Objective To explore the effects and possible mechanism of calcium-sensing receptor(CaSR) in rat myocardial H9c2 cells hypertrophy model using angiotensin Ⅱ (Ang Ⅱ).Methods Cardiac hypertrophy model was established by treating cultured H9c2 cells with Ang Ⅱ in vitro.Hypertrophic H9c2 cells were treated with gadolinium chloride (GdCl3,a specific agonist of CaSR) and/or with Calhex231 (a specific inhibitor of CaSR) and 3-methyladenine (3-MA,a specific inhibitor of autophagy) to divided into 5 groups (six in each group):control,Ang Ⅱ,GdCl3 + Ang lⅡ,GdCl3 + Calhex231 + Ang Ⅱ,GdCl3 + 3-MA + Ang Ⅱ groups.To evaluate the status of H9c2 cells hypertrophy,protein content was determined through a coomassie brilliant blue protein kit and the expression of Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) and the phosphorylation form (pCaMK Ⅱ/CaMK Ⅱ) was analyzed by Western blotting.The protein expression of CaSR,autophagy maker [Beclin-1,micmtubule-associated protein 1 light chain 3(LC3)Ⅱ/LC3 Ⅰ,P62] and Ca2+/calmodulin-dependent-protein kinase-kinase-β (CaMKKβ)-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway was analyzed by Western blotting.Results ①GdCl3 further increased H9c2 cells protein content [control group:(2.52 ± 0.84) g/L,Ang Ⅱ group:(8.72 ± 3.60) g/L GdCl3 + Ang Ⅱ group:(14.17 ± 4.49) g/L,all P ＜ 0.05] and the expression of CaSR (control group:0.22 ± 0.04,Ang Ⅱ group:0.43 ± 0.02,GdCl3 + Ang Ⅱ group:0.63 ± 0.08,all P ＜ 0.05) and pCaMK Ⅱ/CaMKⅡ (control group:0.25 ± 0.05,AngⅡ group:0.51 ± 0.03,GdCl3 + AngⅡ group:0.77 ± 0.06,all P＜ 0.05) induced by Ang Ⅱ.Calhex231 suppressed the increasing of hypertrophy indicators induced by GdCl3 [GdCl3 + Calhex231 + AngⅡ group,CaSR:0.41 ± 0.16,protein content:(9.92 ± 2.54) g/L,pCaMK Ⅱ/CaMKⅡ:0.58 ± 0.08,all P ＜ 0.05].②GdCl3 promoted the effect of Ang Ⅱ in regulation of autophagy such as Beclin-1 protein increased (control group:0.31 ± 0.06,AngⅡ group:0.55 ± 0.09,GdCl3 + AngⅡ group:0.74 ± 0.08,all P ＜ 0.05),LC3 Ⅱ/LC3 Ⅰ increased (control group:0.28 ± 0.06,Ang Ⅱ group:0.56 ± 0.10,GdCl3 + Ang Ⅱ group:1.00 ± 0.15,all P ＜ 0.05) and P62 protein decreased (control group:0.54 ± 0.03,AngⅡ group:0.34 ± 0.02,GdCl3 + AngⅡ group:0.15 ± 0.03,all P ＜ 0.05).Moreover,Calhex231 suppressed autophagy induced by GdCl3 (GdCl3 + Calhex231 + Ang Ⅱ group,Beclin-1:0.53 ± 0.14,LC3 Ⅱ/LC3 Ⅰ:0.57 ± 0.12,P62:0.28 ± 0.05,all P ＜ 0.05).③GdCl3 increased pCaMKKβ/CaMKKβ (control group:0.43 ± 0.09,AngⅡ group:0.76 ± 0.12,GdCl3 + AngⅡ group:1.19 ± 0.21,all P ＜ 0.05),pAMPK/AMPK (control group:0.38 ± 0.11,AngⅡ group:0.68 ± 0.08,GdCl3 + AngⅡ group:1.18 ± 0.08,all P ＜ 0.05) and decreased pmTOR/mTOR (control group:0.90 ± 0.10,Ang Ⅱ group:0.54 ± 0.04,GdCl3 + AngⅡ group:0.29 ± 0.09,all P ＜ 0.05).Furthermore,Calhex231 blocked the effect of GdCl3 on the above-mentioned proteins changes (GdCl3 + Calhex231 + Ang Ⅱ group,pCaMKKβ/CaMKKβ:0.75 ± 0.06,pAMPK/AMPK:0.57 ± 0.05,pmTOR/mTOR:0.51 ± 0.08,all P ＜ 0.05).Conclusion Inhibiting calcium-sensing receptor expression has reversed H9c2 cell hypertrophy induced by Ang Ⅱ,which may be related to suppressing autophagy and suppressing CaMKKβ-AMPK-mTOR pathway.

Head ; surgery ; Humans ; Neck ; surgery ; United States

OBJECTIVEThe objective of this study was to compare the biodistribution and PET imaging of (11)C-PDT and (18)F-FDG in a mouse model of lung adenocarcinoma, and to evaluate the value of (11)C-PDT as a new tracer for PET imaging of lung cancer.

METHODSTwenty four lung adenocarcinoma-bearing mice were randomly divided into two groups, 12 each. The mice received (11)C-PDT or (18)F-FDG injection i.v. respectively. The biodistribution of (11)C-PDT or (18)F-FDG in the mice was measured with a well-gamma detector at 60 min after injection. The PET imagings of mice were performed using either of the two tracers.

RESULTSConsiderable uptake of the both radioactive tracers in the tumors was observed. The tumor uptake of (11)C-PDT [(0.65 +/- 0.20)%ID/g] was significantly lower than that of (18)F-FDG [(7.44 +/- 1.56)%ID/g, P < 0.01]. In the (11)C-PDT group, the highest uptake was observed in the liver, kidney and blood in a successively declining order, while the highest uptake of (18)F-FDG was seen in a order of heart, tumor and kidneys. The tumor/muscle ratio of (11)C-PDT uptake was relatively high (2.02 +/- 0.56), but still lower than that of (18)F-FDG (2.95 +/- 0.49, P < 0.01). All values of other tumor/organ ratios (T/NT) of (11)C-PDT uptake were < 2. High radioactive uptake was showed in the tumor and abdominal organs on PET images in the tumor-bearing mice injected with (11)C-PDT, and (18)F-FDG uptake was showed in the heart, tumor and abdominal organs. The tumor PET images with (11)C-PDT and (18)F-FDG were all clear.

CONCLUSIONThe uptake of (11)C-PDT in lung cancer is higher than that in muscle tissues, and pulmonary cancers can be detected by PET imaging. (11)C-PDT may be a promising PET tracer for lung cancers.

Adenocarcinoma ; diagnostic imaging ; metabolism ; pathology ; Animals ; Carbon Radioisotopes ; pharmacokinetics ; Cell Line, Tumor ; Fluorodeoxyglucose F18 ; pharmacokinetics ; Kidney ; diagnostic imaging ; metabolism ; Liver ; diagnostic imaging ; metabolism ; Lung Neoplasms ; diagnostic imaging ; metabolism ; pathology ; Mice ; Myocardium ; metabolism ; Podophyllotoxin ; pharmacokinetics ; Positron-Emission Tomography ; Tissue Distribution

OBJECTIVETo investigate the feasibility of repairing bone defect with methods of tissue-engineering and human bone morphogenetic protein-2 (hBMP-2) gene transfection in osteoporotic rats.

METHODSTwenty-four 6-month-old female Sprague-Dawley rats underwent ovariectomy, while 8 rats received sham-operations. Three months later, bone mesenchymal stem cells (BMSC) harvested from osteoporotic rats were divided into two groups randomly. Experimental group were transfected by recombinant plasmid carrying hBMP-2 gene, and control group left untreated. All BMSC were seeded into coralhydroxyapatite scaffolds. Then the cell/scaffold constructs were implanted into the defect site created in the ramus of mandible of osteoporotic rats respectively.

RESULTSPositive results were confirmed by immunohistochemistry and in situ hybridization in experimental group. New bone formation was found at the margin of the defect treated with the BMSC modified by hBMP-2 gene transfer at 4 weeks after implantation and appeared mature 8 weeks after the treatment. However, the amount of newly formed bone was much less and there was some adipose tissue at defect margins 8 weeks after implantation in control group.

CONCLUSIONSThe results of this experiment indicate that BMSC-mediated rhBMP-2 gene therapy in conjunction with bone tissue engineering may allow for successful treatment of large bone defects in osteoporosis rats.

Animals ; Bone Marrow Cells ; cytology ; Bone Morphogenetic Protein 2 ; genetics ; Female ; Genetic Therapy ; Humans ; Mandibular Diseases ; surgery ; Mesenchymal Stromal Cells ; cytology ; Osteogenesis ; physiology ; Osteoporosis, Postmenopausal ; therapy ; Rats ; Rats, Sprague-Dawley ; Tissue Engineering ; methods ; Transfection

Objective To study the values of clinicopathological features and expression of thyroid transcription factor 1 (TTF-1) in predicting the mutation status of epidermal growth factor receptor (EGFR) gene in patients with non-small cell lung cancer (NSCLC). Methods Mutation status of exons 18, 19, 20 and 21 in EGFR, and expression of TTF-1 protein in 283 cases of NSCLC diagnosed in Shanxi Provincial Cancer Hospital from January 2013 to December 2014 were analyzed by using amplification refractory mutation system (ARMS) and immunohistochemical method. The correlation of EGFR mutations with the clinicopathological features and TTF-1 expression were studied to explore the values of them in the prediction of EGFR mutations. Results Among 283 cases of NSCLC, the rate of EGFR gene mutation was 30.0 ％(85/283), including 3 cases with double mutations(exon 18 and exon 20 double mutations in one case, exon 19 and exon 21 double mutations in one case, exon 20 and exon 21 double mutations in one case). The EGFR gene mutations were associated with gender, histological type, history of smoking, and expression of TTF-1 (all P<0.001), but not related to age and tumor location (P= 0.785, P= 0.138). The combination of factors with high mutation rates (women, adenocarcinoma, no smoking, and TTF-1 positive) made the positive predictive value of EGFR mutations up to 57.6 ％. And the combination of factors with low mutation rates (male, nonadenocarcinoma, smoking history, TTF-1 negative) made the EGFR negative predictive value up to 90.3％. Conclusion The combination of clinicopathological features and TTF-1 expression status in patients with NSCLC has a great predictive value for EGFR mutations, which can provide a useful reference for clinical treatment decision-making.

AIM: To observe the clinical features of non-posterior retinal multiple-tear detachment and to explore the outcomes of vitreoctomy and scleral buckling in this type of retinal detachment.

METHODS: A retrospective clinical comparative study. Totally 40 eyes of 40 patients with retinal multiple-tear detachment were included in the study. According to surgical methods, the patients were divided into vitrectomy group(PPV group, group A, 18 eyes)and scleral buckling group(SB group, group B, 22 eyes). All patients were followed up for 3 to 6mo to observe the postoperative outcomes of the two groups.

RESULTS: At the end of follow-up, the rate of retinal reattachment in the A group was 100%(18/18). Retinal reattachment rate after removal of silicone oil-filled eyes was 56%(10/18).The rate of complete retinal reattachment in the B group was 86%(19/22), respectively. The difference was not statistically significant in the final retinal reattachment rate comparison(including silicone oil filled eyes)(P>0.05). There was a statistically significant difference in retinal reattachment rate after removal of silicone oil-filled eyes(P<0.05).

CONCLUSION: Non-posterior retinal multiple-tear detachment are mostly caused by extensive retinal degeneration or combined with vitreous traction. Vitrectomy should be chosen in complex cases, but multiple operations are required, while the long-term result of scleral buckling is stable. The choice between vitrectomy and scleral buckling needs comprehensive consideration, and it should be avoided to enlarge the indications for vitrectomy blindly. However, scleral buckling should be preferred if possible, for younger or special groups such as those with one eye.

OBJECTIVETo study the biological features and osteoblast/adipocyte phenotypes of bone marrow stromal cells (BMSCs) of Sprague-Dawley (SD) rats with Type I osteoporosis by induced culture.

METHODSSix-month-old SD rats were used in this study. 16 female rats were randomly divided into two groups. Eight rats were ovariectomied as experimental group to establish the modle of Type I osteoporosis, while other rats received sham-operation. Three month later BMSCs of 16 rats were isolated by discontinueous gradient centrifugation and then plated in alpha-MEM medium as primary culture. Secondary harvested cells were cultured for 14 days in alpha-MEM medium supplemented with dexamethasone, ascorbic acid, vitamin D3, beta-glycerophosphate or dexamethasone, 3-isobutyl-1-methylxanthine, insuline, and indomethine. The cells were screened by inverted microscope each day and cell growth was studied with cell counting. The osteoblast and adipocyte phenotypes were verified by cytochemistry staining, counted the percentage of positive stained cells.

RESULTSThe weight and bone mineral density of rats were statistically different between experimental group and control group. Gomori and Von Kossa's staining demonstrated positive osteoblast phenotypes of alkaline phosphatase and mineralized nods by osteogenic inducer, while Oil Red O staining identified BMSCs treated with adipogenic medium resulted in adipocyte formation and there was no significant difference in the percentage of positive stained cells between two groups.

CONCLUSIONThe model of Type I osteoporosis has been established successfully. BMSCs from SD rats with osteoporosis maintain their differentiation potential.

Adipocytes ; Animals ; Bone Density ; Bone Marrow Cells ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Female ; Mesenchymal Stromal Cells ; Osteoblasts ; Osteoporosis ; physiopathology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo isolate human adipose tissue-derived stromal cells and study the potential of osteogenic differentiation after inductive culture.

METHODSLiposuction human adipose tissues were minced and digested with collagenase type I. The obtained stromal cells were plated in BGJb medium as primary culture for ten days. The second passage cells were harvested and plated in DMEM/F12 medium supplemented with 10% FBS, 5% horse serum and 50 micromol/L hydrocortisone for myogenic induction culture. The cell-anchored slips were removed and fixed in 4% formaldehydam polymerisatum. Toluidine blue, Mallory's phosphotungstic hematoxylin staining and monoclonal antibody to human skeletal muscle myosin heavy chain immunocytochemical methods were used to assay the differentiation of cells.

RESULTSIt was observed that the size and shape of induced cells were much different from those of non-induced cells. Toluidine blue, Mallory's phosphotungstic hematoxylin staining demonstrated there were many basophilic striations within cytoplasm and multinucleated myotubes were formed. Immunocytochemical stain indicated that characterastic skeletal myosin heavy chain was positive in myogenic induced cells.

CONCLUSIONIt seems that human adipose tissue represents an abundant reservoir of adult stem cells that have multi-germline potential to differentiate into myoblasts. Adipose tissue derived stromal cells will be another alternative source for cell-based tissue engineering in skeletal muscle reconstruction.

Adipose Tissue ; cytology ; Adult Stem Cells ; cytology ; Cell Differentiation ; Cell Separation ; Cells, Cultured ; Culture Media ; Humans ; Myoblasts ; cytology ; Myosin Heavy Chains ; metabolism ; Stromal Cells ; cytology

The limitation of traditional Ad vectors result in wide application of capsid-incorporation of antigens into adenovirus capsid proteins, but usually it can't rescue virus successfully when we engineered the hypervariable regions (HVRs) of hexon in adenovirus serotype 3(Ad3) vector. So we deleted or retained some amino acids in HVR1, HVR2, HVR5, HVR7 predicted by bioinformatics, constructed recombinant Ad3 vector pBRAddeltaE3GFP-mHexon, and transfected it into AD293 cell to confirm the influence on the virus rescue. These data of amino acids that can be deleted or retained in the HVRs of Ad3 vector should provide operating foundation for antigen capsid-incorporation strategy in human adenovirus serotype 3, and also lay the groundwork for application of expressing foreign antigens in the hexon of human adenovirus serotype 3 as a platform of multivalent vaccine vectors.
Adenoviruses, Human ; genetics ; immunology ; Amino Acid Sequence ; Cell Line ; Computational Biology ; DNA, Recombinant ; genetics ; Genetic Engineering ; methods ; Genetic Vectors ; genetics ; Humans ; Models, Molecular ; Molecular Sequence Data ; Plasmids ; genetics ; Protein Conformation ; Species Specificity ; Viral Envelope Proteins ; chemistry ; genetics ; immunology

BACKGROUNDAmong various treatments preventing vein graft restenosis, external stent is receiving more and more attention. This study aimed to investigate the effect of non-restrictive external stent on the prevention of vein graft restenosis and the potential mechanisms of platelet-derived growth factor (PDGF) in the process of restenosis.

METHODSThirty-six "New Zealand white rabbits" were randomly divided into two groups, stented group (group S) and control group (non-stented group, group NS). Each rabbit underwent a reversed autologous external jugular vein into common carotid artery bypass grafting. In group S, the vein grafts were surrounded by a non restrictive stent which was 6 mm in diameter (a kind of Dacron vascular prosthesis); and in group NS, there was no stent to support the vein grafts. The grafts were harvested at the first week (1W), second week (2W) and fourth week (4W) after surgery respectively. The dimensions (including the thickness and area of the intima and media, luminal area) were measured by computer-aided image analysis system, and the intimal hyperplasia ratio was defined as the percentage of the area enclosed by the internal elastic lamina occupied by the intima.

RESULTSAt 1W, the difference of the thickness and area of the intima between groups S and NS was not significant (P > 0.05); at 2W and 4W, the thickness and area of the intima and the intimal hyperplasia ratio in group S were less significant than those in group NS (P < 0.05); from 1W to 4W, the thickness and area of the media in group S were smaller than those in group NS (P < 0.05). Immunocytochemistry staining of PDGF-B showed that the percentage of positive cells of intima in both two groups was peaked at 2W, and a significantly smaller percentage was detected in group S compared with that in group NS at 2W and 4W (P < 0.05); the percentage of PDGF-B positive cells of media in both two groups was also peaked at 2W, and that in group S was smaller than that in group NS from 1W to 4W (P < 0.05); and the percentage of PDGF-B positive cells of adventitia in group S was peaked at 4W, whereas the percentage of adventitia in group NS peaked at 2W, and the percentage of adventitia in group S was greater than in group NS at 4W (P < 0.05).

CONCLUSIONSNon-restrictive external stenting inhibits the hyperplasia of the intima and media of the vein grafts and reduces the thickness and area of the intima and media; Non-restrictive external stenting inhibits the synthesis of PDGF and changes its distribution, and then inhibits the hyperplasia of the intima.

Animals ; Female ; Graft Occlusion, Vascular ; prevention & control ; Image Processing, Computer-Assisted ; Immunohistochemistry ; Jugular Veins ; transplantation ; Male ; Models, Animal ; Platelet-Derived Growth Factor ; physiology ; Proto-Oncogene Proteins c-sis ; Rabbits ; Stents