BACKGROUND: Bone morphogenetic proteins (BMPs) are involved in the formation of various tissues including bone, cartilage, tendon, and ligament. Vascular endothelial growth factor (VEGF) promotes angiogenesis by increasing the permeability and migration of endothelial cells.OBJECTIVE: To construct a co-expressing vector of human bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor 165 (VEGF165), and observe the expression of BMP2 and VEGF165 in human bone marrow mesenchymal stem cells (hBMSCs).DESIGN: Observation control trail.SETTING: Department of Plastic Surgery, Affiliated Hospital of Luzhou Medical College and Plastic Surgery Hospital of Peking Union Medical College, Chinese Academy of Medical Sciences.MATERIALS: The MSCs derived from the healthy adult volunteers of marrow donors. pIRES-EGFP-hVEGF165 containing total length of cDNA sequence of human VEGF165 gene was provided by Dr. Cheng Ting from Plastic Surgery Hospital of Peking Union Medical College. pSP65-hBMP2 containing total length of cDNA sequence of human BMP2 gene was provided by Dr. Guo Ximin from the Academy of Military Medical Sciences. Enkaryotic expression vector pIRESneo (Clontech Company) Pyrobest DNA Polymerae, restriction enzyme, DNA ligase and plasmid extraction kit, DNA Fragment Purification Kit (TaKaRa Company), LiorfectamineTM liposome transfection kit, DMEM medium, trypase, TRIzoIRNA extraction kit (Gibco BRL), Omniscript RT kit (Qiagen), TaqplusDNA polymerase (Promega), PMSF, leupeptin, aprotinin, chymostatin (Sigma), protease inhibitor, PVDF membrane (Amersham-Pharmacia Biotech), rabbit anti-human BMP2 antibody and VEGF monoclonal antibody (Santa Cruz Company), goat anti-rabbit lgG-peroxydase (Wuhan Boster), G418 (Ameresco Company in U.S).METHODS: The experiment was conducted in the Affiliated Hospital of Luzhou Medical College and the Plastic Surgery Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences between June 2005 and April 2006.hBMP2 and hVEGF165 cDNA were directional cloned into multiple clone sites of the eukaryotic expression vector pIRESneo. The recombinant plasmid was identified by restrictive enzyme Xho Ⅰ/Bgl Ⅱ digestion analysis and DNA sequencing. Liposome-mediated gene transfer method was used to transfect hBMSCs. For observation, the transfected cells were divided into IRES-hBMP2-VEGF165 group, pIRES-hBMP2 group, pIRES-VEGF165 group and empty vector group, which were transfected with pIRES-hBMP2-VEGF165, pIRES-hBMP2, pIRES-VEGF165 and pIRES-neo. Meanwhile, the same number nontransfected cells were selected as blank control group. The reverse transcription polymerase chain reaction (RT-PCR) and Weszern blot were employed to observe the expression and secretion of hBMP2 and hVEGF165 gene and protein.expression vector hBMP2 and hVEGF165 gene sequence were the same as reported after restrictive enzyme EcoRI and Bgl Ⅱ digestion analysis and pIRES-BMP2 gene sequencing, which showed that the recombinant plasmid pIRES-hBMP2-VEGF165 highly expressed hBMP2-mRNA and VEGF165-mRNA, but the non-transfected or transfected with pIRES-hBMP2-VEGF165 or pIRES-hBMP2 secreted a great quantity of hBMP2, but that non-transfected or transfected with pIRES-VEGF165 or empty vector secreted only little.CONCLUSION: The co-expressing vector of hBMP2 and hVEGF165 can be expressed stably in hBMSCs.

Phosphoinositide 3-kinase/serine-threonine kinase (PI3K/AKT)has been found playing an important role in the pathogenesis of severe acute pancreatitis (SAP)in recent years,but the underlying mechanism has not been clarified.Aims:To investigate the role of PI3K/AKT in regulating the inflammatory response in SAP by evaluating the effect of insulin-like growth factor-Ⅰ (IGF-Ⅰ)and wortmannin,the agonist and inhibitor of PI3K/AKT on Toll-like receptor 4 (TLR4)signaling pathway in macrophage cell line RAW264.7.Methods:RAW264.7 cells were treated with different concentrations of lipopolysaccharide (LPS ), IGF-Ⅰ and wortmannin, respectively, and cell viability was determined by CCK-8 assay.RAW264.7 cells were divided into blank control group (no treatment),LPS group (LPS 1 μg/mL),IGF-Ⅰ group (IGF-Ⅰ 100 ng/mL +LPS 1 μg/mL),wortmannin group (wortmannin 100 nmol/L +LPS 1 μg/mL)and IGF-Ⅰ +wortmannin group (wortmannin 100 nmol/L +IGF-Ⅰ 100 ng/mL +LPS 1 μg/mL).Protein expressions of tumor necrosis factor-α(TNF-α)and interleukin-6 (IL-6)were detected by ELISA;mRNA expressions of TLR4,myeloid differentiation factor 88 (MyD88),AKT,PI3K,p38 mitogen-activated protein kinase (p38MAPK)and nuclear factor-κB (NF-κB)were determined by real-time PCR.Results:After treated with LPS,IGF-Ⅰand wortmannin, respectively,no differences in cell viability of RAW264.7 cells were found between different concentrations groups (P>0.05).Protein expressions of TNF-αand IL-6 in LPS,IGF-Ⅰ,wortmannin and IGF-Ⅰ +wortmannin groups were significantly higher than those in blank control group (P<0.05 ).Protein expressions of TNF-αand IL-6 in wortmannin group were significantly lower than those in LPS and IGF-Ⅰ groups (P<0.05),and those in IGF-Ⅰ+wortmannin group were significantly lower than those in IGF-Ⅰ group (P<0.05).In LPS group,mRNA expressions of AKT and PI3K as well as TLR4 and its downstream molecules MyD88,p38MAPK and NF-κB were significantly higher than those in blank control group (P <0.05 ).Expressions of all above-mentioned mRNAs in IGF-Ⅰ group were further increased and significantly higher than those in LPS group (P<0.05).Expressions of all above-mentioned mRNAs in wortmannin group were significantly lower than those in LPS and IGF-Ⅰ groups (P<0.05 ),and those in IGF-Ⅰ+wortmannin group were significantly higher than those in wortmannin group (P<0.05),but significantly lower than those in IGF-Ⅰ group (P<0.05).Conclusions:PI3K/AKT might regulate TLR4 signaling pathway and its downstream molecules in macrophages, thereby affects the expressions of inflammatory cytokines and being involved in the pathogenesis of inflammatory response in SAP.

Objective To observe the change of serum smooth muscle myosin heavy chain (smMHC) level in the patients with aortic dissection (AD),and evaluate the effect of smMHC in the early diagnosis and prognosis of AD.Methods Forty-two patients with AD were selected as AD group,30 healthy subjects were selected as control group.Blood samples were collected at four time periods (within 3 h of onset,6 h,12 h,24 h),and serum smMHC level were measured by enzyme-linked immunosorbent assay.Results Serum smMHC level of AD group,which collected (within 3 h of onset,6 h,12 h) were significantly higher than that of control group [(88.6 ±21.7),(59.4 ± 18.7),(41.3 ± 10.7) ng/L vs.(17.2 ± 8.3) ng/L,P ＜ 0.01].There was no significant difference between the serum smMHC level of AD group and control group at 24 h after onset [(18.9 ±9.5) ng/L vs.(17.2 ±8.3) ng/L,P ＞ 0.05].Serum smMHC level of Stanford A type group (25 cases) was higher than that of Stanford B type group (17 cases) within 3 h of onset [(95.4 ± 17.8) ng/L vs.(78.5 ± 18.3) ng/L,P＜0.01],and there was no significant difference bewteen the two groups which collected at 6,12 h and 24 h after onset (P ＞ 0.05).Preoperative serum smMHC level was significantly higher than that after intracavitary isolation operation [(58.6 ± 15.9) ng/L vs.(30.1 ± 12.5) ng/L,P ＜ 0.01].Serum smMHC level decreased rapidly after the operation,and there was no significant difference between the two grougs when 12 h after operation [(18.7 ± 8.9) ng/L vs.(17.2 ± 8.3) ng/L,P ＞ 0.05].The serum smMHC level of the deaths (7 cases),which collected within 3 h of onset,6 h,12 h,was significantly higher than that of the survivors (35 cases) [(101.2 ± 20.7) ng/L vs.(86.1 ± 18.9) ng/L,(65.2 ± 16.7) ng/L vs.(58.2 ± 14.2) ng/L,(50.4 ± 10.8) ng/L vs.(39.5 ± 8.3) ng/L,P ＜ 0.05],and there was no significant difference at 24 h after onset (P ＞ 0.05).Detecting serum smMHC level within 3 h of onset,the area under the receiver operating characteristic curve was 0.913,with 51.7 ng/L as a diagnostic critical value,sensitivity and specificity respectively was 88.1％ (37/42) and 96.7％ (29/30).When detecting at 6 h after onset,the area under the curve was 0.865,with 38.5 ng/L as a diagnostic critical value,sensitivity and specificity respectively was 90.4％(38/42) and 90.0％ (27/30).Conclusions The level of serum smMHC in patients with AD increase rapidly after onset,and detecting serum smMHC level within 6 h of onset have important clinical significance in early diagnosis and prognosis of AD.

Alternative splicing of pre-mRNA is an important mechanism for regulating gene function at the post-transcription level and for producing proteomic diversity in higher eukaryotes. The alternative splicing is regulated by the interaction between diverse cis-acting elements and trans-acting factors. Alternative splicing events of oncogenes, tumor suppressor genes and metastasis suppressor genes are associated with the initiation and development of human neoplasms. The protein isoforms sourced from alternative splicing take part in regulating the gene transcription, cell cycle, apoptosis of cells, and playing a role in tumor growth. It is possible for molecular therapy to target directly isoforms of protein produced by alternative splicing or to interfere with the process of alternative splicing.
Alternative Splicing ; genetics ; Humans ; Neoplasms ; genetics ; RNA Precursors ; metabolism ; RNA, Neoplasm ; analysis ; Transcription, Genetic

BACKGROUND: Enophthalmos deformity is the most common complication caused by orbital blow-out fractures, and others are diplopia and worsening of visual acuity. Since the therapeutic result of orbital blow-out is not satisfactory and many complications exist after operation, it is still a dispute to select implantation materials and therapeutic regimens.OBJECTIVE: To observe the therapeutic effect and assess the improvement of visual function by surgical reconstruction with porous high-density polyethylene (Medpor) for the correction of enophthalmos deformity caused by orbital blow-out fractures.DESIGN: A pre-and postoperative controlled study.SETTING: Beauty Center for Trauma Repair,Plastic Surgery Hospital,Peking Union Medical College, Chinese Academy of Medical Science .PARTICIPANTS: Totally 56 patients with orbital blow-out fractures who had enophthalmos deformity caused by fists or traffic accidents, treated at Beauty Center for Trauma Repair,Plastic Surgery Hospital, Peking Union Medical College, Chinese Academy of Medical Science, were selected in this study from December 1996 to March 2004. Final diagnosis were made with case history, X-ray film, two-demensional and three-dimensional CT before operation. 24 cases were accompanied with other areas fractures such as zygoma and nasal bone, 34 cases with diplopia, 35 cases with visual acuity worsening after injured.METHODS: ①Material implantation: Exposure of the orbital floor, inferior and medial walls could be performed through a 2 mm inferior subciliary incision of 3 cm long. To approach the orbital rim via a dissection plane anterior to the orbital septum, sub-periosteal dissection was then performed over the orbital rim, and along the orbital floor to the orbital apex. Mobilized the soft tissue from the bone throughout the entire area of fractures and re-position it to its proper position. Took Medpor (Type 6331) sheets as the implantation materials, trimmed Medpor sheets according to the radian and anatomic form, and 2 mm larger than the defect rim was needed.If other operations were needed during the operation, they could be done.Mannitol and dexamethasone should be used just postoperatively to decrease edema of the orbital contents and reduce inner orbital excessive pressure. ②Functional evaluation standard: Diplopia: completely disappear meant recovered, less diplopia residual meant improvement, no improvement meant inefficacy. Enophthalmos: marked improvement meant the degree of enophthalmos stabilizated at below 2 mm, less improvement meant stabilizated at above 2 mm.MAIN OUTCOME MEASURES: ①Improvement of enophthalmos; ②Improvement of diplopia ; ③Improvement of visual acuity.RESULTS: ①All 56 cases of enophthalmos deformities caused by orbital blow-out fractures improved greatly. ② Of all the 34 patients with diplopia, 27 recovered. ③ 9 patients' visual acuity of 35 improved with different degrees. No diplopia or visual acuity worsening occurred. With a follow-up ranging from 2 months to 5 years, the degree of enophthalmos stabilizated at below 2 mm, and no relapse and other complications occurred.CONCLUTION: Medpor has such advantages as better histocompatibility,fewer complications and better visual function improvement, so it is the preferred implantation material for correcting enophthalmos deformity caused by orbital blow-out fractures.

Objective To explore the risk and treatment of laparoscopic cholecystectomy(LC) in patients with liver cirrhosis and cholelithiasis.Methods To summarize the clinical data of 28 patients with liver cirrhosis and cholelithiasis.The patients were repeated right upper quadrant pain,including 4 cases of gallbladder neck stones incarcerated,gallbladder effusion.Among them,there were 20 cases of hepatitis B cirrhosis,5 cases of schistosomiasis cirrhosis,3 cases of alcohol.Results There were 6 patients with hemorrhage during operation.Among them,5 patients were treated with gelatin sponge,hemostatic gauze and bio-glue spray to stop bleeding,1 case was transferred to open surgery because of bleeding.The remaining 27 cases of LC were successful.Complications occurred in 8 patients,3 cases of increased liver function abnormalities,1 case of upper gastrointestinal bleeding,1 case of mild hepatic encephalopathy,2 cases of significant ascites formation,1 patient underwent subtotal resection of the gallbladder with Hartmanns bag,and bile was found in the ascites after surgery,but the bile disappeared after five days.All patients with complications after symptomatic treatment were cured,no death,no major bleeding and liver failure,bile duct injury,severe biliary fistula and other serious complications.Conclusion Although the risk of LC in patients with liver cirrhosis is higher than that in the general population,with the help of current high-tech surgical instruments,we can safely accomplish this with an improved surgical procedure.As these patients require high technical requirements of surgery,surgeons must have a wealth of experience and familiar laparoscopic liver and gallbladder anatomy.

BACKGROUND: Fibular fixation and tantalum rod implantation are two commonly used methods for the treatment of early osteonecrosis of the femoral head (ONFH), both of which can effectively delay or even reverse the progress of ONFH. However, further comparative evaluation on their mechanical properties and therapeutic efficacy is required.OBJECTIVE: To compare the clinical efficacy of fibular fixation and tantalum rod implantation on ONFH at early stage.METHODS: Fifty-eight patients (81 hips) suffered from ONFH with ARCO stage 1 and stage 2, and underwent fibular fixation (30 cases, 41 hips) or tantalum rod implantation (28 cases, 40 hips). Postoperatively, both groups were followed up for over 2 years. The Harris scores of the hip were compared between two groups before and after treatment. With femoral head collapse and the collapse distance > 4 mm as observation points, the survival rate of the femoral head was compared between two groups.RESULTS AND CONCLUSION: The postoperative Harris scores of the two groups were significantly improved than before (P < 0.05). With the appearance of femoral head collapse as the observation point, the Kaplan-Meier survival curve showed that the overall survival rate of the hip was 83% in the fibular fixation group and 65% in the tantalum rod implantation group. After examined by log-rank (Mantel-Cox), there was a significant difference in the survival rate of the hip at Stage IIC between two groups (P=0.0431). With > 4 mm collapse as the observation point, the Kaplan-Meier survival curve showed that overall survival rate of the hip was 95% in the fibular fixation group and 83% in the tantalum rod implantation group. After examined by log-rank (Mantel-Cox), there was a significant difference in the survival rate of the hip at Stage IIC between two groups (P=0.0418). To conclude, both fibular fixation and tantalum rods implantation applied to ONFH at early stage can effectively improve the hip function, and the survival rate of the hip at ARCO Stage IIC is better in patients undergoing fibular fixation than tantalum rod implantation.

Objective To explore the safety of mobilization and collection as well as the feasibility of selection of autologous peripheral blood stem cells (auto-PBSC) from patients with juvenile severe autoimmune diseases (AID) for autologous hematopoietic stem cell transplantation (auto-HSCT). The clinical significance of these procedure is evaluated. Methods Eight patients with AID, including four patients with systemic lupus erythematosus(SLE),two patients with dermatomysoitis, one patient with juvenile rheumatoid arthritis (JRA), one patient with multiple sclerosis(MS),underwent auto-HSCT. Auto-PBSCs were mobilized in 8 patients using cyclophosphamide(CTX) and granulocyte colony-stimulating factor (G-CSF), and their PBSCs were collected by CS-3000 Blood Cell Separator, then the CD34+cells were selected and purified by CliniMACS CD34+cell selection device. The CD34+ cells were frozenand preserved under -80 ℃ ALL patients received non-myeloablative or lymphoablative conditioning regimens which consisted of CTX/Mel/ATG or CTX/ATG or BEAM/ATG. All patient received CD34+ cells transplantation. The safety of mobilization and collection process of auto-PBSC as well asthe feasibility of selection and purification of CD34+cells were recorded and hematopoietic reconstruction were evaluated. Results All patients tolerated the collection process well, and there was no mobilization-related mortality. The number of collected MNCs and CD34+ cells were 8.35×108/kg and 7.92×106/kg respectively. The number of CD34+ and CD3+ cells after purification was 6.28×106/kg and0.71 ×105/kg respectively. The mean granulocytes and platelet engraftment occurred on days 11 and 15 after G-CSF regimen, and they can be collected using CS-3000 instrument. PBSC mobilization and collection from patients with juvenile severe AID is safe. The CD34+ cell can be highly purified. The auto-PBSC CD34+cell transplantation is an alternative therapy for severe AIDs that do not respond to conventional treatments.

OBJECTIVE: To standardize and simplify the drug receving and preparing model. METHODS: Reasonable pharamcy inventory parameters were set up based on our experience; second order drug storeroom dynamic inventory system was designed and applied, and the indexes of drug storeroom inventory and workload of the new and the old models were reported and analyzed. RESULTS: The new warning system provided an optimal inventory, and in which the incidences of overstock or stock shortage, the frequency of daily drug receving, drug kinds and the time used with checking stock and filling receipt were all significantly less than in the traditional model. CONCLUSION: Due to the second order drug storeroom dynamic inventory warning system, the drug receiving was more scientific, the drug stock amount was reduced and the drug receiving time was saved, and the mangament level of hospital drug storeroom inventory was enhanced.

Objective To investigate the effect of hepatitis B virus core protein (HBc) dimer interfaces amino acids mutation on nucleocapsid assembly and HBV DNA replication. Methods Based on HBc three dimension structure, four HBc dimer interfaces domain mutation plasmids, pHBc14-18M,pHBc120-135M,pHBc23-39M and pHBc122-139M were constructed in pcDNA3.1 vector by PCR site-directed mutagenesis, there was a flag-tag at the C-terminal of all mutants for easy detection. Wild type core protein plasmid 1-183flag was also constructed as a positive control. The 4 mutants were cotransfected HepG2 cells with pHBV1.2 core negative plasmid (pHBV1.2-core-) ,which contained 1.2 copies of HBV whole genome but the core protein would not express due to a stop codon. The capsid formation, HBV pregenome(pgRNA) and HBV DNA replication mediate were analyzed by native agarose gel electrophoresis and Western blot, Northern blot and Southern blot , respectively. The 4 mutants were also cotransfected HepG2 cells with HBV wild type plasmid pHBV1.2 and examined by Southern blot. Virions in the medium were determined by native agarose gel electrophoresis and Western blot. Results Cotransfecting HepG2 cells with pHBV1.2-core- plasmid, pHBc14-18M,pHBc120-135M and pHBc122-139M mutant groups formed nucleocapsid-like structure but pHBc23-39M could not, Northern and Southern blot displayed no signal in all mutants except 1-183flag conrol group. In pHBV1.2 cotransfection experiment, HBV DNA replication was blocked in pHBc14-18M, pHBc120-135M and pHBc122-139M mutant groups, sharply decreased in pHBc120-135M and pHBc122-139M groups, correspondingly virons production in medium were also inhibited. pHBc23-39M mutant exerted no influence on HBV replication. Conclusion pHBc23-39M mutant can neither form nucleocapsid-like structure nor interact with wild type HBc dimmer to interfere HBV replication.On the contrast, pHBc14-18M, pHBc120-135M and pHBc122-139M mutants can form nucleocapsid-like structure by themselves, but this structure does not support HBV DNA synthesis. Besides, they can effectively inhibit wild type HBV DNA replication by contacting with wild HBc dimmers resulting in nucleocapsid dysfunction.