Effect of conditioned medium of mesenchymal stem cells on proliferation, migration and adhesion of human umbilical vein endothelial cells / 中国实验血液学杂志

The aim of this study was to explore the effect of mesenchymal stem cell (MSC) conditioned medium (MSC-CM) on proliferation, migration and adhesion of human umbilical vein endothelial cell (CRL1730) and its mechanism. Isolation and purification of MSC were performed with the classic adhering method, the surface markers (CD29, CD90, CD45 and CD34) in MSC were detected by flow cytometry. MSC were treated and cultured for 3 d, the MSC-CM or MSC overexpressing stem cell-derived factor-1 (SDF-1) conditioned medium (Ad-SDF-1-MSC-CM) were collected. Subsequently, CRL1730 cells were treated respectively with 2% FBS-DMEM, 15% FBS-DMEM (control group), MSC-CM or Ad-SDF-1-MSC-CM for 24 h, the proliferation of CRL1730 cells was detected by MTT method. CRL1730 cell migration in vitro was performed by using wound healing system. The adhesion ability of CRL1730 cells was analyzed by microscope. The results indicated that the CRL1730 cells treated with Ad-SDF-1-MSC-CM showed greater proliferative capacity than CRL1730 cells treated with MSC-CM. While adding with AMD3100 5 µmol/L, the blocker of CXCR4, the CRL1730 proliferation mediated by Ad-SDF-1-MSC-CM was significantly reduced. Meanwhile, compared with MSC-CM, Ad-SDF-1-MSC-CM had greater effects for promoting CRL1730 migration and enhancing adhesion ability of CRL1730 cells, these effects were significantly inhibited by AMD3100. It is concluded that MSC-CM promotes the migration and adhesion ability of CRL1730 cells through SDF-1 expressed by MSC.

Effect of vascular endothelial growth factor on bone marrow-derived mesenchymal stem cell proliferation and the signaling mechanism / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of vascular endothelial growth factor (VEGF) on bone marrow-derived mesenchymal stem cell (MSC) proliferation and explore the signaling mechanism involved.</p><p><b>METHODS</b>MSC culture was performed following the classical whole bone marrow adhering method. The characteristics of MSC were identified by induction of multi-lineage differentiation and flow cytometry for surface marker analysis (CD34, CD45, CD29, and CD90). Following the addition of 50 nmol/L wortmannin, 50 µmol/L PD98059, 30 µmol/L SB203580, 10 µmol/L H89, 20 µmol/L Y27632, 1 µmol/L rapamycin, 10 µmol/L straurosporine, 6 nmol/L Go6976, or 50 µmol/L Pseudo Z inhibitors in the cell culture, the MSC were treated with 20 ng/ml VEGF and the changes of the cell proliferation rate was measured with MTT assay.</p><p><b>RESULTS</b>Cultured MSC were capable of multi-linage differentiation and did not express VEGF-R, CD29 or CD90. Treatment with 20 ng/ml VEGF obviously promoted MSC proliferation, and this effect was inhibited partially by p38 mitogen-activated protein kinase (MAPK) inhibitor rapamycin, PD98059, SB203580, Go6976, and straurosporine.</p><p><b>CONCLUSIONS</b>VEGF promotes MSC proliferation in close relation to the AKT-PKC pathway, in which PKC signal pathway may play the central role.</p>

VEGF promotes the proliferation of bone marrow derived mesenchymal stem cells through ERK1/2 signal pathway / 中国实验血液学杂志

In order to explore the effect of VEGF on mesenchymal stem cell (MSC) proliferation and its possible signal transduction mechanism, MSC culture was performed with the classical bone marrow adhering method; characteristics of passage 3 rat MSC (P3MSC) was identified through multi-differentiation and surface marker assay (CD34, CD45, CD90, CD29); P3MSC were treated with 20 ng/ml VEGF, and the effect of VEGF on the MSC proliferation was measured during 12, 36 and 72 hours by MTT assay. Subsequently, P3MSC were treated with extracellular-signal regulated kinase (ERK1/2) inhibitor PD98059 (50 µmol/L) or p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580 (30 µmol/L) for 30 minutes, the culture medium was replaced with new medium including 20 ng/ml VEGF. After 72 hours, the effect of PD98059 or SB203580 on MSC proliferation mediated by VEGF was measured by MTT assay. The result showed that the cultured MSC expressed PDGFR-α, PDGFR-β and NRP1, but did not express VEGF-R (Flk1 and Flt1). The MSC had the multi-differentiation ability and displayed the characteristics of CD90+ (96.7%), CD29+ (94.6%), CD34- (0.79%) and CD45- (0.84%). The MSC proliferation rate increased gradually with prolonging of the functioning time of 20 ng/ml VEGF, and MSC proliferation rate may reach to maximum value after treating with 20 ng/ml VEGF for 72 hours. The effect of VEGF on MSC proliferation was found to be abolished, even was under level of control group after treating with PD98059 or SB203580 for 30 minutes. Furthermore, the inhibitory effect of PD98059 on MSC proliferation was obviously higher than that of SB203580. It is concluded that the VEGF can promote MSC proliferation, and its possible mechanism may relate to ERK1/2 pathway.

Effect of adenovirus-mediated stromal cell-derived factor-alpha gene transfer on ventricular remodeling in rats with myocardial infarction / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the effect of adenovirus-mediated human stromal cell-derived factor-1alpha (hSDF-1alpha) on ventricular remodeling in rats with myocardial infarction.</p><p><b>METHODS</b>A recombinant adenoviral plasmid containing hSDF-1alpha cDNA was constructed using homologous recombination in bacteria and the recombinant adenovirus particles expressing hSDF-1alpha (AdV-SDF-1) were prepared. In rat models of myocardial infarction induced by left anterior descending artery occlusion, 1x10(10) PFU AdV-SDF-1 or PFU AdV-LacZ were injected at multiple sites into the infarcted myocardium 1 h after the operation, using 200 l cell-free PBS as the control. Four weeks after the injection, the cardiac function of the rats was analyzed, and the heart tissues were taken after the measurement of hemodynamics. On serial frozen sections, histological observation and morphometric measurement were carried out using a microscopic image analysis system, and the expression of hSDF-1alpha was detected by immunocytochemistry.</p><p><b>RESULTS</b>Four weeks after AdV-SDF-1 injection, the myocardium in the infracted area showed significantly higher expression rates of hSDF-1alpha. The injection resulted in a obvious reduction in the infarct size and collagen content and a marked increase in the left ventricle wall, and the rats showed improved cardiac functions.</p><p><b>CONCLUSION</b>SDF-1alpha can improve the cardiac structure and function in rats with myocardial infarction by inhibiting collagen synthesis and deposition in the infarcted area.</p>

Comparison of migration characteristics of MSCs in different assay systems / 中国实验血液学杂志

The aim of this study was to explore the difference of MSC migration mediated by SDF-1/CXCR4 axis through Boyden chamber in vitro migration assay. The SDF-1 density-dependence of MSC migration was observed. Subsequently, the effects of different blocking agents on hSDF-MSC migration were observed after MSC were treated with 50 nmol/L wortmannin, 10 micromol/L LY294002, 50 micromol/L PD98059, 10 micromol/L U73122, 126 micromol/L AMD3100 and 50 nmol/L verapamil respectively. The results showed the efficiency of MSC migration increased gradually with the increasing of hSDF-1 density. And after MSCs treatment with 50 nmol/L wortmannin, 10 micromol/L LY294002, 50 micromol/L PD98059, 10 micromol/L U73122 and 126 micromol/L AMD3100 respectively, the ability of MSC migration decreased. The ability of MSCs migration obviously decreased when MSCs were treated with U73122, AMD3100. It is concluded that the SDF-1/CXCR4-mediated MSC migration may be related to mitogen-activated protein kinase (MAPK), phosphatidylinositol phospholipase C (PI-PLC) and protein kinase (PKC) signal pathways.

The role of PEP-1-SOD1 fusion protein on ischemia-reperfusion injury in isolated perfused rat hearts / 中华心血管病杂志

<p><b>OBJECTIVE</b>The transduction efficiency of the purified PEP-1-SOD1 fusion protein and the effects of PEP-1-SOD1 fusion protein on ischemia reperfusion injury in the isolated perfused rat hearts were investigated.</p><p><b>METHODS</b>The constructed pET15b-SOD1 and pET15b-PEP-1-SOD1 were transformed into BL21 (DE3) for expression and purification of SOD1 and PEP-1-SOD1, respectively. Isolated perfused rat hearts were subjected to 60 min of global ischemia and 30 min of reperfusion and treated with vehicle, 100 micromol/L SOD1 and 25, 50, 100 micromol/L PEP-1-SOD1, respectively. The transduction efficiency was evaluated with immunofluorescent microscopy and Western blot. The enzyme activity of the transduced PEP-1-SOD1 was measured with commercial SOD detection kit. The MDA content in myocardial tissue and the CK activity in coronary exudate at 15 min after reperfusion were also measured. Cardiomyocyte apoptosis was detected with TUNEL. The infarct size was determined in isolated hearts 60 min after reperfusion with TTC staining.</p><p><b>RESULTS</b>Immunofluorescent microscopy and Western blot demonstrated PEP-1-SOD1 was transduced into myocardial tissue in a dose-dependent manner, whereas SOD1 could not be detected in SOD1 group. SOD activity in control, SOD1 group, 25, 50, 100 micromol/L PEP-1-SOD1 groups was (10.06 +/- 0.77) U/mg prot, (10.59 +/- 0.71) U/mg prot, (32.29 +/- 1.42) U/mg prot, (43.16 +/- 1.16) U/mg prot, (55.14 +/- 1.59) U/mg prot, respectively. MDA content in corresponding groups was (1.48 +/- 0.19) nmol/mg prot, (1.39 +/- 0.11) nmol/mg prot, (1.01 +/- 0.14) nmol/mg prot, (0.73 +/- 0.13) nmol/mg prot, (0.50 +/- 0.06) nmol/mg prot, respectively. CK activity in corresponding groups was (1.73 +/- 0.58) U/mg prot,(1.68 +/- 0.14) U/mg prot,(1.40 +/- 0.28) U/mg prot,(0.97 +/- 0.39) U/mg prot, (0.61 +/- 0.56) U/mg prot, respectively. Cardiomyocyte apoptotic index in corresponding groups was (17.25 +/- 0.75)%, (16.63 +/- 1.07)%, (11.50 +/- 0.57) U/mg prot, (6.50 +/- 0.63) U/mg prot, (4.13 +/- 0.52)%, repectively. The percentage of myocardial infarction area was (55.13 +/- 2.18)%, (52.13 +/- 2.59)%, (33.88 +/- 2.06)%, (25.50 +/- 2.16)%, (15.38 +/- 1.14)%, respectively. Compared with control group and SOD1 group, all P < 0.01 These results demonstrated the enzyme activity of the transduced PEP-1-SOD1 was significantly increased in a dose-dependent manner and the MDA content, CK activity, the cardiomyocyte apoptotic index and the infarct size was decreased siginificantly in PEP-1-SOD1 pretreatment groups compared with SOD1 group.</p><p><b>CONCLUSION</b>The native, biologically active form of PEP-SOD1 fusion protein could be effectively transduced into the isolated rat hearts subjecting ischemia reperfusion injury in a dose-dependent manner. The transduced PEP-1-SOD1 has protective effects on ischemia reperfusion injury in the isolated rat hearts.</p>

Protective effect of preconditioning with PEP-1-CAT fusion protein against myocardial ischemia-reperfusion injury in rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the transduction efficiency of purified PEP-1-CAT fusion protein into rat heart and the protective effect of the fusion protein against myocardial ischemia-reperfusion injury.</p><p><b>METHODS</b>PEP-1-CAT or CAT (500 microg) was injected in SD rats via the caudal vein, using normal saline as the control, and the hearts were harvested at 0.5, 1, 2, 4, 8, and 24 h after the injection. The transduction efficiency was evaluated by immunofluorescence technique, and the CAT activity was measured. Forty rats were randomized into 5 groups, namely the sham-operated group, ischemia-reperfusion group, and 3 PEP-1-CAT -treated groups (100, 300, and 500 microg). The left main coronary artery was occluded for 1 h followed by a 2-h reperfusion, and at the end of reperfusion, serum LDH and CK and MDA content in the myocardium were measured.</p><p><b>RESULTS</b>No green fluorescence was observed in saline group or CAT group. Bright green fluorescence was observed in PEP-1-CAT groups at different time points, most conspicuous at 8 h. No significant difference in CAT activity was found between CAT group and saline group (P>0.05); with the lapse of time, CAT activity in PEP-1-CAT group increased gradually, reaching the peak level at 8 h, which was 4.2 folds of that in the saline group. LDH ,CK and MDA were significantly lower in PEP-1-CAT- groups than in ischemia-reperfusion group (P<0.01).</p><p><b>CONCLUSION</b>PEP-1 can mediate the transduction of CAT in rat heart in a time-dependent manner, and PEP-1-CAT preconditioning provides a protective effect against ischemia- reperfusion injury in rats.</p>

Adenovirus-mediated stromal cell-derived factor-1 alpha gene transfer promotes mesenchymal stem cell migration / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the role of stromal-derived factor-1 (SDF-1) in the migration of mesenchymal stem cells (MSCs) and the underlying signal transduction mechanism.</p><p><b>METHODS</b>Rat bone marrow-derived MSCs were infected with 100 ml recombinant adenovirus containing human SDF-1alpha gene (Ad-hSDF-1alpha), and the cell migration changes were observed at 1, 2, and 3 days after the infection. Twelve hours after Ad-hSDF-1alpha transfection, the MSCs in separate cultures were treated with wortmannin (50 nmol/L), LY294002 (10 mmol/L), PD98059 (50 mmol/L), U73122 (10 mmol/L), AMD3100 (0.1 g/L), or verapamil (50 nmol/L), respectively, and the signal transduction pathways involved in MSC migration were analyzed.</p><p><b>RESULTS</b>The MSCs grew in colonies after transfection with Ad-hSDF-1alpha, but this growth pattern was substantially attenuated after treatment with wortmannin, LY294002, PD98059, U73122, AMD3100 and verapamil, among which U73122 and AMD3100 treatments resulted in the most conspicuous inhibitory effects.</p><p><b>CONCLUSION</b>The effect of SDF-1 in promoting MSC migration is related to mitogen-activated protein kinase, phosphatidylinositol phospholipase C, and protein kinase signal pathways.</p>

Effects of different doses of thrombopoietin on proliferation of bone marrow mesenchymal stem cells in mice / 中国实验血液学杂志

To explore the effect of different doses of thrombopoietin on proliferation of bone marrow mesenchymal stem cells (MSCs) in mice, 20 Kunming mice (35 +/- 5 g) were divided randomly into 4 groups: low-dose TPO group, moderate-dose TPO group, high-dose TPO group and normal control group (n = 5). The experimental groups were subjected to intraperitoneal injections of TPO at a dose of 25, 50, 100 microg/kg, respectively, and normal control group were treated with saline at a dose of 0.1 ml/g per day for 5 days. The bone marrow was harvested on 12 hours after the final administration. The bone marrow nucleated cells (BMNCs) were counted and seeded at a density of 10(6) cells/cm(2). The colony-forming unit-fibroblast (CFU-F) of MSCs was cultured and evaluated. The CFU-F of MSCs underwent osteo-genic induction and adipogenic induction, and cytochemical and immunocytochemical staining were performed to verify their multipotential. CFU-F and the cell percentage of CD90(+), CD105(+), CD34(+) in BMNCs were analyzed by flow cytometry. The results showed that the number of BMNCs and the cell percentage of CD90(+), CD105(+), CD34(+) and CFU-F increased obviously in TPO groups as compared with the normal control group (p < 0.05). The number of BMNCs increased most obviously in the 50 microg/kg TPO group. However, there was no significant difference in number of CFU-F between 50 microg/kg and 100 microg/kg TPO group (p > 0.05). The CFU-F of MSCs in bone marrow had their osteogenic and adipogenic differentiation potentials in vitro. It is concluded that the number of BMNCs and the cell percentage of CD90(+), CD105(+) and CFU-F increased after administration with TPO. It means that TPO can enhance MSCs to proliferate in bone marrow. However, the number of BMNCs and CFU-F can not increase with the increase of TPO dose.