AIM:To construct eukaryotic expression vector expressing double shRNA sections targeting Survivin gene.METHODS: Eukaryotic expression vector expressing double shRNA sections targeting Survivin gene were designed and chemically synthesized.They were directionally inserted into plasmid pGenesil-1 with respectively U6 promoter and termination code,the common green fluorescence protein(EGFP) gene and Neo gene. In this way,the vector of pGenesil-1 shRNA containing 2 sections of Survivin shRNA were constructed and they were transfected into the pancreatic cancer cell Bx-PC3.Transfection was detected by fluorescence microscope.The inhibition expression of Survivin mRNA was measured by RT-PCR.RESULTS: HE1 and HE2 plasmids were identified by the biocatalyst cut which confirmed the exactitude and were analyzed by the sequence analysis which verified the perfect clone plasmid inserted by them.CONCLUSION: A eukaryotic expression vector of double short hairpin RNA for Survivin gene is successfully constructed.The pancreatic cancer cells Bx-PC3 succeed to be transfected and expression of Survivin mRNA is inhibited obviously.

To assess the variability of adhesin gene hpaA in differene H pyloristrains with PCR restriction fragment length polymorphism (RFLP) A 710 bp gene hpaA , obtained from 9 different H pylori strains, were digested by Hha Ⅰand Hae Ⅲ individually and analyzed by agarose gel electrophoresis Four different polymorphic types were found in hpaA digested with Hae III and five types with Hha I Clinical isolates of H pylori from Chongqing showed difference among them and remarkably distinguished from foreign standard strains Mongolia gerbil adapted H pylori strain,which were obtained from Mongolia gerbil infected with clinical isolate, also showed inconsistence in hpaA RFLP The hpaA gene from different H pylori strains revealed 1 variability, and this might provide an effective method for developing molecular epidemiology of H pylori

Objective To investigate the effect of olfactory entheath cells (OECs) transplantation on neural function and synaptic density of rats with traumatic brain injuries (TBI) and the possible mechanism. Methods Rats were divided into sham operation group, TBI group and OECs engrafted group. The brains of the rats were injured by Feeney percussion device through free falling. After cultured and identified by using specific marker (known as P75), OECs were transplanted into the area around the injured brain. Cortical somatosensory evoked potential (CSEP) and motor evoked potential ( MEP) were evaluated at day 14 after cell transplantation to determine the neurophysiologic function following TBI. Moreover, the synaptic densities around the injured brain were determined by using immunohistochemical method. One-way ANOVA test was used for statistical analysis. Results The transplanted OECs could survive and migrate around the injury site in the host brain 14 days after OECs transplantation. In addition, rats subjected to OECs implantation showed a marked neurophysiologic improvement and a significant increase of synaptic densities compared with the control group. Conclusion OECs transplantation can improve the neurophysiologic function and increase the synaptic density, which provides experimental basis for treatment of TBI with OECs.

Objective To establish contusion brain injury model in rats, and investigate the efficacy of intravenous administration of neural stem cells(NSC) on posttraumatic neurocognitive function recovery and NGF expression in rats. Methods Cerebral contusion model in motor-sensory cortex of the right parietal cortex in rat was established by a 50 g-weight hammer falling respectively from 30 cm height along guide stick to impact collision pole by improved trauma device for model of contusion brain injury based on Feeney method. And the NSC isolated from GFP transgenic mice were injected intravenously via the tail vein 24 h after the brain trauma, and 1 week later neurocognitive function scores and NGF immunostaining were performed to explore the efficacy of NSC transplant. Results The NSCs from the GFP transgenic mice gathered at the injury site 1 weeks after transplants.Neurocognitive function scores and NGF-positive cells measurement(226 ±27,23 ±4 ) in the treatment group revealed significant increase than in the brain trauma group(300 ±36;15 ±3 )(P＜0.05). Conclusion The intravenous NSC injection in rats can survive and migrate to the injured brain region and promote the post-injury neurocognitive function restoration. The increase of NGF expression may underline one of most important mechanisms in NSC treatment' s rats after brain injury.

A strain was separeted from the Yueqing bay using pine pollen baiting.The vegetative thallus of the separated strain is oval and unincleate.It possesses a cell wall composed of many compact layers of closely pressed scales, which can be resolved where the cell wall is disrupted.The radiating branched extensions of the thallus, the ectoplasmic net, emerges from the sagenogenetosome.Asexual reproduction is by conversion of the vegetative thallus to many biflagellate zoospores, during which tetrads of cells are formed.It was identified with Schizochytrium sp.based on the features mentioned above.

OBJECTIVETo observe the effects of Danlou Tablet (DT) on inflammatory reaction, and expressions of lipoprotein-associated phospholipase A2 (LP-PLA2), secretory phospholipase A2 (sPLA2), and to analyze potential mechanisms.

METHODSForty male Wistar rats were randomly and equally divided into five groups, i.e., the normal control group, the model group, the Western medicine (WM) group, the low dose DT group, the high dose DT group, 8 in each group. Rats in the normal control group were fed with basic forage for 12 successive weeks, while AS rat model was established in rats of the other four groups by feeding high fat and sugar forage plus intraperitoneal injection of vitamin D₃. Normal saline, atorvastatin calcium suspension (at the daily dose of 1.8 mg/kg), low dose DT suspension (at the daily dose of 450 mg/kg), and high dose DT suspension (at the daily dose of 900 mg/kg) were administered to rats in the model group, the WM group, the low dose DT group, the high dose DT group respectively by gastragavage for 8 successive weeks. The general condition of all rats was observed. Rats were sacrificed after gastric administration and their serum collected. Serum levels of lipids (TC, TG, HDL-C, LDL-C) and inflammatory factors [IL-6, TNF-α, monocyte chemoattractant protein 1 (MCP-1), oxidized low-density lipoprotein (ox-LDL), lipoprotein-associated phospholipase A2 (LP-PLA2), secretory phospholipase A2 (sPLA2)] were detected. Pathological changes of thoracic aorta were observed by HE staining. Protein and gene expressions of LP-PLA2 and sPLA2 in thoracic aorta were measured by Western blot and real-time fluorescent quantitative PCR respectively.

RESULTSCompared with the normal control group, rats in the model group were in low spirits and responded poorly. Typical atherosclerotic plaque could be seen in thoracic aorta of rats in the model group. Serum levels of TC, TG, LDL-C, IL-6, TNF-α, MCP-1, ox-LDL, LP-PLA2, and sPLA2 significantly increased (P < 0.05); protein and gene expressions of LP-PLA2 and sPLA2 in rat thoracic aorta increased (P < 0.05) in the model group. After 8 weeks of intervention, rats in 3 medication groups appeared active, and HE staining showed subsidence of plaque in rat thoracic aorta. Compared with the model group, serum levels of TC, TG, LDL-C, IL-6, TNF-α, MCP-1, ox-LDL, and LP-PLA2 decreased in 3 medication groups (P < 0.01, P < 0.05); serum sPLA2 level decreased, protein and mRNA expressions of LP-PLA2 and sPLA2 in rat thoracic aorta decreased in the WM group (P < 0.01, P < 0.05); protein and mRNA expressions of LP-PLA2 in rat thoracic aorta significantly decreased in the low dose DT group (P < 0.01, P < 0.05), and those of LP-PLA2 and sPLA2 decreased in the high dose DT group (P < 0.01, P < 0.05).

CONCLUSIONDT could fight against inflammatory reaction and AS possibly through inhibiting LP-PLA2 expression and reducing ox-LDL production.

1-Alkyl-2-acetylglycerophosphocholine Esterase ; blood ; Animals ; Aorta, Thoracic ; pathology ; Atherosclerosis ; drug therapy ; Chemokine CCL2 ; blood ; Drugs, Chinese Herbal ; pharmacology ; Inflammation ; drug therapy ; Interleukin-6 ; blood ; Lipids ; blood ; Lipoproteins, LDL ; blood ; Male ; Phospholipases A2 ; blood ; Plaque, Atherosclerotic ; Random Allocation ; Rats ; Rats, Wistar ; Tablets ; Tumor Necrosis Factor-alpha ; blood

ObjectiveTo explore the feasibility of bone marrow stromal cells (BMSC) of rats differentiating into endothelial cells.MethodsBone marrow cells were obtained by an aseptic technique. Afterwards, the obtained cells were divided into two groups: cells in test group were propagated in 1640 medium with recombinant human GM-CSF (rhGM-CSF) (400ng/ml), and that in control group were propagated in medium with 1640 medium only. The differentiated cells were detected by specific immunology marker by immunocytochemistry and immunofluorescence at day 8.ResultsThe differentiated cells demonstrated the characters of endothelial cells under phase contrast microscopy. Cells of the test groups demonstrated specific characters by immunocytochemistry stain.ConclusionBone marrow stromal cells can multiply vigorously and differentiate into cells with endothelial cells characters in the medium with high concentration rhGM-CSF.

Objective To investigate the effect of BPI-1095 on caspase-3 protein expression in middle artery occlusion(MCAO) rats.Methods Cerebral ischemia was induced with MCAO in adult male SD rats.Rats were randomly subjected into 6 groups with 15 rats in each group.Each rat has been given tested medication of different dosage and was sacrificed 24 h after treatment.The area of infarction was measured on each slice by image analysis system.Meanwhile,immunohistochemistry staining was used to identify caspase-3 expression in ischemic brain tissue.Results The infarcted area were significantly decreased in big and moderate dose treated rats(P<0.05,vs the placebo group).The expression of caspase-3 protein decreased in contralateral and ipsilateral hemisphere areas.The caspase-3 positive cell was significantly decreased in rats treated with big doses compared with placebo-or ASA-treated rats.Conclusion BPI-1095 shows neuroprotection in MCAO rats,which may related with the inhibition of caspase-3 expression resulting in apoptosis in penumbra.

ObjectiveTo investigate the neuroprotective effects of different doses of BPI-1095 on infarct volume and neurological outcome in middle cerebral artery occlusion(MCAO) rats.MethodsCerebral ischemia model was induced with MCAO in adult male SD rats. 10 minutes after surgery, rats were randomly subjected into six groups with 15 rats in each group. Each rat has been given different dosage tested medication and was sacrificed 24 h after treatment. Neurological functional behaviour tests were performed 4 h and 24 h after treatment. After the final behaviour test, 7 or 8 rats (remain 5 rats for brain tissue stain) were randomly picked up from each group. Their infarction volume was measured with image analysis system after 2% triphenyl-tetrazolium chloride (TTC) stain. ResultsHigh dose (240 mg/kg) and moderate dose (80 mg/kg) of BPI-1095 were able to improve the neurological deficit in MCAO rats (P<0.05, vs vehicle-treated group), as well as they decrease the infracted volume (P<0.05, vs the vehicle-treated group ) 24 h after ischemia.Conclusion80～240 mg/kg BPI-1095 is able to improve neurological deficit effectively and reduce infarct volume significantly.

Objective To evaluate the clinical effects of color Doppler ultrasound in perforating vein closure by foam sclerotherapy for the treatment of venous leg ulcer.Methods Choosing the patients with venous ulcers (classified as C6) that underwent superficial and perforating veins closure by ultrasound-guided foam sclerotherapy in the Department of Vascular Surgery,First Affiliated Hospital of Chongqing Medical University,during December 2014 to August 2016.All patients were followed up by color Doppler scanning for veins closure,and the postoperative healing of skin ulcers were observed at the same time.Results A total of 114 patients with 119 limbs were enrolled,which were confirmed 1-3 perforating vein lesions by ultrasound.An average of 2.1 perforators were closed per leg (1-3 perforators).The average follow-up period was 11.3 months (1-21 months).The healing rate is 100％ and all the insufficient perforating veins were occlusion 1 month postoperative.Three cases of ulcer recurrence,and 8 cases of insufficient perforator recanalization were found.Serious complications did not appear.Conclusions Color doppler ultrasound can improve the safety and efficacy of perforating vein closure by foam sclerotherapy for the treatment of venous leg ulcer,and it has a high clinical value.