Differentiation Of Positive Cells Research Articles

Substitution of antelope horn in Danqi Piantan capsule with artificial bezoar in vitro

The in vitro cell culture experiment was conducted to study the effect of Danqi Piantan capsule (DPC) and DPC dislodge the antelope horn with artificial bezoar double (DPCBD) on nerve regeneration and blood vessel regeneration and preliminarily investigate the possibility of substituting antelope horn in DPC with artificial bezoar. In this experiment, rats were randomly divided into 5 groups: the blank serum control group, the model group, DPC groups (0.306 g x kg(-1) x d(-1), the same below), DPC remove of antelope horn (DPCRA) groups and DPCBD groups. Brain microvascular endothelial cells cultured in vitro (BMEC), astrocytes and neural stem cells (NSC) were co-cultured to simulate neurovascular unit, label neurons with microtubule associated protein III (β-tubulin III) antibody and lable astrocytes with glial fibrillary acidicprotein (GFAP). ELISA was used for the detection of the content of BMEC lactate dehydrogenase instrument method (LDH), the inverted phase contrastmicroscope was adopted to observe the formation of BMEC tube like structure, the number of leukocytes and leukocytes adherent to BMEC were counted under the microscope, the expression levels of β-tubulin III and the ratio of GFAP positive cells was detected with inimmunofluorescence, and RT-PCR method was used to detect NGF, BDNF, VEGF and VEGFr-2 mRNA. According to the result, compared with the model group, both DPC and DPCBD can reduce LDH leakage, promote the formation of BMEC tube like structure, inhibit leukocytes and their adhesion to BMEC, increase the β-tubulin III positive cell differentiation proportion (P < 0. 01), reduce the proportion of GFAP positive cells (P < 0.01), increase the expressions of co-cultured NGF, VEGF, BDNF and VEGFr-2 mRNA to a certain extent, with the most significant difference on NGF and VEGF mRNA expressions (P < 0.05) and the same efficacy in both groups. DPCRA groups showed less impact on all indexes than that of DPCBD and DPC groups. The same efficacy of DPCBD and DPC on nerve regeneration and angiogenesis suggested that antelope horn in DPC can be substituted by artificial bezoar.

Expression of CD133 during differentiation of AC133 positive glioma stem cells

Objective To explore the correlation between the expression of CD133 and undifferentiated state of glioma cells.Methods Differentiation was induced for 2 weeks in glioma stem cells enriched with AC133 expression.Real-time quantitative polymerase chain reaction (PCR) was performed to study CD133 mRNA expression.Western blotting was used to detect the CD133 protein expression.By using flow cytometry,extracellular and intracellular AC133 expression was examined.Enzyme linked immunosorbent assay (ELISA) was applied to measure the AC133 expression in the culture supernatant.Results The extracellular AC133 expression in glioma stem cells was significantly down-regulated upon differentiation (P＜ 0.05 ). Quantitative CD133 mRNA expression in NCH421k cells before differentiation was 1.08 ±0.39,1.08 ± 0.51 in serum upon differentiation,and 1.07 ± 0.54 in serum plus 1 μmol/L alltrans retinoic acid (P ＞0.05),and that in NCH441 cells was 2.61 ± 1.72,2.53 ± 1.18 and 2.18 ± 1.73respectively (P ＞ 0.05). No significant difference in the CD133 protein expression was observed before and after differentiation.Conclusion Extracelluar AC133 more sensitively mirrors undifferentiated state of glioma cells than CD133 mRNA and protein. Key words: CD133; AC133; Glioma; Brain tumor stem cell; Differentiation

The effect of 5-fluorouracil on enriching cancer stem cells of hepatoma cell line BEL-7402

To investigate the effect of 5-FU (5-fluorouracil) on enriching cancer stem cells of HCC cell line BEL-7402 and the biological characteristics of enriched cells. The enriching concentration of 5-FU was determined by CCK-8 (cell counting kit-8). Flow Cytometry was used to determine the changes in cell cycle and positive expression ratio of surface marker CD56, CD54, EpCAM and CD133. The self-renewal and differentiation of positive cells were tested by colony formation assay, and were compared with the control group. Enriching concentration of 5-FU was determined as 10 μg/ml with 48 h incubation. After enrichment, G0/G1 phase cells increased from 57.50 %+/-0.98% to 68.70%+/-3.41% (P<0.05). Whereas S phase cells decreased from 40.26%+/-4.12% to 31.80%+/-4.15% (P<0.01); G2/M phase cells disappeared in experimental group, and was 5.80%+/-1.87% in control group (P<0.01). The proportion of the cell cycle changed with significant statistical differences. Meanwhile, positive rate of cell surface makers CD56, CD54, EpCAM and CD133 increased from 0.57%+/-0.12%, 8.10%+/-6.79%, 0.3%+/-0.01% and 3.20%+/-0.99% to 4.13%+/-0.06%, 50.08%+/-1.69%, 0.55%+/-0.07% and 10.51%+/-1.13%, respectively. The difference was significant (P<0.05). The colony forming ratio of CD56, CD54, EpCAM and CD133 negative cells and positive cells were 2.11%+/-0.21%, 3.32%+/-0.31%; 0.86%+/-0.101%, 2.40%+/-0.52 %; 7.19%+/-0.56%, 7.73%+/-0.71%; 2.70%+/-0.26%, 5.75%+/-0.81%, respectively, and significant differences were found between (P<0.05). 5-fluorouracil enriched the cancer stem cell population in HCC cell line BEL-7402. CD56 and CD54 can be used as important surface markers in research of liver cancer stem cells.