Protective effect of nicotinic acid amide on human umbilical cord mesenchymal stem cells

AIM: To investigate the effect of nicotinic acid amide (NAA) on the infusion damage of human umbilical cord mesenchymal stem cells (hUC-MSCs) under the condition of instant blood-mediated inflammatory reaction (IBMIR). METHODS: Normal peripheral blood without anticoagulant at volume of 2.7 mL was mixed with 0.3 mL physiological saline (as blank group), CFSE labeled hUC-MSCs (1×10⁶ cells in 0.3 mL as MSC group) and CFSE labeled hUC-MSCs (1×10⁶ cells in 0.3 mL) preprocessed with NAA at concentration of 10 mmol/L for 24 h (as MSC+NAA group), respectively. The mixture was immediately injected into the improved Chandler Loop model, placed in 37 ℃ water bath, and then started the peristaltic pump at the speed of 20 mL/min for 1 h. The number of CFSE labeled hUC-MSCs, platelets, white blood cells were counted and the concentration of complement C3a was measured before and after cycling, respectively. RESULTS: After 1 h circulation, the platelet dissipation rate were (29.96±10.88)% in blank group, (77.76±19.29)% in MSC group all and (50.13±18.10)% in MSC+NAA group; and the leukocyte counts were (37.82±13.81)% in blank group, (64.57±17.08)% in MSC group and (41.52±17.26)% in MSC+NAA group. Compared with blank group, the differences of the dissipation rates in MSC group and MSC+NAA group all had statistical significance. The hUC-MSCs relative survival rate in MSC+NAA group was higher than that in MSC group. C3a concentrations in blank group, MSC group and MSC+NAA group were (206.27±58.10), (230.47±39.61) and (208.37±40.66) μg/L, respectively. CONCLUSION: Co-circulating the mixture of hUC-MSCs with normal peripheral blood without anticoagulant in the improved Chandler Loop for 1 h depletes a large number of hUC-MSCs and blood components, and increases C3a, suggesting that this model can induce IBMIR. NAA has a protective effect on the hUC-MSCs in the infusion damage by inhibiting IBMIR, reducing the wastage of the blood components and enhancing the survival rate of the hUC-MSCs.     

Effects of cytokines on renovation of acute renal failure in mouse with bone marrow derived mesenchymal stem cell transplantation

AIM: To observe the effects of cytokines on renovation of acute renal failure (ARF) in mouse with bone marrow derived mesenchymal stem cell (MSC) transplantation. METHODS: ARF animal model was induced in mouse by subcutaneous injection of cisplatin. Mice were randomly assigned into 3 groups: normal control group, ARF group and MSC group. After 24 h cisplatin injection, animals were injected intravenously with MSC in MSC group. Animals were sacrificed at 1 d, 4 d, 7 d, 14 d and 28 d after cisplatin injection. The blood urea nitrogen (BUN) and serum creatinine (Scr) were measured. The renal morphologic changes were scored with Paller’s criterion on hematoxylin and eosin (HE) stained sections. The mRNA and protein expressions of HGF, BMP-7, TNF-α and IL-10 were detected by RT-PCR and immunohistochemistry method. RESULTS: After 4 d of cisplatin injection, the BUN and Scr values in MSC group were significantly lower than those in ARF group (P<0.01). After 7 d and 14 d, the values of BUN and Scr in MSC group were still lower than those in ARF group (P<0.01, P<0.05). The renal morphologic scores of MSC group were also lower than those of ARF group. After 7 d, the expressions of HGF, BMP-7 and IL-10 were higher in MSC group than those in ARF group, the expression of TNF-α in MSC group was lower than that in ARF group. CONCLUSION: MSC promotes the recovery of acute renal failure induced by cisplatin. The mechanism may partly depend on paracrine of growth factor and amelioration of inflammatory.     

Effects of IL-6 and IL-11 on differentiation of cord blood CD34+ cells towards megakaryocytes

AIM: To investigate the effects of interleukin-6 (IL-6) and interleukin-11 (IL-11) on differentiation of cord blood CD34+ cells towards megakaryocytes and platelet production in vitro.METHODS: The CD34+ cells from fresh umbilical cord blood samples were cultured in serum-free culture medium with thrombopoietin (TPO) 50 μg/L,IL-3 10 μg/L,stem cell factor (SCF) 50 μg/L as control groups,then 10 μg/L IL-6 or IL-11 or IL-6＋IL-11 respectively was added as treatment groups.Mononuclear cells (MNCs) in cultured cells were detected by cell counter,megakaryocytes (CD41+ cells) and platelets were measured by flow cytometry,respectively.Platelet agglutination after thrombin induced was observed by microscopy and flow cytometry.RESULTS: Compared with the control group,the number of MNCs was not significantly different（P>0.05）,but the numbers of CD41+ cells and platelets were increased significantly (P<0.05) in treatment groups.There were more platelet particles in treatment groups than those in control group by microscopy and the results also showed that the cytoplasmic fragments from the cultures responded to thrombin induction.CONCLUSION: It is concluded that both IL-6 and IL-11 induce the cord blood CD34+ cells to differentiate towards megakaryocytes and produce platelets.     

Effects of cotransplantation of umbilical cord blood and mesenchymal stem cells by intra-bone marrow injection on hematopoietic reconstitution in a rat model

AIM: To investigate the effects of cotransplantation of mesenchymal stem cells (MSCs) and umbilical cord blood (UCB) by intra-bone marrow (IBM) injection on the hematopoietic reconstitution and recovery of bone marrow MSCs in the recipients. METHODS: Wistar female rats were transplanted with fetal and neonatal peripheral blood (FNPB) and BrdU-labeled MSCs separated from BMNCs of F344 rats. The MSCs were infused by IBM injection in bilateral tibiae or intravenous injection (IV), while the FNPB was all via IBM route. The survival rate, reconstitution of hematopoietic and immunological function, engraftment level of HSCs and recovery of bone marrow (BM)-MSCs in recipients were monitored. The origins of BM-MSCs of recipients were examined by immunofluorescence assay. RESULTS: (1)The survival rate in the two cotransplantation groups was 100% at day 60, while that in FNPB group was only 66.7%. (2)The counts of peripheral blood cells and BM hematopoietic stem/progenitor cell colonies of the recipients were better in cotransplantation groups than those in FNPB group, especially in the FNPB (IBM)+MSC (IBM) group. (3)No significant difference between of engraftment level of HSCs in the two cotransplantation groups was observed. The percentage of RT1A1 cells subset in FNPB (IBM)+MSC (IBM) group was much higher than that in FNPB group (P<0.05). (4)At day 30, the growth characteristic of recipient BM-MSCs was still below normal, but that in FNPB (IBM)+MSC (IBM) group was the best of all the experiment groups (P<0.05). (5)The donor MSCs coexisted with host MSCs in only a few recipient rats. CONCLUSION: The cotransplantation of MSCs and FNPB can accelerate the recovery of recipient BM-MSCs and hematopoietic reconstitution, promote the engraftment level of HSCs. Cotransplantation by IBM route is safe and has better effects on hematopoietic reconstitution than by IV route.     

Effect of proliferating cell nuclear antigen specific antisense oligonucleotide on differentiation of cord blood CD34+ cells

AIM:To study the effect of proliferating cell nucler antigen antisense oligonucleotide on ex vivo expansion of cord blood CD34+ hematopoietic stem/progenitor cells. METHODS:CD34+ cells were purified from fresh cord blood by immunomagnetic beads. CD34+ cells were incubated in liquid culture system with different concentrations of PCNA-ASODN. Using flow cytometry, the number of different kinds of stem/progenitor cells and PCNA expression were measured after CD34+ cell incubation. RESULTS:PCNA was lowly expressed in low experiential group, with a positive rate of (27.2±3.6)% and (19.0±1.5)%, the positive rate of control group was (53.8±8.3)% (P<0.01), a high significant difference was observed. Just in low concentration of PCNA-ASODN, the percentage of CD34+cells were increased to (33.4±3.2)%, CD34+CD38-cells expanded (57.8±9.9) folds, and the percentage of CD34+cells were (25.2±2.6)% (P<0.01), the CD34+CD38-cells expanded (43.5±7.4) folds (P<0.05), it has significant difference compared with the control group. CONCLUSION:Low concentration of PCNA-ASODN decreases PCNA expression effectively, slows down differentiation of CD34+cells during ex vivo expansion procedure, and improves the expansion efficiency.     

Adult human mesenchymal stem cell differentiates into adipocytes

AIM:To investigate the differentiation from human mesenchymal stem cells (hMSC) to adipocytes.METHODS: hMSC were separated from rib marrow and expanded in culture medium. To detect the surface antigens, the labeled cells were analysed on a FACScan flow cytometer. hMSC were induced with dexamethasone, insulin, 1-methy1-3-isobutylxanthine and indomethacin which acted as adipocyte differentiation inducer. The cells were stained with Oil Red O. The number of adipocytes were counted on a phase-contrast microscope.RESULTS: hMSC were expanded as undifferentiated cells in culture for more than 5 passages. The isolated cultured MSC comprised a single phenotypic population and displayed a fibroblast-like morphology. These expanded, attached MSC were uniformly positive for CD29, CD44, CD90, CD105, CD166 and didn't express CD14, CD34, CD45, CD11a. After induced with induction medium, lipid vacuoles were first detectable within the cells at 48 hours. Two weeks later, more than 85% MSC differentiated into adipocytes which displayed a perinuclear accumlation of lipid vacuoles, as detected by Oil Red O. CONCLUSION:hMSC can be induced to differentiate into adipocytes.     

Biological characterics of human fetal cord blood mesenchymal stem cells

AIM: To investigate the biological characterics of human second-trimester fetal cord blood mesenchymal stem cells (MSC) and its application prospects in utero gene transfer/therapy (IUGT). METHODS: Nuclear cells separated from cord blood were cultured in DMEM medium. Surface antigens of the MSC were analyzed by the FACScan flow cytometry. Adipogenic and osteogenic mediums were used to assess the differentiation ability of the cells. Adenovirus vector deliver green fluorescent protein gene (Ad-GFP) was used to transfected the MSC and the expressing of GFP was detected by fluorescent microscope. The MSC were injected into the liver of newborn rat. The immunofluorescence analysis was conducted to determine the presence of double-positive CD105⁺/CD166⁺ cells in different organs of rats. MSC were subcutaneous injected into the human-nonobese diabetes/severe combined immunodeficiency disease (NOD/SCID) mice and carcinogenesises of the MSC in vivo were detected by pathological diagnosis. RESULTS: MSC could be separated from fetal cord blood. These cells were uniformly positive for CD29, CD44, CD59, CD105, CD166 and negative for CD34, CD45, CD80, CD86, HLA-DR. The cells had the abilities to differentiate into adipogenic and osteogenic cells in vitro, expressed the GFP at high levels (56.32%±3.28%). The MSC were located at different organs after injected into the newborn rats and didn't have carcinogenicity in vivo. CONCLUSION: Human second-trimester fetal cord blood MSC is an promising target cells in fetal IUGT.     

Biological characteristics of JAK2 transduced CD34+ cells from cord blood during ex vivo expansion

AIM: To explore the feasibility and biological characterization of long-term regulated expansion of JAK2 transduced human CD34⁺ cord blood cells in vitro.METHODS: A retrovirus (RV) vector which contains JAK2 catalytic domain and two binding sites for a chemical inducer,dimerization (AP20187),was cloned (designated MGI-F₂JAK2).CD34⁺cells were enriched from cord blood with a MiniMACS system.The purified CD34⁺ cells were transfected with supernatant from the retrovirus packaging cell line that expressed JAK2.Following transduction,cells were expanded into four groups: AP20187 alone,FL alone,TPO,alone,AP20187+FL+TPO,respectively.The expanded cells were monitored by GFP expression,immunophenotyping,progenitor colony assay,karyotype analysis as well as tumorigenesis in nude mice.RESULTS: The purity of selected CD34⁺ cells was over 91% and gene transfer rate was 49.32%±6.21%.Only the group of AP20187 +FL+ TPO was obtained a significant sustained outgrowth of the transduced CD34⁺ cord blood cells.The percentage of GFP+ cells consistently produced a rise to the 90% peak level by the end of 8th week of culture.Flow cytometry analysis showed that the phenotype of the expanded cells was CD33⁺,CD61⁺ and Gly-A⁺ partial positive;CD38⁺ and HLA-DR⁺ strong positive,while CD2,CD7 and CD19 were almost negative.Colony assays performed in methycelluos,which can give rise to BFU-E,CFU-GM and CFU-Mix,the CFU-GM was predominantly in all colonies.The tumor was not observed in nude mice and the karyotype analysis was normal from expanded cells.CONCLUSION: The results demonstrate that AP20187-mediated activation of JAK2 signaling is capable of stimulating expansion JAK2 transduced CB CD34⁺ cells in combination with FL and TPO.This system may have applications for studies in signaling transduction,hematopoiesis,and for gene and cell therapy.     

Supportive and expansive effects of aorta-gonad-mesonephros (AGM)-derived stromal cells on hematopoietic stem in vitro

AIM: To explore the supportive and expansive effects of aorta-gonad-mesonephros(AGM) region derived stromal cells on hematopoietic stem cells (HSC) in vitro.METHODS: The murine stromal cells were separated and cultured from AGM region of a 11 day postcoitum (dpc) mouse embryo and 6 week mouse. After identification by Wrights staining and flow cytometry, the stromal cells were co-cultured with the embryonic stem cell(ESC)-derived, cytokine-induced HSCs, and the maintenance and expansion of HSCs were evaluated by detecting CD34+，CD34+Sca-1+cells with flow cytometry.Blast colony-forming cell (BL-CFC) and high proliferative potential colony-forming cells(HPP-CFC) were determined by semi-solid medium clonal culture.RESULTS: AGM-derived and bone marrow(BM)-derived stromal cells were similar in morphology and phenotype, and had common character of stromal cells. Supported by AGM stromal cells or by BM stromal cells, more primitive progenitor cells HPP-CFC were expanded, but BL-CFC expansion was only detected in AGM-derived stromal cells. In the supporting of BM stromal cells CD34+ hematopoietic stem/progenitor cells were expanded 3-4 times, but no significant expansion in CD34+Sca-1+ cells was observed. While in the supporting of AGM stromal cells, both CD34+ hematopoietic stem/progenitor cells and CD34+Sca-1+ cells were expanded significantly from 4 to 5 times, respectively (P<0.05).CONCLUSION: AGM-derived stromal cells significantly support the expansion of HSC, and also maintain the self-renewal activity and multi-lineage differentiation of HSC in vitro.     

Characters of the fibroblast-like cells cultured from the mobilized peripheral blood cells

AIM: To explore the characters of fibroblast-like (F-L) cells cultured from granunocyte clony stimunating factor (G-CSF)-mobilized peripheral blood cell (PBC) harvests. METHODS: The adherent cells in the PBC harvests were cultured for 2 week in the mediums of RPMI-1640/L-DMEM/G-CSF or interleukin-3 (IL-3) plus RPMI-1640, the cultured F-L cells were analyzed by flow cytometry (FC). RESULTS: The adherent non-confluent F-L cells obtained from the four groups were similar in their phenotypes: CD33+, CD11c+, CD64+, CD14+, CD45+, HLA-DR+, CD86+, CD34-, CD38-, CD3-, CD19-, CD56-, CD29-, CD44-, CD105-. The F-L cells are similar to monocytes except CD38- and were distinct from dendritic cells (DC) or mesenchymal stem cells (MSC). CONCLUSION: The cultured F-L cells are macrophages rather than DC or MSC. G-CSF, rhIL-3 enhances their numbers.     

The ex vivo expansion characteristic of endothelial progenitor cells

AIM: To explore the ex vivo expansion characteristics of the endothelial progenitor cells (EPCs). METHODS: CD34⁺ cells were selected from umbilical cord blood mononuclear cells (MNC) by MiniMACS system, expanded at the same conditions as that for total MNC, coincubation of CD34⁺ and CD34^- from the same donation for EPCs. In addition, we tested the effect of vessel endothelial growth factor (VEGF) and passage on cell differentiation, expansion kinetics and apoptosis. EPCs were determined and quantified by immunocytochemistry and flow cytometry. RESULTS: Coculture of CD34⁺ and CD34^-,total MNC led to a significant increase in the expansion of CD34⁺ cells compared with CD34 enrichment (P＜0.05). There was a trend toward decreased apoptosis in cultures when early passage was performed once the linear cord like structures appeared. There was no significant effect on apoptosis between with VEGF and without VEGF group (P＞0.05). These differentiated EPCs were stained positive for CD34⁺, von Willebrand factor (vWF), KDR, CD31 and incorporate acetylated low-density lipoprotein (LDL). CD34⁺ and AC133⁺cells accounted for 68.2%±6.3% (n=6) and 57.2%±9.8% (n=6) of attaching (AT) cells at day 7 of culture, respectively. CONCLUSIONS: Coculture of CD34⁺ and CD34^- or culture of MNC enhances ex vivo expansion of EPCs. Early passage decreases apoptosis rate, VEGF has no significant effect on ex vivo expansion of EPCs.     

Human umbilical cord mesenchymal stem cells migrate to Raji cells and promote their proliferation

AIM：To investigate the interaction between human Burkitt lymphoma cell line Raji and human umbilical cord mesenchymal stem cells (hUC-MSC). METHODS：The direct and indirect effects of MSC on Raji cells were investigated. The viability of Raji cells was tested by CCK-8 assay, and the cell cycle was determined by flow cytometry. The importance of vascular endothelial growth factor (VEGF) in the migration of MSC to Raji cells was analyzed by blocking VEGF expression in Raji cells with small interfering RNA (siRNA). VEGF level in the supernatant was detected by ELISA, and the mRNA expression of VEGF was measured by quantitative real-time PCR (qRT-PCR). RESULTS：Both MSC and MSC-conditioned medium (MSC-CM) promoted the growth of Raji cells. The viability of Raji cells co-cultured with MSC-CM was 0.99±0.05 at 72 h, and that in control group was 0.71±0.07. Both direct co-culture with MSC and MSC-CM turned the Raji cells from G0/G1 phase to S phase. The number of Raji cells co-cultured with MSC-CM in S phase was increased from 16.33±1.37 to 28.50±1.41, and the number in G0/G1 phase was decreased from 77.70±1.57 to 54.40±1.57. The expression of VEGF was down-regulated either at mRNA or protein level after transfection with siRNA. The ability of MSC migrated to Raji cells was significantly declined (96.00±5.28 vs 143.00±7.20). CONCLUSION：Raji cells recruit MSC by secreting VEGF, and MSC promote the proliferation of Raji cells by turning the cells from G0/G1 phase to S phase.     

Effect of simvastatin on expression of integrin-linked kinase and renal tubulointerstitium injury in rats induced by high fat diet

AIM: To explore the effect of simvastatin on expression of integrin-linked kinase (ILK) and the injury of renal tubulointerstitium in the rats induced by high fat diet. METHODS: Fifty-four 6-8 week-old female Wistar rats were randomly assigned to the following three groups: high fat diet group, simvastatin group (rats were fed with high fat diet plus 10 mg·kg-1·d-1 simvastatin) and control group. Six rats in each group were sacrificed at 4th week, 10th week, and the others at 20th week. The injury of renal tubulointerstitium was observed under microscope with HE staining and the expression of renal ILK was determined by Western blotting analysis and immunohistochemistry. Levels of total cholesterol (TC) and triglyceride (TG) in serum were measured by enzymatic colormetric methods. RESULTS: The serum TC and TG levels and the expression of renal ILK significantly increased in both high-fat diet group and simvastatin group, compared to control group at 4th week, reaching a maximum at 20th week (P<0.01). Tubulointerstitium injuries including vacuolar degeneration, syncytial change, clody swelling, necrosis and atrophy of renal tubular epithelial cells, thinning of tubal wall, lumens compensational expansion or even abolition, and inflammatory cell infiltration, interstitial fibrosis were found in both high fat diet group and simvastatin group, compared to control group at 4th week, worsened to a maximum at 20th week. However, all of these ameliorated in simvastatin group, compared to high fat diet group at each time point. CONCLUSION: The results indicate that high-fat diet induces significant lesion of renal tubulointerstitium and increased expression of renal ILK. Simvastatin may play an important role in protecting against tubulointerstitium injury induced by hyperlipoidemia by down-regulating the expression of renal ILK.     

Effect of nimesulide on ligature-induced periodontitis in rats

AIM: The purpose of this study was to observe the effect of nimesulide on periodontitis. METHODS: The gingival index (GI) was measured before the rats were sacrificed at the ends of week 4, 5 and 8. The periodontal tissues were stained with hematoxylin and eosin. Histological changes were observed by microscope. The periodontal attachment loss (AL) was measured by Tiger cell image analyzer. RESULTS: (1) Experimental periodontitis was successfully induced in rats by placing a piece of 3/0 braided silk around the cervix of the lower incisors at week 4 after the ligature. (2) In ligature-induced periodontitis group, at week 4 after the ligature, the GI and AL were significantly higher than those in control group (P<0.01). The histopathologic changes of periodontium in periodontitis group showed obvious inflammation, and the severity of destruction for periodontium was increased as time passed. (3) In the nimesulide prevention group, the GI and AL were significantly lower than those in periodontitis group (P<0.01). The histopathologic examination showed less inflammatory responses, and no obvious alveolar bone resorption was observed. (4) In the nimesulide treatment group, the GI and AL were significantly lower than those in periodontitis group at the end of week 5 and 8 after the ligature (P<0.01).CONCLUSION: (1) In ligature-induced periodontitis, nimesulide inhibits effectively its progress. (2) In the developing periodontitis, a significant improvement is observed in GI and AL following the treatment with nimesulide.     

Therapeutic efficacy of Ang-1 gene-modified MSCs in cerebral infarction

AIM: To observe the therapeutic efficacy of Ang-1 gene-modified mesenchymal stem cells (MSCs) in cerebral infarction. METHODS: The constructed lentiviral vector carrying the Ang-1 gene was used to infect the rat mesenchymal stem cells (rMSCs) to establish the Ang-1 gene-modified rMSCs (Ang-rMSCs). Adult male F344 rats were subjected to transient (2 h) middle cerebral artery occlusion (MCAO) with modified Zea Longa method. Phosphate buffered saline (PBS, 1 mL 0.1 mol/L, for control group), or Ang-rMSCs suspension (1 mL, for Ang-rMSCs group), or rMSCs suspension (1 mL, for pNL-rMSCs group), were infused into tail vein of rat respectively at 24 h after MCAO (n=8 in each group). Functional recovery measurements using the modified neurological severity scores (mNSS) were performed at 24 h post-MCAO and 1 week, 1 month and 3 months post-transplantation, respectively. The quantitative evaluation of blood-brain barrier permeability was performed at 1 week post-transplantation. The distribution, differentiation and malignant sign of grafted rMSCs were observed with immunofluorescence staining and histological method. RESULTS: Significant neurological function improvement was observed in groups treated with Ang-rMSCs or pNL-rMSCs at 1 week, 1 month post-transplantation compared with that in control group, as evidenced by mNSS (P<0.01). Better neurological function improvement was also found in Ang-rMSCs group than that in pNL-rMSCs group (P<0.01). The results of quantitative evaluation of blood-brain barrier permeability showed that the permeability in Ang-rMSCs group was the lowest compared to those in other two groups (P<0.01), and in the pNL-rMSCs group was the lower than that in control group (P<0.01). The grafted rMSCs survived in Ang-rMSCs and pNL-rMSCs groups, most were localized around the ischemic focus, and a few of them expressed NSE, NF and GFAP. The grafted rMSCs expressed BDNF abundantly in Ang-rMSCs group. These grafted rMSCs survived up to 3 months at least. No malignant sign was observed in these grafted cells. CONCLUSION: Ang-1 gene-modified MSCs transplantation has better therapeutic efficacy in cerebral infarction than that of MSC transplantation. The transplantation of cells with gene engineering is an effective therapeutic method for stroke patients.     

CPPU对葡萄果实生长发育促进效应的解剖学观察

对CPPU处理的全球红葡萄果实进行组织形态动态生长研究,结果表明:CPPU处理能促进早期子房膨大生长,子房壁的加厚生长和输导组织的生长.经CPPU处理比对照能提早使幼果进入迅速细胞分裂期,细胞层数增多.10 mg/L的CPPU处理不仅显著增加细胞层数,而且增大了细胞体积,刺激果实膨大的效果较好;15 mg/L的CPPU处理则抑制果实的细胞分裂和体积增大,其主要差异存在于中果皮,在外果皮上差异不明显.在幼果生长期,CPPU处理的细胞体积膨大幅度小于对照;在果实生长中、后期,其膨大幅度则大于对照.     

Human mesenchymal stem cells differentiate into osteoblasts

AIM:To investigate the differentiation from human mesenchymal stem cells (hMSC) into osteoblasts. METHODS:MSC were separated from human marrow with Ficoll-Paque reagent and expanded in cuture medium. To detect the surface antigens, The labeled cells were analysed on a FACScan flow cytometer. hMSC were induced to differentiate from mesenchymal stem cells into osteoblasts with dexamethasone, vitamin C, β-GP. Cell morphology、AP activity、calcium deposition and osteopontin were detected. P10 MSC were compared to P3 MSC in the tendency of osteoblastic differentiation. RESULTS:The cultured MSC comprised a single phenotypic population and displayed a fibroblast-like morphology. hMSC showed a strong self-renewal capacity. After primary culture, approximately (5-6)×10⁵ cells were obtained. These expanded attached MSC were uniformaly positive for CD29,CD44,CD59,CD105,CD166 and didn’t express CD11a, CD14, CD33, CD34, CD45, CD38, CD80, CD86, CD117. After osteoblasts induction, the cells changed from spindle-shape to cuboidal and polygonal in cell morphology. The AP activity increased gradually and many scattered calcium nodes were observed. The expression of osteopontin was positive. CONCLUSION:hMSC can be induced to differentiate into osteoblasts.     

Effect of hyperbaric oxygen on periodontitis with psychological stress in rats

AIM: To investigate the role of chronic psychological stress on periodontitis and the effects of hyperbaric oxygen (HBO) on periodontitis with psychological stress in rats. METHODS: Male special pathogen-free Wistar rats (n=80) were randomly divided into 4 groups: (1)normal control group; (2)experimental periodontitis group: the periodontitis model was induced by wrapping 3/0 silk ligature inoculated with Porphyromonas gingivalis around the left maxillary second molar of the rats; (3)psychological stress stimulation group; (4)periodontitis model with stress stimulation group. Psychological stress was removed at the 9th week after ligature, and 4 rats from each experimental group were randomly chosen for HBO treatment. The rats were sacrificed at the 2nd, 4th, 8th and 10th weeks after ligature. The levels of blood glucose, adrenocorticotropic hormone (ACTH), corticosterone and adrenaline were measured as the stress markers. The histological changes of periodontal tissues were observed under microscope with HE staining. RESULTS: The levels of blood glucose, ACTH, corticosterone and adrenaline in psychological stress stimulation group and periodontitis with stress group were significantly higher than those in control group and experimental periodontitis group at the 2nd and 4th weeks after ligature (P<0.05). The levels of the stress markers were significantly lower than those in untreated groups in the 10th week after HBO (P<0.01). The sites of gingival attachment were normal in control group and psychological stress stimulation group. Periodontal pocket, and periodontal attachment loss (AL) were observed in experimental periodontitis group. The tissue damage was much heavier in periodontitis model with stress stimulation group as the furcation of tooth was exposed and the tissue damage was observed on both sides of the adjacent teeth. No significant difference of AL between psychological stress stimulation group and normal control group during the experiment was observed. The AL in periodontal model with stress stimulating group was significantly higher than that in experimental periodontitis group at the 2nd, 4th and 8th weeks (P<0.01). The level of AL was attenuated at the 10th week after HBO (P<0.01). No difference of histological change in periodontal tissues was observed between control group and psychological stress stimulation group. Severer inflammatory changes and alveolar bone destruction were observed in periodontitis with stress group than those in experimental periodontitis group. The levels of inflammation reduced at the 10th week after HBO. CONCLUSION: Stress stimulation is one of the inducing factors of periodontitis in rats, which aggravates periodontitis. HBO may represent a useful way in treating psychological stress periodontitis.