OBJECTIVETo study the overexpression of Sox9 gene on rabbit bone marrow mesenchymal stem cells for repairing articular cartilage injury in vivo.

METHODSRabbit bone marrow mesenchymal stem cells (BMSCs) were transduced with lentivirus vector containing Sox9 gene and then cartilage specific molecule was detected by RT-PCR in vitro. Total 48 knee joints of 24 mature New Zealand white rabbits were randomly divided into 3 groups according to different defect treatment. After animals anesthesia,a full-thickness cylindrical cartilage defect of 4 mm diameter and 3 mm deep was created in the patellar groove using a stainlesssteel punch. Meanwhile, the transfected cells were implanted to repair the rabbit model with full-thickness cartilage defects. Cartilage defects tissue was observed with light microscope, electron microscope, HE and immunohistochemistry staining to assess the repair of defects by the complex at 6 weeks or 12 weeks after the implantation.

RESULTSAt 3 days after the transfection, Sox9 gene expression was highest and Sox9 gene expression decreased with the increase of time. At 3 days after the transfection, the expression of collagen type II began and reached the peak at 14 days. It showed that the bone marrow mesenchymal stem cells went into chondrogenic differentiation after transfected by Sox9 gene. Histological observation showed that at 6 weeks after the operation, the defects in the experimental group was filled with hyaline like cartilage tissue, 12 weeks after operation,the defects of cartilage and subchondral bone had satisfactory healing. Both at 6 and 12 weeks postoperatively, the defects were filled with fibrous tissues in control groups. Meanwhile, immunohistochemical staining of sections with type II collagen antibodies showed the proteins in the regenerated tissue stained positive for type II collagen and stronger than the control groups. The histological scoring system indicated that the cartilage repair of experiment groups were better than the two control groups with statistical significances.

CONCLUSIONOverexpression of Sox9 gene on rabbit bone marrow mesenchymal stem cells (BMSCs) promote the repair of cartilage defect.

Animals ; Bone Marrow Cells ; metabolism ; Bone Marrow Transplantation ; Cartilage, Articular ; injuries ; metabolism ; Cell- and Tissue-Based Therapy ; Female ; Genetic Vectors ; genetics ; metabolism ; Humans ; Lentivirus ; genetics ; metabolism ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; metabolism ; Osteoarthritis ; genetics ; metabolism ; therapy ; Rabbits ; SOX9 Transcription Factor ; genetics ; metabolism ; Tissue Engineering

OBJECTIVETo construct one lentiviral vector containing mouse SRY-related high mobility group-box gene 9 (SOX9) and transfect the murine bone mesenchymal stem cells (mBMSCs) in vitro and observe the expression of target gene.

METHODSRNA from the vectors containing mouse SOX9 gene were extracted and SOX9 genes were amplified by reverse transcription-Polymerase Chain Reaction (RT-PCR). The SOX9 genes were connected into lentiviral vectors pGC-FU. Then pGC-FU-SOX9 transduced into 293T cells to produce recombinant lentivirus called as Lenti-SOX9-EGFP. mBMSCs were transfected. The expression of target gene was detected by immunofluorescence, RT-PCR and Western Blot.

RESULTSLenti-SOX9-EGFP was recombined successfully and transduced efficiently into mBMSCs. The expression of SOX9 gene was confirmed by RT-PCR and Western Blot.

CONCLUSIONLentiviral vector of mouse SOX9 gene can transfect successfully into mBMSCs. Meanwhile, SOX9 gene may be expressed in mBMSCs. This will provide the target cells for the following study about SOX9 gene repairing cartilage injury.

Animals ; Female ; Gene Expression ; Genetic Therapy ; Genetic Vectors ; Lentivirus ; genetics ; Male ; Mesenchymal Stromal Cells ; metabolism ; Mice ; Osteoarthritis ; therapy ; Reverse Transcriptase Polymerase Chain Reaction ; SOX9 Transcription Factor ; genetics ; Transduction, Genetic ; Transfection

OBJECTIVETo evaluate the safety and efficacy between endoscopic papillary balloon dilatation (EPBD) and endoscopic sphincteropapillotomy ( EST) for common bile duct stones using meta-analysis method.

METHODSRandomizd controlled trials comparing EPBD with EST for common bile duct stones and published from January 1990 to July 2012 were recruited. This meta-analysis was conducted to estimate short-term and long-term complications. Fixed random effect model or random effect model was established to analyze the data.

RESULTSTwelve randomizd controlled trials were included in this analysis. These studies included 1865 patients, 925 of them were treated with EPBD and 940 were treated with EST. The analysis of basic characteristics of these included studies showed that: compared to EST, patients in the EPBD group were younger (OR = -1.16, 95% CI: -1.49 to -0.84, P = 0.00), while in two groups, there were no significant difference (P > 0.05) in gender proportion, average size of stones, number of gallstones, previous cholecystectomy, the number of merged duodenal diverticulum, common bile duct diameter, the total follow-up time. Also, compared to EST, the overall stone clearance in the EPBD group was lower (OR = 0.64, 95% CI: 0.42 to 0.96, P = 0.03), pancreatitis incidence was higher (OR = 2.67, 95% CI: 1.61 to 4.43, P = 0.00), incidence of bleeding (OR = 0.12, 95% CI: 0.04 to 0.34, P = 0.00), acute cholecystitis (OR= 0.39, 95% CI: 0.18 to 0.84, P = 0.02), total long-term complication rate (OR = 0.53, 95% CI: 0.36 to 0.77, P = 0.01), stone recurrence rate more than a year were lower (OR= 0.48, 95% CI: 0.26 to 0.90, P = 0.02). While in two groups, there were no significant difference (P > 0.05) in the stone removal on 1 '' attempt, the total near-term complications and acute cholangitis.

CONCLUSIONSOn the basis of lower rates of bleeding, EPBD seems to be preferred strategy over EST for endoscopic remove of common bile duct stones in patients who have coagulopathy. Although stone recurrence rate more than a year of EPBD is lower, but the overall stone clearance rate is lower and the risk of pancreatitis is higher than that of EST.

Dilatation ; Gallstones ; surgery ; Humans ; Postoperative Complications ; epidemiology ; Randomized Controlled Trials as Topic ; Sphincterotomy, Endoscopic ; Treatment Outcome

OBJECTIVETo investigate transient expression of fusion protein with a chimeric HBsAg-HSP70 construct in HepG2 cells.

METHODSEnkaryotic expression plasmids inserted HBsAg gene or chimeric HBsAg-HSP70 gene were prepared and transfected into HepG2 cells by means of cationic liposome. mRNA were detected by RT-PCR and proteins expressed in the cells were detected by immunocytochemistry 48 hours later. HBsAg in cultured supernatants and cell lysates were assayed by ELISA.

RESULTSFusion protein (HBsAg-HSP70) transient expression in HepG2 cells were confirmed by RT-PCR, immunocytochemistry or ELISA, but fusion protein was not assayed in cell cultured supernatants by ELISA.

CONCLUSIONSTransfection of HepG2 cells with a chimeric HBsAg-HSP70 construct leads to express fusion protein, but it does not secrete into cell cultured supernatants.

Bacterial Proteins ; Enzyme-Linked Immunosorbent Assay ; HSP70 Heat-Shock Proteins ; genetics ; Hepatitis B Surface Antigens ; genetics ; Hepatitis B Vaccines ; immunology ; Humans ; Immunohistochemistry ; Plasmids ; Recombinant Fusion Proteins ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Vaccines, DNA ; immunology

Objective To observe the anatomic and imaging morphology ofjugnlar bulb and its relationship with the surrounding structures, and to investigate the classification ofjugnlar bulb and its clinical significance. Methods We dissected 30 human temporal bones and studied multi-slice spiral CT imaging data of temporal bone of 120 cases and blood vessel cast mould specimen of the jugular bulb of 6 cases, to observe the morphology of jugnlar bulb and its spatial relationship with the surrounding structures. We made an imagined sagittal plane on the medial well of the tympanic cavity, with a horizontal tangent line of the proximal wall of the tympanic cavity and a vertical tangent line of the posterior wall of the tympanic cavity as coordinate axes (X axis and Y axis), respectively, so the 4 quadrants ( Ⅰ , Ⅱ, Ⅳ, Ⅳ) were formed. The jugular bulb was classified intro 4 types according to the quadrant where its top was projected and subtyped according to its position on the inner or outer side of the plane. The operation via mastoid approach was simulated on specimen to observe the effect of jugnlar bulb on the operation route. Results Some jugular bulbs were flat type and others were prominent types. The classification in the group of CT image: type Ⅰ , 11 case (9%);type Ⅱ, 63 cases (53%);type Ⅲ, 25 cases (21%);type Ⅳ, 21cases (17%). The classification in the group of specimen: type Ⅰ, 1 case (3%);type Ⅱ, 11 cases (37%);type Ⅲ, 8 cases (27%);type Ⅳ, 10 cases (33%). Each type of the jugular bulb had different effects on the operative approach. Conclusions The classification method with the 4 quadrants is a simple and three-dimensional way to describe the position of the jugular bulb for imaging diagnosis or operative scheme design.

Objective To examine the expression of tumor necrosis factor in placenta of pregnant rats with chronic periodontitis.Methods Twenty Wistar female rats were randomly divided into two groups,control(n=8)and experimentalgroup(n=12).The periodontitis model was established in the experimental group.The females and males in the two groups got together four weeks later.Nineteen days after pregnancy all rats were executed and placenta collected.The delivery time and neonatal birth weight were recorded and the pathological changes of periodontal tissue observed.Tumor necrosis factor(TNF)expression was examined in placenta by real-time quantitative polymerase chain reaction analysis.Results The animal model of chronic periodontitis was successfully established.Experimental group delivered 30 offspring and the control group 56 offspring.The average number of pups born alive per liner in experimental group(4.1±2.2)Was significantly lower than that in control group(9.2±2.2),P<0.05.The birth weight of pups in experimental group[(5.01±0.43)g]wag significantly lower than that in the control group[(5.79±0.53)g],P<0.05.The relative quantitative expression of TNF was(1.807±0.265)in experimental group the and (1.003±0.021)in the control group(P=0.001).Conclusions Chronic periodonfitis may be related to preterm low birth weight.

Objective:To study the overexpression of Sox9 gene on rabbit bone marrow mesenchymal stem cells for re?pairing articular cartilage injury in vivo. Methods:Rabbit bone marrow mesenchymal stem cells(BMSCs)were transduced with lentivirus vector containing Sox9 gene and then cartilage specific molecule was detected by RT-PCR in vitro. Total 48 knee joints of 24 mature New Zealand white rabbits were randomly divided into 3 groups according to different defect treat?ment. After animals anesthesia,a full thickness cylindrical cartilage defect of 4 mm diameter and 3 mm deep was created in the patellar groove using a stainlesssteel punch. Meanwhile,the transfected cells were implanted to repair the rabbit model with full thickness cartilage defects. Cartilage defects tissue was observed with light microscope,electron microscope,HE and im?munohistochemistry staining to assess the repair of defects by the complex at 6 weeks or 12 weeks after the implantation. Re?sults:At 3 days after the transfection,Sox9 gene expression was highest and Sox9 gene expression decreased with the increase of time. At 3 days after the transfection,the expression of collagen typeⅡbegan and reached the peak at 14 days. It showed that the bone marrow mesenchymal stem cells went into chondrogenic differentiation after transfected by Sox 9 gene. Histologi?cal observation showed that at 6 weeks after the operation,the defects in the experimental group was filled with hyaline like cartilage tissue,12 weeks after operation,the defects of cartilage and subchondral bone had satisfactory healing. Both at 6 and 12 weeks postoperatively,the defects were filled with fibrous tissues in control groups. Meanwhile,immunohistochemical stain?ing of sections with typeⅡcollagen antibodies showed the proteins in the regenerated tissue stained positive for typeⅡcolla?gen and stronger than the control groups. The histological scoring system indicated that the cartilage repair of experiment groups were better than the two control groups with statistical significances. Conclusion:Overexpression of Sox9 gene on rab? bit bone marrow mesenchymal stem cells(BMSCs)promote the repair of cartilage defect.

Acute spinal cord injury(ASCI)can be divided into primary injury and secondary injury.Spinal cord edema is important for the development of secondary injury after ASCI.Spinal cord edema can be mainly divided into cytotoxic edema and angioedema.The application of dehydrating agents in the treatment of acute spinal cord injury is obvious.This article de-scribed the application of mannitol,hypertonic saline,glycerol fructose,furosemide,human serum albumin,resveratrol and other dehydrating agents in the treatment of ASCI.

OBJECTIVETo study the role of dendritic cells (DCs) and macrophages, differentiated from the same individual peripheral blood monocytes, in tumor antigen- presenting.

METHODSDCs and macrophages were differentiated from human peripheral blood monocytes by adding both Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) or GM-CSF only. Then they were loaded with tumor antigen at different concentrations and cocultured with autologous T cells in 96-well flat-bottomed microtiter plates for five days at 37 degrees C, 5% CO(2). (3)H-thymine was added before the culture terminated, and twelve hours later, the cells were gathered to test the cpm value.

RESULTSBoth DCs and macrophages chased with tumor antigen could strongly stimulate the proliferation of autologous T cells, especially DCs. The stimulation effect with 20 microl/ml antigen was the most remarkable and the cmp values were 11,950.3 +/-1621.8, 8,708.5 +/-176.1, 402.5+/-43.1 in DCs group, Macrophages group, and lymphocytes group, respectively.

CONCLUSIONThe antigen presenting role of DCs is stronger than that of macrophages from the same individual.

Antigen Presentation ; immunology ; Antigen-Presenting Cells ; immunology ; physiology ; Antigens, Neoplasm ; immunology ; Carcinoma, Hepatocellular ; immunology ; Dendritic Cells ; immunology ; physiology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Liver Neoplasms ; immunology ; Macrophages ; immunology ; physiology ; Tumor Cells, Cultured

OBJECTIVETo determine whether human prostate cancer cell lines undergo epithelial-mesenchymal transition (EMT) and become more invasive when induced by HIF-1alpha, and to explore the underlying molecular mechanism.

METHODSThe cell line LNCaP, appropriate for the HIF-1alpha induction test, was screened out from 4 different EMT-negative prostate cell lines through vimentin gene detection by RT-PCR. The recombinant plasmid pCDNA3. 1(-)/HIF-1alpha was constructed and transfected into LNCaP with the Lipofectamine 2000 system. The control plasmid pCDNA3.1 (-) was transfected by the same method. The positive clone cells were selected by G418 and confirmed by Western blot and immunofluorescence staining. Then a Transwell polycarbonate filter, coated with 100 micol Matrigel at 1:20 dilution in the serum-free medium, was used to analyze the invasive potency. The expression of E-cadherin and vimentin was detected by Western blot.

RESULTSAmong the 4 different EMT-negative cell lines, LNCaP was the only one that expressed the vimentin gene but not protein. The expression of HIF1alpha was obviously higher in LNCaP/HIF1alpha than in LNCaP/pCDNA3. 1 (- an LNCaP. The number of the LNCaP/HIF1alpha cells that penetrated through the Transwell polycarbonate filter was significantly larger than that of the LNCaP and LNCaP/pCDNA3. 1(-) cells. Compared with the LNCaP/pCDNA3.1(-) and LNCaP cells, the expression of vimentin was up-regulated, while that of E-cadherin down-regulated, in LNCaP/HIF1alpha.

CONCLUSIONThe over-expression of HIF-1alpha could induce EMT in the human prostate carcinoma cell line LNCaP and enhance its invasiveness through E-cadherin and vimentin regulation.

Cadherins ; biosynthesis ; Cell Line, Tumor ; metabolism ; pathology ; Epithelium ; pathology ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Male ; Neoplasm Invasiveness ; Prostatic Neoplasms ; metabolism ; pathology ; Reverse Transcriptase Polymerase Chain Reaction ; Vimentin ; biosynthesis