ObjectiveTo explore the role of BTBD10 overexpression in the proliferation of insulinoma cell line INS-1and its mechanism. MethodsThe recombined expression plasmid of pcDNA4.0-BTBD10 was constructed by gene cloning technique and was transfected into INS-1 cell by lipofectamine 2000. The stable overexpression BTBD10 of INS-1cell was selected at 48th hour after transfection.INS-1 cell proliferation activity was measured by MTT method.The expression of BTBD10,protein kinase B(Akt),phospho-Akt(p-Akt),mammal target of rapamycin (mTOR) and phospho-mTOR (p-mTOR) were determined by Western blot.ResultsThe stable overexpression BTBD10 of INS-1 cell wassuccessfullyconstructed.OverproductionofBTBD10promotedbetacellproliferation.The phosphorylation of Akt and mTOR was increased and the ratio of p-Akt/Akt and p-mTOR/mTOR was enhanced in the INS-1 overexpressed by BTBD10.But the expression of total Akt and mTOR presented no obvious changes. Conclusion The overexpression BTBD10 of INS-1 cell could activate of Akt/mTOR signalling pathway via stimulating phospho-mTOR and Akt,and enhance overall cell protein translation,so as to promote proliferation of INS-1 cell.

BACKGROUND: Pathophysiological mechanism of local microenvironment is complex after central nerve injury; especially,both inflammatory reaction at an acute phase and formation of secondary glial scar have tremendous effects on effective regeneration of axon, regeneration and arrangement of local nerve cells, proliferation and migration of local stem cells;therefore, it becomes a basic reason for blocking nerve repair in an early period. Thus, how to effectively resist inflammatory factors in injured region at an acute phase and how to optimize microenvironment of neural regeneration are the most important strategies for repairing spinal cord injury in recent years.OBJECTIVE: To design, establish and screen the best expression of interleukin-6 receptor (IL-6R) α to inhibit shRNA adenovirus expression vector by using spinal cord injury models.DESIGN: Duplicative measurement study.SETTING: Department of Spine Surgery, the Second People's Hospital of Shenzhen.MATERIALS: A total of 40 healthy Wistar rats, either gender, 8-10 weeks old, were selected in this study. Rabbit-anti-rat glial fibrillary acidic protein (GFAP) antibody Ⅰ was provided by Santa Cruz Compan; siRNA eukaryon expression plasmid pGenesil (cohtaining green fluorescent expression system) was provided by Wuhan Jingsai Bioengineering Company.METHODS: The experiment was carried out in ImmuneOpening Laboratory, Basic Medical Faulty, Tongji Medical College, Huazhong University of Science and Technology, and Medica Laboratory Center, the Second People's Hospital of Shenzhen in November 2006. Three pairs of shRNA template which composed of 19 bp reverse repeated motif of IL-6 receptor (IL-6R) α target sequence with 9 bp spacer were designed and synthesized, then the recombinant adenovirus expression vectors with green fluorescence protein were constructed in vitro respectively. The acute spinal cord injury models were completed, and the adenovirus recombinants were regionally injected post 12 hours after spinal cord injury;in addition, the inhibitory effect of RNA interference (RNAi) on expression of IL-6R in local region after spinal cord injury were detected by using real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot so as to screen adenovirus expression vector which had the best inhibitory effect on expression of IL-6R.MAIN OUTCOME MEASURES: Inhibitory effect of RNAi on expressions of IL-6R RNA and protein in local region after spinal cord injury.RESULTS: Sequence analysis showed that IL-6R-shRNA recombinant adenovirus expression vector was successfully constructed, and optimal IL-6R-shRNA recombinant adenovirus expression vector was screened by using real-time fluorescence quantitative RT-PCR and Western blot. The IL-6R expressions were 49% and 56% at the levels of mRNA and protein, respectively.CONCLUSION: The IL-6R--shRNA recombinant adenovirus expression vector is successfully constructed and screened.The gene expression of IL-6R can be highly inhibited after acute spinal cord injury.

BACKGROUND: Recently biodegradable hydrogel has been extensively used to delivery anticancer drug and bioactive macromolecule. However, to protect the activity of the bioactive macromolecule, we need to obtain series of hydrogel which have milder hydrogelation conditions and shorter hydroglation time.OBJECTIVE: To prepare enantiomeric poly(L-Lactic acid) (PLLA)-poly(ethylene glycol (PEG)-PLLA/ poly(D-Lactic acid) (PDLA)-PEG-PDLA stereocomplex hydrogel which has shorter hydroglation time, to physically encapsulate a model drug-lysozyme and sustained release it from the hydrogel. METHODS: Triblock copolymers of PLLA-PEG-PLLA and PDLA-PEG-PDLA were synthesized by ring-opening polymerization of L(D)-lactide using PEG as the initiator and Sn(Oct)2 as the catalyst. The triblock copolymers were characterized by 1H nuclear magnetic resonance, FT-IR and X-Ray diffractometry. A hydrogel was prepared from an aqueous mixture of PLLA20-PEG227-PLLA20 and PDLA21-PEG227-PDLA21 (10 wt% concentration) at room temperature for 12 hours. X-Ray diffractometry test was used to research the gelation mechanism. The release profile of the lysozyme as a model drug from the hydrogel was tested. The morphology of the freeze-dried hydrogel was investigated by scanning electron microscope. The cytotoxicity of the hydrogel was evaluated by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide) assay.RESULTS AND CONCLUSION: Triblock copolymers of PLLA-PEG-PLLA and PDLA-PEG-PDLA were obtained. Both the PEG and PLA blocks in the copolymers could crystallize, but the crystallization of the PEG block was predominant. The stereocomplex formation between the PLLA and PDLA blocks within the hydrogel was confirmed by the X-Ray diffractometry analysis. The release profile of the lysozyme from the hydrogel exhibited a sustained-release pattern with a duration period of 7 days. The hydrogel exhibited a 3D interconnected porous structure with 50-100 μm pore size after being freeze-dried. The mouse fibroblast cell viability percentage was 99.3% after the cells contacted with the 100% extracted liquid for 72 hours.

Objective To identify histopathologic correlates for the various MRI appearances of breast fibroadenomas.Methods Thirty-eight fibroadenomas in 33 patients(aged 24—57 years)examined with gadolonium-enhanced MR imaging were observed for signal intensity on T_2-weighted images,contrast enhancement,shape,and internal septation,and these findings were correlated with histopathologic findings.All cases underwent surgery and were proved by pathology.Results(1)The lesion shape was lobular,or round in 34 of 38 fibroadenomas(89.5%).(2)The signal intensity on T_1-weighted images was less than or equal to that of fibroglandular tissue in all cases.The signal intensity on T_2-weighted images was highly varible:high T_2 signal intensity was associated with more myxomatous stromal(mean myxoid-sclerotic index value of 1.9),higher stromal cellularity(mean stromal cellularity index value of 2.2); Fibroadenomas with low T_2 signal intensity had stromal that was nearly uniformly sclerotic(mean myxoid- sclerotic index values of 2.8)and low stromal cellularity(mean stromal-cellularity index value of 1.2). Significant differences were found between these two groups,x~2=11.267 and x~2=10.415(P0.05).The degree of contrast enhancement was proved to be related to ages of patients.The enhancement was more intensely in younger patients.(5)Internal septations were identified within nine of 33 enhancing fibroadenomas (27.3%)and appeared to correlated with collagenous bands at histopatholigic analysis.Conclusions Fibroadenomas demonstrate marked histopathologic variability.The resultant variability in the MR appearance correlated with the degree of myxomatous or sclerotic and stromal cellularity.Lobulation and internal septation,which appear to reflect intrinsic growth patterns of fibroadenomas,may provide more reliable information for distinction.Familiarity with the diagnostic features would facilitate to make the differential diagnosis correctly.

Objective To discuss the feasibility and short-term clinical effectiveness of DSA-guided percutaneous vertebroplasty (PVP) for the treatment of painful osteoblastic metastatic spinal lesions. Methods During the period from Jan. 2010 to Dec. 2011 at authors’ hospital PVP was carried out in 23 patients with osteoblastic spinal metastases (34 lesions in total). Coexisting osteoblastic pathological fracture was found in twelve patients. The WHO standards, visual analogue scale (VAS) and karnofsky-KPS score were used to evaluate the therapeutic results. Results Technical success was achieved in all patients. All patients were followed up for at least 3 months. Of 20 patients who had complete clinical data, complete remission (CR) was obtained in 6, partial remission (PR) in 10, mild remission (MR) in 3 and no remission (NR) in one. The clinical effectiveness (CR+PR) was 80%. The mean VAS scores dropped from preoperative (7.0 ± 1.6) to (2.2 ± 1.9) at 24 hours after the treatment, and to (2.4 ± 2.1) and (2.5 ± 2.1) at one and three months after the treatment respectively. The mean KPS scores rose from preoperative (76.5 ± 10.4) to (86.5 ± 11.8), (88.0 ± 12.0) and (89.0 ± 10.8) at 24 hours and one, three months after the treatment respectively. Small amount leakage of PMMA was observed in 4 cases (17.4%) with no obvious clinical symptoms. Conclusion DSA-guided PVP is a feasible and effective treatment for painful osteoblastic spinal metastases. This therapy can immediately relieve pain and reinforce spine, besides, it can remarkably improve the living quality and
decrease the incidence of paraplegia.

Objective To evaluate the metabolic characteristics of insulin secretion and insulin sensitivity in isolated postchallenge hyperglycemia(IPH) and to clarify the factors responsible for the development of IPH. Methods(Eight hundred) and fifty subjects were classified into the following three groups based on the results of a 75-g oral glucose tolerance test(OGTT): normal glucose tolerance(NGT),n=557;isolated impaired glucose tolerance(iIGT),n=146;and IPH,n=147.Insulin secretion(insulinogenic index) and insulin sensitivity(insulin sensitivity index) were identified in the three groups. Results From NGT to iIGT and IPH in these subjects,the insulinogenic index and insulin sensitivity index were gradually decreased(P

Objective To study the effect of amylin on the secretion of glucagon and insulin in isolated rat islets. Methods The models of isolated rat islets were established by collagenase digestion and dextran gradient centrifugation. The influence of various concentrations of amylin on both glucagon and insulin secretion was studied. Results Various concentrations of glucose increased the insulin secretion and decreased the glucagon secretion.Compared with the control, islets exposed to amylin (10~ -10 to 10~ -5 mol/L) for 1 hour showed decreasing glucagon secretion as the glucose increased (0, 5.6 and 11.2 mmol/L) (P

Human tumstatin(hTumstatin)cDNA was amplified from recombinant plasmid pET-3c-tum, cloned in frame with the signal sequence in yeast vector pPICZalphaA and transformed into Pichia pastoris GS115 by electroporation. The expression of hTumstatin in GS115(pPICZalpha-tum)was then induced by methanol and secreted into the culture medium, with a yield of 25mg/L as shown by SDS-PAGE and Western blotting. The expressed hTumstatin was purified to more than 85% purity using a simple one-step SP-Sepharose cation exchange chromatography. The MTT and chick chorioallantoic membrane assay showed that the yeast produced hTumstatin could inhibit the proliferation of human umbilical vein endothelial cells and the neovascularization induced by bFGF. Hoechst 33258 fluorescent staining also demonstrated the apoptotic change in endothelial cellular nuclear morphology.
Angiogenesis Inhibitors ; metabolism ; Autoantigens ; genetics ; metabolism ; Cell Proliferation ; Cells, Cultured ; Collagen Type IV ; genetics ; metabolism ; DNA, Complementary ; genetics ; Electroporation ; Endothelial Cells ; cytology ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Umbilical Cord ; cytology

OBJECTIVETo investigate the effect of tumor necrosis factor alpha (TNFalpha) on radiosensitivity of nasopharyngeal carcinoma (NPC) in relation to TNFalpha-induced cell cycle synchronization.

METHODSThe radio-resistance of a NPC cell line subclone CNE-2Z-S1 was verified by in vivo experiments and flow cytometry was performed to evaluate cell cycle synchronization in TNFalpha-treated CNE-2Z-S1 cells. The radiosensitivity of the cell synchronized CNE-2Z-S1 cells was determined by clone formation in vitro and in vivo experiment in nude mice.

RESULTSTNFalpha was capable of inducing cell cycle arrest and synchronization of CNE-2Z-S1 cells. Pretreatment with TNFalpha remarkably enhanced the radiosensitivity of CNE-2Z-S1 in vitro, and in vivo experiments with nude mice also suggested the role of TNFalpha in enhancing the radiosensitivity of NPC.

CONCLUSIONTNFalpha can enhance the radiosensitivity of NPC cells by inducing cell cycle synchronization.

Animals ; Cell Cycle ; drug effects ; radiation effects ; Cell Line, Tumor ; Humans ; Mice ; Mice, Nude ; Nasopharyngeal Neoplasms ; drug therapy ; pathology ; radiotherapy ; Radiation-Sensitizing Agents ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate the effects and mechanisms of panaxoside Rg1 on the new vessel formation in acute myocardial infarction (AMI) rats.

METHODSThe AMI model of male Sprague-Dawley (SD) rats was established, and rats were randomly divided into the AMI model group, the treatment group of panaxoside Rg1, the placebo group and the treatment group of panaxoside Rg1 plus rapamycin. Cardiac creatases were determined with 1 mL blood drawn from vena caudalis of the rats 48 h after the model was successfully made. After 4 weeks, Evans blue was injected into the aorta roots of the rats, and then, red tetrazoline was dyed again and the myocardial infarction area was evaluated. The microvessel density (MVD) of infarction area was determined by the immunohistochemistry of CD31; enzyme-linked immunosorbent assay (ELISA) was used to detect the protein content of CD31 and hypoxia inducible factor-1alpha (HIF-1alpha) of the infarction area.

RESULTSThe MVD in the infarction area and the contents of CD31 and HIF-1alpha in the Rg1 treatment group were higher than those in the AMI model group significantly (P<0.05). The cardiac creatase and infarction area were lower in the Rg1 treatment group than those in the AMI model group significantly (P<0.05). The above effects, however, disappeared when rapamycin, the antagonist of mammalian target of rapamycin (mTOR), was administered simultaneously.

CONCLUSIONSPanaxoside Rg1 could increase the expression of HIF-1alpha and CD31 of myocardium and stimulate the angiogenesis. The above mentioned role of panaxoside Rg1 might be related to the excitation of mTOR receptor.

Animals ; Cell Count ; Collateral Circulation ; drug effects ; Drug Evaluation, Preclinical ; Ginsenosides ; administration & dosage ; pharmacology ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Intracellular Signaling Peptides and Proteins ; metabolism ; physiology ; Male ; Microvessels ; pathology ; Myocardial Infarction ; drug therapy ; metabolism ; pathology ; Neovascularization, Physiologic ; drug effects ; Placebos ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Protein-Serine-Threonine Kinases ; metabolism ; physiology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Sirolimus ; administration & dosage ; pharmacology ; TOR Serine-Threonine Kinases