BACKGROUND: Compared with normal physiological flap, main advantage of venous skin flap is that it throws off the limitation of arterial vascular territory on donor site and recipient site of traditional axial skin flap. However, its survival rate is unstable. OBJECTIVE: To explore effects of basic fibroblast growth factor (bFGF)-poly(lactic-co-glycolic-acid)] (PLGA)-sustained release microspheres on the survival of rabbit venous flaps.DESIGN, TIME AND SETTING: A randomized controlled animal study was performed at the University of South China from May to October 2008.MATERIALS: A total of 24 healthy New Zealand rabbits were equally randomly assigned to bFGF-PLGA sustained release microsphere, blank microsphere and blank control groups.METHODS: The formulation of bFGF microspheres was optimized by orthogonal design. bFGF-PLGA microspheres were prepared by optimized method. Lateral abdominal wall skin flap was created in rabbits from 3 groups. Five days before operation, 28.85 g/L bFGF-PLGA microspheres 3 mL (containing bFGF 20 μg) was intradermally injected into rabbits from the bFGF-PLGA sustained release microsphere group. An equal volume of blank microsphere + bFGF was injected in the rabbits of the blank microsphere group. Rabbits from the blank control group were infused with the same volume of saline. MAIN OUTCOME MEASURES: Morphology and particle distribution of bFGF-PLGA microspheres, drug loading volume, encapsulation efficiency, in vitro drug release characteristics were measured. After seven days, the survival area of skin was determined. Rabbit skin samples received CD34~+ immunohistochemical staining to detect the expression of CD34~+ and average number of blood vessels. RESULTS: The bFGF microsphere prepared based on optimized formulation exhibited well-defined properties, with the even and uniform sphere in appearance, regular particles without adhesion, about 98% of particles with a size distribution between 12.50 to 43.49 μm, with a mean particle size of 26.93μm and size span of (0.611 ± 6.60). The drug loading volume and encapsulation efficiency of bFGF microsphere reached [(23.11 ±0.44 )x10~3]% and (86.51±0.83)%, respectively. In the burst release phase, the rate of in vitro drug release amounted to 27.78%, but rose to 81.56% accumulatively 30 days later. The in vitro drug release of bFGF microsphere corresponded with Higuichi equation (r= 0.997). The sustained-release microspheres, blank microspheres and normal saline group, the average survival of the flap and the average number of blood vessels were similar (P=0.597, P=0.336), but still significantly lower than the bFGF-PLGA sustained release microsphere group (P=0.000). Results of immunohistochemical staining revealed that bFGF-PLGA promoted blood supple between flap and surroundings, improved flap survival and abundant CD34~+ expression. CONCLUSION: bFGF microsphere with good morphology, high drug loading volume and encapsulation efficiency can be obtained using W/O/W multiple emulsion evaporation method. The bFGF microsphere can promote the survival of rabbit venous flaps through a long period due to sustained release of bFGF.

BACKGROUND: The soft tissues would shrink with nasal framework collapse following surgical trauma, which cause aseptic inflammation, lead to parts of cartilage resorption. Accordingly, long-term saddle nose deformity usually accompanied by short nasal columella. Complications such as skin perforation or ulceration would appear if corrected the deformity using medical silicone rubber with large tension.OBJECTIVE: To explore the effectiveness of repairing post-traumatic saddle nose deformity with autologous costal cartilage transplantation.METHODS: A total of 21 cases with post-traumatic saddle nose deformity accompanied by short nasal columella were selected, including 6 males and 15 females, aged 16 45 years. All of the cases had trauma history and agreed with the treatment. The costal cartilage was obtained from the seventh rib and formed to babylon weeping willow leaf shape and columella nasi stent to repair post-traumatic saddle nose deformity. The "V-Y" progradation suture was used in the philtrum introcession and the botton of nasal columella to extend the nasal columella. The recovery of saddle nose deformity after transplantation, discharge of transplanted cartilage, as well as the incision scar status was observed.RESULTS AND CONCLUSION: The results were satisfactory and there were no complications after transplantation. All the cases were followed up from 6 months to 2 years. No case suffered costal cartilage grafts discharge or chondral deformation. The scar was little at the bottom of nasal columella. It is an ideal method for repairing post-traumatic saddle nose deformity using transplantation of autologous costal cartilage with "V-Y" progradation suture.

Objective To examine the changes in the expression of voltage-gated sodium channel?subunit mRNA in the dorsal root ganglion(DRG)and the role it plays in the neuropathie pain.Methods Thirty- two male SD rats weighing 250-400g were randomly divided into 2 groups:groupⅠneuropathic pain(SNL,n= 20)and groupⅡsham operation(n=12).Neuropathic pain was produced by ligation of right sciatic nerve according to Seltzer.Paw withdrawal latency to noxious thermal(PWHL)and mechanical(PWML)stimulation were measured before(baseline)and 1,2,3,5,8,11,14,28 day after sciatic nerve ligation(SNL).DRG at L_(4,5) was isolated on the 14th day after SNL in 8 SNL and 4 sham-operated animals for determination of sodium channel?subunit mRNA expression(by in-situ hybridization).Results PWHL and PWML were significantly decreased on the 2nd-28th day after SNL as compared to the baseline in SNL group.There was no significant difference in?_1 subunit mRNA expression between the 2 groups.The?_2 subunit mRNA expression in DRG was hardly detectable.The?_3 subunit mRNA expression in DRG on the operated side was significantly higher in SNL group than in sham-operation group.Conclusion The up-regulation of sodium channel?_3 subunit mRNA expression in DRG may play an important role in neuropathic pain.

BACKGROUND:Compared with normal physiological flap,main advantage of venous skin flap is that it throws off the limitation of arterial vascular territory on donor site and recipient site of traditional axial skin flap.However,its survival rate is unstable.OBJECTIVE:To explore effects of basic fibroblast growth factor (bFGF)-[poly(lactic-co-glycolic-acid)] (PLGA)-sustained release microspheres on the survival of rabbit venous flaps.DESIGN,TIME AND SETTING:A randomized controlled animal study was performed at the University of South China from May to October 2008.MATERIALS:A total of 24 healthy New Zealand rabbits were equally randomly assigned to bFGF-PLGA sustained release microsphere,blank microsphere and blank control groups.METHODS:The formulation of bFGF microspheres was optimized by orthogonal design.bFGF-PLGA microspheres were prepared by optimized method.Lateral abdominal wall skin flap was created in rabbits from 3 groups.Five days before operation,28.85 g/L bFGF-PLGA microspheres 3 mL (containing bFGF 20 ?g) was intradermally injected into rabbits from the bFGF-PLGA sustained release microsphere group.An equal volume of blank microsphere + bFGF was injected in the rabbits of the blank microsphere group.Rabbits from the blank control group were infused with the same volume of saline.MAIN OUTCOME MEASURES:Morphology and particle distribution of bFGF-PLGA microspheres,drug loading volume,encapsulation efficiency,in vitro drug release characteristics were measured.After seven days,the survival area of skin was determined.Rabbit skin samples received CD34+ immunohistochemical staining to detect the expression of CD34+ and average number of blood vessels.RESULTS:The bFGF microsphere prepared based on optimized formulation exhibited well-defined properties,with the even and uniform sphere in appearance,regular particles without adhesion,about 98% of particles with a size distribution between 12.50 to 43.49 ?m,with a mean particle size of 26.93 ?m and size span of (0.611 ? 6.60).The drug loading volume and encapsulation efficiency of bFGF microsphere reached [(23.11?0.44)?10-3]% and (86.51?0.83)%,respectively.In the burst release phase,the rate of in vitro drug release amounted to 27.78%,but rose to 81.56% accumulatively 30 days later.The in vitro drug release of bFGF microsphere corresponded with Higuichi equation (r=0.997).The sustained-release microspheres,blank microspheres and normal saline group,the average survival of the flap and the average number of blood vessels were similar (P=0.597,P=0.336),but still significantly lower than the bFGF-PLGA sustained release microsphere group (P=0.000).Results of immunohistochemical staining revealed that bFGF-PLGA promoted blood supple between flap and surroundings,improved flap survival and abundant CD34+ expression.CONCLUSION:bFGF microsphere with good morphology,high drug loading volume and encapsulation efficiency can be obtained using W/O/W multiple emulsion evaporation method.The bFGF microsphere can promote the survival of rabbit venous flaps through a long period due to sustained release of bFGF.

Objective To investigate the effects of different degrees of hemodilution with the artificial plasma substitutes most commonly used in clinical practice on coagulation in vitro. Methods Ten male ASA physical status I volunteers aged (28.8?1.6) yr and weighing (66?8) kg were enrolled. Venous blood samples obtained from each volunteer were diluted with 10% HES (200 000/0.4-0.55), 4 % succinylated gelatin (gelofusine GEL), 3.5 % polygeline (Haemaccel HAE) and lactated Ringer's solution (RL) by 33% [blood: diluent(B:D) =2:1], 50% (B:D=1:1) and 66% (B:D=1:2). Coagulation of the undiluted blood (control) and diluted blood was measured with sonoclot coagulation and platelet function analyzer (SCT) and by routine coagulation tests. The parameters measured included activated clotting time (ACT), clot rate, platelet function (PF), Hct, platelet count (Pit), plasma fibrinogen concentration (Fig) and activated partial thromboplastin time (APTT) . Results (1) At 33 % dilution ACT was significantly shortened in all the four groups as compared with control value; at 50 % dilution ACT was significantly shorter than the control in RL, HES and HAE groups; at 66 % dilution ACT was significantly prolonged in HES group. (2) Clot rate was significantly decreased at 33 % dilution only in HES group but was significantly decreased in all the four groups at 50 % and 66 % dilution. (3) PF decreased significantly only in GEL group at 33 % dilution but was significantly decreased in GEL and HES groups at 50 % and 66 % dilution. (4) With increasing dilution Hct, Pit and Fig gradually decreased and APTT was prolonged. Conclusion Coagulation changes are closely related to the degrees of dilution. At 33 % dilution, the three artificial plasma substitutes tested do not significantly affect hemostasis. At 50 % and 66 % dilution coagulation is badly impaired, but there are differences among the substitutes used for dilution. HAE impairs oagulation least as it contains higher calcium

N-carbamoylase is a part of hydantoinase operon which can transform N-carbamoylamino acid to corresponding ammo acids. The L-N-carbamoylase of Arthrobacter BT801, codied by the HyuC gene, is the rate-limiting and the only stereoselective enzyme. HyuC DNA fragment was amplified by PCR from the plasmid of pUC18-169. The target fragment was introduced into pPIC3. 5K plasmid to construct the pPIC3. 5K-hyuC expressing vector which was then transduced into Pichia pastoris GS115 cells after being linearized by BglⅡ digestion. Multi-copies insertion transformants were screened on G418 plates. The recombinant protein was proved to have biological activity of hydrolyzing N-carbamoylphenylalanine into phenylalanine through enzyme activity determination.

AIM: To explore the role of phosphatidylinositiol 3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3K/Akt/eNOS) signaling pathways in the inhibitory effects of puerarin on oxidized low-density lipoprotein (ox-LDL)-induced tissue factor (TF) expression in vascular endothelial cells.METHODS: The mRNA expression of TF was detected by real-time fluorescent quantitative PCR.The protein levels of TF and Akt was determined by Western blot.The content of the nitric oxide (NO) was measured by nitrate reduction method.RESULTS: Compared with control group, incubating endothelial cells with ox-LDL significantly induced TF expression at mRNA and protein levels and the dephosphorylation of Akt protein, and decreased NO production.Incubation of the endothelial cells with puerarin for 1 h and then treatment of the cells with ox-LDL decreased the TF expression at mRNA and protein levels, increased Akt protein phosphorylation and intracellular NO content.Co-incubation of the endothelial cells with PI3K inhibitor LY294002 and puerarin for 1 h and then treatment of the cells with ox-LDL augmented the TF expression at mRNA and protein levels and the Akt protein dephosphorylation, and decreased NO production.Co-incubation of the endothelial cells with eNOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) and puerarin significantly decreased the inhibitory effect of puerarin on ox-LDL-induced TF expression at mRNA and protein levels in the endothelial cells, and reduced Akt protein phosphorylation and NO production.CONCLUSION: Puerarin inhibits ox-LDL-induced TF expression at mRNA and protein levels in the human umbilical vein endothelial cells via activation of PI3K/Akt/eNOS signaling pathway.

Objective To investigate the effects of puerarin on the expression of human umbilical vein endothelial cells (HUVECs) tissue factor (TF) and tissue factor pathway inhibitor (TFPI) induced by oxidized low-density lipoprotein (ox-LDL).Methods After HUVECs were incubated with different concentrations of puerarin and 50 mg/L ox-LDL,the expression of TF and TFPI mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blot respectively.Results Compared with control,treatment with ox-LDL caused the augment of TF mRNA and protein expression (P<0.01),and the decrease of TFPI mRNA and protein expression.However,50,100,and 200 μmol/L puerarin blunted the augment of TF mRNA and protein expression and weakened the inhibition of TFPI mRNA and protein expression induced by ox-LDL(P<0.01).Conclusions Puerarin reduces HUVECs TF and TFPI mRNA and protein induced by ox-LDL.

Background Neuroglobin (Ngb) is a newly discovered member of globin superfamily.It is thought to regulate cell survival under hypoxia or oxidative stress condition.Ngb is expressed at a high level in retinal neuron,suggesting that retina may be one of important functional sites of Ngb.Objective The aim of this study was to investigate the protective role of endogenous Ngb on retina ganglion cells (RGCs) following chronic high intraocular pressure(IOP)in mice and the underlying mechanisms.Methods This study included the in vitro and in vivo experiment.RGCs derived from adult C57BL/6J wild type(WT) mice and Ngb-transgenic(Ngb-Tg) mice which cultivated by our laboratory were incubated with 5.0,7.5,10.0 mmol/L glutamic acid for 3 days.RGCs survival rate was calculated for the ration of dead and survival cells using a double labeling kit to evaluate the influence of Ngb on RGCs survival rate in the addition of glutamic acid.Chronic ocular hypertension models were established by injection of fluorescent microballon(MB) (10 μm)into the anterior chamber of WT mice and Ngb-Tg mice,The mice were divided into WT control group(n=18),Ngb-Tg control group(n=30),WT+MB single injection group(n=38),Ngb-Tg+MB single injection group (n =38),WT+MB twice injection group (n =6) and Ngb-Tg + twice injection group (n=6).In addition,WT+PBS injection group (n =6) and Ngb-Tg+ PBS injection group (n =6) were designed as negative controls to identify if it can affect IOP or not.The mice were sacrificed on 0 day(control group),3 days and 1,4,8 weeks followed the MB anterior chamber injection.Real-time PCR,Western blot and immunoytochemistry were used respectively for the analysis of the expressions of Ngb mRNA and protein in mouse retina,and the survival rates of RGCs were compared between the two types mice.Dihydroethidium (DHE) in retina was detected after cardiac perfusion and ATP level in mouse retina homogenate was analyzed.Results The RGCs survival rate was significantly higher in Ngb mice compared with WT mice in 5.0,7.5 and 10.0 mmol/L glutamic acid treatcd groups (t =2.810,3.020,3.110,P＜ 0.01).IOP of WT+ MB single injection group and Ngb-Tg+ MB single injection group were elevated in comparison with the WT control group and WT+PBS injection group and the high IOP remained for 4 weeks.Ngb level was raised in the WT+MB single injection group on the third day following injection,but the Ngb concentration remained a high level in the Ngb-Tg mice during the period of the experiment duration.The RGCs apoptosis rate was elevated both in the WT mice and Ngb-Tg mice 1,4,8 weeks after injection of MB,however,the cell apoptosis rate was higher in WT mice than that of Ngb-Tg mice(P＜0.05).DHE content in retina in the Ngb-Tg+MB single injection group was significantly lower than that in the WT+MB single injection group (t =3.212,P=0.008),and ATP content in retina was elevated in the Ngb-Tg+MB single injection group compared with WT+MB single injection group(t =2.864,P＜0.01).Conclusions It is suggested that Ngb might be a neuroprotective molecules against RGCs death by decreasing oxidative stress and improving mitochondria function.

Objective:To explore effects of trehalose as a cryopreserve agent on survival rate of fatty tissue after cryopreservation.Methods:The liposuction was used on the abdomen of adult female.After centrifugation and purification,adipose was randomized into the following three groups,the trehalose group,the fetal bovine serum (FBS)+ 10％DMSO group and the physiological saline group.The specimens were cryopreserved at-196 ℃ for 3 months and then the HE staining,glucose transfer method and CK method were used to detect the cell survival rate in each group.Results:The activity of adipose in the trehalose group and FBS+10％DMSO group adipose was higher than that in the physiological saline group (P＜0.05);while there was no significant difference between the trehalose group and FBS+10％DMSO group (P＞0.05).Conclusion:As cryoprotectant,trehalose could keep fat cell viability,and adipose tissue can be used for clinical transplantation after 3 months' freezing.