BACKGROUND: Osteosarcoma cell OS-9607 is a cell line cultured from human osteosarcoma tissue. To construct cDNA library of this cell line so as to clone target gene from it.OBJECTIVE: To construct cDNA library of osteosarcoma cell OS-9607 by switching mechanism at 5' end of RNA transcript (SMART) technique and analyze its quality.DESIGN: Verified experimental study SETTING: Department of Orthopaedics, Changhai Hospital, Second Military Medical University of Chinese PLA MATERIALS: This experiment was carried out at the Department of Orthopaedics, Changhai Hospital, Second Military Medical University of Chinese PLA in May 2005. Osteosarcoma cell OS-9607 was cultured in CO2 incubator. Cells were pre-treated with DM for 30 minutes, recovered for 24 hours in standard culture medium. The whole RNA of osteosarcoma cell OS-9607 was isolated by RNAease reagent kit. The quantity and integrity of total RNA was detected by ultraviolet spectrometer and electrophoresis on a denaturing formaldehyde agarose gel stained by ethidium bromide (EtBr). METHODS: Total RNA was extracted from human osteosarcoma cell OS9607 and mRNA was isolated, cDNA of OS-9607 cells was acquired by in situ polymerase chain reaction (PCR) and long-distance PCR (LD-PCR),then the cDNA was ligated with dephosphorylated λ phage vector. The recombinants were constructed with SMART cDNA library construction kit.The size of cDNA inserts and the diversity of library were detected by PCR.MAIN OUTCOME MEASURES: The quality and integrity of total RNA, the titer of un-amplified and amplified library and recombination fraction; analysis of cDNA length of library RESULTS: ①The concentration of the isolated total RNA was determined and the measuring absorbance was between 260 nm and 280 nm. Its absorptance was about 1.9 A. 0.8％ denaturing formaldehyde agarose gel electrophorosis showed clear 28S rRNA and 18S rRNA,and the brightness rate of 28S rRNA and 18S rRNA (28S/18S) was 2:1. ②After linked to λ phage in vitro, packed and transfected host bacteria, the titer of unamplified library was 2.2×107 pfu/mL, and the titer of amplified 1 ibrary arrived to 4.5×1010 pfu/mL. The recombination fraction analysis showed that there were 8 blue plaques and 1 360 colorless plaques in one plate, so the recombination fraction was 99.5％. ③ The obtained cDNA library consisted of 1.6×106 recombinant bacteriophages and the average exogenous inserts of the recombinants was 1.5 kb.CONCLUSION: These results suggest that the obtained osteosarcoma cell OS-9607 cDNA library has high quality, which sets up a firm foundation for the further research on osteosarcoma cell OS-9607 and its elated genes screen.

Objective:To analysis the N-terminal amino acid sequence of 5D3Ag purified by mAb5D3 immunoaffinity column.Methods:SDS-PAGE and Western Blot were used to study the characteristics of 5D3Ag.From SDS-PAGE gel,5D3Ag was transferred to PVDF membrane and its N-terminal amino acid sequence was analysised with an auto amino acid sequencer.Results:SDS-PAGE of 5D3Ag showed a band on the gel with molecular weight of 43 kD.Western Blot study showed that mAb5D3 can recognize 5D3Ag.The N-terminal amino acid sequence was NQGSHGSEEQ.Conclusion:It is sure that 5D3Ag is unique to Osteosarcoma because of the purification method itself and the characteristics of mAb5D3.Compared with other reported Osteosarcoma associated antigen,5D3Ag is different from them in molecular weight and may be a novel Osteosarcoma associated antigen.With the help of N-teminal amino acid sequence,some primers may be designed to clone the whole length gene of 5D3Ag.The identification of 5D3Ag gene may be useful in understanding the biologic mechanics of Osteosarcoma and developing new diagnostic and therapeutic strategies.

HIV-1 integrase (IN) is a key enzyme for the viral replication. The protein-protein interaction (PPI) between HIV-1 IN and a cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is a validated target for anti-HIV drug discovery. In order to build the platform for screening inhibitor against PPI between IN and LEDGF/p75, the vector containing the LEDGF/p75 protein cDNA was constructed and expressed in Escherichia coli and the function of the LEDGF/p75 protein was assayed. The LGDGF/p75 encoding gene optimized according to the preference codon usage of E. coli, was synthesized and cloned into the expression vector pGEX-4T-1 to form a recombined plasmid, then transformed into host cell E. coli BL21 (DE3). The recombined clones were identified and confirmed by BamH I/Sal I digestion and sequencing, the successfully recombined plasmid in the host cell was induced by IPTG and the condition of the expression was optimized. The expressed protein was purified by the Ni2+ affinity chromatography column and SDS-PAGE was used to analyze the molecular weight and specificity. In addition, ELISA assay was used to analyze the function of the recombinant protein. The recombinant LGDGF/p75 was soluble, and expressed highly and stably in E. coli. The protein was proved to enhance HIV-1 IN strand transfer activity in vitro by ELISA. It will be helpful to build the platform of screening inhibitors against PPI between IN and LEDGF/p75.

Objective: To find out the effect of mineralization condition medium on bone marrow stromal cells(MSCs) proliferation,differentiation and the secretion of extracellular matrix. Methods: MSCs of the third passage were induced by minerlization condition medium (?=10%FBS,10 nmol/L dexamethasone,50 mg/L L-vitamin C and 10 mmol/L ?-sodium glycerophosphate in DMEM). The control cells were cultured in DMEM with ?=10% FBS. The cells were observed by inverted microscope. The proliferation status and alkaline phosphatase level of cells were investigated with MTT assay. Mineralization potential was studied by Von-Kossa staining. TypeⅠ,type Ⅲ collagen and BMP synthesis were examined by immunohistochemistry stains. Results:The proliferation of MSCs in mineralization condition medium was decreased, but ALP level was increased(P

Objective:To investigate the feasibility of marrow stromal cells/ceramic bovine bone/hydrogel in the repair of autogenous calvarial defects. Methods:The complex of ceramic bovine bone,hydrogel,rhBMP-2,TGF-? and marrow stromal cells(MSCs) induced by minerlization inducing medium was prepared and applied in the repair of autogenous calvarial defects in 8 SD rats(group A).Ceramic bovine bone /MSCs was used as control in another 8 SD rats(group B). 4 and 8 weeks after operation 4 rats in each group were sacrificed, the osteogenesis was examined by HE staining,modified Mallory's trichrome staining and image analysis.Results:4 weeks after operation, osseous plerosis can be seen in both groups, the quantity of bone-like tissues formation(?m~2) in group A and group B was 6 813.09?96.32 and 3 839.25?104.52(P

Objective: To investigate the effect of ceramic bovine bone (CBB) combined with hydrogel complex (CBB/HG), ?-tricalcium phosphate (?-TCP) combined with hydrogel complex(?-TCP/HG) on the osteogenesis of bone marrow stromal cells (MSCs) in vitro. Methods: MSCs(induced by 50 mg/L ascorbic acid,10 -8 mol/L dexamethasone and 0.01 mol/L ?-sodium glycerylphosphate) were cultured on CBB/HG , ?-TCP/HG,CBB and ?-TCP respectively. 5 and 10 days after set up, marrow stromal cell attachment and morphology were observed by scanning electronic microscope, the proliferation of the cells was evaluated by cell counting, Alkaline phosphatase(ALP) activity was tested,typeⅠcollagen synthesis were examined by immunohistochemistry stainning. Results: On day 5 and 10 the marrow stromal cells on CBB/HG and on ?-TCP/HG were significantly more than those on CBB or ?-TCP. TypeⅠcollagen synthesis and ALP activity of the cells on CBB/HG and ?-TCP /HG were not different but significantly higher than those on CBB or ?-TCP. Conclusion: CBB/HG and ?-TCP /HG may stimulate bone marrow stromal cell proliferation, differentiation and extracellular matrix synthesis.

Objective To evaluate the second generation micro-endoscopic discectomy (MED) system in the treatment of lumbar disc herniation. Methods Eighty-five patients with single or double level lumbar disc herniation were treated with MED under epidural anesthesia from May 2000 to October 2001.Single disc herniation was in 72 cases, double disc herniation in 13 cases; lateral recess stenosis in 15 cases. Results The average following-up period was 14 months and the successful therapeutic results achieved 92 9%(79/85). the patients were allowed to walk 3 d and discharged 3～5 d after operation. The length of incision was only 1.6 cm. Conclusions MED has short-term satisfactory therapeutic efficacy in the treatment of lumbar disc herniation. The suitable indication and precise technique are the key for the operation.

Objective To explore the application effect on theInternet plusused in basis surgical operation and formative evaluation. Methods The students of Chongqing Medical University who planned to study the basis of surgical operation in the 2015 academic year were randomly divided into two groups, with 166 students in the experimental group and 165 students in the control group.Internet plusmodel was used in the experimental group in surgery based teaching and the formative evaluation system; the control group used the traditional teaching mode and traditional formative assessment method. Indicators of forma-tive evaluation system include animal operation score, small group global impression, the homework score (small group overall form), the final exam results and operation examination results. After class, the ques-tionnaire was used to investigate the effect of students' learning. The t test or rank sum test of two groups of students were performed by using SPSS 17.0. Results Average scores of animals operation and homework score of the students in the experimental group were respectively (9.23 ±0.65) and (9.53 ±0.66), higher than in the control group (8.56 ±0.62) and (8.96 ±0.52), and the difference between the two groups had statistical significance (P<0.05). The results of the questionnaire showed that according to the students' self evaluation the students in the experimental group were superior to the control group (P=0.03) in the aspect of professional knowledge learning. Conclusion Internet plus used in surgery teaching and formative evaluation, can significantly improve the effect of teaching and promote students' learning.

Many pediatric surgeons have little training in laparoscopic application.The major problem in clinical practice of laparoscopic technician is without regularly training,unsuitable indication,and ignoring peroperation management.so that there are much more serious complications.For these reasons,it is a prerequisite that surgon and anesthetist must be trained before clinical practice,and the qualification of admission should be strictly controlled.

Teaching method of grouping and numbering the experimental animals refers to management of laboratory animals by marking them with numbers. In an experimental group, the same marked experimental animal is performed surgery operation by each group member. As a whole, each group member gets the same score according to the evaluation of operation performance of that group and the score is recorded as usual performance score. This method is simple and easy to implement. It is the optimizing of traditional basic operative surgery teaching methods which is conducted not only to improve medical students' sense of responsibility and animal protection awareness, to make full use of teaching resources and reduce waste, to cultivate medical students' team cooperation awareness, but also to deepen medical students' knowledge about surgical complications and improve the quality of teaching.