Bone marrow mesenchymal stem cells modified by adenoviral vector containing CTLA4Ig inhibit immune response ex vivo

AIM: To investigate the feasibility and infection efficiency of MSCs as the target cells of gene delivery mediated by adenoviral vector carrying CTLA4Ig gene, and to study the mechanism of transgenic MSC to inhibit immune response ex vivo. METHODS: The recombinant adenovirus containing CTLA4Ig gene was constructed, by which rat MSCs with various multiplicity of infection (MOI) were conducted. The infection efficiency was analyzed with FACS and fluorescence microscope. The expression of CTLA4Ig protein in transgenic MSCs was detected by FACS and western blot. Co-culturing the transgenic MSCs with mixed lymphocytes, the inhibitory effect of transgenic MSCs on lymphocyte proliferation was also observed. RESULTS: The adenoviral vector delivered CTLA4Ig gene with high efficiency to MSCs. The expression of CTLA4Ig protein was detected in transgenic MSCs. The gene modified MSCs inhibited the proliferation of mixed lymphocytes and maximal inhibition rate was observed on day 4 of MLR. The inhibition induced by CTLA4Ig was donor-specific. CONCLUSION: MSCs is a promising target cell for gene delivery. The expressed CTLA4Ig specifically inhibited the lymphocyte proliferation ex vivo.     

Construction and identification of recombinant adenovirus vector containing CTLA4Ig gene

AIM:To construct a recombinant adenovirus expression vector containing CTLA4Ig gene.METHODS:The CTLA4Ig gene derived from the plasmid PCDNA3.0/CTLA4Ig by using polymerase chain reaction (PCR) was inserted into the backward position of cytomegalovirus (CMV) immediate early promoter of the shuttle plasmid (pAdTrack-CMV). After being identified by endonuclease, PCR and sequencing, the recombinant shuttle plasmid pAdTrack-CTLA4Ig was co-transformed into E.coli. BJ5183 cells with the adeoviral backbone plasmid pAdEasyl-1 to obtain the homologous recombination. The adenovirus was generated in 293 cells. A series methods such as PCR and fluorescence microscope was employed to identify the generated recombinant adenovirus.RESULTS:Recombinant CTLA4Ig adenoviruses were constructed and the titer of virus was generally up to 1.65×10¹² phaque forming units per liter (PFU/L).CONCLUSION:Success in constructing recombinant pAdTrack-CTLA4Ig will be the base of the further research on its expression in the mammalian cells, and be potenially used in the prevention of transplant rejection and autoimmunity diseases.     

Oxidized low density lipoprotein induces the expression of macrophage migration inhibitory factor

AIM: To examine expression of macrophage migration inhibitroy factor (MIF) gene and protein in macrophages induced by oxidized low density lipoprotein (ox-LDL). METHODS: Macrophages were incubated with ox-LDL at the concentration of 150 mg/L for time course (0-36 h) and with ox-LDL at the different concentrations (0-300 mg/L) for 24 h, expression of MIF mRNA and protein were detected by RT-PCR and ELISA. RESULTS: The results showed that ox-LDL increased MIF gene and protein expression in macrophages in a dose and time-dependent manner. After the exposure of macrophage to ox-LDL, the expression of MIF mRNA level increased consistently with protein. CONCLUSION: MIF may play an important role in atherosclerosis.     

Sodium butyrate induces mouse embryonic stem cells to differentiate into hepatocytes in vitro

AIM:To explore mechanism by which sodium butyrate induces mouse embryonic stem cells (ES) to differentiate into hepatocytes in vitro. METHODS:E14 mouse ES cells were cultivated in a routine way, and then cultivated in suspension to form embryonic bodies (EBs). EBs were transferred into 6-well culture dishes and 3 mmol/L sodium butyrate was added into the culture medium. Morphological changes were investigated by phase contrast microscopy. α-fetoprotein (AFP), albumin (ALB) and cytokeratin 18 (CK18) were examined by immunofluorescence staining. AFP, ALB, α1-antitrypsin (AAT) and TTR mRNA were assayed by RT-PCR. Proportion of ALB positive cells was analyzed by flow cytometry. Periodic acid Schiff (PAS) reaction and indocyanine green (ICG) uptake assay were performed to assess the characteristic hepatocyte function of the differentiated cells. RESULTS:In the presence of sodium butyrate, parts of ES cells differentiated into a population with epithelial morphology similar to mouse hepatocytes. AFP and TTR mRNA expression were observed at 7 d, and ALB and AAT mRNA expressed at 14 d. Hepatocytes specific markers, ALB, AFP and CK18 were positive expression in immunofluorescence staining at 14 d. PAS reaction and ICG uptake were positive for the hepatocyte-like cells. CONCLUSION:Mouse ES cells can be induced into hepatocyte-like cells by sodium butyrate efficiently, and these ES cells-derived hepatocytes possess characteristic hepatocytic function.     

Foam cells can be induced by oxidized low density lipoprotein in human monocyte-derived dendritic cells

AIM: The purpose of this study is to investigate the mechanisms related to oxidized low-density lipoprotein (ox-LDL) and dendritic cells (DCs) in the process of atherosclerosis.METHODS: Human DCs were prepared from human CD14+ peripheral blood monocytes using rhGM-CSF (100 μg/L) and rhIL-4 (40 μg/L).Cells were incubated with 100 mg/L native or oxidized LDL for 72 h.The formation of foam cells was investigated by electron microscopy and oil red O staining.Phenotypic and immune functional assays were used with FACS, FITC-dextran phagocytosis, allogeneic mixed T lymphocytes reaction and secretion of Th1/Th2 (IL-12/IL-2) cytokines were also conduced.RESULTS: DCs treated with ox-LDL, but not native LDL were induced into foam cells after cultured for 72 h.Compared with native LDL, ox-LDL-treated DCs were less potent in FITC-dextran phagocytosis.ox-LDL promoted allogeneic T cells proliferation.Moreover, ox-LDL upregulated CD80 (72.4± 9.6 vs 89.5±10.1, P<0.01), CD86 (67.2±8.8 vs 80.2±11.6, P<0.01), HLA-DR (80.6±9.8 vs 86.6±10.8, P<0.01) and CD1a (40.2±10.3 vs 60.2±9.3, P<0.01) expressions, increased IL-12 secretion ［(44.3±8.9)ng/L vs (65.1±10.4)ng/L, P<0.05］ in DCs.However, the secretion of IL-2 was decreased ［(43.6±7.8)ng/L vs (10.0±4.5 )ng/L, P<0.01］ significently.CONCLUSION: DCs were induced into foam cells by ingesting ox-LDL with some functional characteristic of mature DC.DCs seem to be a new source of foam cells and play a key role in immunopathogenesis of atherosclerosis.     

Role of B cells in CD45RB antibody-induced transplantation immune tolerance

AIM: To investigate the role of B cells in CD45RB antibody-induced transplantation immune tolerance. METHODS: Single cell suspension was made from the spleen of BALB/c nude mice disposed by CD45RB antibody, then mixed cultured with T cells of BALB/c mice and spleen cells of C57BL/6 mice. The Th1, Th2, Treg and Tm cells were monitored by flow cytometry during the culture process. The skin graft model was set up with B6.μMT^-/- mice as receptors and BALB/c mice as donors. CD45RB antibody was intraperitoneally injected into the receptors after transplantation and then CD3⁺CD45RB^hi cells were detected by flow cytometry. In another mixed lymphocyte culture, CD45RB antibody was added, and then B cells were isolated and injected into B6.μMT^-/- mice through the tail vein. The heart transplantation model was established with B6.μMT^-/- mice as receptors and BALB/c mice as donors, and then the survival and the migration of B cells to the thymus were observed. RESULTS: When T lymphocytes were co-cultured with B lymphocytes treated with anti-CD45RB monoclonal antibody(mAb) in vivo, the percentages of Th2 and Treg cells were up-regulated and Th1 cells were down-regulated, but Tm cells were not altered as compared with the control. In vivo without B lymphocytes, anti-CD45RB mAb also down-regulated the expression of CD45RB in T lymphocytes. The reduction was faster and the percentage of CD3⁺CD45RB^hi T cells was not altered as compared with the control. The B lymphocytes treated with anti-CD45RB mAb in vitro prolonged the lifetime of receptor in heart transplantation model but failed to induce complete tolerance. After recieving B cells treated with anti-CD45RB mAb and allogeneic heart transplantation, B cells migrated to the thymus in B6.μMT^-/- mice. CONCLUSION: B lymphocytes play a definite role in the transplantation immune tolerance induced by anti-CD45RB mAb through their affection on T-cell subgroups and also in the central tolerance. However, the induction of immune tolerance can not only rely on B cells.     

Effect of vitamin C on expansion of CD4+ effector memory T cells in vitro

AIM：To investigate the effect of vitamin C (VC), a small-molecule compound, on the expansion of CD4+ effector memory T cells (TEM) in vitro and to improve the effect of adoptive immunotherapy. METHODS：Normal human peripheral blood CD4+ T-lymphocytes were isolated and randomly divided into 2 groups: control group and experiment group. VC was added into experimental group. Thereafter, the expansion of CD4+ TEM in the 2 groups was detected by cell counter and flow cytometry. RESULTS：VC did not significantly affect the total number of CD4+ T cells, while it raised the ratio of TEM in CD4+ T cells at an optimal concentration of 100 mg/L. After 10 days of the expansion of CD4+ T cells, the number of CD4+ TEM in experiment group was (3.56±0.35)×106, significantly larger than that in control group ［(1.22±0.15)×106, P<0.01］. CONCLUSION: VC can effectively promote the expansion of CD4+ TEM in vitro, which provides a simple, safe and effective expansion method for adoptive immunotherapy.     

Doxorubicin induces stemness of mouse TNBC 4T1 cells through Stat3-Oct-4/CD44 signaling pathway

AIM:To investigate the stemness of mouse triple-negative breast cancer (TNBC) 4T1 cells induced by doxorubicin (DOX) and the underlying mechanism. METHODS:The 4T1 cells and MDA-MB-468 cells were treated with DOX at different concentrations (0, 0.05, 0.1 and 0.5 μmol/L) for 24 h, and the shape and viability of the cells were observed. The concentration of DOX at 0.1 μmol/L was chosen as the optimal concentration for the following experiments. The 4T1 cells and MDA-MB-468 cells resistant to DOX were established by continuous stimulation with DOX for 4 weeks, and named as 4T1-DOX and MDA-MB-468-DOX. Sphere formation assay was used to detect the stemness of 4T1 cells and MDA-MB-468 cells. The expression of CD133 was observed by immunofluorescence staining. The expression of CD44 was analyzed by flow cytometry. The protein levels of Stat3, phosphorylated Stat3 (p-Stat3) and Oct-4 were determined by Western blot. RESULTS:The sphere formation ability of the 4T1-DOX cells was stronger than that of the 4T1 control cells. The 4T1-DOX cells expressed high levels of the stemness markers CD133 and CD44 as compared with the 4T1 cells (P<0.05). Furthermore, the 4T1-DOX cells exhibited enhanced activation of Stat3 (p-Stat3) and increased expression of Oct-4 (P<0.05), while the expression of total Stat3 had no obvious variation. In addition, when activation of Stat3 was inhibited by WP1066, the protein levels of p-Stat3, Oct-4 and CD44 were down-regulated (P<0.05). Furthermore, inhibition of Stat3 phosphorylation reduced the sphere formation ability of the 4T1-DOX cells (P<0.05). CONCLUSION:DOX induces the stemness of mouse TNBC 4T1 cells through Stat3-Oct-4 signaling pathway.     

Low density lipoprotein from patients of diabetes mellitus induces apoptosis of murine macrophages via activating caspase-12 pathway

AIM:To explore the effect of low density lipoprotein from the patients of diabetes mellitus (DM-LDL) on the activation of caspase-12 an important molecule in endoplasmic reticulum stress (ERS)-associated apoptotic pathway, in the murine macrophages, and to clarify the underlying molecular mechanisms of apoptosis. METHODS:Murine macrophage RAW264.7 was exposed to DM-LDL (25, 50 and 100 mg/L), normal low density lipoprotein (n-LDL, 50 mg/L), or tunicamycin (TM, 4 mg/L) for 24 h. Additionally, RAW264.7 macrophages were precultured with 4-phenylbutyric acid (PBA, 5 mmol/L) for 1 h and then exposed to DM-LDL (100 mg/L) for 24 h. The cell viability and apoptosis were detected by MTT assay and flow cytometry with Annexin V-FITC/propidium iodide staining, respectively. Lactate dehydrogenase (LDH) activity in the media was measured by assay kit. The protein level of caspase-12 was determined by Western blot. RESULTS:Similar to TM (an ERS inducer), treatment with DM-LDL caused significantly decrease in the viability and increase in LDH activity in the media and apoptotic rate of the RAW264.7 macrophages (P<0.05). Additionally, DM-LDL induced activation of caspase-12 especially at the dose of 50 and 100 mg/L (P<0.01). However, the ERS inhibitor PBA protected RAW264.7 macrophages from DM-LDL-induced decrease in viability and increase in LDH activity and apoptosis (P<0.05). Furthermore, PBA attenuated DM-LDL-induced activation of caspase-12 (P<0.05). CONCLUSION:DM-LDL may induce apoptosis in RAW264.7 macrophages, and the mechanism may be related to the activation of caspase-12.     

Proliferation of CD4+CD25+ T cells from PBMCs of the gastric cancer patients and inhibitory effect on CD4+CD25- T cells in vitro

AIM:To investigate the proliferation of CD4+CD25+ T cells from PBMCs of the gastric cancer patients and the inhibitory effect on CD4+CD25- T cells in vitro. METHODS:Magnetic activated cell sorting (MACS) method was used to separate CD4+CD25+T and CD4+CD25-T cells from peripheral blood monocytic lymphocytes in the gastric cancer patients, and then the purity and activity of CD4+CD25+T cells were analyzed with flow cytometer. After stimulated with anti-CD3 Ab, anti-CD28 Ab and rh IL-2, CD4+CD25- and CD4+CD25+ T cells were cocultured. The inhibitory effect of CD4+CD25+T on CD4+CD25-T cells was assayed by ［3H］ thymidine proliferation experiment. RESULTS:(1)After sorting, CD4+CD25+ T cells purity in healthy control and gastric cancer patients were 83.80%±1.84% and 84.13%±2.77%, respectively. No significant difference between the two groups (P>0.05) was observed. (2)The activity of CD4+CD25+ and CD4+CD25- T cells in healthy control and the gastric cancer patients after sorting were 98.52%±0.72% and 97.80%±0.95%. There was no significantly difference between the two groups (P>0.05). (3) CD4+CD25+ T cells obviously inhibited the CD4+CD25-T cell proliferation in vitro. The inhibition achieved to maximum in coculture of CD4+CD25+ T cells together with CD4+CD25- T cells (ratio of 1∶〖KG-*2〗1). CONCLUSION:The MACS system can effectively isolate CD4+CD25+ and CD4+CD25- T cells. After sorting, CD4+CD25+T cells obviously inhibit the proliferation of CD4+CD25- T cells in vitro and the inhibitory effect display an effect-target ratio relationship.     

Role of CCL17 and CCL22 expression in dendritic cells in maternal-fetal immune tolerance

AIM: To determine the expression of CCL17 and CCL22 in dendritic cells (DC) from human decidua and endometria. METHODS: The decidua were collected from normal pregnant women undergoing induced abortion and recurrent spontaneous abortion (RSA) women undergoing early abortion.The endometria were cllected from non-pregnant women undergoing abdominal hysterectomy.The mononuclear cells in the decidua and endometria were isolated. DC were induced by GM-CSF and IL-4, cultured in vitro and identified. The expression of CCL17 and CCL22 in DC at mRNA and protein levels was analyzed by real-time PCR and ELISA. RESULTS: The mRNA levels of CCL17 and CCL22 in decidual DC in normal pregnancy group were 3.04?0.40 and 1.83?0.24, respectively, significantly higher than those in endometrial DC in non-pregnancy group (0.85?0.24 and 0.31?0.08, respectively, P<0.01) and those in decidual DC in RSA group (1.65?0.14 and 0.96?0.09,respectively,P<0.01). Decidual DC continually and strongly secreted CCL17 and CCL22. The levels of CCL17 and CCL22 in normal pregnancy group were significantly higher than those in non-pregnancy group and RSA group at the same culture time point (P<0.01). CONCLUSION: The expression of CCL17 and CCL22 in decidual DC in pregnant woman increases. This may attract more CD4⁺CD25⁺ regulatory T cells to decidua and play an important role in the establishment of maternal-fetal immune tolerance.     

XBP-01 accelerated apoptosis induced by oxidized low density lipoprotein in vascular smooth muscle cells

AIM: Previous studies performed with XBP-01 in vitro indicated that XBP-01 could inhibit vascular smooth muscle cells from being transformed into foam cell and could eliminate the atherosclerotic plaque in C57BL/6J mouse. This experiment is to investigate its mechanism of eliminating plaques in vitro. METHODS: The cultured porcine artery smooth muscle cells incubated with XBP-01 of 0.1 mg/L for 24 h after preincubated with oxidized low density lipoprotein of 15 mg/L for 72 h in vitro. The samples were analyzed by fluorescence microscope, confocal microscope system and flow cytometry. RESULTS: Apoptosis was triggered by being incubated with oxidized low density lipoprotein and this process was accelerated additionally by being incubated with XBP-01. CONCLUSION: XBP-01 can be effective in eliminating atherosclerotic plaque by accelerating the process in which oxidized low density lipoprotein induced smooth muscle cell apoptosis.     

TNF-α induces PIP3-mediated necroptosis in MLO-Y4 cells

AIM: To explore whether tumor necrosis factor-α (TNF-α) induces necroptosis in murine long bone osteocyte-like cell line MLO-Y4 and the possible mechanism. METHODS: The MLO-Y4 cells were divided into control group, TNF-α group, TNF-α+necrostatin-1 (Nec-1) group, TNF-α+Z-VAD group and TNF-α+receptor-interacting protein 3 (RIP3)-siRNA group. The death rate of MLO-Y4 cells was assessed by flow cytometry with Annexin V-FITC/PI staining. The morphological features of the cells were observed under transmission electron microscope (TEM). The protein levels of RIP1, RIP3 and cleaved caspase-3 were determined by Western blot. Finally, the numbers of total cells and RIP1-RIP3-positive cells were observed under laser scanning confocal microscope. The production of reactive oxygen species (ROS) in the cells was measured by DCFH-DA staining. RESULTS: Compared with control group, the apoptotic or necroptotic rate of the cells induced by TNF-α was increased significantly (P<0.01). The increased apoptotic or necroptotic rate was dramatically reduced by treating with Nec-1, Z-VAD or RIP3-siRNA transfection (P<0.01). In TNF-α group and TNF-α+Z-VAD group, a lot of MLO-Y4 cells with typical necroptotic morphological features were observed under TEM. However, obvious necroptotic cells were not found in Nec-1 or RIP3-siRNA treatment group. The protein level of RIP1 in the cells treated with Nec-1 was sharply lower than that in TNF-α group (P<0.01). However, Z-VAD did not reduce the elevated levels of RIP1 and RIP3. RIP3-siRNA effectively down-regulated the protein level of RIP3 compared with TNF-α group (P<0.01). Nec-1 effectively down-regulated the protein levels of RIP1 colocalized with RIP3 compared with TNF-α group (P<0.01). However, Z-VAD did not reduce the levels of RIP1 colocalized with RIP3. Nec-1, Z-VAD and RIP3 siRNA significantly decreased the ROS levels (P<0.01). CONCLUSION: TNF-α induces the necroptosis of MLO-Y4 cells. RIP3 play vital roles in the cell necroptotic signal pathway. ROS may be the executor of necroptosis of MLO-Y4 cells.     

Proliferation and apoptosis of human umbilical vein endothelial cells induced by oxidized low density lipoprotein

AIM:To investigate proliferation and apoptosis of cultured endothelial cell (ECV-304 cell line) induced by varied concentrations of oxidized low density lipoprotein (ox-LDL). METHODS:Cell morphology, Typan blue test, MTT test, LDH release test, flow cytometry and micro-molecular weight DNA fragment gel electrophoresis of apoptosis were used for the detection of the cytotoxic effects of ox-LDL on ECV-304 cell line.RESULTS:0.1, 1, 10 mg/L ox-LDL could promote proliferation of ECV-304 cells. When the concentration of ox-LDL reached up to 100 mg/L and above, the distinct cytotoxic effect appeared. Further study showed that the apoptosis rate of endothelial cells, induced by ox-LDL of 150 mg/L and 200 mg/L for 12 hours, are 15.86% and 21.89%, respectively. 18 h and above hours after incubation, the apoptosis rate began to decrease and rate of necrosis increased. CONCLUSION:ox-LDL has strong cytotoxic effects on endothelial cells and could give rise to different pathologic process, such as proliferation, apoptosis prophase, apoptosis and necrosis.     

Native and oxidized low density and very low density lipoprotein enhance the expression of MIP-1α mRNA in aortic smooth muscle cells

AIM: To understand whether native and oxidized low density and very low density lipoprotein (n-LDL, n-VLDL, ox-LDL, ox-VLDL) enhance the expression of macrophage inflammatory protein (MIP)1α mRNA in cultured aortic smooth muscle cells (SMCs). METHODS: Native low density and very low density lipoprotein were isolated from normal blood donors by density gradient ultracentrifugation, and were oxidatively modified by adding CuCl2. After a 24 h-exposure of the cultured SMCs to n-LDL， n-VLDL， ox-LDL and ox-VLDL, respectively, the expression of MIP-1α mRNA was determined by in situ hybridization and RT-PCR. RESULTS: Cultured aortic SMCs expressed MIP-1α mRNA at low level. N-LDL， n-VLDL， ox-LDL and ox-VLDL enhanced the expression of MIP-1α mRNA in SMCs, ox-LDL and ox-VLDL showed stronger effect than n-LDL and n-VLDL, respectively. The effect of ox-VLDL was most striking. There was a significant difference between groups (P<0.01). CONCLUSION: N-LDL, n-VLDL, especially ox-LDL and ox-VLDL, may play an important role in the formation of early atherosclerotic lesion by inducing SMCs to express MIP-1α.     

Role of Toll-like receptor 4/MAPKs pathway on monocyte chemoattractant protein-1 secretion induced by oxidized low density lipoprotein in vascular smooth muscle cells

AIM: To investigate the role of Toll-like receptor 4/MAPKs pathway on the secretion of monocyte chemoattractant protein-1 (MCP-1) induced by oxidized low density lipoprotein (ox-LDL) in the vascular smooth muscle cells (VSMCs). METHODS: mRNA and protein expressions of MCP-1 in VSMCs stimulated with oxidized low density lipoprotein were determined by RT-PCR and ELISA, respectively. The phosphorylated forms of ERK1/2 and p38MAPK were determined by Western blotting. TLR4 neutralizing antibodies (a specific TLR4 inhibitor), PD98059 (ERK1/2 specific inhibitor), SB23015 (p38MAPK specific inhibitor) and SP600125 (JNK specific inhibitor) were used to investigate the underlying mechanisms. RESULTS: The mRNA and protein expressions of MCP-1 in VSMCs were up-regulated by ox-LDL (P<0.05), while those were inhibited by TLR4 neutralizing antibodies, PD98059 or SB23015 (P<0.05), but not by SP600125 (P>0.05). TLR4 had regulatory effect on the phosphorylation of ERK1/2 and p38MAPK. CONCLUSION: ox-LDL is an endogenous ligand of TLR4. The secretion of MCP-1 induced by ox-LDL in VSMCs is at least in part via TLR4/ERK1/2 and TLR4/p38MAPKs pathways.     

Icaritin induces differentiation of MC3T3-E1 subclone 14 cells via estrogen receptor and BMP/Smad signaling pathway in vitro

AIM: To observe the effects of icaritin (ICT) on the proliferation and differentiation of MC3T3-E1 subclone 14 cells (a pre-osteoblast cell line) and to observe the role of estrogen receptor (ER) and bone morphogenetic protein(BMP)/Smads signaling pathways in the differentiation of the cells. METHODS: The methods of WST-8 and BrdU were used to observe the viability and proliferation of MC3T3-E1 subclone 14 cells after treatment with different concentrations of ICT. The effects of ICT and noggin on the levels of alkaline phosphatase(ALP), type I collagen (Col I) and bone Gla protein (BGP) in MC3T3-E1 subclone 14 cells were observed after ER was blocked by ICI182780. The relative mRNA levels of BMPs (2, 4, 7) were detected by real-time PCR. The protein phosphorylation of Smad1/5/8 was determined by Western blotting after ER signaling pathway was blocked by ICI182780. RESULTS: ICT at concentrations of 0.1 μmol/L and 1 μmol/L increased the levels of ALP, Col I and BGP, and the numbers of mineralized nodules in MC3T3-E1 subclone 14 cells, indicating that ICT-promoted the differentiation, but did not affect the cell viability and proliferation. After the ER receptor signaling was blocked, ICT-promoted differentiation was significantly decreased. ICT improved the mRNA expression of BMP-2, 4, but did not affect the mRNA expression of BMP-7. After the ER receptor signaling was blocked, ICT-promoted phosphorylation of Smad1/5/8 was significantly decreased. Blockage of BMP/Smad signaling inhibited the effect of ICT on the differentiation. CONCLUSION: Icaritin induces the differentiation of MC3T3-E1 subclone 14 cells by activating BMP/Smad signaling pathway through ER.     

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.