BACKGROUND: It has been verified vascular endothelial cells can propagate and differentiate into vessel by planted in the tissue-engineered material in vivo, but it may cause severe trauma when obtain vascular endothelial cells, and it has limited sources. OBJECTIVE: To investigate whether bone marrow mesenchymal stem cells (BMSCs) can differentiate into vascular endothelial cells by combined induction of vascular endothelial cell growth factor (VEGF), basic fibroblast growth factor (bFGF) and insulin-like growth factor (IGF)-1 by experimental observation. DESIGN, TIME AND SETTING: The control cell observation experiment was conducted at the Central Laboratory of First Affiliated Hospital, Liaoning Medical University from November 2006 to June 2007. MATERIALS: An 8-week-old Japanese big ear rabbit was used to isolate BMSCs. VEGF, bFGF and IGF-1 were purchased from Peprotech Company. METHODS: After anaesthesia, bone marrow was extracted from the rabbit. Enough BMSCs were harvested by adherence and trypsinization and randomized into two groups. BMSCs in the induction group were inoculated in DMEM supplemented with 10% fetal bovine serum (FBS), 10 ?g/L VEGF, 1 ?g/L bFGF and 2 ?g/L IGF-1 for 2 weeks. BMSCs in the control group were inoculated in DMEM containing 10% FBS. MAIN OUTCOME MEASURES: Morphological observation, nitric oxide (NO) content detected, immunohistochemistry staining of Ⅷ factor-related antigen and Weibel-Palade body under a electron microscope in BMSCs. RESULTS: Five days after induction, BMSCs were distributed in cluster, showing round. Non-induced BMSCs were evenly distributed, showing spindle or triangle. Fourteen days after induction, NO content in supernatant was significantly higher in the induction group than in the control group (t=3.75, P

Bone defect repair is a complicated process of connective tissue repair under the control of various cytokines.It is the ample blood supply which is a necessary condition to promote regeneration of fracture,especially bone defect and nonunion.Hypoxia inducible factor 1 can induce formation of neovascularization with perfect physiological function in the lesion of bone defect by regulating various gene expression,and thus provide nutritional support and favorable conditions to metabolism for different cells in the process of osteogenic differentiation and osteogenic activity to promote the healing of fracture.Both gene therapy and using hypoxia inducible factor 1 directly have an ability to promote neovascularization in the lesion of fracture.It is a hotspot at present that hypoxia inducible factor 1 in the field of bone tissue engineering is used to treat bone defect.Hypoxia inducible factor 1 transgenic therapy needs further research in the repairing process of bone defect.

With the emergence of tissue engineering techniques, using tissue engineering methods and tools to repair cartilage defects become a new treatment model, while looking for a suitable carrier of chondrocytes is the current focus of the study in cartilage tissue engineering. In this paper, synthetic biodegradable polymers and natural extracellular matrix substitutes (natural polymers) make progress in two areas reviewed.

BACKGROUND: Anterior cruciate ligament (ACL) is the kernel of articular genu, for the past few years, the incidence rate of cruciate ligament injury increased gradually.OBJECTIVE: To review on the mechanism of the injury of ACL, the method of operation, the election of transplant, the reconstruction of equilong, the reconstruction of ligament and so on, and to look forward to providing some references on treating the ACL injury.METHODS: A computer-based research was conducted in Medine database (1996-01/2009-03), with the key words of "Anterior cruciate ligament, implant, reconstruction, repair". Chongqing VIP database and Qinghua Tongfang database (1996-01/2009-03) were retrieved with the same key words.RESULTS AND CONCLUSIONS: 200 literatures on arthroscopic ACL reconstruction were collected, excluding older, repeated and similar research, and the 18 standard literatures were included. ACL is a key structure to stabilize knee joint, and exercises and various factors during daily life could induce its injury. Unsuitable treatment would severely affect movement ability or movement loss. To recover structure and function of knee joint, damaged ACL should be reconstructed. With the development of technique and innovation of surgical instrument, arthroscopic reconstruction has presently been a popular method to treat ACL. There are many methods to treat ACL injury, but none of them can duplicate ACL anatomical relationship. It is still in doubt whether the operation can reduce the secondary damage. We have much more work to research on modus operandi, implant, postoperative rehabilitation and other adjunctive therapies.

The vaseularization of tissue-engineered bone is the key problem which the development and employment of large sized tissue-engineered bone.The vascular endothelial cell has a great effect on promoting vascularization in tissue-engineered bone.Vascularizations fall into two modes of vaseulogenesis and angiogenesis according to differences in source of endothelial cells.Co-culture of osteoblasts and vascular endothelial cells has better result than single culture of each kind of cells.Different ways of improving the vascularization,such as searching for new source of vascular endothelial cell,co-culture and in vivo experiment are investigated to meet the challenge of bone tissue engineering.

BACKGROUND: Whether bone morphogenetic protein 2 (BMP-2) can be transduced into marrow stromal cells (MSCs) and produce osteogenic effects by viral or non-viral vector remains unclear? OBJECTIVE: To observe the expression of cultured rabbit MSCs transfected with pcDNA3-hBMP2 in vitro. Simultaneously, the MSCs were transfected but not screened and then transplanted into autologous muscle to investigate the osteogenic capability by X-ray. DESIGN, TIME AND SETTING: A controlled observation experiment was performed at the Department of Orthopedics, Liaoning Medical University between November 2004 and April 2005. MATERIALS: Six adult New Zealand rabbits, of either gender, weighing 2.0-3.0 kg, were included for this study. BMP2 antibody was the product of Sanaka Company, USA. pcDNA3-hBMP2 was provided by Professor Pu Qin from the Department of Biochemistry, Fourth Military Medical University of Chinese PLA). Restriction enzyme was purchased from Takara biotechnology (Dalian) CO., LTD., China. METHODS: Super-purified plasmid pcDNA3-hBMP2 was extracted from E. coli. Bone marrow was taken from the adult rabbit femur for harvesting MSCs by density gradient separation. The MSCs were divided into the following 4 groups: Group A, cells were transfected and screened by G418; Group B, cells were transfected by pcDNA3-hBMP2; Group C, cells were transfected by empty vector pcDNA3; Group D, only transfection reagent Fugene 6 was added.MAIN OUTCOME MEASURES: Transient BMP2 expression was analyzed by immunohistochemistry. Expression of osteocalcin and collagen I was examined by immunohistochemistry and in situ hybridization, respectively. Two weeks after transfection, MSCs from the group B were autologously transplanted into the muscle. Four weeks later, X-ray assay was used to observe bone formation.RESULTS: pcDNA3-hBMP2 was successfully transduced into MSCs and transiently expressed BMP2 100%. Four weeks after gene transfection, expression levels of osteocalcin and collagen I were significantly higher in the group A than in the groups C and D. X-ray results demonstrated new bone formation four weeks after MSCs transplanted into the muscle.CONCLUSION: pcDNA3-hBMP2 can safely and efficiently transfect MSCs and induce them to differentiate towards osteoblasts by secreting BMP2.

BACKGROUND:The self-renew and regeneration capacity of the injured spinal cord is thought to be limited. Accordingly, cel transplantation is one potential strategy for promoting functional recovery after spinal cord injury. OBJECTIVE:To explore the effects ofPTEN silencing on the biological properties of bone marrow mesenchymal stem cels, hoping to offer better seed cels for tissue engineering. METHODS:Bone marrow mesenchymal stem cels were transfected with specific siRNA-silencedPTEN gene using the liposome method, and then RT-PCR was used to detect the mRNA expression ofPTEN. Variation of biological properties ofPTEN-transfected cels were detected by the way of MTT assay, cel cycle analysis, and Transwel assay. RESULTS AND CONCLUSION:PTEN is expressed highly in bone marrow mesenchymal stem cels, which is successfuly interfered by siRNA.PTEN-silenced cels have stronger survival, proliferation and migration abilities, which become a kind of better seed cels for tissue engineering.

Employed staffs in military hospital are the basic power in the development of hospital, and they are playing an im-portant role in the military medical security.The article gives a detailed elaboration on the innovation and practice of employed staff man-agement in our hospital.In order to improve the efficiency and scientific management level of employed staffs, the innovative measure-ments in employment pattern, post management, education development, policy system, promotion passage are put forward in combination with current situation of employed staff in order to promote the construction of employed staffs and the development of the hospital.

BACKGROUND: Vascular endothelial growth factor (VEGF) can promote angiogenesis, and has been extensively used in treatment of bone defect. However, few studies have addressed its isomer VEGF121. OBJECTIVE: To construct adenovirus vector carrying VEGF121-FLAG and humanized Renilla reniformis green fluorescent protein 1(hrGFP-1) and observe its expression in bone marrow stromal stem cells (BMSCs). METHODS: Using polymerase chain reaction technique, VEGF121 gene contained in the plasmid of pTG19T-VEGF121 was used to remove termination codon. NotI and Xho I restriction sites were added before and after gene sequence. Obtained gene subclone was moved onto pMD19-T plasmid. The pMD19-T-VEGF121 and pShuttle-CMV-IRES-hrGFP-1 plasmids underwent double enzymatic digestion. Small fragment and big fragment were retrieved utilizing gel. Subsequently, coupled reaction was conducted to complete the construction of shuttle plasmid. After measuring virus titer, BMSCs were transfected and the fluorescence intensity was observed under fluorescence microscope.RESULTS AND CONCLUSION: Recombinant adenovirus plasmid was successfully constructed by enzymatic digestion determination and gene sequence. Fluorescence microscope has shown that BMSCs transfected with recombinant adenovirus presented significantly green fluorescence expression. Thus, adenovirus vector carrying VEGF121-FLAG and hrGFP-1 gene can express in eukaryotic cells, which can be used for gene therapy for ischemic disease.

BACKGROUND: Hypoxia-inducible factor-1 (HIF-1) can regulate the co-expression of various genes, and can induce angiogenesis with integrated physiological function.OBJECTIVE: To construct a novel adenoviral eukaryotic expression vector that can co-express mutant hypoxia-inducible factor-1 alpha (HIF-1a) target protein and humanized Renilla reniformis green fluorescent protein (hrGFP) reporter molecule under normoxic conditions.METHODS: The human HIF-1α gene carried by target gene donor plasmid pCMV6-XL5-HIF1α was sequenced and the site of restriction enzyme in above gene was analyzed. Site-directed mutagenesis of three amino acids including the 402 location, the 564 location, and the 803 location in gene coding region in HIF-1α were performed by polymerase chain reaction and sequencing was also done for monitoring mutation. The HIF-1α gene mutated correctly (HIF-1αmu) was coupled to adenoviral shuttle vector pShuttle-CMV-IRES- hrGFP-1. The recombinant adenovirus shuttle vector carrying HIF-1αmu gene was transferred to BJ5183-AD-1 electroporation competent cells after sequencing identification and Pme I restriction enzyme linearization.HIF-1αmu and hrGFP gene as well as hemeo-expression elements of hrGFP gene were reconstructed into adenoviral genome plasmids using homologous recombination mechanism in bacterium. Recombinants were obtained by Pac I restriction enzyme digestion and sequencing identification.RESULTS AND CONCLUSION: Amino acids including the 402 location, the 564 location and the 803 location in gene coding region in HIF-1α had become alanine after site-directed mutagenesis. Recombinant adenoviral expressing vector was successful as confirmed by restriction enzyme digestion and sequencing. These findings demonstrate that a novel recombinant adenoviral mutant eukaryotic expression vector pAd-HIF1αmu-IRES-hrGFP-1 was successfully constructed.