Objective:To construct the pIRES2-enchanced green flurescent protein expression vector carrying BMP_2 gene in order to provide an ideal reporter gene for the expression and identify the location of portein in vitro and vivo.Methods:The BMP_2 cDNA sequence was excised,and subcloned on pUC18 vector.And then the BMP_2 sequence on the pUC18 plasmid was excised again and ligated into Sal Ⅰ site of pIRES2-EGFP treated by phosphorylase in sense and antisense direction.The correct recombinant plasmid was transfected into 293-T cells,the expression of BMP_2 was determined by FCM,LCM and RT-PCR.Results:The recombinant expression vector was verified correctly by enzyme digestion,sequence analysis.This vector was expressed in cells.Conclusion:A pIRES2 green fluorescent protein reporter gene vector containing BMP_2 has been constructed successfully,thus providing an important and convenient tool to study intracellular localization of BMP_2.

BACKGROUND: It has been proved that vascular endothelial growth factor 165(VEGF165) and bone morphogenetic protein 2(BMP2) can accelerate the vascularization synergistically.OBJECTIVE:To construct the vectors, pDsVEGF165Red1-N1 and pIRES2-BMP2-enhanced green fluorescent protein(EGFP) followed by co-transfected into HEK 293-T cells,and study their expression and location of VEGF165and BMP2 in the cells.DESIGN: A randomized and controlled experiment.SETTING: National Key Laboratory,Institute of Surgery Research,Daping Hospital,Third Military Medical University.MATERIALS: The experiment was conducted at National Key Laboratory,Institute of Surgery Research,Daping Hospital,Third Military Medical University from September 2002 to March 2004. pcDNA3.1\BMP2 ( gift of Dr.Bostrom, UCLA School of Mediine, Los Angeles,USA).pDsRed1-N1(gift of Pro. Roger Y.Tisen,University of California,San Diego,USA). pUC18/VEGF165,293-T cells(preserved by our Laboratory).METHODS: According to the nucleotide sequence of hVEGF165, the primers were designed.The hVEGF165 gene without stop codon was amplified by polymerase chain reaction (PCR).The fragment digested was cloned into the expression vector pDsRed1-N1.Meantime,the pIRES2-BMP2-EGFP expression vector was constructed.The two plasmids were co-transfected into HEK 293-T cells.The co-expression and distribution of the VEGF165and BMP2 were observed with confocal laser microscopy (CLSM) and detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blotting.MAIN OUTCOME MEASURES: Identification of the recombinant plasmids and the expressing mRNA and protein in 293-T cells.RESULTS: The recombinant plasmids were verified correct construction by restriction enzyme analysis, PCR and sequencing. The two genes which were co-transfected could express in HEK 293-T cells.The co-expression of the report genes,RFP and EGFP, were found over the cytoplasm and in the nuclei by CLSM.CONCLUSION: Two report gene expression vectors contain VEGF165 and BMP2 have been constructed successfully, which can be co-expressed in HEK 293-T cells. Thus, they can provide important and convenient tool to study intracellular interaction of VEGF165 and BMP2.

[Objective]To analyze the causes of anterior knee pain after intramedullary(IM) nailing treatment of tibial shaft fractures.[Method]A total of 256 patients with tibial shaft fractures were treated with intramedullary nailing from 2000 to 2007 in our hospital.There were 183 males and 73 females with a mean age of 45 years.All patients were followed up at least once after fracture healing.[Result]Among 256 patients,166 experienced anterior knee pain,accounting for 65%.Ninety-six(58%) treated with transtendinous nailing complained of and 70(42%) treated with paratendinous nailing complained of anterior knee pain.It was worsened after activity but relieved only in 33(20%) by resting or taking analgesic drugs.The anterior knee pain disappeared one year after the IM nailing was taken out.[Conclusion]Anterior knee pain can not be reduced by different approaches of operation for tibial shaft fractures.However,the removal of intramedullary nailing and the muscle exercises around the knee joint can reduce the occurrence of anterior knee pain.

Forty-eight male Wistar rats,weighing 280 to 320 g,were selected and prepared by fasting but free drinking for 12 hours before the experiment.They were randomly divided into four groups by random number table (12 rats in each group):control group,ischemic group,reperfusion group and propofol group.Control group:normal saline infusion[10 ml /(kg?h)]for 1 h in caudal vein without any other treatment.Ischemic group:on the basis of control group,supply with 20 mmHg(1 mmHg =0.133 kPa)pneumoperitoneum pressure for 1 h. Reperfusion group:on the basis of ischemic group,keep pneumoperitoneum pressure open for 30 min.Propo-fol group:infusion propofol in caudal vein [10 ml /(kg?h),saline 10-fold dilution],given 20 mmHg pneu-moperitoneum pressure for 1 h,then kept this pressure open for 30 min.The expression changes of blood urea nitrogen(BUN),creatinine(Cr),superoxide dismutase(SOD),malondialdehyde(MDA)and nuclear factor E2-related factor 2(Nrf2)were detected individually.Results The levels of BUN,Cr and MDA gradually increased,while the level of SOD decreased in control group,ischemic group and reperfusion group (P ﹤0.05,respectively).Compared with ischemic group and reperfusion group,the levels of BUN,Cr and MDA decreased significantly,and the level of SOD increased markedly in propofol group(P ﹤0.05,respectively). The expression of Nrf2 protein and mRNA gradually increased in the control group,ischemia group,reperfu-sion group and propofol group (P ﹤0.05).Conclusion Carbon dioxide pneumoperitoneum can cause renal IRI in rats.Propofol has a protective effect of IRI by regulating the expression of Nrf2 protein.

BACKGROUND: To date, it has been confirmed that etomidate pre-treatment can reduce the damage of remote organs caused by limb ischemia-reperfusion, but whether etomidate post-treatment has protective effect on remote organs and its mechanism has been rarely reported. OBJECTIVE: To investigate the influence of etomidate post-treatment on limb ischemia-reperfusion lung injury. METHODS: A rat model of limb ischemia-reperfusion lung injury was prepared by clamping the bilateral femoral arteries for 2 hours and reperfusion for 3 hours. After 2 hours of limb ischemia, I/R group experienced the process of limb ischemia-reperfusion; I/R+ETO group, I/R+Dex 0.2 group, I/R+Dex 0.5 group and I/R+Dex 1.0 group, besides the model of limb ischemia-reperfusion, were injected with etomidate 1.0 mg/kg and dexamethasone 0.2, 0.5 and 1.0 mg/kg respectively through tail vein. At 3 hours of reperfusion, blood samples were extracted from the carotid artery, blood gas analysis was performed and the partial pressure of blood oxygen (PaO2) was recorded. The pathological changes were detected by immunohistochemistry. Apoptotic index was detected by Hoechst 33258 staining and wet/dry weight ratio was detected. Fas protein and Fasl mRNA of lung tissue were detected by western blot and RT-PCR respectively. Tumor necrosis factor-α and interleukin-1β levels were detected by ELISA. RESULTS AND CONCLUSION: Compared with the I/R group, PaO2 increased (P < 0.05), Apoptotic index, wet/dry weight ratio, Fas and FasL mRNA, tumor necrosis factor-α and interleukin-1β decreased in the I/R+ETO group, and the number of apoptotic lesions decreased in the I/R+ETO group (P < 0.05). Compared with the I/R+ETO group, the change of each index was not statistically significant in the I/R+Dex 0.5 group (P > 0.05). To conclude, etomidate post-treatment can reduce lung injury caused by limb ischemia-reperfusion in rats, and its mechanism may be related to the down-regulation of Fas/FasL. In the statistical point of view, etomidate 1.0 mg/kg has the potency intensity of reducing lung injury, almost equivalent to dexamethasone 0.5 mg/kg.

BACKGROUND: Curcumin pre-conditioning can alleviate liver Injury induced by limb Ischemia/reperfusion (l/R), but whether curcumin post-conditioning has protective effect against liver cold l/R Injury and its mechanism are still poorly studied. OBJECTIVE: To Investigate the effects of curcumin post-conditioning on hepatocyte apoptosis in rats with liver cold l/R Injury in rats. METHODS: Eighty adult male Sprague-Dawley rats were randomly divided Into four groups (n-20 per group) by using a random number table: Sham group, l/R group, curcumin post-conditioning group (l/R+Cur group), and dexamethasone group (l/R+Dex group). The liver blood flow was completely blocked. Then the splenic vein and the adrenal vein were used as the inflow and outflow tracts to inject 0 °C compound Ringer lactate solution followed by cold perfusion for 30 minutes. After stopping cold perfusion, the proximal splenic vein and the right adrenal vein were ligated to remove the spleen, and then the blood flow In the liver restored. The cold l/R model was successfully established. After 30 minutes of cold ischemia, 60 mg/kg curcumin was injected Into the rat tail vein In the l/R+Cur group, 0.5 mg/kg dexamethasone was injected Into the rat tail vein In the l/R+Dex group, and the same amount of saline was Injected in the other groups. Blood sample was taken from the carotid artery at 6 hours after reperfusion. Serum levels of aspartate aminotransferase and alanine transferase were detected. Then the rats were sacrificed to detect malondlaldehyde level In liver tissue, observe liver pathological changes by hematoxylin-eosin staining, measure hepatocyte apoptosis Index by Hoechst 33258 staining, detect expression of Bcl-2 and Bax protein by western blot, expression of caspase-9 mRNA by RT-PCR, and levels of tumor necrosis factor-a and interleukin-1 ß by ELISA. RESULTS AND CONCLUSION: Compared with the sham group, aspartate aminotransferase, alanine transferase, malondialdehyde levels and apoptosis Index in the l/R group increased significantly (P < 0.05). Hematoxylin-eosin staining showed a large number of inflammatory cells infiltrated in the hepatic sinusoids, eosinophilic changes of hepatocytes, cytoplasmic loosening, balloon-like changes of hepatocytes, occasional patchy necrosis, and scattered punctate necrosis foci in the l/R group. The l/R group had significantly decreased expression of Bcl-2, and increased expression of Bax (P < 0.05), Caspase-9 mRNA, tumor necrosis factor-a and interleukin-1 ß (P < 0.05). Compared with the l/R group, aspartate aminotransferase, alanine transferase, malondlaldehyde levels and apoptosis index in the l/R+Cur group decreased significantly (P < 0.05). Hematoxylin-eosin staining showed that Inflammatory infiltration In the hepatic sinusoids decreased significantly, eosinophilic and balloon-like hepatocytes decreased significantly, but occasionally a small amount of scattered punctate necrosis was observed in the l/R+Cur group. There was significantly increased Bcl-2 expression, and significantly decreased Bax (P < 0.05), Caspase-9 mRNA, tumor necrosis factor-a and interleukin-1 ß (P < 0.05). No significant differences were observed between l/R+Cur group and l/R+Dex group (P > 0.05). In a word, curcumin-post conditioning can alleviate liver injury induced by cold l/R in rats. Its mechanism may be related to down-regulation of Bcl-2/Вах ratio, inhibition of caspase-9 mRNA expression, and reduction of the release of tumor necrosis factor-a and interleukin-1 ß, therefore playing an antl-apoptotic role in liver protection.

Objective To detect the expression of COX-2 and p53 in breast carcinoma tissue and investigate their associations with clinical prognosis.Methods The expression of COX-2 and p53 was carried out in 16 cases of normal epithelial tissue and 152 cases of breast carcinoma tissue using immunohistochemistry SP method.The correlation of their expression with clinical characteristics was analyzed using SPSS software 16.0.Survival analysis was used to investigate their effects on tumor prognosis.Results No positive COX-2and p53 expressions were observed in normal epithelial tissue.Among 152 patients,89 (58.6 ％) were positive staining rete for COX-2 and 93 (61.18 ％)were shown p53 expression, with a statistically significant associations between expressions of COX-2 and p53 and breast cancer (r =0.426,P ＜ 0.01).The COX-2 and p53 expressions were significantly correlated with pathological grade, clinical stage, lymph node or distant organ metastasis.There was a statistically significant correlation between COX-2 and p53 expression and pathological grade Ⅰ / Ⅱ.The 5-year progress-free survival (PFS) rate in patients with COX-2 over-expression was 61.3 ％, which was remarkably lower than that in those with low COX-2 expression.There was no statistically significant difference of 5-year PFS between positive and negative p53 expression.A shorter 5-year PFS was seen in patients with co-expression of COX-2 and p53 than in those with either COX-2 or p53positive expression alone and also than in those with both COX-2 and p53 negative expression.Conclusion Detection of the expressions of COX-2 and p53 can be used to predict the prognosis of breast cancer.

Objective To evaluate the effects of interferon therapy after curative surgical intervention on improving prognosis of patients with hepatitis C-related hepatocellular carcinoma (HCC).Methods We searched randomized clinical trials from 1990 to 2015 on interferon therapy after curative surgical intervention in patients with hepatitis C-related HCC from the Cochrane Library,the Cochrane Database of Systematic Reviews,MEDLINE and Embase.A Meta-analysis was carried out using Revman 5.Results There were 7 studies included in this research.The results showed that interferon therapy after curative surgical intervention in patients with hepatitis C-related HCC reduced the recurrence rate of HCC at 3 years (RR =0.84,95％ CI 0.73 ～0.97,P ＜0.05).The therapy could not improve the 3-year survival rate in these patients (RR =1.04,95％ CI 0.90 ～ 1.21,P ＞ 0.05).Stratified subgroup analyses showed interferon therapy after liver resection reduced the recurrence rate (RR =0.62,95 ％ CI 0.39 ～ 1.00,P =0.05).For patients with tumors less than 3 cm,interferon therapy reduced the recurrence rate (RR =0.82,95％ CI 0.69 ～ 0.98,P ＜ 0.05).Conclusion Interferon therapy after curative surgical intervention improved prognosis in patients with hepatitis C-related HCC.

ObjectiveTo study the clinicopathological features of endometrial cancer patients diagnosed during or after tamoxifen (TAM)treatment for breast cancer and to evaluate the relevance between the clinicopathological features and the cessation time of TAM treatment. Methods42 TAM-related endometrial cancers patients in the Affiliated Hospital of Medical Collage of Qingdao University were divided into 2 groups according to the cessation time of TAM.Group B were 20 past TAM users who were diagnosed as endometrial cancer 12 or more months since the cessation of TAM.Group A included the rest 22 patients.The clinicopathological features of the 2 groups were analyzed retrospectively.ResultsThe age of patients was ranging from 32 to 85 years when they were diagnosed endometrial cancer.There was no significant difference in terms of the age at which breast cancer was diagnosed, BMI, HRT, single dose of TAM, duration and cumulative dose between the 2 groups.However, the difference of the age at which endometrial cancer was diagnosed, menopausal status, the interval between diagnostic date of the 2 cancers, histological types, pathological stage, myometrial invasion, and cervical invasion had statistical significance between the 2 groups ( P ＜ 0.05 ). Conclusion Compared with group A, group B have a higher pathological stage and more invasive histological features, which may be because group B include more elderly and postmenopausal patients.

Background: The objective of this study was to investigate the impact of robot-assisted surgery (RA) on the risk of new vertebral compression fracture (NVCF) and bone cement leakage in patients with osteoporotic vertebral compression fractures (OVCF) after percutaneous vertebral augmentation (PVA), including percutaneous kyphoplasty (PKP) and percutaneous vertebroplasty (PVP). Methods: A meta-analysis was performed to evaluate the clinical outcomes and adverse effects of RA-PVA versus fluoroscopy-assisted (FA)-PVA in patients with OVCF. A validation cohort of 385 patients who underwent PVP or PKP was retrospectively analyzed.In addition, we attempted to create well-calibrated nomograms to estimate the risk of NVCF and bone cement leakage. Results: The meta-analysis revealed that the incidence of NVCF and bone cement leakage was significantly lower in RA-PVA than in FA-PVA. The validation cohort confirmed that RA-PVA provided better results than FA-PVA in terms of NVCF and bone cement leakage. Conclusions The meta-analysis and the validation cohort suggest that RA reduced the risk of NVCF and bone cement leakage in patients with OVCF after PVA. The nomograms are accurate and easy-to-implement methods for clinicians to estimate the risk of NVCF and bone cement leakage after PVA.