Objective To study the effects of deoxycytidine (5-aza-2 deoxycytidine, DAC) on DNA Methylation state and expression of mRNA and protein of pl6 gene in human squamous lingual carcinoma SCC-9 cells in vitro. Methods The SCC-9 cells were divided into four groups, group 0, 1, 2 and 3 which processed using three gradients concentration of DAC. The group 0 without DAC was as the control group. Q-MSP was used to detect the state of methylation of the p16 in SCC-9 cells treated by DAC after 48 hours. Real-time fluorescence quantitative PCR was used to detect mRNA expression level changing of the p16 in SCC-9 cells treated by DAC. Immunohistochemical method was used to detect the expression of p16 protein. Results The hypermethylation and non-methylated p16 gene in SCC-9 was mixed with the results of Q-MSP. The results of Real-time PCR showed that mRNA expression of p16 in SCC-9 cells which treated by the different concentration of DAC for 48 hours was higher the control group. And difference was statistically significant (P < 0.05). The high expression of p16 protein was found in the experimental group with immunohistochemical method. Conclusion The p16 gene methylation states of SCC-9 may be suppresses and the recovery of mRNA and protein expression of p16 gene must be prompted by DAC.

DNA from 32 cases of tongue squamous cell carcinoma(TSCC)specimens and the neck lymph node metastasis specimens were processed by bisulfite treatment.The methylation of the specimens was examined by Q-MSP amplification.The consistency of p1 6 methyla-tion of primary TSCC with that of lymph node metastasis tumor was 90.62%(P <0.05).We assumed that p1 6 gene promoter CpG island methylation rates in primary TSCC and metastatic lymph nodes are consistent,p1 6 gene methylation status can be a methylation predictor be-tween premary and lymph node metstasized TSCC.

Aim To investigate the effects of midazolam on porcine isolated coronary artery rings pre-contracted by potassium chloride(KCl)and the possible mechanism.Methods The vessel tension recorder system was used.Isotonic tension of porcine isolated coronary artery rings precontracted by KCl(30 mmol·L-1)was recorded.The vasorelaxing action of midazolam and effects of various drugs were observed in the rings.Results Midazolam(3×10-6～1×10-4 mol·L-1)respectively concentration-dependently reduced the contraction induced by KCl,and there was significant difference between the rings with intact and denude endothelium(P<0.05).On KCl-induced precontraction,midazolam′s relaxation was depressed by L-NAME and the blend of L-NAME and L-Arg(P<0.05),but was not affected by Indo,L-Arg and 1400W.The contraction was not prevented by pretreatment with the inhibitor of Na+/Ca2+ exchanger(KB-R7943).The inhibitor of KATP(Gli)restrained the diastolic function of midazolam(P<0.05),while the inhibitor of BKCa(TEA),Kir(BaCl2),KV(4-AP)had no obvious effect.Conclusions Midazolam produces remarkable vasodilatation on KCl pre-contracted porcine isolated coronary artery rings.Its relaxtion effect is via concentration-dependent and endothelium-dependent mechanisms and relevant to the production of NO.Na+/Ca2+ exchanger is not involved midazolam′s vasodilatation on KCl pre-contracted porcine coronary artery rings.The relaxant mechanism of midazolam may be concerned with KATP.The Kir,BKCa and KV may be not involved.

OBJECTIVETo investigate the promotive effect of transplantation of bone morphogenetic protein-2 (BMP-2) gene transfected bone mesenchymal stem cells (BMSCs) compounded with injectable bone tissue engineering scaffold material Pluronic F-127 on bone regeneration in rabbit mandibular distraction osteogenesis(DO).

METHODSForty-eight New Zealand's white rabbits were randomized into four groups with twelve in each. All the objects were prepared into DO surgical model. On the 2nd day of consolidation, group A, B, C and D were injected with the same amount of 200 microL of the compound of BMP-2 gene transfected BMSCs with Pluronic F-127, the solution with BMP-2 gene transfected BMSCs, the solution of BMSCs and physiological saline at distraction zone, respectively. Two halves of the objects of all groups were sacrificed at the end of 2nd and 6th week consolidation, respectively. And the specimens of right mandible were prepared for radiological, histomorphological and immunohistochemical examinations to evaluate bone regeneration.

RESULTSBoth radiological and immunohistochemical images were analyzed and processed with professional software. At the end of 2nd and 6th week consolidation, the bone mineral density and the expression of BMP-2 protein in distraction area of group A were significantly higher than those of B, C, D group (P<0.01). Group B was significantly higher than that in group C and D (P<0.01). There was no significant difference between group C and D (P>0.05). And the regeneration quality of distraction zone in group A and B were better than those in group C and D, just as that of group A better than group B. Conclusion The transplantation of BMP-2 gene transfected BMSCs compound with Pluronic F-127 could effectively promote bone regeneration in rabbit mandibular DO.

Animals ; Bone Density ; Bone Morphogenetic Proteins ; Bone Regeneration ; Mandible ; Mesenchymal Stromal Cells ; Osteogenesis ; Osteogenesis, Distraction ; Poloxamer ; Rabbits ; Tissue Engineering ; Tissue Scaffolds ; Transfection

BACKGROUND: Mandibular distraction osteogenesis (MDO) is a kind of endogenous tissue engineering technology that lengthens the jaw and opens airway so that a patient can breathe safely and comfortably on his or her own. Endothelial progenitor cells (EPCs) are crucial for MDO-related angiogenesis. Moreover, emerging evidence suggests that heat shock protein 20 (Hsp20) modulates angiogenesis under hypoxic conditions. However, the specific role of Hsp20 in EPCs, in the context of MDO, is not yet known. The aim of this study was to explore the expression of Hsp20 during MDO and the effects of Hsp20 on EPCs under hypoxia. METHODS: Mandibular distraction osteogenesis and mandibular bone defect (MBD) canine model were established. The expression of CD34, CD133, HIF-1α, and Hsp20 in callus was detected by immunofluorescence on day 14 after surgery. Canine bone marrow EPCs were cultured, with or without optimal cobalt chloride (CoCl2 ) concentration. Hypoxic effects, caused by CoCl2 , were evaluated by means of the cell cycle, cell apoptosis, transwell cell migration, and tube formation assays. The Hsp20/KDR/PI3K/Akt expression levels were evaluated via immunofluorescence, RT-qPCR, and western blot. Next, EPCs were incorporated with either Hsp20-overexpression or Hsp20-siRNA lentivirus. The resulting effects were evaluated as described above. RESULTS: CD34, CD133, HIF-1α, and Hsp20 were displayed more positive in the callus of MDO compared with MBD. In addition, hypoxic conditions, generated by 0.1 mM CoCl2 , in canine EPCs, accelerated cell proliferation, migration, tube formation, and Hsp20 expression. Hsp20 overexpression in EPCs significantly stimulated cell proliferation, migration, and tube formation, whereas Hsp20 inhibition produced the opposite effect. Additionally, the molecular mechanism was partly dependent on the KDR/PI3K/Akt pathway. CONCLUSION In summary, herein, we present a novel mechanism of Hsp20-mediated regulation of canine EPCs via Akt activation in a hypoxic microenvironment.

A male patient, with diagnose of Madelung disease, was admitted in September 2009. He has been a heavy drinker for decades before onset of the disease. This patient was characterized by the large amount of symmetrical deposits of adipose tissue in the subcutaneous layer around neck, and without obesity on other sites. The excessive adipose tissue was surgically removed by three steps. Appearance almost returned to normal. No recurrence happened after 8 years of follow-up.