OBJECTIVE:To develop a method for concentration determination of voriconazole in human plasma and apply it in the clinic. METHODS:UPLC-MS/MS method was adopted. Using ketoconazole as internal standard,the determination was per-formed on Shim-pack VP-ODS column with mobile phase consisted of water(containing 1‰ formic acid and 2 mmol/L ammonium acetate)-acetonitrile(gradient elution)at flow rate of 0.3 ml/min and column temperature of 40℃.The electrospray ion source,pos-itive ionizing pattern and multiple reaction monitoring were used;the mass transition ion-pairs of voriconazole and internal standard were m/z 351.2→282.2 and m/z 532.1→490.2. RESULTS:The linear range of voriconazole were 1-10 000 ng/ml (r=0.999 5,n=5),and the limit of quantitation was 1 ng/ml;RSDs of inter-day and intra-day were all lower than 10%;method recovery was higher than 90%(RSD<8%),and extraction recovery was higher than 70%(RSD<8%). The plasma concentrations of voriconazole in 10 patients with invasive fungal infection determined by this method were 507.33-7 011.24 ng/ml,and those of 3 patients were outside the recommended treatment concentration range. CONCLUSIONS:The established method is fast,accurate and sensitive,and can be applied for the therapeutic drug monitoring of voriconazole.

As a therapeutic strategy for ischemic stroke,neuroprotective agents are used to antagonize a series of harmful molecular biological events in cerebral ischemia.This article reviewes the current status of neuroprotective agents in the treatment of acute ischemic stroke,and the challenges from pre-clinical evidences translating into the clinical trials.The vascular endothelial cells,glial cells and neurons should be studied as a whole.

Iatrogenic gastrointestinal perforation is one of the severe adverse events of endoscopic therapeutic procedure. For acute iatrogenic perforation,management by endoscopic techniques is a simple and rapid modality to close the perforation with minimal invasiveness and avoiding the traditional surgical trauma. Endoclips,suture with special instruments,covered stents,degradable sheets combined with tissue adhesive,and combined endoscopic techniques such as snares combined with endoclips,are the major endoscopic therapeutic modalities for closure of iatrogenic gastrointestinal perforation. In this article,the current status and progress of endoscopic management for acute iatrogenic gastrointestinal perforation were reviewed.

Objective To investigate the expression of AIB1 protein in bladder cancer and its significance.Methods 30 cases of bladder cancer and 30 adjacent normal tissues were collected from 30 patients underwent radical or TURBt.The level of AIB1 protein expressed in these tissues were detected by immunohitochemical method.Results The positive rate of AIB1 protein in bladder carcinoma was higher than that in the normal tissues [43.3 ％ (13/30) vs 3.3 ％ (1/30),x2 =4.316,P ＜ 0.05].The positive rate of AIB1 in T3 stage was higher than that in T1 or T2 stage [62.5 ％ (5/8) vs 25.0 ％ (2/8),42.9 ％ (6/14),x2 =4.623,P ＜ 0.05].No correlation was found between the expression of AIB1 protein and the sex,age,multiplicity or pathological grade (P ＞ 0.05).Conclusions There is a close relationship between AIB1 and bladder cancer.AIB1 might have great value on the prognosis of patients with bladder cancer.

BACKGROUND: Research has shown that the vascular endothelial growth factor (VEGF) of the ends of the fractured bone is heavily expressed 72 hours to 3 weeks after the fracture and it is supposed that it has a promoting effect on fracture healing. Inducing angiogenesis through VEGF gene transfection has gradually attracted the attention of the researches.OBJECTIVE: To find an efficient way of exogenous VEGF gene in vivo transfection through injecting the mixture of positive ion liposome transfection agent and plasmid and to study the promoting effect of extra VEGF gene expression on bone fracture healing.DESIGN: A randomly grouping, blank control trial.SETTING: Animal Laboratory of Huaxi Medical Center of Sichuan University MATERIALS: Totally 40 adult male SD rats, weighing 230 to 250 g,were involved. All the animals were randomly divided into the experiment group and the control group with 20 rats in each group.METHODS: The experiment was conducted at the Animal Laboratory of Huaxi Medical Center of Sichuan University from April to December 2003.Altogether 40 rats were involved to establish fractured models of right shaft of femur. Cut the bone in the middle of bone stem, retroplanted a Kirsh' nail with 1 mm diameter through intercondylar part and the fractured bone was fixed. In the experimental group, a mixture of 100 μL of liposome transfection agent and 100 μg of pBLAST49-mVEGF plasmid was injected in multiple points into the subperiosteum of the both sides of the ends of the fractured bone. The same volume of normal saline was injected into the rats in the control group. Then, 2 rats in each group were put to death 3,7,14,28,42,56,70 days after the operation and femoral bone specimen was collected.MAIN OUTCOME MEASURES: Observation of right femoral fractured staining results of VEGF, with the apperance of brown granules as positive.RESULTS: Two rats were selected at 7 time points separately, and altogether 28 rats entered the stage of result analysis. The other 12 rats were fracture at different time points: For the experimental group, 28 days after the operation, cartilage callus appeared and replaced fibrocallus gradually,and the fracture line disappeared. Fifty-six days after the operation, the bone healed completely. For the control group, 28 days after the operation , fibrocallus was observed, and the fracture line was still clear. 56days after the operation, much callus appeared, and the fracture line beof fractured bone was stained with hemotoxylin eosin (HE). In the experiment group, 56 days after the operation, the bone healed completely and trabecular like bones were rebuilt. The bone marrow cavity of the fractured region was open again. In the control group, Fifty-six days after the operation, no mature bone was formed, and the bone marrow cavity was not different time points: The expression in the two group reached to the peak on day 14 and began to decrease on day 28. The expression of VEGF in the experimental group was obviously higher than that in the control group.CONCLUSION: Injection of the mixture of positive ion liposome transfection agent into the subperiosteum of rats is an effective approach for in vivo transfection and pBLAST49-mVEGF gene transfection can effectively facilitate the bone fracture healing of rats.

BACKGROUND: Ectogenesis vascular endothelial growth factor (VEGF) could enhance the activity of alkaline phosphatase (AKP) and concentration of cycli adenosine monophosphate (cAMP) fivefolds in cultured osteoblast cell. What' s the effect of ectogenesis VEGF gene transfection on osteoblasts is still by no means clear.OBJECTIVE: To investigate the effect of gene transfection and expression of ectogenesis VEGF on the osteogenesis activities of osteoblast cell.DEDIGN: A completely randomized controlled study.SETTING: Laboratory of Transplantation and Immunity, West China Hospital, Sichuan University MATERIALS: Cranial osteoblasts of newborn two or three-day male SD rat.METHODS: The experiment was conducted in the Laboratory of Transplantation and Immunology of Huaxi Hospital, Sichuan University from April to December 2003 The cranial osteolasts of newborn rat were separated and cultured with enzyme digestion method then were identified by teoblasts cultured in vitro with cation liposomes transfection as gene transations, immunohistochemical staining was performed on VEGF and collagen type I and osteocalcium were detected.collagen I and secretion of osteocalcium of osteoblasts.RESULTS: The concentration of osteocalcium and expression of type I collagen of the 1- 5 generation osteoblast cell in pBLAST49-mVEGF gene transfer group were significantly higher than that in the control group (P ＜0.05).CONCLUSION : It is found in this experiment that the synthesis of collagen I was enhanced obviously after sussceful transfection of pBLAST49-mVEGF plasmid. Compared with the control group, the diffence of intergrated optical density gained by Mias image analysis system was significant( P ＜ 0.05),indicating that pBLAST49-mVEGF plasmid transfection can improve the synthesis of type I collagen and secretion of osteocalcium of osteoblasts.

Objective To examine cerebrovascular reactivity to CO2 inhalation in mice. Methods In vivo Two-Pho?ton imaging technique was used to record the reaction of cerebral cortical vessels including penetrating artery, surface vein and capillary in 5 male C57 mice after CO2 inhalation under a thinned-skull cranial window. Nitric oxide syntheses inhibitor L-NAME and Prostaglandin syntheses inhibitor Indomethacin were used to block different vasodilator pathways, respectively. Results Different mouse cortical vessels displayed different degrees of dilation to 1-minute 5%CO2 inhala?tion. The penetrating artery exhibited the most obvious dilation (45.01%±4.45%). L-NAME intervention significantly di?minished cerebravascular CO2 reactivity(P<0.05). Indomethacin significantly attenuated the dilation of artery but not capillary comparing with L-NAME intervention(P<0.05). Conclusions Different vessels react differently to CO2 inhala?tion in which postaglandins and NO signal pathways are involved.

Objective To conduct a hiomechanical comparison of the four common internal fixation methods for the anterior eruciate ligament(ACL)avulsion fractures of displaced tibial eminence.Methods Sixteen fresh cadaver knee joints were used and randomized into four equal groups of four fixation methods:antegrade wiring group, retrograde wiring group,suturation group,and intramedullary screw group.The knee joint specimens were fixed at flexion of 30?and subject to continuous stretch stresses of 30 N,60 N and 90 N respectively on a material testing machine(MTS 858 Bionix test system,USA)which conducted a simulated Lachman test.The specimens were scanned at different angles by a three dimensional laser scanner.Data were recorded and processed by image software to es- tablish three-dimensional structure models of femur,tibia and knee joint.The test results were analyzed statistically on a computer.Results There were no obvious differences between each fixation group in the length change of ACL when the stresses were 30 N and 60 N(P＞0.05).Under 90 N stress,however,the mean length change between the femoral and tibial attachments of ACL was the smallest(4.8?1.7)mm(2.5 to 6.2 mm)in the suturation group(P＜0.05). There were no distinct differences between the intramedullary screw group and the retrograde wiring group in the changes of A CL shift(P=0.214).The average front shift in the retrograde wiring group was(6.2?1.2)mm(4.8 to 8.2 mm) and significantly smaller than that in the antegrade wiring group(P＜0.05).The antegrade wiring group made the largest average front shift under different stresses and its average front shift was(7.2?1.3)mm(5.6 to 8.7 mm). Conclusions The knee joint stability provided by the suturation fixation is distinctly better than that by the other three fixation methods.The antegrade wire fixation provides the poorest knee joint stability.There is hardly any difference between intramedullary screw fixation and retrograde wiring fixation.

Objective To examine outcomes of superficial vein stripping for vein varicosis caused by combination of superficial and deep venous reflux and the effect on deep vein reflux. Methods From January 2004 to December 2006, twenty patients (22 lower extremities) with combined superficial and deep venous reflux were enrolled for evaluation, patients with superficial venous reflux alone worked as controls.All patients underwent superficial vein stripping and followed up for 2 years. Venous clinic severity score (VCSS) and color duplex ultrasound test were used to evaluate the change of clinic symptoms and valve reflux of deep veins. Results Preoperative VCSS of superficial and deep vein reflux group and superficial vein reflux group was 7.1 ± 2. 9 and 6. 6 ± 2. 0 respectively (P = 0. 44). At two years after operation the VCSS was 2. 3 ± 1.2 and 1.8 ± 0. 8 respectively without significant statistic differences, whereas comparing postoperative VCSS with individual preoperative parameters there was a significant decrease (P <0. 01). In combined superficial and deep vein group, deep venous reflux reduced in 7 limbs, progressed in 4 limbs, no change in 12 limbs. While in superficial reflux alone group, deep venous reflux occurred in 4 limbs.Conclusions Superficial vein stripping effectively improves symptoms of great saphenous vein varicosis of lower extremity caused by combination of superficial and deep vein reflux without a significant effect on the stares of deep vein reflux.

Objective To evaluate the different value of color Doppler ultrnsonography and Doppler vascular examinations in diagnosis for deep venous thrombosis(DVT)in the lower extremities.Methotis Imaging of color Doppler ultrasound scanning was employed as diagnostic criteria for DVT on 178 lower extremities of 146 suspected patients,as compared to the result by Doppler vascular examinations.Results Color Doppler ultrasonograph showed hish accuracy in diagnosis for DVT,as compared to that by Doppler vascular examination with 97.9 percent(142/145)positive for the femoral and popliteal veins and relatively lower positive diagnostic vallie for thrombosis in the inferior vena cava,iliac vein,anterior tibial vein,posterior tibial vein and calf veins.Conclusions Color Doppler ultrasonography is superior to Doppler vascular examination in determining DVT of the lower extremities and can be used as a main diagnostic method for it.Doppler vasculiar examination can be used as an initial screening method for DVT and deep venous angiography should not be used as a routine diagnostic measure for it.