Effects of maxadilan on proliferation of human induced pluripotent stem cells

AIM: To investigate the promoting effect of maxadilan, which specifically activate the type I receptor for pituitary adenylate cyclase-activating polypeptide (PACAP), on the proliferation of human induced pluripotent stem cells (IPSCs). METHODS: PACAP type I (PAC1) receptor in IPSCs was detected by RT-PCR and Western blotting. maxadilan at various concentrations was added to the medium of IPSCs as experimental groups. The medium in control group was without maxadilan treatment. The effect of maxadilan on theproliferation of IPSCs was measured with Cell Counting Kit-8 (CCK-8). The changes of cell cycle caused by maxadilan in IPSCs were analyzed by flow cytometry. The analysis of karyotype was carried out in IPSCs treated with maxadilan. Proteins and gene expression levels of both Nanog and OCT4 in IPSCs treated with maxadilan were detected by real-time quantitative polymerase chain reaction (real-time-qPCR) and immunofluorescence. The gene expression levels of Nestin and PAX6 in both IPSCs treated with maxadilan and cells of embryonic body, which was birthed from IPSCs with maxadilan treatment, were detected by real-time qPCR. The ability of IPSCs treated with maxadilan differentiating into 3 embryonic layers was evaluated by analyzing the component of embryo using RT-PCR. RESULTS: The PAC1 receptor in IPSCs was identified by RT-PCR and Western blotting. Viability of the IPSCs with 100 nmol/L maxadilan treatment was increased by 16% compared with control group. The differences with statistical significance were found in the cell viability between 100 nmol/L maxadilan treatment group and control group (P<0.05). The average values of proliferation index (PI) in IPSCs with 100 nmol/L maxadilan treatment for 3 h, 6 h and 9 h were 47.23%, 59.70% and 55.67%,respectively, while that in control group was 37.00%. The differences with statistical significance were found in PI between 100 nmol/L maxadilan treatment for 3 h group, 6 h group, 9 h group and control group (P<0.05). Normal karyotype and unchanged pluripotent state in IPSCs treated with maxadilan were observed. Compared with control group, the gene expression levels of Nestin and PAX6 were not significantly different in both IPSCs and the cells of embryonic body birthed from IPSCs with maxadilan treatment. The ability of differentiation into 3 embryonic layers in IPSCs treated with 100 nmol/L maxadilan was found. CONCLUSION: PAC1 receptor presents in IPSCs. Maxadilan promotes the proliferation of IPSCs but does not affect their pluripotent state and karyotype.     

Protective effect of sodium selenite on human keratinocytes from ultraviolet-B irradiation

AIM: To investigate the protective effect of sodium selenite (Na₂SeO₃) on human keratinocytes under ultraviolet-B (UVB) irradiation. METHODS: The cultured HaCaT cells were divided into 4 groups: (1) normal control group; (2) Na₂SeO₃ group: pretreated with Na₂SeO₃ at doses of 10 nmol/L, 50 nmol/L, 100 nmol/L, 200 nmol/L and 1 μmol/L for 24 h; (3) UVB group: irradiated with UVB at doses of 300, 600 and 900 J/m² ; (4) Na₂SeO₃+UVB group: after pretreated with Na₂SeO₃ for 24 h, irradiated with UVB at doses of 300, 600 and 900 J/m² . The cell proliferation was detected by MTT assay. The apoptotic rates of HaCaT cells treated with UVB at dose of 300 J/m² were assessed by flow cytometry. RESULTS: Compared with normal control group, the cell proliferation activity in UVB group decreased significantly (P<0.05). The cell activity was inversely correlated with the irradiation intensity. No significant difference of the cell activity between Na₂SeO₃ group and normal control group was observed. The cell proliferation in Na₂SeO₃+UVB group was higher than that in UVB group significantly (P<0.05). Na₂SeO₃ at concentration of 100 nmol/L showed the strongest activity to promote cell proliferation. After 300 J/m² UVB irradiation, the apoptotic rate in Na₂SeO₃+UVB group decreased significantly (P<0.05) compared with UVB group. The inhibitory effect of Na₂SeO₃ at concentration of 100 nmol/L on apoptosis was the strongest.CONCLUSION: The damage of human keratinocytes by UVB irradiation is in a dose-dependent manner. The photoprotection performance of Na₂SeO₃ reduces the damage of human keratinocytes induced by UVB irradiation.     

Inhibitory effect of IGF-1 overexpression on oxidative damage in umbilical cord mesenchymal stem cells

AIM: To analyze the inhibitory effect of insulin-like growth factor-1 (IGF-1) overexpression in umbilical cord mesenchymal stem cells (UC-MSCs) on oxidative damage and to develop new application model for UC-MSCs. METHODS: UC-MSCs were isolated from human umbilical cord with enzymatic digestion, and further characte-rized with flow cytometry. IGF-1-overexpressing UC-MSCs (UC-MSCs-IGF-1) were established by retrovirus infection. IGF-1 expression of UC-MSCs-IGF-1 was evaluated by real-time quantitative PCR and flow cytometry, and its surface markers, as well as osteogenic and adipogenic differentiation ability, were further analyzed. The proliferation, anti-oxidative damage and anti-apoptosis abilities of UC-MSCs-IGF-1 were evaluated when treated with H₂O₂ at different concentrations (0 μmol/L, 10 μmol/L, 50 μmol/L and 100 μmol/L). RESULTS: UC-MSCs showed positive expression of CD29, CD90 and CD105, but negative expression of CD34, which coincided with the normal phenotype of mesenchymal stem cells. UC-MSCs-IGF-1 established with retrovirus infection showed much higher expression of IGF-1 compared with normal UC-MSCs, and expressed the same surface markers as UC-MSCs.The osteogenic and adipogenic differentiation abilities were also observed. With the oxidative damage by H₂O₂ treatment, UC-MSCs-IGF-1 showed more strong proliferation, anti-oxidative damage and anti-apoptosis abilities as compared with normal UC-MSCs. In addition, the activity of SOD in UC-MSCs-IGF-1 was a little higher than that in control group. CONCLUSION: IGF-1 overexpression in UC-MSCs inhibits oxidative damage and cell apoptosis. UC-MSCs-IGF-1 may have more advantagies in clinical application.     

Aged bone marrow stem cells can be rejuvenated by cell fusion with young bone marrow stem cells

AIM: To investigate the function of aged bone marrow mesenchymal stem cells (BMSCs) fused with young BMSCs in mice. METHODS: The cell fusion model, which was made by C57BL/6 mouse BMSCs labeled with PKH26 membrane red fluorescence (young cells, age of 2-3 months, Y) and (old cells, age of 18-24 months, O), and young and old BMSCs of green fluorescent protein (GFP) transgenic C57BL/6 mouse, was established by the induction of polyethylene glycol 1500 (PEG 1500). The cell fusion rate and cell surface markers were detected by flow cytometry. The morphology and nuclear characteristics of the fused cells were observed under fluorescence microscopy. In this study, the age dependent changes in BMSCs proliferation and differentiation potential in Y group, O group, and another three fusion groups (Y-Y group, Y-O group, O-O group) were examined. The proliferation potentials in 5 groups were compared by counting cell numbers at days 2, 4, 6, and 8. The osteogenic and adipogenic differentiation potentials of the cells in 5 groups were determined by using standard differentiation procedures. RESULTS: The fusion rate of 30.45%±4.13% was obtained by PEG 1500 induction. No significant difference of the fusion rates in Y-Y, Y-O and O-O groups was observed. Fused BMSCs coincided with the common BMSCs were reactive to the BMSCs lineage-specific CD44, Sca-1 surface markers and negative for the hematopoietic stem cells (HSCs) lineage-specific surface markers such as CD34, CD117, CD31, and CD45. The percentage of increasing cell numbers in Y-O group was significantly higher than that in O-O group at days 2, 4, 6, and 8. The positive rate of the area stained with Alizarin red, which represents osteogenic differentiation potential of BMSCs, was significantly higher in Y-O group than that in O-O group ［(25.46%±1.52%) vs (13.85%±1.69%), P<0.01］. In Y-O group, the higher rate of the positive area stained with oil red O, which represents adipogenic differentiation potential of BMSCs, was observed as compared to that in O-O group ［(12.99%±2.61%) vs (6.03%±1.71%), P<0.05］. CONCLUSION: Aged bone marrow stem cells can be rejuvenated by cell fusion with young bone marrow stem cells, particularly the proliferation and differentiation potentials.     

Effect of miR-21 over-expression on proliferation,migration and diffe-rentiation of c-Kit+ cardiac stem cells

AIM: To explore the effect of microRNA (miR)-21 on proliferation, migration and differentiation abilities of c-Kit⁺ cardiac stem cells (CSCs). METHODS: c-Kit⁺ CSCs were cultured and selected by the methods of enzyme digestion and magnetic bead separation. miR-21 mimics (50 nmol/L) and mimics negative control (MNC) were transfected into c-Kit⁺ CSCs with Lipofectamine^® 2000. The cells was divided into 3 groups:control group:c-Kit⁺ CSCs without any pretreatment; MNC group:the cells were transfected with MNC for 48 h; mimics group:the cells were transfected with miR-21 mimics for 48 h. qPCR was used to assess the expression of miR-21 in each group. CCK-8 and EdU assays were used to determine the cell proliferation. qPCR and immunofluorescence were used to detect the differentiation in each group. Scratch assay was adopted to explore the migration ability of the cells. RESULTS: The expression of c-Kit in the c-Kit⁺ CSCs were 90.8%, with 0.6% of CD45 and 0.5% of CD34. A significant increase in miR-21 expression was observed when the cells were transfected with miR-21 mimics for 48 h (P<0.05). CCK-8 and EdU assays showed that miR-21 significantly increased cell proliferation as compared with MNC group and control group (P<0.05). No difference in the expression of Nkx2.5, CD31 and α-SMA at mRNA and protein levels was observed, and no difference of the migration ability in 3 groups of the c-Kit⁺ CSCs was found. CONCLUSION: Over-expression of miR-21 significantly promotes the proliferation of c-Kit⁺ CSCs, without any effect on the cell migration and differentiation.     

Interleukin-4 regulates phonotype,proliferation and hematopoietic supporting capacity of human umbilical cord mesenchymal stem cells

AIM: To explore the effects of interleukin-4 (IL-4) on the biological characteristics and hematopoietic supporting effects of umbilical cord mesenchymal stem cells (UC-MSC). METHODS: The phenotype of UC-MSC was detected by flow cytometry after IL-4 stimulation, and the proliferation ability of UC-MSC was measured by BrdU-ELISA. Oil red O and alizarin red were used to observe the ability of differentiation. The mRNA expression in UC-MSC was determined by real-time fluorescence quantitative PCR. The culture medium isolated from UC-MSC was used to analyze the ability in promoting colony formation.RESULTS: After IL-4 stimulation, the expression of CD11b, CD19, CD34, CD45, CD73, CD90, CD105, HLA-DR and HLA-ABC was unchanged. IL-4 inhibited the proliferation of UC-MSC, but no difference was detected on osteogenic and adipogenic differentiation. The culture medium from IL-4-induced UC-MSC possessed strong ability for promoting CD34+ colony formation ability. CONCLUSION: IL-4 inhibits the proliferation of UC-MSC and enhances its hematopoietic supporting ability.     

Proliferation and differentiation of bone marrow mesenchymal stem cells in process of bone loss in ovariectomized rats

AIM: To study the function of proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs) for bone loss in the pathogenesis of osteoporosis (OP) in ovariectomized rats. METHODS: Animal model of OP was established by ovariectomy (OVX,bilateral ovarian resection) in 10-week-old healthy female Sprague-Dawley (SD) rats.BMSCs were isolated, cultured and purified by the combination of density gradient centrifugation, adhesion separation and limited dilution method, and cultured in vitro to the 3rd~4th passage in all experiments. The BMSCs phenotype appraisal was studied by flow cytometry. Colony-forming assay was applied to detect the BMSCs proliferation ability. The MTT method was used to analyze the growth curves of BMSCs. After adipogenic induction (ADI), lipid drops were observed by oil red O staining to compare the adipogenic potential between the 2 kinds of BMSCs. After osteogenic induction (OSI), calcium nodules were observed by alizarin red staining (ARS). The mRNA expression levels of BMSCs osteogenesis-related proteins, for instance, Runx2, osteocalcin (OCN) and osteopontin (OPN) were measured by RT-PCR. RESULTS: Compared with sham group, the colony-forming ability of BMSCs in OVX group became decreased, the proliferation capacity was declined, the osteogenic potential was decreased, and the adipogenic potential was increased(P<0.05). CONCLUSION: In ovariectomized OP rats, the proliferation and osteogenesis of BMSCs decrease, and the adipogenesis of BMSCs increases, which may cause rapid bone loss and play an important role in the pathogenesis of OP.     

Effects of basic fibroblast growth factor on proliferation and collagen production in human umbilical cord mesenchymal stem cells

AIM：To observe the growth pattern and surface markers of human umbilical cord mesenchymal stem cells(hUCMSCs) and to explore the influence of basic fibroblast growth factor (bFGF) on the proliferation and collagen production in hUCMSCs cultured in vitro. METHODS：hUCMSCs were isolated by enzyme digestion method and adherent culture. The surface markers CD45, CD34, CD105, CD29 and HLA-DR of the cells were analyzed by flow cytometry. The osteogenic ability and adipogenic differentiation were confirmed with oil red O and alizarin red staining. The optimal concentration of bFGF to promote the proliferation of hUCMSCs was 20 μg/L. The cells in control group were cultured in the growth medium consisting of DMEM/F12 and 10% volume fraction of fetal bovine serum. The cells in experiment group were cultured under the same condition of control group but plus 20 μg/L bFGF. The proliferation of hUCMSCs was analyzed by MTT assay. The expression of type I and III collagens at mRNA and protein levels was determined by RT-PCR and Western blotting. RESULTS：The growth curves indicated that bFGF promoted the proliferation of hUCMSCs. The hUCMSCs expressed CD29, but did not express CD34, CD45 or HLA-DR in the presence or absence of bFGF unanimously. The cells were alizarin red staining-positive and oil red O staining-positive. Compared with control group, the expression of type I and III collagens significantly decreased at mRNA and protein levels in experiment group. CONCLUSION：bFGF promotes the proliferation of hUCMSCs and does not change the expression of the surface markers. bFGF inhibits the expression of type I and III collagens at mRNA and protein levels, indicating that bFGF enhances the healing of wound without inducing scar hyperplasia.     

Effects of Arg-Gly-Asp-Ser tetrapeptide on proliferation and apoptosis of hepatic stellate cells in vitro

AIM:To investigate the effects of Arg-Gly-Asp-Ser (RGDS) tetrapeptide on proliferation, apoptosis and caspase 3 expression in FN-stimulated HSCs in vitro. METHODS:[³H]-thymidine incorporation, Annexin-V/Propidium Iodide double-labeled flow cytometry(FCM), TUNEL, scanning electron microscope and transmission electron microscopy were employed to estimate the influence of RGDS on proliferation and apoptosis of HSCs. The adhesion rates were observed by toluidine blue colorimetric assay. The expression of caspase-3 protein was detected by FCM. RESULTS:①Compared with control and FN groups, RGDS tetrapeptide at concentrations of 25 mg·L^-1, 50mg·L^-1 and 100 mg·L^-1 inhibited the proliferation of HSCs (P<0.01), and the inhibition rates of 100 mg·L^-1 at 12 h, 24 h and 48 h were 62.73%, 74.23%, 80.22%, respectively.②RGDS tetrapeptide induced the HSC apoptosis in dose-dependent and time-dependent manners(P<0.01). Observed with scanning electron microscope, the cell bodies and cellular processes of HSCs exposed to RGDS tetrapeptide were seen to be diminished. Microvilli on the cell surface decreased, became short even disappeared. Observed with transmission electron microscopy, the chromatins condensed, shrunk and aggregated along inside of nuclear membrane to exist in the form of ball, petal and crescent. Sometimes, apoptotic bodies formed. ③After exposure of HSCs to RGDS tetrapeptide for 2 h, the inhibition rates of adhesion were 8.82%, 29.41% and 45.59%, respectively, but that of RGES group was only 4.41%, P<0.01. ④ The expression of caspase 3 was obviously higher in RGDS tetrapeptide group than that in FN group, RGES tetrapeptide. CONCLUSION: These results suggest that RGDS tetrapeptide may inhibit proliferation and induce apoptosis of HSCs in both dose- dependent and time- dependent manners in vitro, which may be related to the abrogation of cell adhesion and caspase 3.     

Effects of survivin inhibitor YM155 on mitochondrial apoptotic pathway of retinoblastoma Y79 cells

AIM: To investigate the effects of survivin inhibitor YM155 {4,9-dihydro-1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(2-pyrazinylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium bromide} on the apoptosis, mitochondrial membrane potential (Δψ_m) and cytochrome C (Cyt C) of retinoblastoma Y79 cells, and to analyze the mitochondrial mechanisms of apoptosis.METHODS: Y79 cells were cultured in vitro and treated with YM155 at concentrations of 0, 0.5, 1, 2, 4 and 8 nmol/L. The cells in control group were treated without YM155. The proliferation of Y79 cells were measured by CCK-8 assay and bromodeoxyuridine (BrdU) labeling assay. Y79 cells were randomly divided into 4 groups:control group (with equal volume of RPMI-1640 nutrient medium), positive control group (10 nmol/L topotecan), low-dose (1 nmol/L) YM155 group and high-dose (2 nmol/L) YM155 group. The effects of YM155 on the apoptosis, the changes of Δψ_m, the mitochondrial distribution and the protein level of Cyt C in the Y79 cells were evaluated by flow cytometry with Annexin V-FITC/PI staining, JC-1 staining, immunofluorescence analysis and Western blot, respectively. RESULTS: Compared with control group, YM155 significantly inhibited the proliferation of Y79 cells and induced apoptosis (P<0.05). YM155 significantly reduced Δψ_m of the Y79 cells, promoted Cyt C which released from mitochondria to the cytosol and reduced the protein level of Cyt C in the mitochondria (P<0.05). CONCLUSION: YM155 inhibits Y79 cell proliferation and induces apoptosis, and the possible mechanisms may be involved in the mitochondrium-mediated apoptotic pathway.     

《中国蔬菜品种志》

AIM: To characterize the gene expression of sortilin on adipogenic and osteogenic differentiation in mesenchymal stem cells (MSCs) in vitro and explore its significance.METHODS: MSCs derived from human bone marrow were isolated and cultured in vitro, then were stimulated in osteogenic medium and adipogenic medium, respectively. Osteopontin and lipoprotein lipase were detected by RT-PCR. Sortilin expression was analyzed by semiquantitative RT-PCR. RESULTS: 1.MSCs displayed the potential of differentiation into osteoblast and adipocyte. 2.Sortilin was upregulated one day after osteogenic induction and remained upregulated for a week. The expression of sortilin was significant increased on day 3(P＜0.01). 3. No significant changes of sortilin expression was found in adipogenic differentiation (P＞0.05).CONCLUSION: Sortilin may be useful to modulate the osteogenic differentiation and may not be necessary for adipocyte commitment in MSCs. The regulation of sortilin expression may provide new protocal and strategy for the treatment of osteoporosis and osteopenic disease.     

Changes of miR-155-5p in serum of cervical cancer patients and effects of miR-155-5p on cervical cancer cells

AIM:To detect the serum level of miR-155-5p in the patients with different cervical diseases, and to analyze its effects on the proliferation, cell cycle and apoptosis of cervical cancer cells. METHODS:SYBR Green I real-time quantitative PCR was used to detect the level of miR-155-5p in the serum of the patients with different cervical diseases. miR-155-5p mimic or inhibitor was used to increase or decrease the expression of miR-155-5p in cervical cancer cells. The proliferation, cell cycle and apoptosis were measured by CCK-8 assay and flow cytometry. RESULTS:The serum level of miR-155-5p in cervical cancer group was higher than that in cervicitis group and healthy group. No statistical difference of the serum miR-155-5p level between cervical intraepithelial neoplasia group and cervical cancer group was observed. Compared with blank group, liposome group and negative control group, the proportion of S-phase cells increased and apoptotic cells decreased in SiHa cells transfected with 100 nmol/L and 200 nmol/L miR-155-5p mimic. The proportion of G2/M-phase cells increased significantly in SiHa cells transfected with 100 nmol/L and 200 nmol/L miR-155-5p inhibitor. CONCLUSION: Compared with healthy controls, the serum level of miR-155-5p in the cervical cancer patients increases, and may act as a novel tumor molecular marker for diagnosis of cervical cancer. miR-155-5p has no significant effect on the proliferation, cell cycle and apoptosis of HeLa cell. miR-155-5p may promote SiHa cells to enter S phase and inhibit the apoptosis of SiHa cells.     

Effect of camptothecin on the activation,proliferation and cell cycle distribution of the mouse T lymphocytes in vitro

AIM: To study the effects of camptothecin (CPT) on the activation, proliferation and cell-cycle distribution of the mouse T lymphocytes stimulated by concanvalin A (ConA) in vitro. METHODS: A model of T cell activation and proliferation was established by stimulated the cells with Con A. T cells were treated with different concentrations of CPT. The expression of CD69, the early marker of CD3+ T cell activation, was measured by FACS. The proliferation index was determined by carboxyl fluorescin diacetate succinmidyl ester by flow cytometry. The cell-cycle distribution was analyzed by propidium iodide staining. RESULTS: After stimulation with Con A for 6 h, the activation rate of CD69+ T cell in Con A group was (58.88±0.55)%. The percentages of CD69 positive cells were (55.48±0.98)%, (54.67±1.05)%, (50.40±0.82)%, (42.47±1.32)%, correspond to the treatments with different concentrations of CPT (10 nmol/L, 20 nmol/L, 50 nmol/L, 100 nmol/L), respectively. After 48 h treatment with Con A, the proliferation index in different concentrations of CPT treatment (10 nmol/L, 20 nmol/L, 50 nmol/L and 100 nmol/L) exerted a definite inhibitory effect on the proliferation (P<0.01). Moreover, the cell-cycle distribution analysis showed that apoptosis peak was observed in different concentrations of CPT treatment after 48 h cultured with Con A. CONCLUSION: CPT significantly inhibits the early stages of the Con A-induced T cell activation and proliferation, and detents the T lymphocytes in G₀/G₁ phase.     

Effect of CD151 on biological characteristics of human umbilical cord mesenchymal stem cells

AIM：To study the effect of CD151 on the biological characteristics of human umbilical cord mesenchymal stem cells (hUC-MSCs). METHODS：CD151 expression on hUC-MSCs was interfered by siRNA. The cells were divided into siRNA-CD151 group and negative control group (treated with siRNA-NC). The efficiency of interference after 72 h and the changes of other surface markers were detected by flow cytometry. The ability of differentiation was assessed by oil red O and von Kossa staining. The cell cycle was analyzed by flow cytometry. The mRNA expression of CD151, hepatocyte growth factor (HGF), transforming growth factor β1 (TGF-β1), cyclooxygenase 2 (COX-2) and indoleamine 2, 3-dioxygenase (IDO) in hUC-MSCs was detected by real-time PCR. The secretion of HGF by hUC-MSCs was measured by ELISA. RESULTS：The results of flow cytometry showed that the expression of CD151 (11.97±2.63 vs 95.66±1.56, P<0.01) and CD105 (93.66±0.21 vs 83.37±0.71, P<0.05) on hUC-MSCs in siRNA-CD151 group was lower than that in negative control group. The consistent results were also achieved by using the method of real-time PCR. Treatment with siRNA-CD151 down-regulated the progress of the cell cycle as the G1 phase increased and the S phase decreased. The mRNA expression levels of HGF and TGF-β1 in hUC-MSCs in siRNA-CD151 group were lower than those in negative control group, and opposite result of COX-2 mRNA expression was observed. The IDO mRNA in hUC-MSCs was unchanged with IFN-γ stimulation for 24 h. HGF concentration in siRNA-CD151 group was decreased as compared with negative control group. CONCLUSION：Interfering CD151 expression on hUC-MSCs doesn’t change other surface markers except CD105, and maintains the capacity of adipogenic differentiation. However, it changes the osteogenic differentiation, proliferation and the expression of immunomodulatory cytokines.     

Effect of sinapine on H2O2-induced adipogenic differentiation of bone marrow mesenchymal stem cells

AIM:To investigate the effects of sinapine, an effective monomer of Chinese medicine, on hydrogen peroxide (H₂O₂)-induced adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).METHODS:The undifferentiated rat BMSCs were identified and screened by flow cytometry. The adipogenic differentiation of BMSCs was induced by H₂O₂, and the toxicity of sinapine on BMSCs was tested by CCK-8 assay. After the modeling method and the concentration range of sinapine were determined, the lipid droplets in the cells were detected by Oil Red O semi-quantitative assay, and the optimal drug concentration was selected. Finally, Oil Red O assay was observed 24 h after drug intervention, and the expression of adipogenic differentiation-related proteins, adipocyte protein 2 (aP2), peroxisome proliferator-activated receptor γ (PPARγ) and glucose transporter 4 (Glut4), at mRNA and protein levels in the BMSCs was determined by qPCR and Western blot.RESULTS:Treatment with H₂O₂ at 200 μmol/L for 1 h induced BMSCs to differentiate into adipocytes. Below the concentration of 40 μmol/L, sinapine had no toxicity to BMSCs. The best inhibitory concentration of sinapine on adipogenic differentiation was at 15 μmol/L. The number of lipid droplets in sinapine (15 μmol/L) group was significantly lower than that in model group. In sinapine group, the expression of aP2, PPARγ and Glut4 at mRNA and protein levels was lower than that in model group (P<0.01).CONCLUSION:Sinapine inhibits H₂O₂-induced adipogenic differentiation of rat BMSCs. The mechanism may be related to the PPARγ/AMPK signaling pathway.     

Effects of lipoxin A4 on proliferation and IL-2/IL-2 receptor expression in Jurkat T cells

AIM: To investigate the effect of lipoxin A4 on the proliferation and IL-2 production in Jurkat T cells. METHODS: Jurkat T cells were activated in vitro with anti-CD3 (2 mg/L) and anti-CD28 (2 mg/L) antibodies in the absence or presence of lipoxin A4 (0.1 nmol/L-100 nmol/L) for 24 h, then ［3H］-TdR was added into the medium and radioactivities were measured by scintillation counting. The concentrations of IL-2 in the supernants were determined by ELISA. Cells were harvested and the expression of CD25 was assessed by FCM. For analysing the cell cycle, the cells were stained with PI and DNA contents were detected by FCM. RESULTS: Lipoxin A4 suppressed the proliferation of anti-CD3 and anti-CD28 antibodies activated Jurkat cells in a dose-dependent manner, which was associated with reduced proportion of S phase cells. Furthemore, lipoxin A4 significantly inhibited the production of IL-2 but had no obvious effect on CD25 expression. CONCLUSION: Lipoxin A4 can suppress proliferation of activate Jurkat cells and IL-2 production, through which lipoxin A4 might negatively regulate immune response.     

Mechanism of 13-methyltetradecanoic acid in inducing bladder cancer T24 cell apoptosis in vitro

AIM: To study the effect of 13-methyltetradecanoic acid (13-MTD) on bladder cancer T24 cell apoptosis and the underlying mechanisms. METHODS: T24 cells were treated with 13-MTD at different concentrations. MTT cell proliferation assay was used to observe the inhibitory effect. The cell cycle and cell apoptosis were displayed by FCM. Apoptosis index was investigated by TUNEL assay. Western blotting was used to detect the expression of apoptosis related proteins. RESULTS: p38 and JNK phosphorylation were presented 2 h after stimulated by 13-MTD, but AKT phosphorylation were inhibited. Cytochrome C was released from the intermembrane space of mitochondria 8 h later. No change of FADD and p-FADD was observed. Several downstream caspase substrates (PARP, lamin B, RB) were cleaved in 12 h. CONCLUSION: In the course of T24 cell apoptosis induced by 13-MTD, JNK/SAPK and p38MAPK signaling pathway are activated and the PI3K/AKT signaling pathway is inhibited. The substrates of caspase: PARP, Rb, lamin B are cleavaged by activated caspase enzymes to promote T24 cell apoptosis.     

Effects of rhTGF-β1 on proliferation and osteogenic differentiation of MSCs in SD rats

AIM：To investigate the effects of recombinant human transforming growth factor β1 (rhTGF-β1) on the ability of proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (MSCs), as well as its effects on the expression of bone morphogenetic protein 2 (BMP-2), Smad4 and core binding factor α1 (Cbfa1). METHODS：SD rat MSCs were isolated and purified by the differential time adherent method. MTT assay was used to confirm the optimal concentration of rhTGF-β1 for the proliferation of MSCs. The optimal concentration for differentiation of MSCs into osteoblast was also determined by observing the activity and positive staining of alkaline phosphatase. According to the different induction conditions, MSCs were divided into 4 groups:control group, classic group, rhTGF-β1 group, and rhTGF-β1+classic group. Alkaline phosphatase, type I collagen, bone Gla protein and calcium nodes were detected to evaluate the osteogenic differentiation. BMP-2 was detected by ELISA and the mRNA expression of Smad4 and Cbfa1 was analyzed by real-time quantitative PCR. RESULTS：The optimal concentrations of rhTGF-β1 for the proliferation of MSCs and for the osteogenic differentiation of MSCs were 10 and 5 μg/L, respectively. The MSCs in classical group and rhTGF-β1 group were promoted to osteogenic differentiation, and the mRNA expression of BMP-2, Smad4 and Cbfa1 was increased. rhTGF-β1 induced osteogenic differentiation of MSCs in the early and middle terms. However, in rhTGF-β1+classic group, the osteogenic differentiation of MSCs was more obvious in the late term. CONCLUSION:The induction conditions of classical group, rhTGF-β1 group and rhTGF-β1+ classical group promote the differentiation of MSCs by increasing BMP-2 secretion and starting the TGF-β superfamily/Smads signaling pathway to regulate the differentiation of MSCs.     

Adipose-derived stem cells differentiate to neuron-like cells in vitro

AIM: We isolated and purified adipose-derived stem cells (ASCs) from adipose tissue in order to study their characters and examine their neuron differentiation capacity in vitro. METHODS: ASCs were isolated and cultured. The morphology of ASCs was observed under microscope and their phenotype was examined by flow cytometry. All-trans retinoic acid (ATRA) was used to induce ASCs into neuron-like cells. RT-PCR was used to detect the expression of nestin. Immunohistochemistry and Western blotting were used to detect the expression of neurofilament (NF) and neuron specific enolase (NSE), respectively. RESULTS: ASCs displayed a fibroblast-like morphology adhering to the culture plate. The cells expressed several surface antigens such as CD29 and CD105, while did not express CD31, CD34, CD45 and HLA-DR. Under suitable conditions, various passages of ASCs all had the capacity of neuron differentiation. They expressed nestin, NF and NSE 10 days after adding ATRA in the culture system. CONCLUSION: ASCs isolated from adipose tissue can differentiate into neuron-like cells in vitro. ASCs may be used as an alternative source of cells for neural disease therapy.     

Purification and differentiation potentiality of fetal liver mesenchymal stem cells from mouse

AIM: To purify and investigate the differentiation potentials of fetal liver mesenchymal stem cells (flMSCs) from murine in vitro.METHODS: flMSCs from mouse fetuses at embryonic and fetal day (ED)13.5 or ED14.5 were isolated by adhering to plastic and passaged by modified method.Cell cycle and phenotype were analyzed by flow cytometry.The cell differentiation was induced by special induction media.The cells differentiated to adipose,cartilaginous and osteoid tissues were identified with oil red O,Toluid blue,alkaline phosphatease (ALP) and von Kossa’s staining.The cells differentiated to neural-like cells were detected by RT-PCR and immuno-staining.RESULTS: Fibroblast-like cells predominated in culture.(83.76±2.88)% of flMSCs stayed in the G₀/G₁ phases.Homogenous cells were positive for mesenchymal lineage markers CD44,CD29,but not for markers of hematopoietic cells CD45,CD11b.flMSCs were able to differentiate into adipogenic,chondrogenic,osteogenic and neurogenic cells.CONCLUSION: flMSCs can be purified by modified plastic-attachment method and have multiple differentiation,which is available to stem cell therapy for various diseases.