cell cycle in the HL 60 cell line

Objective To investigate the effects of arotinoid acid (Ro13 7410) on the morphological and functional alterations of leukemia HL 60 cell line and compared with those of RA Methods Differentiation of HL 60 cells was assessed by morphology and by NBT reduction Trypan blue exclusion was used to determine viability Apoptosis was assessed by changes in cell morphology and by measurement of fragmented DNA using the PCD assay kit Telomerase PCR ELISA kit tested telomerase activity The cell cycle was analyzed by flow cytometry Results Incubation of the HL 60 cells with 10 -6 10 -8 ?mol/L Ro13 7410 resulted in suppression of cell growth Apoptotic cells were detected following exposure to 10 -6 ?mol/LRo13 7410 for 3 hours by measurement of the “in situ” enzymatic labeling of DNA breaks with biotinylated dUTP Ultrastructural examination of Ro13 7410 treated samples showed cells with chromatin compaction and cytoplasm condensation and the presence of “apoptotic bodies” Cells induced into apoptosis were accompanied by Department of Hematology, the First Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 400016, China (Liu XS, Lou LS, Zeng SR and Tang ZH) Department of Clinical Biochemistry, Chongqing University of Medical Sciences, Chongqing 400046, China (Jiang JK, Zhang Y, Xu XG, Liu BZ, He YJ and Kang GF) increase of intracellular free Ca 2+ Percentage of HL 60 cells reduced NBT following incubation with Ro13 7410 was lower than with all trans retinoic acid (RA) (27% vas 85%) Telomerase PCR ELISA assay showed that HL 60 cells cultured in the absence of inducing agents had significant telomerase activity Telomerase activity declined gradually after 10 -6 ?mol/L Ro13 7410 treatment, and changes becoming evident at 1 day The inhibition of telomerase activity at day 5 of treatment with Ro13 7410 was less effective than with RA DNA flow cytofluorimetric analysis revealed that Ro13 7410 caused partial cell arrest in the G 2/M phase after a 2 day treatment and the percentage of cells arrested in the G 2/M phase increased after 4 days treatment With RA treated cells, a reduction in the percentage of cells in the G 2/M phase was observed after 2 day of treatment Conclusion Our study shows that Ro13 7410 suppresses HL 60 cells growth mainly via the induction of apoptosis and is less effective than RA in induction differentiation Ro13 7410 dramatically inhibits telomerase activity during the course of induction and results in G 2/M arrest within 2 days These findings suggest that Ro13 7410 is worthy of further study for its effects on leukemic cells