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 evaluate the effects of propofol on apoptosis in the hippocampal neurons of fetal rats in vitro.Methods The isolated hippocampal neurons were seeded into 96-well plates or 24-well plates at a density of 5 × 104 cells/ml.The cells were randomly divided into 5 groups (n =18 each) using a random number table:control group (group C),in tralipid group (group Ⅰ) and propofol 1,10,100 μmol/L groups (P1-3 groups).In group Ⅰ,10％ intralipid was added to the culture media until the final concentration reached 100 μmol/L.In groups P1-3,propofol was added to the culture media until the final concentration reached 1,10 and 100 μmol/L,respectively,and the cells were then incubated for 3 h.The cell apoptosis was assessed by flow cytometry.The expression of Bcl-2 mRNA and caspase-3 mRNA was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).The expression of Bcl-2 and actived-caspase-3 protein was determined by Western blot analysis.The apoptosis rate was calculated.Results Compared with group C,the apoptosis rate was significantly increased,the expression of Bcl-2 mRNA and protein was down-regulated,and the expression of caspase-3 mRNA and actived-caspase-3 protein was up-regulated in P1-3 groups (P ＜ 0.05).There was no significant difference in the parameters mentioned above between group Ⅰ and group C (P ＞ 0.05).Conclusion Propofol induces apoptosis in isolated hippocampal neurons by inhibiting Bcl-2 expression and enhancing caspase-3 activity in fetal rats.

Objective To explore the prognosis of elderly patients suffered from hospital-acquired pneumonia (HAP) by T cell subsets and clinical pulmonary infection score (CPIS).Methods A cohort of 125 elderly patients admitted in ICU & ED (Emergency Department) from Aug,2012 to Jul,2013 were enrolled for a prospective and observational study.The patients were divided into 3 groups:HAP survival group (n =50,group A),HAP death group (n =40,group B) and non-HAP group (n =35,control group).The criteria of exclusion were patients with auto-immune diseases,immunodeficiency,allergic disorders,malignancies,diabetes,trauma,surgical diseases,or patients with recent use of immunosuppressive agents or cyclooxygenase-inhibitors (Aspirin etc.).In the control group,patients with nosocomial pneumonia and other diseases afecting the CPIS were excluded.APACHE Ⅱ scores of all patients were recorded.Blood T cell subsets (including values of CD3,CD4 +,CD8 +,and CD4 +/CD8 +)were measured on the admission day,the 1st day of HAP onset and the 5th day after onset of HAP in HAP patients whereas these measurements were tested only on the admission day in controls.Meanwhile,the CPISs were recorded on the admission day,the 1st day of HAP onset and the 5th day after onset of HAP in HAP patients.Flow cytometer (FCM) was used to detect T cell subsets.Data of statistical analysis were represented as Mean ± SD.The significant differences in T cell subsets and CPIS between survival group and death group were analyzed by independent t test.The paired samples t test was employed in survival group and death group.Linear correlation analysis was made between CD4 +/CD8 + ratio and CPIS in survival and death groups,respectively.Results There were no significant differences in demographics and clinical features (including age,sex,length of stay,APACHE Ⅱ scores) of patients in survivors and non-survivors (P ＞ 0.05).The values of CDs (CD3,CD4 + and CD4 +/CD8 + ratio) between patients of control group and patients of HAP groups were not significantly different on the admission day (P ＞ 0.05).The values of CDs on the admission day were much lower than those on the 1 st day of HAP onset in both survivors and nonsurvivors (P ＜ 0.05).The values of CDs on the 5th day after onset of HAP were higher than those on the 1 st day of HAP onset in the survival group (P ＜ 0.05),while there were no significant differences in CDs between different intervals after HAP onset in the death group (P ＞ 0.05).There were no significant changes in values of CD8 + in any group (P ＞ 0.05).Both survivors and non-survivors had much higher CPIS values on the 1st day of HAP onset than those on the admission day (P ＜0.01).The survival group had higher CPIS on the 5th day after onset of HAP compared to the 1st day of HAP onset (P ＜0.01),while there was no significant change in the death group.Linear correlation analysis showed negative correlation between CD4 +/CD8 + ratio and CPIS on both the 1 st day of HAP onset (survival group:R =-0.740,P =0.004 ; death group:R =-0.613,P =0.035) and the 5th day after onset of HAP (survival group:R =-0.639,P =0.009; death group:R=-0.686,P=0.021).Conclusions The hospital-acquired pneumonia appears as an immune imbalance disorder.The difference in CDs is a promising objective tool,aiding in prediction of prognosis of HAP in the elderly,the lower the CDs,the higher severity.The CD4 + / CD8 + ratio showed a negative correlation with CPIS.Monitoring of T cell subsets and CPIS may provide clinical value for the treatment of hospital-acquired pneumonia in the elderly.

Objective To evaluate the effect of dexmedetomidine on the activity of glycogen synthase kinase-3 beta (GSK-3β) during propofol-induced apoptosis in hippocampal nerve cells of newborn rats.Methods Sixty male 7-day-old Sprague-Dawley rats,weighing 10-15 g,were divided into 6 groups (n=10 each) using a random number table:normal saline group (group NS),fat emulsion group (group F),propofol group (group P) and different doses of dexmedetomindine groups (group D25,group D50 and group D75).Normal saline and fat emulsion 100 μl were injected intraperitoneally in group NS and group F,respectively.In group P,propofol 50 mg/kg was intraperitoneally injected,and an increment of propofol 50 mg/kg was added after righting reflex completely recovered,with the total amount of 100 mg/kg.In group D25,group D50 and group D75,dexmedetomidine 25,50 and 75 μg/kg were intraperitoneally injected,respectively,and 30 min later propofol 100 mg/kg was administered.At 2 h after emergence,the rats were sacrificed,their brains were removed for determination of apoptosis in hippocampal nerve cells (by TUNEL),and the hippocampi were isolated for detection of the expression of GSK-3β and phosphorylated GSK-3β (p-GSK-3β) by Western blot analysis.The apoptosis index (AI) and ratio of p-GSK-3β/GSK-3β were calculated.Results Compared with group NS,AI was significantly increased,the expression of p-GSK-3β was down-regulated,and the p-GSK-3β/GSK-3β ratio was decreased in P,D25,D50 and D75 groups (P＜0.05).Compared with group P,AI was significantly decreased,the expression of p-GSK-3β was up-regulated,and the p-GSK-3β/GSK-3β ratio was increased in D25,D50 and D75 groups (P＜0.05).Compared with group D25,AI was significantly decreased (P＜0.05),and no significant change was found in the expression of p-GSK-3β or ratio of p-GSK-3β/GSK-3β in D50 and D75 groups (P＞0.05).Compared with group D50,AI was significantly decreased (P＜0.05),and no significant change was found in the expression of p-GSK-3β or ratio of p-GSK-3β/GSK-3β in group D75 (P＞0.05).Conclusion The mechanism by which dexmedetomidine attenuates propofol-induced apoptosis in hippocampal nerve cells may be related to inhibition of GSK-3β activity in newborn rats.

Objective To explore the relationship between serum 25-hydroxy vitamin D [25(OH)D] concentrations and the risk of Henoch-Schonlein purpura (HSP).Methods A case control study was designed.Serum 25 (OH)D concentrations were measured by radioimmunoassay in 214 participants,including 53 H SP patients and 161 status-matched healthy controls.Information concerning demographic data,genetic,background,and environmental exposures was collected using questionnaire.The study participants were divided into four groups according to quartile range of 25(OH)D concentration and logistic regression modeling was used to evaluate the relation with HSP risk by estimating odds ratios(OR)and 95％confidence intervals(CI).Results The HSP group had a significantly lower concentration of 25(OH)D than the control group (the median in the HSP group was 11.4 ng/mL;controls:15.36 ng/mL,P＜0.05).When the first interval was set as the reference level,the OR (95 ％ CI) of the second,third,and fourth intervals were:0.468(0.341-0.771),0.442(0.302-0.627),0.339 (0.199-0.501).After adjusting the analysis for the presence of pathogenic related confounding fact OR,the OR(95％CI)of the second,third,and fourth intervals were:0.459(0.333-0.741),0.408(0.317-0.611),0.387 (0.221-0.517).The 25 (OH) D level was inversely correlated with the risk of HSP(P＜ 0.05).Conclusion The risk of HSP was decreased with the increase of serum 25 (OH) D concentration,25 (OH) D may be a protection factor in the pathogenesis of HSP.

Objective To evaluate the role of opioid receptors in fentanyl-induced inhibition of proliferation and migration of human gastric cancer cell line MGC-803.Methods The human gastric cancer cell line MGC-803 was cultured in DMEM liquid culture medium.The cells were seeded in 6-well or 96-well plates and then randomly divided into 4 groups (n =54 each):control group (group C),fentanyl group (group F),naloxon group (group N) and naloxon + fentanyl group (group NF).The cells were exposed to 0.1 μmol/L fentanyl and 10 μmol/L naloxon in F and N groups,respectively.The cells were incubated with 10 μmnol/L naloxon for 30 min and then O.1 μmol/L fentanyl was added to the culture medium in group NF.The viability of the cells was detected by MTT assay after being incubated with fentanyl for 12,24,36,48,60 and 72 h.The cell apoptosis was assessed by flow cytometry after being incubated with fentanyl for 24 h.The migration of the cells was detected by wound healing assay after being incubated with fentanyl for 48 h.The proliferation of the cells was determined by colony formation assay at 7 day of incubation with fentanyl.Results Compared with group C,no significant changes in the viability of the cells,rate of colony formation,apoptotic rate and rate of cell wound healing were found in group N (P ＞ 0.05),and the viability of the cells,rate of colony formation and rate of cell wound healing were significantly decreased,and the apoptotic rate was increased in F and NF groups (P ＜ 0.05).There was no significant difference in the viability of the cells,rate of colony formation,rate of cell wound healing and apoptotic rate between group NF and group F (P ＞ 0.05).Conclusion Opioid receptors are not involved in fentanyl-induced inhibition of proliferation and migration of human gastric cancer cell line MGC-803 in vitro.

OBJECTIVETo investigate the effect of knocking-down microRNA-221 (miR-221) expression on the radiosensitivity of human colorectal carcinoma cells.

METHODSHuman colorectal carcinoma-derived cell line Caco2 was transfected with miR-221 antisense oligonucleotides (anti-miR-221) via Lipofectamine 2000. Real-time quantitative PCR was performed to detect the expression of miR-221 and PTEN mRNA in Caco2 cells. The changes in the protein expression of PTEN in the transfected cells were detected by Western blotting. The cell death after transfection and irradiation was detected by flow cytometry.

RESULTSTransfection with anti-miR-221 caused a significant reduction in miR-221 expression (P<0.05) and up-regulated PTEN protein expression (P<0.05) in Caco2 cells. The percentage of cell death was significantly increased in anti-miR-221 group and anti-miR-221 with irradiation group (P<0.01). Anti-miR-221 significantly enhanced the radiosensitivity of Caco2 cells, which was partially reversed by PTEN-siRNA.

CONCLUSIONAnti-miR-221 can enhance the radiosensitivity of colorectal carcinoma cells by up-regulating the expression of PTEN.

Caco-2 Cells ; radiation effects ; Colorectal Neoplasms ; genetics ; metabolism ; Humans ; MicroRNAs ; genetics ; metabolism ; PTEN Phosphohydrolase ; metabolism ; RNA, Messenger ; genetics ; Radiation Tolerance ; Transfection ; Up-Regulation

Objective To explore rationale and clinical application of simplified modified radical thyroideetomy for differentiated thyroid carcinoma.Methods From Jan.2007 to Jun.2010,349 cases of differentiated thyroid carcinoma received simplified operative procedure based on standard modified radical thyroidectomy.The simplified procedure took a low small collar incision(about 10-12 cm).In separating upper and lower skin flaps,subcutaneous tissues covering posterior triangle of neck and posterior edge of sternoeleidomastoid muscle were spared to protect sensory nerves.Subtotal thyroidectomy Was performed to resect the affected lobe,isthmus,and the majority of opposite lobe without considering the size of primary tumor or whether metastasis to the neck lymph nodes happened.Soft tissues of the mainly metastatic areas(Ⅱ a、Ⅲ、Ⅳ、Ⅴb)were cleared.The accessory nerve was not exposed routinely to avoid stimulation.Lymph nodes metastasis in different areas was recorded respectively.Complications in different operative modes were compared.Results Compared with standard modified radical thyroidectomy,the simplified mode had shorter scar-and no limit of neck mobility.Because of muscles and nerves pemervation,movement dysfunction and abnormal sensation of neck and shoulder decreased obviously.The operation duration was shortened.Cervical lymph node status Was evaluated,which provided basis for prognosis judgment and comprehensive treatment.Conclusions The simplified modified radical procedure has the benefit of decreased trauma while maintains the similar recurrence rate compared to modified radical thyroidectomy.It improvs the life quality of patients.This procedure fits the principle of functional radical neck dissection better.

Objective To investigate the effects of intrathecal injection of Roscovitine on the pain behavior and the expression of pERK1/2 and pPPARγ receptor in spinal cord in rats with neuropathic pain.Methods Fifty-two male adult Sprague-Dawley rats of 220～ 250 g were randomly divided into 4 groups:Sham+DMSO group (dimethyl sulfoxide,S+D group),Sham+Roscovitine (S+R) group,CCI+ DMSO group (I+D),CCI+ Roscovitine (I+R) group.The corresponding drugs were administered by intrathecal injection from the seventh day after CCI once a day for 14 days.The right hind paw mechanical threshold value (PMWT) was measured by Von Frey filament and paw thermal withdrawal latency(PWTL) was measured by Hargreaves methods before 1 day and 3 d,7 d,10 d,14 d after surgery.The spinal cord lumbar enlargement was taken at 14 d after surgery,and Cdk5,p35,phosphorylated ERK protein (pERK),total ERK protein (ERK),phosphorylated PPARγprotein (pPPARγ) and total PPARγ protein (PPARγ) were detecteded by Western blot.Results Roscovitine was administered by intrathecal injection alleviated mechanical allodynia and thermal hyperalgesia of CCI rats.Compared with I+D group,PWMT in I+R group at 7 d,10 d,14 d after administration((4.65±0.08)g,(6.47±0.12)g,(7.90±0.19)g,) vs ((3.71 ±0.06)g,(2.45±0.17)g,(2.31±0.15)g) (Fgroup =505.71,P＜0.05,Ftime×group =15.33,P＜0.05),PWTL((9.22±0.33) s,(13.52±0.43) s,(12.63±0.88) s) vs ((8.02±0.20) s,(5.90±0.28) s,(5.40±0.38) s) (Fgroup =355,P＜0.05,F time×group =8.589,P＜0.05) were significantly increased.Compared with I+D group(p35 (0.58±0.02),pERK (1.12±0.13),pPPARγ (0.77±0.16)),p35 (0.46±0.04,F=11.06,P＜0.05) and pERK(0.79±0.11,F =22.91,P＜ 0.05) in I+ R group,were significantly dereased,and the expression of pPPARγ(0.99±0.13,F =17.62,P＜0.05))was significantly increased.Conclusion Intrathecal injection Roscovitine can alleviate both mechanical allodynia and thermal hyperalgesia in rats with neuropathic pain,and its mechanisms may be related to the inhibition of Cdk5/p35 and ERK activity,enhancement of PPARγ activity in the spinal dorsal horn.