Lycium barbarum polysaccharides attenuate oxidative stress injury of hu-man endothelium-like EA. hy926 cells induced by hydrogen peroxide / 中国病理生理杂志

AIM:To study the effect of Lycium barbarum polysaccharides (LBP) on oxidative stress injury of human endothelium-like EA. Hy926 cells induced by hydrogen peroxide (H2O2). METHODS:The EA. Hy926 cell model of oxidative stress injury was established by H2O2 treatment. The EA. Hy926 cells were divided into 5 groups:control group, damage (H2O2 at 50 mmol/L) group, LBP (100 mg/L) group, anti-damage groups (LBP at 50 mg/L, 100 mg/L or 200 mg/L+50 mol/L H2O2), and LY294002 (20 μmol/L) group. The effect of LBP at different concentrations on the cell viability of EA. Hy926 cells was measured by CCK-8 assay, and the optimum concentration of LBP was screened out. The apoptotic of EA. Hy926 cells was analyzed by flow cytometry. Acridine orange/ethidium bromide ( AO/EB) staining was used to observe the morphological characteristics of the apoptotic cells. The cell migration ability was detected by scratch method. The levels of nitric oxide (NO) and vascular endothelial growth factor (VEGF) in the cell culture medium were examined. The protein levels of cleaved caspase-3, Bax, Bcl-2, endothelial NO synthase (eNOS), p-eNOS and p-Akt were determined by Western blot. RESULTS:LBP at concentration of 100 mg/L significantly attenuated the injury of EA. Hy926 cells induced by H2O2, as indicated by improved cell viability ( P <0.05 ) and decreased apoptosis ( P <0.05). Pretreatment with LBP elevated the levels of NO and VEGF (P<0.05), and promoted the migration ability of EA. Hy926 cells. LBP also increased the Bcl-2/Bax ratio, down-regulated the protein level of cleaved caspase-3, and up-regulated the protein levels of eNOS and p-eNOS. The protective effect of LBP were abolished by pretreatment of the EA. Hy926 cells with the inhibitor of PI3K (P<0.05). As a result, the protein level of p-Akt was down-regulated, and the level of NO was also significantly reduced. CONCLUSION:LBP has protective effect on H2O2-induced EA. Hy926 cells by attenuating apoptosis of the cells. The mechanism is closely related to the activation of PI3K/Akt/eNOS signaling pathway.

Effect of cladribine on growth inhibition and autocrining cytokines in hu-man umbilical vein endothelial cell line EA.hy926 / 中国病理生理杂志

AIM: To study the effects of cladribine on growth and secretion activity of human umbilical vein endothelial cell line EA.hy926, and to investigate the mechanism of its anti-tumor effect by inhibiting endothelial cells. METHODS:The effects of cladribine at different concentrations on the cell viability were detected by CCK -8 assay.Apop-tosis and cell cycle distribution were examined by flow cytometry.The protein expression levels were determined by Western blot.The levels of tumor necrosis factor-α(TNF-α), transforming growth factor-β1(TGF-β1)and vascular endothelial growth factor(VEGF)secreted by EA.hy926 cells with cladribine treatment for 48 h were analyzed by ELISA.The nitric oxide(NO)production was measured by Gries method.RESULTS:Cladribine at 0.4~1 μmol/L inhibited the viability of EA.hy926 cells in time-and dose-dependent manners.The IC50was about 3.644 μmol/L.The results showed 43.74% cells in S phase when the concentration of cladribine was 0.4 μmol/L,and 77.23 % cells in S phase when the concentra-tion of cladribine was 1 μmol/L.The apoptosis was not induced by cladribine at 0.4~10 μmol/L.The protein expression of Bax and caspase-3 did not change.The expression of p21 increased and the p53 decreased(P<0.05).The levels of TNF-αand TGF-β1 secreted by EA.hy926 cells increased after cladribine treatment for 48 h.The levels of VEGF and NO decreased.CONCLUSION:Cladribine obviously inhibits the viability of EA.hy926 cells.The mechanism is related to the cell cycle arrest.Cladribine promotes the secretion of TNF-αand TGF-β1 by EA.hy926 cells and inhibits the secretion of VEGF and NO.

CCL3 promotes hBMSC proliferation and inhibits exosome secretion / 中国病理生理杂志

AIM:To explore the regulatory effect of chemokine CCL 3 on exosome secretion from human bone marrow mesenchymal stem cells(hBMSCs).METHODS: hBMSCs were stimulated with chemokine CCL 3 at different concentrations in vitro.The proliferation of hBMSCs was measured by CCK-8 assay and viable cell counting.Exosome se-cretion from hBMSCs was qualitatively analyzed by transmission electron microscope(TEM)and flow cytometry, and the quantitative analysis was carried out by flow cytometry and nanoparticle tracking analysis(NTA).RESULTS:Compared with control group,the viability of the hBMSCs detected by CCK-8 assay was increased when hBMSCs were treated with CCL3(P<0.05).The results of viable cell counting demonstrated that the number of hBMSCs was raised in CCL 3 group in a dose-dependent manner(P<0.05).The results of flow cytometry showed that hBMSCs expressed 3 CCL3-related spe-cific receptors,CCR1,CCR5 and CCR9.Compared with control group,the fluorescence intensity of CCR9 in CCL3 group was obviously enhanced.However,no significant difference of fluorescence intensity for CCR 5 and CCR1 was observed be-tween the 2 groups.The results of NTA demonstrated that the secretion capacity of CCL 3-induced hBMSCs was far less than that in control group(P<0.05).However, the microvesicles larger than 100 nm in CCL3 groups were increased(P<0.05).The above results indicated that the higher concentration of CCL 3 induced the lower secretion of exosomes.In addi-tion,the results of flow cytometry demonstrated that CCL 3-induced hBMSCs showed lower quantity of CD 9 +exosomes than those in control group(P<0.01).CONCLUSION:CCL3 promotes the proliferation of hBMSCs but depresses the secre-tion of exosomes in a dose-dependent manner.CCL3 affects the size distribution of exosomes and increases the number of nonfunctional microvesicles of larger than 100 nm in size.CCL3 induces the expression of CCR9 in hBMSCs.

Salinomycin Enhances the Apoptosis of T-cell Acute Lymphoblastic Leukemia Cell Line Jurkat Cells Induced by Vincristine / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was aimed to investigate the effect of salinomycin combined with vincristine on the proliferation and apoptosis of Jurkat cells and its possible mechanisms.</p><p><b>METHODS</b>The proliferation of Jurkat cells was examined by CKK-8 assay. Flow cytometry was used to assess cellular apoptosis. Levels of BCL-2, caspase-3, and caspase- 8 were measured by Western blot.</p><p><b>RESULTS</b>The salinomycin or vincristine, either alone or in combination, inhibited the proliferation of Jurkat cells in a dose-dependent manner. Salinomycin combined with vincristine produced more obveous inhibition of cell proliferation than either compound used alone (P<0.05). Western blot analysis showed that the combined use of Sal and VCR reduced the expression of BCL-2 protein, and increased expression of caspase 3 and caspase 8 protein, more significantly. Furthermore, combination of Sal and VCR synergistally promoted apoptosis of the Jurkat cells (P<0.05).</p><p><b>CONCLUSION</b>The combination of salinomycin and vincristine synergistically inhibits proliferation and promotes apoptosis of T-cell acute lymphoblastic leukemia Jurkat cells.</p>

Apoptosis and expression of Fas/FasL in tumor infiltrating dendritic cells in human endometrioid adenocarcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate apoptosis of tumor infiltrating dendritic cells (TIDC) and their expression of Fas/FasL (CD95/CD95L) in human endometrioid adenocarcinoma.</p><p><b>METHODS</b>The apoptotic rate of TIDC was measured in 45 cases of endometrioid adenocarcinoma and 20 cases of normal endometrium tissues (control) by double-label immunohistochemistry using the monoclonal antibody S-100 protein and TUNEL technique. The expressions of Fas and FasL in TIDCs were detected using double-label immunohistochemistry and imaging analysis.</p><p><b>RESULTS</b>The apoptotic rate of TIDCs in endometrioid adenocarcinoma were significantly higher than that in normal endormetrium [(13.02∓0.64)% vs (6.82∓0.53)%, P<0.05]. The expression levels of Fas in the TIDCs were significantly lower, whereas FasL expression significantly higher in endometrioid adenocarcinoma than in normal endormetrium (7.88∓1.05 vs 19.25∓3.03, P<0.05; 12.95∓2.25 vs 7.51∓1.14, P<0.05).</p><p><b>CONCLUSION</b>Increased apoptosis of the TIDCs and abnormal expression of Fas/FasL in TIDCs in endometrioid adenocarcinoma may lead to tumor immune escape.</p>

Anti-aging effect of transplantation of mouse fetus-derived mesenchymal stem cells / 生理学报

To determine the role of allogeneil graft of mesenchymal stem cells in mammalian longevity, mesenchymal stem cells were isolated from BALB/c mouse uterine-incision delivery fetus by two successive density gradient centrifugations, and then were purified and amplified by adherent culture. Identified P1 mesenchymal stem cells were injected (i.v.) through vena caudalis into the 15-month-old female BALB/c mice three times. The mice were evaluated with ultrasoundcardiogram, autopsy, score of cardiac, skin, lung, kidney, colon histopathology and serum total superoxide dismutase activity, maleic dialdehyde content, glutathione peroxidase activity. The results showed that after transplantation, the long-term surviving stem cells were found to be located in many organ tissues with in situ Y chromosomal hybridization dyeing. Median life span was increased in these animals after transplantation. Skin, cardiac, lung, kidney and colon pathology development were delayed. The retrogradation of heart function was attenuated, the increase of heart mass index (the mass of heart/the mass of the body), and serum maleic dialdehyde content, the decrease of spleen mass index (the mass of spleen/the mass of the body), serum total superoxide dismutase activity and glutathione peroxidase activity were reduced three months after transplantation (all P<0.05). These results support the idea that longevity can be enhanced by transplantation of mesenchymal stem cells and reinforce the hypothesis of mesenchymal stem cell as antiager.

PI3K activity is associated with expression of neural specific genes in mouse fetal liver cells enhanced by butylated hydroxyanisole / 中国应用生理学杂志

<p><b>AIM</b>To study the mechanism of butylated hydroxyanisole-induced neural differentiation of fetal liver cells in vitro.</p><p><b>METHODS</b>14.5-day-old mouse fetal liver-derived cells were cultured, and were induced by 200 micromol/L butylated hydroxyanisole (BHA) combined with PI3K inhibitor LY294002 (20 micromol/L), and then were incubated in serum-free medium. Expression of genes in treated or untreated cells were assayed by Western blotting or RT-PCR.</p><p><b>RESULTS</b>There was low level of neurofilament-L (NF-L) and brain factor-1 (BF-1) but no neurofilament-H (NF-H) and tyrosine hydroxylase (TH) in fetal liver cells. BHA promoted significantly expression of neuron-specific NF-L, NF-H, BF-1, and TH in fetal liver cells. NF-L mRNA increased 5.8 fold, NF-H mRNA 8.0 fold, BF-1 mRNA 2.68 fold, and TH mRNA 30 fold, respectively (all P < 0.01 vs untreated cells). NF-L protein increased 11.29 fold, NF-H 5.5 fold, BF-1 2.53 fold, TH 4.76 fold. Moreover, expression of these BHA-induced genes were inhibited by PI3K inhibitor LY294002.</p><p><b>CONCLUSION</b>BHA induced neural differentiation of fetal liver cells through PI3K.</p>

CALM-AF10 fusion transcripts in primary leukemia with t(10;11) and in vitro chemotherapy sensitivity of leukemic cells with t(10;11) / 中国实验血液学杂志

In order to determine the involvement of CALM-AF10 fusion transcripted in primary leukaemias with t(10;11) and its chemotherapy sensitivity in vitro, the AF10-CALM fusion transcripts were detected by reverse transcription-polymerase chain reaction (RT-PCR), and the chemotherapy sensitivity testing in vitro was undergone by MTT assay in five t(10;11) leukemia samples from patients with ALL, AML and lymphoblastic lymphoma. The results showed that five different-sized AF10-CALM product and four different-sized CALM-AF10 products were detected. The chemotherapy sensitivity of leukemic cells with t(10;11) in vitro to drugs is lower than that of leukemic cells without t(10;11). 3 out of 5 cases of t(10;11) leukemia were sensitive to chemotherapeutic drugs, while 31 out of 36 cases of leukemia without t(10;11) were sensitive at same condition. There were significant differences (P < 0.01), consistent with clinical features of patients. Apoptosis rate of leukemic cells with t(10;11) induced by chemotherapeutic drugs was lower than that of leukemic cells without t(10;11), (16.37 +/- 2.56)%, and (33.75 +/- 5.59)%, respectively (P < 0.01). It is concluded that the CALM-AF10 fusion transcripts are a common features and are involved in the pathogenesis of haematological malignancies with t(10;11), and are associated with a poor prognosis.

Differentiation of human fetal liver CD34+ cells into neuronal cells induced by beta-ME and BHA in vitro / 中国应用生理学杂志

<p><b>AIM</b>To establish model of differentiation of fetal liver stem cells induced by beta-ME + BHA into neural cells in vitro;</p><p><b>METHODS</b>CD34+ cells from naturally aborted human fetal liver were isolated with MACS Kit, and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). After confluent more than 80%, the 5 passage cells were induced by 10(-3) mol/L beta-mercaptoethanol (beta-ME) and 2 x 10(-4) mol/L BHA for 24 hours, and washed with PBS, and then incubated in serum-free medium for 5 hours to 5 days. The characteristics of treated cells were assayed by immunocytochemistry staining analysis.</p><p><b>RESULTS</b>Cells treated by beta-ME+ BHA exhibited neuronal phenotype, and expressed neuronal specific proteins such as nestin, NeuN, TrnJ-1, and NF-M, which were not found in control cells. Statistic analysis showed that 81% cells were NeuN-positive, 75% cells TuJ-1-positive, 47% cells NF-M-positive, 90% cells NSE-positive.</p><p><b>CONCLUSION</b>beta-ME + BHA can induce human fetal liver CD34+ cells to produce neuronal specific antigens and proteins in vitro and become neuronal cells. CD34+ cells from human fetal liver possess potentials of differentiation into neural cells.</p>