Objective Human immunoglobulin combinatorial library was generated by using phage surface display expression system, and phage antibodies (Fabs) to platelet were screened.Methods:PBMC were separated from a patient who Were transfusion refractory to platelet. mRNA was isolated and cDNA was synthesized by reverse transcriptase.The immunoglobulin heavy chain Fd and the light chain ? genes were amplified by half nested PCR and the PCR products were cloned into the expression vector pCome3 respectively.The immunoglobulin combinatorial library was constructed and screened by 3 rounds of affinity selection.Results Human Immunoglobulin Combinatorial Library was successfully constructed.The specific phage antibodies were highly enriched after 3 rounds of biopanning selection against platelet membrane proteins.Conclusion The antibody library and human monoclonal antibodies to platelet may be useful as molecular tools to study the anti platelet drugs, platelet related diseases, epitope of human platelet antigens.

[Objective]To observe the histological characteristics of autologous chondrocytes/human articular cartilage derived scaffold (HACDS) composites for repairing chondral defects in rabbit models. [Methods]A full-thickness articular-cartilage defect (diameter 4 mm,thickness 2 mm) was created in the femoral condyle of rabbits. The rabbits were divided into two groups: control group,HACDS scaffold only,and experimental group,chondrocytes/HACDS scaffold. Fifteen months after implantation,the specimens were observed by inverted phase contrast microscopy,and assessed by staining with haematoxylin-eosin,alcian blue,toluidine blue,safranin O,as well as by the immunohistochemistry of collagen type Ⅱ.[Results]Histologically,the generated neo-cartilage integrated well with its surrounding normal cartilage and subchondral bone in the defects of experimental group at 15 months post-implantation,whereas only fibrous tissue was filled in the defects of control group. Histochemical and immunohistochemical analysis revealed that alcian blue,toluidine blue,safranin,and were obviously positive in the experimental groups. However,alcian blue,toluidine blue,and safranino O were negative in the control group.[Conclusion]The current histological examination demonstrated that an engineered cartilage composed of autologous chondrocytes on HACDS scaffold could be successfully obtained and further applied to repair an articular cartilage defect in a rabbit model.

Objective: To confirm the structure and preferential conformation of the Schiff base of gossypol with 1, 3, 4, 6-tetra-O-acetyl-β-D-glucosamine and explore its anti-HIV-1 activity.Methods: The Schiff base of gossypol with 11, 3, 4, 6-tetra-O-acetyl-β-D-glucosamine was synthesized and identified by FT-IR, NMR spectroscopy and the PM6 semi-classical calculation.The inhibitory activity of the novel compound against the laboratory-adapted HIV-1IIIB strain was examined using the HIV-1IIIB/TZM-bl indicator cell culture system.Results: The 1H and 13C-NMR signals of the new Schiff base were assigned.The PM6 semi-classical calculation indicated that enamine-enamine tautomeric form of the new Schiff base was more stable,which was stabilized by the intramolecular hydrogen bonds.The anti-HIV-1 test showed that the compound could block the entry of HIV-1IIIB into the target cells.Conclusion: The Schiff base of gossypol with 1, 3, 4, 6-tetra-O-acetyl-β-D-glucosamine exhibits enamine-enamine tautomeric form in solution, which shows potential anti-HIV-1 activity.

BACKGROUND:Because human cells for culturing alveolar bone cell line are from alveolar bone, which is in oral cavity,and easily polluted, so laboratory study is often unsuccessful. Because the samples are from adults, so cell division index and the successful rate of culture are low.OBJECTIVE: To compare the biological characteristics of survived cell line established through passage,cryopreservation and revitalization following in vitro culturing the alveolar bone tissue obtained from normal persons and patients with chronic periodontitis accompanied with osteoporosis in aseptic operation; To compare the biological characteristics of two kinds of cells so as to provide theoretical and related experimental evidence for defect, repair and treatment of alveolar bone.DESIGN: Controlled observation.SETTING: Department of Stomatology, Beijing Chaoyang Hospital, Capital Medical University; Institute of Orthopaedics,General Hospital of Chinese PLA.MATERIALS: Alveolar bone tissue obtained from normal persons and patients with chronic periodontitis confirmed in clinic was used in aseptic operation.METHODS: Alveolar bone tissue from normal persons and chronic periodontitis accompanied with osteoporosis were cultured in vitro. In the four cell lines (H-171, H-258, 261, 262) cultured primarily, cell lines H-171 and H-258 were chosen from periodonitis patients group and normal group respectively, and stained with histochemical and immunohistochemical methods. Cell morphology was observed. Doubling time and division index of two kinds of cells were calculated with cytometry. After several circles of passage, cryopreservation and revitalization, growth and aging rule of cells were compared.MAIN OUTCOME MEASURES: Passage and biological characteristics of two groups of cell lines.RESULTS: ①In the abnormal alveolar bone group, there was one successful primary culture and cells presented short-spindle shape. There were 3 times of cryopreservation and 3 times of revitalization. Its doubling time was 53.4 hours. The average division index was about 4‰. Cells well grew after 20 times of passages. ②In the normal alveolar bone group, there were 26 cases of cell lines cultured primarily, but passage was found in only 3 cases of cell lines due to various causes. There were 10 passages and the cells presented long-spindle shape. After two circles of cryopreservation and revitalization, the survival and growth rate of cells were inferior as compared with cell line H-171.Doubling time was 65.9 hours and the average division index was 3.5‰. ③Both two kinds of cells adhered the wall, with the characteristics of osteoblasts: AKP, toluidine blue, PAS, tetracycline-labeled mineralized nodus, type Ⅰ collagen and BMP-2 immunohistochemical staining all presented positive.CONCLUSION: Both two kinds of cultured cells have the characteristics of osteoblasts. The growth speed of cell line H-171 is faster than that of cell line H-258. No obvious mutation is found in 20 passages. In the 8th generation of H-258,aging appears and growth speed becomes slow.

AIM: To investigate the effect of oral clarithromycin on serum concentrations of aminophylline at steady state and its pharmacokinetics in New Zealand rabbits. METHODS: The serum concentration of aminophylline was determined by FPIA in rabbits given(po) aminophylline or aminophylline combined with clarithomycin. The experiment was divided into 2 stages: (Ⅰ)the subjects only received a four day course of oral aminophylline until steady state;(Ⅱ)aminophylline and clarithromycin were coadministrated from the d 5 to d 10. The dose of aminophylline was 30 mg?kg -1 and the dose of clarithromycin was 50 mg?kg -1 for each rabbit. The two series of pharmacokineticis parameters were tested by statistic analysis. RESAULTS: There was no significant variation in pharmacokinetic parameters between two stages. CONCLUSION: It is not necessary to change the therapeutic dose of aminophylline when the drug is taken in combination with clarithromycin.

BACKGROUND: Elimination of antigenic substances from natural extracellular matrix with the integrity of the tissue structure retained renders the matrix to possess better biocompatibility and provides a cell culture environment close to conditions of the internal environment. Such materials are the primary choice for cell culture scaffold in tissue engineering.OBJECTIVE: To prepare human articular cartilage acellular matrix so as to provide a methodological basis for further study of articular cartilage acellular matrix as cell scaffold materials.DESIGN: A single sample study of bone tissues.SETTING: The experiment was performed in Institute of Orthopedics, General Hospital of PLA, between January and May in 2004. The specimens were obtained from patients requiring joint replacement for femoral neck fracture.MATERIAIS: The experiment was conducted in the Department of Orthopedics, General Hospital of PLA from January to May in 2004. Human articular cartilage specimens were obtained from the femoral head of patients with total hip arthroplasty for femoral neck fracture.METHODS: Totally 10 specimens of fresh articular cartilage(3.5 mm × 4. 5 mm × 2.0 mm) were obtained and freeze-dried for 12 hours. Cartilage acellular matrix was prepared using Triton X-100, Dnase and Rnase and identified by means of hematoxylin-eosin(HE) and safranine O staining and immunohistochemical staining for cartilage proteoglycan.MAIN OUTCOME MEASURES: Histological observation of the articular cartilage acellular matrix and immunohistochemical staining of cartilage proteoglycan.RESULTS: HE and safranine O staining both showed no cellular structure in the matrix with only recesses left by the removed cells. Immunohistochemical staining for cartilage proteoglycan yielded positive results, suggesting the presence of cartilage proteoglycan in the acellular matrix.CONCLUSION: Human articular cartilage acellular matrix can be prepared using the modified four-step procedures with detergent and enzymatic extraction with lyophilization, and the preserved cartilage proteoglycan in the material may retain good pressure resistance.

BACKGROUND:Cartilage extracelular matrix with a large number of signaling molecule proteins and factors is likely to be an ideal material for tissue engineering cartilage.
OBJECTIVE: To investigate the possibility of calcium alginate and cartilage extracelular matrix combined with microencapsulated stem cels derived from human umbilical cord Wharton’s jely to construct ectopic tissue-engineered cartilage in nude mice.
METHODS: Microfilament suspension of the cartilage extracelular matrix was prepared. Human stem cels derived from Wharton’s jely of the umbilical cord were inoculated in to calcium alginate and cartilage extracelular matrix gel microspheres as experimental group. Stem cels derived from human umbilical cord Wharton’s jely were incubated in simple alginate gel microspheres as control group. After in vitro culture, the microspheres wereimplanted into the dorsal subcutaneous tissue of nude mice. Samples were taken after 4 weeks, respectively, for gross and histological observation.
RESULTS AND CONCLUSION:The stem cels exhibited paralel-chondrocyte morphology in microspheres, which grew and proliferated quite wel during in vitro culture. A new paralel-cartilaginous tissue was found in the subcutaneous tissue 4 weeks after surgery in the experimental group, and the tissue was positive for hematoxylin-eosin, safranine O, toluidine blue and colagen II. A large number of paralel-chondrocytes and cartilage lacuna-like structures were observed under a microscope with no obvious inflammatory reaction around the microspheres. The control group showed the partial degradation of microspheres, surrounded by only a smal number of inflammatory cels and lymphocytes. Calcium alginate and cartilage extracelular matrix microspheres have a rather good histocompatibility which can be used to construct paralel-cartilaginous tissues by implanting stem cel-microspheric compound into the subcutaneous tissue of nude mice.

OBJECTIVETo investigate the biological characteristics of cell lines of healthy and diseased human dental alveoli.

METHODSPrimary cell lines from either healthy or diseased human dental alveoli were obtained. Two cell lines, H-258 and H-171 derived from healthy and diseased human tissues respectively, were selected for morphological study and research on their growth and aging, using cell counting, and histochemical and immunohistochemical staining.

RESULTSPrimary cell lines were successfully established from innormal dental alveoli. After freezing and thawing for three times, cell growth was continued and no morphological alterations were observed. The doubling time was 53.4 hours and mean division index (MDI) was 4 per thousand. Cells were kept normal after twenty generations with no obvious reduction of doubling time and MDI. Of twenty-six primary cell lines derived from healthy human dental alveoli, only three cell lines achieved generation. After freezing and thawing for twice, cultured cells were still alive at a decreased growth speed, with doubling time of 85.9 hours and MDI of 3 per thousand. Both cell lines, H-171 and H-258, shared the characteristics of osteoblast.

CONCLUSIONSPrimary cell lines of diseased human dental alveoli show greater growth potential. All cell lines of dental alveoli share characteristics of osteoblast. The technique we developed may be put into practice for the treatment of abnormal dental alveoli.

Objective To investigate the incidence of altitude reaction among people who rapidly ascend to highaltitude and the health status of experimental emergency blood donors after ascending to high -altitude areas above 4600meters.Methods The vital signs of 185 subjects were analyzed according to GJB1098-1991 and questionaires were filledout.Then the incidence of altitude reaction in different -altitude areas and the change in health status before and after blooddonation at high-altitude were evaluated.Results There was no significant difference in the incidence of altitude reactionin areas of different altitude.However, the incidence of moderate and severe altitude stress increased significantly .Thesimilar altitude reaction incidence despire different means of transportation (74.40% by railway and 70.00% by air) suggestedthat the mode of transportation had little effect.And our results showed good health status among emergency blood donorsbefore and after donation.Conclusion Emergency blood collection is feasible at high altitude (more than 4600 meter high).

Objective To investigate the optimal centrifugation conditions for preparation of rat and mouse platelet rich plasma (PRP) by single centrifugation.Methods Arterial blood of rats and mice by femoral artery cannulation and cardiac puncture were obtained respectively, anticoagulation with 14％CPDA-1, while white blood cells in the blood were filtered out.Then the blood was divided into sterile EP tubes, while PRP was prepared by centrifugation in different conditions (the centrifugal force was 300×g-600×g, and the centrifugal time was 4-12min).The number of blood cells of the anticoagulant whole blood, the leukocyte-depleted blood sample and PRP were counted by hematology analyzer, and platelet recovery rates were compared between different methods.Results The platelet recovery rate was highest when the blood samples of rats and mice were centrifuged at 400×g and 300×g for 8min respectively.Conclusion It is a key to prepare PRP by single centrifugation that selecting the appropriate centrifugal force and time and reaching a critical state before the formation of the buffy coat.