Fetal liver-derived Sca-1+ cells can differentiate into both neurons and astrocytes in vivo

AIM: To determine whether Sca-1⁺ cells from fetal liver can differentiate into neural cells. METHODS: The sex of 14.5-day-old murine fetuses was determined by PCR analysis of sry gene, and Sca-1⁺ cells from male fetal liver were isolated with a magnetic cell sorting kit, 2×10³ of which were then transplanted into lethally irradiated female mice. The donor cells and their characteristics in recipient brains were identified and detected by FISH and immunohistochemistry double-staining analysis at 60, 120, 180 days after transplantation. RESULTS: There existed many male cells in brains of female recipients, some of them express neuron-specific nuclear protein (NeuN), and some of them express the astrocyte-specific marker, i.e. glial fibrillary acidic protein (GFAP). CONCLUSION: Sca-1⁺ cells from fetal liver, which contain hematopoietic stem cells, can differentiate into neuronal cells and astrocytes in the brains of adult mice.     

The transdifferentiation of Sca-1+ cells from murine fetal liver

AIM:To explore transdifferentiation potential of Sca-1⁺ cells from murine fetal liver. METHODS:2×10³ of Sca-1⁺ cells from male murine fetal liver were transfused into female mouse irradiated lethally with γ ray from ⁶⁰ Co source (10 Gy) via tail vein. Two months later, FISH and immunohistochemistry were used to detect the situation for transdifferentiating of the donor cells (male cells) in tissues of female recipient mouse. RESULTS:The renal tubular epitheliocyte-like and neurocyte-like cells with Y chromosome were found on the sections of renal and brain tissues from female recipient mice. These cells have phenotype characteristics of RCA⁺/CD₄₅^-F_4/80^- and NueN⁺/CD₄₅^-F_4/80^-, respectively. CONCLUSION:The evidence is provided for Sca-1⁺ cells from murine fetal liver to transdifferentiate into both renal and brain tissue cells.     

Effects of β-ME and RA on expression of GFAP in mesenchymal cells derived from mouse fetal liver

AIM: To investigate the effects of β-mercaptoethanol (β-ME) and all-trans rentinal acid (RA) on glial fibrillary acidic protein (GFAP) expression in mesenchymal cells derived from mouse fetal liver in vitro. METHODS: Cells suspension from 14.5-days-old mouse fetal liver were cultured in DMEM/HEPES/F12 supplemented with 20％ FCS and mesenchymal cells were acquired after discarding nonadherent cells. The 5th passage cells were induced by β-ME and RA. The characteristics of treated cells were assayed by immunocytochemistry staining at 5 hours and 5 days after induction. β-actin as an internal control, GFAP gene expression of mesenchyal cells was detected with semi-quantitative RT-PCR. RESULTS: After being inducted by β-ME and RA, 80％ approximately of the cells exhibited typical neural morphology and about 85％ expressed GFAP phenotype. Semi-quantitative RT-PCR showed that mRNA expression of GFAP increased in treated cells versus untreated cells (P<0.01). CONCLUSION: GFAP expression in mesenchymal cells derived from mouse fetal liver in vitro increases after being treated with β-ME and RA.     

Effect of lentiviral vector of microRNA-9-1 on differentiation of mouse bone marrow mesenchymal stem cells into neurons

AIM: To investigate the role of microRNA-9 in inducing bone marrow mesenchymal stem cell(MSCs) differentiation into neurons.METHODS: The lentiviral vector of microRNA-9-1(microRNA-9-1-LV) was constructed and transfected into mouse MSCs. The cells were divided into non-transfected group, transfected group(transfected with microRNA-9-1-LV) and negative control group(transfected with FU-RNAi-NC-LV). MSCs were treated with β-mercaptoethanol(β-ME) as an inducer for triggering the cells to differentiate into neurons. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope. The mRNA expression of microtublin-associated protein 2(MAP-2) was detected by RT-PCR. The expression of neuron-specific markers,neuron-specific enolase(NSE), MAP-2 and glial fibrillary acidic protein(GFAP), were measured by immunocytochemical method. The viability of MSCs was determined by MTT method. RESULTS: The results of PCR confirmed successful construction of mouse microRNA-9-1-LV. The virus titer was 1×10¹² TU/L(TU, transduction unit). The best transfection efficiency(up to 91.3%±4.2%) and survival rate appeared when multiply of infection(MOI)was 20 and on 4th day. β-ME induced MSCs to differentiate into neurons and the best efficiency of the induction was observed in transfected group. The expression levels of NSE and MAP-2 in transfected cells were higher than those in the cells of other group(P<0.05).CONCLUSION: MicroRNA-9-1-LV has high transfection efficiency in mouse MSCs. Higher differentiation rate from mouse MSCs to neurons is induced by β-ME after the cells are transfected with microRNA-9-1-LV.     

Insulin action potentiation by 17β-estradiol in cultured C2C12 mytoblasts

AIM:To investigate the effect of 17β-estradiol on insulin action in cultured C2C12 mytoblasts. METHODS:C2C12 mytoblasts were cultured in 35 mm wells of six-well culture plate in an atmosphere of 5% CO₂ at 37℃ in DMEM supplemented with 10% FBS and penicillin/streptomycin(1×10⁵ U/L) to reach 80% confluence. Insulin-resistance C2C12 mytoblasts were obtained by incubating the cells for 24 hours in the presence of a high concentration (5×10^-7 mol/L) of insulin. After treatmented with 17β-estradiol (1 nmol/L and 10 nmol/L, respectively) for 24 hours, C2C12 mytoblasts were performed to measure insulin-stimulated 2-DG uptake and GS, PFK, PK activities. RESULTS:17β-estradiol enhanced the capacity of insulin-stimulited 2-DG uptake, increased the GS, PFK and PK activities and prevented insulin-induced resistance in cultured C2C12 mytoblasts. CONCLUSION:17β-estradiol potentiates insulin action and preventes insulin-induced resistance in cultured C2C12 mytoblasts.     

Butylated hydroxyanisole promotes expression of neural specific genes in mouse fetal liver cells through extracellular signal-regulated kinase signaling pathways

AIM: To study the mechanism of butylated hydroxyanisole-induced neural differentiation of fetal liver Sca-1+ cells in vitro. METHODS: Sca-1+ cells from 14.5-day-old mouse fetal liver were isolated with a magnetic cell sorting kit. Cultured cells pretreated with or without extracellular signal-regulated kinase (MEK1) inhibitor, PD98059, were induced by 200 μmol/L butylated hydroxyanisole (BHA) for 24 hours, and then incubated in serum-free medium. Expression of genes in treated or untreated cells were assayed by Western blotting and RT-PCR. RESULTS: There was low level of neuronfilament-L (NF-L) and brain factor-1 (BF-1) in fetal liver Sca-1+ cells, but no neuronfilament-H (NF-H) and tyrosine hydroxylase (TH) was observed. BHA significantly promoted the expression of neuron-specific NF-L, NF-H, BF-1, and TH in fetal liver Sca-1+ cells. NF-L, NF-H, BF-1 and TH increased by 6.32 fold, 2.73 fold, 3.37 fold and 2.68 fold, respectively (all P<0.01 vs untreated cells). However, the expression of BHA-induced genes were inhibited by ERK kinase inhibitor PD98059 (25 μmol/L). CONCLUSION: BHA induces neural differentiation of fetal liver cells through ERK.     

Role of caveolin-1 on down-regulation of LPS-induced monocyte chemotactic protein 1 by 17β-estradiol in vascular smooth muscle cells

AIM: To elucidate the effect of caveolin-1 on the down-regulation of LPS-induced monocyte chemotactic protein 1 (MCP-1) by 17β-estradiol (E₂) in vascular smooth muscle cells (VSMCs).METHODS: The primary-cultured VSMCs were exposed to E₂ at concentrations of 10^-9-10^-6 mol/L. LPS-induced MCP-1 production was assayed by ELISA. The protein expression of caveolin-1 was determined by Western blotting and was silenced by β-methyl cyclodextrin(β-MCD) or caveolin-1 specific siRNA. RESULTS: LPS significantly enhanced MCP-1 production. E₂ at concentrations of 10^-9-10^-6 mol/L inhibited LPS-induced MCP-1 production. The use of caveolin-1 inhibitor β-MCD or silencing the protein expression of caveolin-1 by specific siRNA largely impaired LPS-enhanced MCP-1 production, while E₂ markedly inhibited caveolin-1 expression. CONCLUSION: Inhibition of LPS-induced MCP-1 production by E₂ is related to the suppression of caveolin-1.     

Supportive effects of human AGM region and fetal liver stromal cells on differentiation of murine embryonic stem cells into hematopoietic stem cells and in vivo hematopoietic reconstitution

AIM: To investigate the differentiation of murine embryonic stem cells (ESCs) into hematopoietic stem cells (HSCs) by the supportive effects of human aorta-gonad-mesonephros (AGM) region and fetal liver (FL) stromal cells.METHODS: E14 ESCs were induced into embryoid body (EB) first. Then the cells from EB were further co-cultured with human AGM region and FL stromal cells in non-contact system. On day 6, the cells derived from EB were collected for Sca-1⁺c-Kit⁺ cell analysis by flow cytometry, colony forming unit (CFU) assay and teratoma formation checking. BALB/c female mice conditioned with lethal dose of γ-ray irradiation were transplanted with EB cells from different culture systems. The survival rates, engraftment of donor cells, reconstitution of hematopoietic were monitored.RESULTS: Sca-1⁺c-Kit⁺ cells in EB cells co-cultured with human AGM region and FL stromal cells had the value of (21.96±2.54)%, and the total CFU was as (520±52)/10⁵ cells, which were statistically greater than those in EB cells only cultured with human AGM region stromal cells (P＜0.05). No teratoma was found in NOD-SCID mice after subcutaneous injection of EB cells co-cultured with human AGM region and FL stromal cells. In BALB/c female mice transplanted of EB cells co-cultured with human AGM region and FL stromal cells, the survival rate was 77.8%, and the peripheral blood cell count was obviously improved on day 14. PCR results showed the recipients all had sry gene copies from donor in bone marrow. The recipient mice transplanted with EB cells only cultured with human AGM region stromal cells all died within 15 days.CONCLUSION: Stromal cells from human AGM region and FL enhance the directed differentiation of ESCs into HSCs which can reconstruct hematopoiesis in vivo.     

Bio-effects of salusins on isolated rat heart and neonatal cardiomyocytes

AIM: To investigate the bio-effects of salusins on rat heart and cardiomyocytes. METHODS: The cardiac function was determined by multipurpose polygraph in isolated rat heart treated with various concentrations of salusin-α or salusin-β.[⁴⁵Ca²⁺] and[³H]-Leu incorporation were determined in cultured neonatal rat cardiomyocytes with β-liquid scintillation counter. RESULTS: 10^-12-10^-7mol/L salusin-α and salusin-β had no effects on isolated rat cardiac function. However, salusin-α and salusin-β stimulated uptake and[³H]-Leu incorporation. The [⁴⁵Ca²⁺] uptake induced by salusins were inhibited by nicardipine, and were synergistically increased by endothelin-1. The[³H]-Leu incorporation induced by salusin-α and salusin-β was inhibited by nicardipine, FK506 (a special inhibitor of carcineulin), PD₉₈₀₅₉ (inhibitor of MAPK) and chelerthine (inhibitor of PKC). The effects of salusin-β[⁴⁵Ca²⁺] on uptake was stronger than those of salusin-α. But there were no statistical difference in[³H]-Leu incorporation between salusin-α and salusin-β. CONCLUSIONS: Salusin-α and salusin-β did not affect directly cardiac function in rat hearts. But salusins improved calcium uptake and protein synthesize in neonatal rat cardiomyocytes. Those effects of salusins were related with calcium channel, carcinuelin, MAPK and PKC signal pathways. Salusins may be the regulatory factors for myocardium growth and hypertrophy.     

Protective effect of HSF1 on mice with LPS-induced acute lung injury and screening of relevant differentially-expressed genes

AIM: To study the protective effect of heat shock factor1 (HSF1) on the mice with lipopolysaccharide (LPS)-induced acute lung injury (ALI), and to screen the relevant differentially-expressed genes. METHODS: ALI mouse model was established by LPS intracheal instillation. The macroscopic and pathological changes of the lung tissue were observed, and the concentrations of total protein, TNF-α, IL-β, IL-6 and VEGF in the bronchoalveolar lavage fluid (BALF) were analyzed. Differentially-expressed genes in the lung tissues of HSF1^+/+ mice and HSF1^-/- mice with ALI induced by LPS were screened by gene chips. The key gene was verified by real-time qPCR. RESULTS: The macroscopic and pathological changes of the lung injury in HSF1^-/-+LPS mice were more serious than those in HSF1^+/++LPS mice. The concentrations of total protein, VEGF, TNF-α, IL-1β and IL-6 in the BALF of HSF1^-/-+LPS mice were significantly higher than those of HSF1^+/++LPS mice (P<0.05). Compared with the HSF1^+/+ mice, a total of 918 differentially-expressed genes were indentified in the HSF1^-/- mice, among which the expression levels of 65 genes had obvious diffe-rence, with 28 genes up-regulated, including Atg7, ccr1, cxcr2, Tbl1xr1, Mmp9, Pparg, Plcb2, Arrb2, Cntn1, Col4a6, etc, and 37 genes down-regulated, including Fgfr1, Fgfr2, Map4k4, Ddx58, Tfg, Stat3, Smad4, Lamc1, Sdc3, etc. The results of real-time qPCR showed that the mRNA level of CXCR2 in HSF1^-/-+ LPS mice was significantly higher than that in HSF1^+/++ LPS mice, which was consistent with the results of gene chips. CONCLUSION: HSF1 has protective effect on the mice with LPS-induced ALI. CXCR2 may be involved in the protective effect of HSF1 on this process.     

Meglumine cyclic adenylate induces differentiation of bone marrow mesenchymal stem cells into cardiomyocytes in vitro

AIM: To study the effect of meglumine cyclic adenylate (MCA) on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into cardiomyocytes in vitro. METHODS: The whole bone marrow adherent culture method was used to isolate, culture and amplify the BMSCs. The surface markers of BMSCs were determined by flow cytometry analysis. MCA at concentrations of 10^-2 mol/L, 10^-3 mol/L, 10^-4 mol/L, 10^-5 mol/L, 10^-6 mol/L and 10^-7 mol/L was added to the culture medium containing the second generation of BMSCs.5-Azacytidine(5-Aza) was used as a positive control. The cell viability was measured by MTT method.The cAMP content in BMSCs was detected by ELISA. The mRNA expression of GATA-4, Cx43 and β-MHC in MCA group and MCA+H89 (a PKA inhibitor) group was measured by SYBR-RT-PCR. The differentiation effects of MCA and 5-Aza were compared by flow cytometry. RESULTS: Most of the BMSCs expressed CD44 and CD71, and did not express CD45. MCA inhibited the viability of BMSCs in a time-and dose-dependent manner, and MCA atthe concentration of 10^-2 mol/L showed particularly remarkable effect. MCA significantly increased intracellular cAMP level in BMSCs in a concentration-dependent manner. The mRNA expression of GATA-4, β-MHC and Cx43 in MCA group were significantly higher than that in blank group (P<0.05), and the highest effect was under the condition of MCA induction at the concentration of 10^-3 mol/L for 3 days. The mRNA expression of GATA-4, β-MHC and Cx43 in MCA group was higher than that in 5-Aza group and H89+MCA group (both P<0.05). Differentiation rate in MCA group was slightly higher than that in 5-Aza group (20.24%±1.02% vs 18.39%±0.58%, P<0.05). CONCLUSION: MCA stimulates BMSCs to increase intracellular cAMP production and inhibits the viability of BMSCs, thus promoting the mRNA expression of GATA-4, β-MHC and Cx43 through the cAMP/PKA signaling pathway.     

EP2A promotes human CD34+ cell homing in vitro but not proliferation

AIM: To investigate the role of prostaglandin E₂ receptor 2 agonist (EP₂A) in proliferation and homing of human CD34⁺ cells. METHODS: Bone marrow fluid and peripheral blood containing stem cells were collected from healthy donors mobilized by granulocyte colony-stimulating factor in our department. Human CD34⁺ cells were isolated by the method of magnetic-activated cell sorting microbeads. Bone marrow mononuclear cells were isolated by Ficoll-Paque centrifugation, and the bone marrow mesenchymal stem cells (BMMSC) were cultured with L-DMEM. Human CD34⁺ cells and BMMSC were divided into 4 groups, and treated with PGE₂ (as positive control), DMSO (as negative control), EP₂A and EP₂A+prostaglandin E₂ receptor 2 antagonist (EP₂AA), respectively. After exposed to the reagents, human CD34⁺ cell viability was measured by CCK-8 assay, the number of colonies was evaluated by colony-formation assay, the cell cycle distribution was analyzed by flow cytometry, and the protein expression of survivin, β-catenin and CXC chemokine receptor 4 (CXCR4) was detrmined by Western blot. Moreover, the concentration of stromal cell-derived factor-1α (SDF-1α) in the BMMSC was detected by ELISA. RESULTS: The cell viability and the colony number of human CD34⁺ cells in EP₂A group were not higher than those in negative control group. Furthermore, the proportion of human CD34⁺ cells treated with EP₂A in G₂/M phase was not elevated compared with negative control group. The protein expression of survivin and β-catenin did not up-regulated in human CD34⁺ cells exposed to EP₂A, but the protein expression of CXCR4 in human CD34⁺ cells and the concentration of SDF-1α in BMMSC were elevated. CONCLUSION: EP₂A promotes human CD34⁺ cell homing in vitro but not proliferation.     

IL-1β promotes differentiation of naïve T cells into Th22 cells and its expression in non-small cell lung cancer

AIM:To investigate the mechanism of interleukin-1β (IL-1β) promoting the transformation of naïve T cells into Th22 cells and the correlation of its peripheral blood expression in non-small cell lung cancer patients. METHODS:CD4⁺ naïve T cell magnetic bead sorting kit was used to isolate the peripheral blood mononuclear T cells from healthy people. Transforming growth factor-β (TGF-β) and IL-2 were added to promote differentiation and proliferation. IL-1β was used to induce differentiation into Th22 cells. The proportion of CD4⁺ IL-22⁺ T cells was analyzed by flow cytometry, and the expression of IL-22 was detected by ELISA. We selected 60 cases of non-small cell lung cancer patients in our hospital, including 18 in I phase, 20 in Ⅱ phase, 13 in Ⅲ phase and 9 in IV phase, as well as 25 healthy persons. The proportion of Th22 (CD4⁺ IL-22⁺) cells in peripheral blood was detected by flow cytometry, and the serum levels of IL-1β and IL-22 were measured by ELISA. RESULTS:IL-1β induced the transformation of naïve T cells into Th22 cells and promoted the secretion of IL-22 (P<0.05). The proportion of Th22 cells and the IL-22 and IL-1β levels in peripheral blood of the patients with non-small cell lung cancer were higher than those in healthy subjects, and correlated with the clinical stage. CONCLUSION:IL-1β induces the differentiation of Th22 cells and the expression of IL-22. The levels of IL-1β and IL-22 are related to the progression of non-small cell lung cancer, which may be involved in immunosuppression and promote the occurrence of non-small cell lung cancer.     

Effect of urotensin II on cultured pulmonary arterial smooth muscle cells of rabbits

AIM: The effect of urotensin II (U-II) on proliferation of cultured pulmonary arterial smooth muscle cells (PASMCs) of rabbits and its mechanism are investigated. METHODS: PASMCs were isolated using explant technique. RPASMCs were incubated in serum-free medium with different concentrations of nicardipine, calcimodulin antagonist W₇, PKC inhibitor H₇ or MAPK inhibitor (PD₉₈₀₅₉), with or without U-II. RPASMC proliferation was examined by MTT assay and by the increase in [³H]-thymidine incorporation into DNA. RESULTS: U-II (10^-9 mol/L-10^-7 mol/L) increased A value of PASMCs by MTT assay and [³H]-thymidine incorporation in PASMCs in a dose-dependent manner. U-II induced a maximal effect at a concentration of 10^-7 mol/L. A value and [³H]-thymidine incorporation rose 42.9% and 68.5% (P<0.05), respectively. U-II had no effect at a concentration of 10^-10 mol/L. Nicardipine, W₇, H₇, PD₉₈₀₅₉ (10^-7 mol/L-10^-5 mol/L) inhibited the effect of U-II in inducing increase of A value and -thymidine incorporation in a dose-dependent manner, with the maximal inhibitory rate of 42.3%, 19.6%, 23.2%, 10.5% (P<0.05) in A value and 46.6%, 9.8%, 21.7%, 14.7% (P<0.05) in [³H]-thymidine incorporation at concentration of 10^-5 mol/L, respectively. CONCLUSION: Our results suggest that U-II may induce proliferation of PASMCs of rabbits by Ca²⁺, CaM, PKC and MAPK signal transduction pathway.     

Expression of Wnt/β-catenin signaling pathway-related proteins in breast cancer and its clinical significance

AIM: To evaluate the expression of Wnt/β-catenin signaling pathway-related proteins in breast cancer and the significance. METHODS: The patients with breast cancer (n=150) in our hospital from January 2015 to January 2017 were selected as study object. The tumor tissue samples of these patients were obtained from paraffin section of breast cancer by surgical resection with complete clinicopathological data. The corresponding paracancerous tissue sam-ples were taken from the non-tumor tissue samples from the above breast cancer patients, which were 0.5~1 cm away from the tumor tissue. The methods of real-time PCR and Western blot were performed to examine the expression of Wnt-1 and β-catenin at mRNA and protein levels. Human breat cancer MCF-7 cells were divided into 3 groups:control group (MCF-7 cells without treatment), agonist group[MCF-7 cells+Wnt3a (1 mg/L)] and antagonit group[MCF-7 cells+DKK1 (16 μmol/L)]. The expression of Wnt-1 and β-catenin at mRNA and protein levels was detected by real-time PCR and Western blot. RESULTS: Compared with the paracancerous tissues, the expression levels of Wnt-1 and β-catenin were higher in tumor tissues at mRNA and proteins levels (P<0.05). Notably, the positive expression rates of Wnt-1 and β-catenin were significantly higher in tumor tissues than that in the paracancerous tissues. Furthermore, Wnt-1 expression was associated with tumor metastasis (χ²=5.352, P=0.021), tumor stage (χ²=9.412, P=0.002) and tumor size (χ²=9.412, P=0.002). In addition, β-catenin expression was also associated with tumor metastasis (χ²=9.851, P=0.002) and tumor stage (χ²=5.661, P=0.017). Compared with control group, the expression of Wnt-1 and β-catenin at mRNA and protein levels in agonist group was increased (P<0.05),while that in antagonist group was decreased (P<0.05). CONCLUSION: The expression levels of Wnt-1 and β-catenin related with Wnt/β-catenin signaling pathway are increased in the breast cancer, which are closely related to the malignant state of the tumor.     

Morphological study of macrophages infected with constructed recombinant adenovirus carrying gp120 gene of Chinese HIV-1 strain

AIM: To construct a recombinant adenovirus carrying gp120 gene of Chinese HIV-1 strain,which can infect mouse bone marrow-derived macrophages (BMM). METHODS: Co-transfection of shuttle and backbone plasmids of AdMax system into 293Ad5⁺ cells was performed, followed by viral packaging, propagation and purification. These viruses were subject to Karber TCID₅₀ titration. The expression of gp120 protein in 293Ad5⁺ cells was determined by ELISA. The viral titration was validated by a multiplicity of infection (MOI) test with BMM. RESULTS: The titers of the outcome viruses, including AdMax-HIV-1 gp120 (Ad-gp120) and its vector control Ad-GFP, were 10^8.3 and 10^8.1 TCID₅₀/mL, respectively. Both recombinant adenoviruses infected BMM with similar capacity of 293Ad5⁺ cell infection, which validated the TCID₅₀ titration.The gp120 protein was positive in 293Ad5⁺ cell lysates. BMM activation was observed morphologically after Ad-gp120 infection as compared with Ad-GFP-infected cells. CONCLUSION: Functional adenovirus containing HIV-1 gp120 of prevalent strains in China was successfully constructed. Infection of Ad-gp120 causes BMM activation.     

Effects of thyroid hormone on the myosin heavy chain mRNA expression in cardiac myocytes induced by angiotensin Ⅱ

AIM: To study the effect of thyroid hormone on the expressional change of myosin heavy chain(MHC) gene in cardiomyocyte induced by angiotensinⅡ(AngⅡ) and its potential mechanism. METHODS: Cardiac myocyte was cultured according to the method of Simpson. 10^-8 mol/L T₃ and 10^-7 mol/L AngⅡ were added to the culture medium, respectively or synchronously. After 48 h, the expression of α and β-MHC mRNA in myocytes were detected by RT-PCR. The protein kinase C activation were detected by PepTag non-radioactive PKC assay. The incorporation of -Leucine and [³H]-thymine to test the protein and DNA synthesis in myocytes were also performed. RESULTS: AngⅡalone increased the incorporation of [³H]-Leucine of myocytes while it had no effect on the incorporation of [³H]-thy mine. The expression of β-MHC mRNA was increased and the expression of α-MHC mRNA was decreased significantly at the condition of AngⅡ. The enhanced PKC activation was induced by AngⅡalso. When AngⅡand T₃ were added to the culture medium synchronously, though the incorporation of [³H]-leucine and [³H]-thymine were not changed compared with AngⅡ treated alone. The α-MHC mRNA expression was increased and the β-MHC mRNA expression was decreased significantly. The PKC activation of the myocytes also was decreased. CONCLUSIONS: T₃ inhibited the expressional change of myosin heavy chain gene in cardiac myocytes induced by AngⅡ. The effect of T₃ on the change of PKC activation in cardiac myocytes may be one of its mechanisms.     

Effects of interferon-γ, tumor necrosis factor-α and interleukin-1β on apoptosis of human airway smooth muscle cellsin vitro

AIM:To clarify if interferon-γ(IFN-γ), tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)can induce apoptosis of human airway smooth muscle cells (ASMCs) in vitro.METHODS:Human ASMCs were isolated and cultured in DMEM containing 10% fetal bovine serum. Passage 4-6 cell was used in the experiment. IFN-γ,TNF-α and IL-1β, were used separately or together in the treatment of human ASMCs. The effects of IFN-γ,TNF-α and IL-1β on the growth of the cells was detected by MTT method at the hour 0,24,48 and 72. Light microscopy and electron microscopy were used to examine the morphological change. DNA fragmentation was analyzed by agarose gel electrophoresis. SP immunohistological staing method was performed to detect the change of expressions of p 53, bcl- 2 and bax gene. The apoptosis cell percentage were detected by in situ end labeling technique (TUNEL)of fragmental DNA. RESULTS:(1)IFN-γ or IFN-γ together with TNF-α and IL-1β decreased the number of viable cells in a time dependent manner. (2) Light and electron microscopic examination showed cell shrinkage, membrane blebbing, nuclear contraction, chromatin condensation and nuclear fragmentation in human ASMCs. (3) Agarose gel electrophoresis showed a characteristic"ladder"of DNA bands representing integer multiples of the internucleosomal fragments (about 180-200 bp) in cytokine cotreated human ASMCs. (4)The expression of p 53 and bax gene in cytokine cotreated group was significantly higher than in control group, but the expression of bcl-2 gene was lower than in control group. (5)Stimultaneous treatment with IFN-γ(4×10⁵ U/L),TNF-α(4×10⁵ U/L)and /or IL-1β (10×10⁴ U/L) induced apoptosis of human ASMCs. Apoptotic index of human ASMCs in cytokine co-treated group was significantly higher than in control group (P＜0.01).CONCLUSION:Stimultaneous treatment with IFN-γ,TNF-α and /or IL-1β induced apoptosis of human ASMCs. These immune cytokines may play an important role in airway remodeling of asthma and of chronic obstructive pulmonary disease.     

Role of Oct3/4 in differentiation of rat bone marrow mesenchymal stem cells into neurons

AIM: To investigate the role of Oct3/4 in inducing differentiation of rat bone marrow mesenchymal stem cells (MSCs) into neurons in vitro. METHODS: Lentivirus (LV) vector containing Oct3/4 gene was constructed and transfected into rat bone marrow MSCs. The MSCs were divided into non-transfection group, transfection group (transfected with Oct3/4 -LV) and negative control group (transfected with FU-PCG-NC-LV). β-mercaptoethanol (β-ME) was used to induce differentiation of MSCs into neurons. Morphological changes and the fluorescence in transfected MSCs were observed under inverted fluorescence microscope. The expression of Oct3/4 and microtubulin-associated protein 2(MAP-2) at mRNA and protein levels was detected by RT-PCR and Western blotting. The expression of Oct3/4 and the neural cell specific markers neuron-specific enolase(NSE), MAP-2 and glial fibrillary acidic protein(GFAP) were determined by immunocytochemical method. The viability of the MSCs was analyzed by MTT assay. RESULTS: The results of PCR confirmed that the Oct3/4 -LV was successfully constructed and the virus titer was 2×10¹¹ TU/L. The best transfection efficiency and survival rate appeared when multiply of infection(MOI) was 10 and at 48 h, and the fluorescence of MSCs was mostly displayed. The efficiency of transfection was up to 83.4%±2.2%. The shape of the MSCs was changed in transfection group, and the survival rate of the MSCs in transfection group was significant lower than that in other groups (P<0.05). MSCs were induced by β-ME to differentiate into neurons and the best efficiency of induction was observed in transfection group. The typical neuronal morphology was observed in transfection group after induction and the expression levels of NSE and MAP-2 were higher than those in other groups (P<0.05). Compared with other groups, the expression of Oct3/4 in transfection group was significantly increased (P<0.01). Furthermore, the expression of Oct3/4 was time-dependently decreased and there was significant difference between before induction and 5 h after induction (P<0.05). CONCLUSION: Oct3/4 may have an important role in regulating the differentiation of rat MSCs into neurons.