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To construct soluble TNF related apoptosis inducing ligand (TRAIL) expression system and investigate the effect of the expression product on tumor cell. It may provide valuable information for research into the immune system of the finless porpoise. The full-length cDNA of TRAIL (designated fTRAIL) was cloned from the total RNA of the finless porpoises blood using RT-PCR techniques and then the extracellular soluble fragments of fTRAIL (designated fsTRAIL) was ligated into pET43.1a. Recombinant soluble fTRAIL (pET43.1a-fsTRAIL) fused with Nus-his tag was efficiently expressed in Escherichia coli BL21 (DE3) and the Nus-His-fsTRAIL protein was purified. The expression of Nus-His-fsTRAIL was verified by Western blotting. In vitro, the effects of the purified Nus-His-fsTRAIL protein on Jurkat and HeLa cells were etected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) assay, TrypanBlue and Flow Cytometry analysis. The expression system pET43.1a-fsTRAIL was constructed and Nus-His-fsTRAIL protein was expressed successfully. In vitro, the Nus-His-fsTRAIL protein was able to inhibit the proliferation and induce apoptosis of Jurkat and HeLa cells in a dose-dependent manner. The Nus-His-fsTRAIL protein has anti-tumor activity against Jurkat and HeLa cells in vitro.
Subject(s)
Animals , Humans , Apoptosis , Blotting, Western , Cloning, Molecular , DNA, Complementary , Escherichia coli , HeLa Cells , Jurkat Cells , Porpoises , TNF-Related Apoptosis-Inducing LigandABSTRACT
The endothelial-mesenchymal transition (EndMT) is known to be involved in the transformation of vascular endothelial cells to mesenchymal cells. EndMT has been confirmed that occur in various pathologic conditions. Transforming growth factor β1 (TGF-β1) is a potent stimulator of the vascular endothelial to mesenchymal transition (EMT). Aspirin-triggered resolvin D1 (ATRvD1) has been known to be involved in the resolution of inflammation, but whether it has effects on TGF-β1-induced EndMT is not yet clear. Therefore, we investigated the effects of AT-RvD1 on the EndMT of human umbilical vein vascular endothelial cells line (HUVECs). Treatment with TGF-β1 reduced the expression of Nrf2 and enhanced the level of F-actin, which is associated with paracellular permeability. The expression of endothelial marker VE-cadherin in HUVEC cells was reduced, and the expression of mesenchymal marker vimentin was enhanced. AT-RvD1 restored the expression of Nrf2 and vimentin and enhanced the expression of VE-cadherin. AT-RvD1 did also affect the migration of HUVEC cells. Inhibitory κB kinase 16 (IKK 16), which is known to inhibit the NF-κB pathway, had an ability to increase the expression of Nrf2 and was associated with the inhibition effect of AT-RvD1 on TGF-β1-induced EndMT, but it had no effect on TGF-β1-induced EndMT alone. Smad7, which is a key regulator of TGF-β/Smads signaling by negative feedback loops, was significantly increased with the treatment of AT-RvD1. These results suggest the possibility that AT-RvD1 suppresses the TGF-β1-induced EndMT through increasing the expression of Smad7 and is closely related to oxidative stress.
Subject(s)
Humans , Actins , Endothelial Cells , Human Umbilical Vein Endothelial Cells , Inflammation , Oxidative Stress , Permeability , Phosphotransferases , Transforming Growth Factors , Umbilical Veins , VimentinABSTRACT
Objective To evaluate the effects of dezocine on diabetic neuropathic pain (DNP ) and expression of NMDA receptor subunit 2B (NR2B) in the spinal dorsal horns of rats .Methods Forty-eight male Sprague-Dawley rats , aged 4 weeks , weighing 150-170 g , with DNP induced by intraperitoneal injection of streptozocin (STZ) 50 mg/kg (successful induction of diabetes was defined as blood glucose >16.7 mmol/L) , were randomly divided into 2 groups ( n=24 each) using a random number table:DNP group and dezocine group (group D) .Twenty-four normal rats were chosen and served as normal control group (group C) .In group D , dezocine 2.52 mg/kg was injected intramuscularly once a day for 7 consecutive days starting from 2nd week after STZ injection ,while the rats in DNP and C groups received the equal volume of normal saline .Paw withdrawl threshold (PWT) to mechanical stimulation was measured before dezocine injection (T0 ) ,and on 1st ,3rd ,5th and 7th days after dezocine injection (T1-4 ) and on 7th day after the end of dezocine injection (T5 ) .Twelve rats in each group were sacrificed after measurement of PWT at T4 ,and T5 .The lumbar segments of the spinal cord were removed for determination of NR2B protein expression (by immuno-histochemistry and Western blot ) and NR2B mRNA expression (by RT-PCR ) in the spinal dorsal horns .Results Compared with group C ,the PWT at T0-5 in group DNP and at T0 and T5 in group D was significantly decreased , and the expression of NR2B protein and mRNA at T4 ,5 in DNP group and at T5 in D group was up-regulated ( P0.05) . The PWT was significantly lower at T0 and T5 ,and the expression of NR2B protein and mRNA was higher at T5 than at T4 in group D ( P<0.05 ) .Conclusion Dezocine can effectively relieve DNP in rats and inhibition of NR2B expression in the spinal dorsal horns is involved in the mechanism .
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Objective To explore the value of MR imaging-guided percutaneous cervical discectomy and discolysis with oxygen-ozone mixture for treatment of cervical disc herniation.Methods Eight herniated cervical discs in 7 patients were diagnosed by MRI, including 5 discs of lateral protruding type, 2 discs of paramedian protruding type and one disc of central protruding type.All patients underwent MR imaging-guided percutaneous cervical discectomy and discolysis with oxygen-ozone mixture.The procedures were guided by a set of 0.23 T open MR system mounted with iPath 200 optical tracking system.The herniated portion of the disc was punctured with a 14 G MR-compatible needle in the healthy side via anterolateral oblique route.The interventional steps were as follows; firstly, cut herniated part with percutaneous discectomy probe and inject 2ml oxygen-ozone mixture of 60 μg/ml; secondly, retreat the needle to the disc center, resect nucleus pulposus, and inject 2 ml oxygen-ozone mixture of 60 μg/ml.All patients were followed up for 6 months, with 4 patients by telephone and 3 patients in outpatient clinic.The effect of treatment was evaluated according to Williams postoperative assessment standard.Results All procedures were performed successfully.The clinical outcome was evaluated as excellent in 5 cases, good in 1 case and fair in 1 case.The total ratio of excellent and good was 85.7%.No serious complication occurred expect 1 case with intraoperative paroxysmal pain.Conclusion MR imaging-guided percutaneous cervical discectomy and discolysis with oxygen-ozone mixture was a safe, effective and minimally invasive method for the treatment of cervical disc herniation.
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Objective To explore the value of MR imaging-guided percutaneous lumbar discectomy and discolysis with oxygen-ozone mixture for treatment of posterolateral lumbar disc herniation via a new puncture approach of facet joint medial route. Methods All 114 lumbar intervertebral discs in 103 patients were diagnosed as posterolateral lumber disc herniation by CT or MRI, which were located at the levels of L3-4 in 5 cases, LA-5 in 87 cases and L5-S1 in 22 cases. The procedure was guided under 0. 23 T open magnetic resonance with iPath 200 optical tracking system. A 14 G MR-compatible needle was punctured into the disc center via a new puncture approach of facet joint medial route. The therapy steps were as follows: firstly, cut nucleus pulposus and inject 6 ml oxygen-ozone mixture of 60 μg/ml in the disc center;secondly, retreat the needle to the local prominence, cut prominent part and inject 6 ml oxygen-ozone mixture of 60 μg/ml. Thirdly, retreat the needle to the periradicular nerve root, inject 15 ml oxygen-ozone mixture of 40 μg/ml and 4 ml pain-block liquid. All patients were followed up at 3 days, 1 month, 3 months and 6 months after operation, evaluated for the effect of treatment with the modified Macnab criteria, and the results were compared with the χ2 test. Results All procedures were successfully performed. Intraoperative dural injury occurred in 5 cases. Postoperative infection of intervertebral space occurred in 2 cases. The clinical effective rate was 96. 1% (99/103), 84.5% (87/103), 94.2% (97/103), 95.1% (98/103)respectively at 3 days, 1 month, 3 months and 6 months after operation, and the differences were signifieant (χ2 = 12. 942, P = 0. 005 ) . Conclusion MR imaging-guided percutaneous lumbar discectomy and discolysis with oxygen-ozone mixture via facet joint medial route is a minimally invasive, safety and effective method for the treatment of posterolateral lumbar disc herniation.
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Objective To evaluate the feasibility, accuracy and its clinical value of MRI-guided needle biopsy of lung lesions. Methods A total of 137 patients with pulmonary nodules or masses underwent lung biopsy in low-field open MRI equipped with iPath 200 optical tracking systems. Among them, 103 cases had solitary pulmonary lesion; the other 34 cases had multiple foci. The maximum diameter of the lesion was not smaller than 3.5 cm ( ≥ 3.5 cm) in 57 patients, between 1.5 cm and 3.4 cm( 1.5-3.4 cm) in 71 patients, not greater than 1.4 cm ( ≤ 1.4 cm) in 9 patients. Results The puncture success rate was 100.0% (57/57) for lesions ≥3.5 cm, 98.6% (70/71) for lesions 1.5-3.4 cm,77.8% (7/9) for lesions ≤1.4 cm and 97. 8% (134/137) for total cases, respectively. According to the pathological results, pulmonary lesions were malignant in 98 cases and benign in 39 cases. The sensitivity,specificity, accuracy, positive predictive value and negative predictive value of MRI-guided lung biopsy were 94.2%(98/104), 100.0% (33/33), 95.6% (131/137), 100.0% (98/98) and 84.6% (33/39),respectively. Conclusion MRI-guided needle biopsy of lung lesion can be performed precisely in a lowfield open MRI with a low risk of complications. As a supplement to US or CT-guided biopsy, it is worth further promotion and application.