Objective To optimize the PCR primer sets for Simian virus 40 (SV40) detection and establish an assay method for SV40 which is of high sensitivity, strong specificity, broad applicability. Methods Two pairs of PCR primers were designed of based on 21 different SV40 strains genome by Primer Premier 5.00 software, and the features of two pairs of PCR primers were analyzed by Oligo software (version 6.71), conservative nucleotide of two pairs of PCR primers and the PCR amplification product were analyzed by DNAMAN software (version 6.0.40). Two pairs of new-built PCR primers were compared with those derived from China pharmacopoeia (Clip) in these aspects. The detection sensitivity of four pairs of PCR primers were analyzed using different SV40 DNA diluent as PCR template. The detection specificity of four pairs of PCR primers were analyzed using sterile water, Vero cell DNA, SV40 DNA as PCR template, respectively. Results The sequences of the new PCR primer sets VP1 and T are conservative for 21 Strains. The sequences of PCR primer sets GCVP1 and GCT are conservative for SV40 strains whose accession No. is J02400, NC_001669, AF316139 and AF316141. As far as the same diluent SV40 DNA template is concerned, the PCR amplification efficiency of PCR primer set VP1 and T is higher than that of PCR primer set GCVP1 and GCT. There are non-specific band in nucleic acid electrophoresis for amplification products of PCR primer sets GCVP1 and GCT, whereas there are no non-specific band in nucleic acid electrophoresis for amplification products of PCR primer sets VP1 and T. Conclusion The new assay method for SV40 nucleic acid sequence has many better qualities than those in Chp such as high sensitivity, strong specificity, broad applicability, conservation of primers and their amplification products and so on.

Fibroblast growth factor (FGF) is a key regulator of developmental processes. A FGF homolog (vFGF) is found in all lepidopteran baculoviruses. Autographa californica nucleopolyhedrovirus (AcMNPV) and Bombyx mori NPV (BmNPV) vFGFs are chemotactic factors. Here we analyzed the vfgf of Helicoverpa armigera NPV (HearNPV), a group Ⅱ NPV. The HearNPV vfgftranscripts were detected from 18 to 96 h post-infection (hpi) of Hz-AMI cells with HearNPV and encoded a 36 kDa protein, which was secreted into the culture medium. HearNPV vFGF had strong affinity to heparin, a property important for FGF signaling via an FGF receptor. Unlike its AcMNPV homolog, HearNPV vFGF specially chemoattracted Hz-AM 1, but not other insect cells such as Sf9 and Se-UCR and not the mammalian cells 293 and HepG2. HearNPV vFGF is also associated with the envelope of BV but is absent in occlusion-derived virus, which coordinated to the chemotatic activity analysis.

Baculoviruses produce two viral phenotypes, the budded virus (BV) and the occlusion-derived virus (ODV). ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the lack of an in vitro system, the molecular mechanism of ODV infection is still unclear. Here we present data demonstrating that Helicoverpa armigera nucleopolyhedrovirus (HearNPV) ODV infected cultured Hz-AM1 cells in a pH dependent manner. The optimal pH for ODV infection was 8.5, which is same to that in the microvilli of midgut epithelial cells, the ODV native infection sites. Antibodies neutralization analysis indicated that four HearNPV oral infection essential genes p74, pif-1, pif-2 and pif-3 are also essential for HearNPV ODV infection in vitro. Thus, HearNPV-HzAM1 system can be used to analyze the mechanism of ODV entry.

The effect of insulin gene enhancer binding protein (ISL1) on proliferation of HIT-T15 cells was investigated.ISL1 significantly promoted cell proliferation.ISL1 also increased the advance of HIT-T15 cell phase significantly.The results showed that ISL1 promoted proliferation of HIT-T15 cells.

Clinically, atrial fibrillation (AF) is one of the most common cardiac arrhythmia, and patients with arterial fibrillation carry high risk of stroke. Oral administration of anticoagulation such as warfarin for the prevention of stroke has some risks to induce bleeding; moreover, some patients are not able to tolerate the medication. Percutaneous occlusion of the left atrial appendage is safe and effective for the prevention of stroke in patients with atrial fibrillation, although at present it is only used for the patients who have contraindications to anticoagulation medication. This paper aims to review a variety of left atrial appendage occlusion devices and to analyze the relationship between the different shape design of occluder and the left atrial appendage morphology.

BACKGROUND:Bone marrow derived mesenchymal stem cells (BMMSCs) have multi-differentiation potentials, possessing a repairing capability for the sectional bone defects if combined with degradable porous β-tricalcium phosphate china. This provides a new idea in clinical repair of various bone defects.OBJECTIVE: To explore in radiology the curative effect of implanting porous β-tricalcium phosphate and autogenous BMMSCs compound to treat bone defects.DESIGN: A randomized controlled study with callus growth at various healing periods as subjects for observation. SETTING: Department of Orthopaedics, Renmin Hospital, Wuhan University; Tissue Engineering Center, Research Institute of Basic Medicine,Academy of Military Medical Sciences of Chinese PLAMATERIALS: This experiment was carried out at the Tissue Engineering Center, Research Institute of Basic Medicine, Academy of Military Medical Sciences of Chinese PLA between March and September 2002. Twenty China healthy adult sheep were randomized into the groups of blank control group (4 sheep), simple implantation group (8 sheep) and complex implantation group (8 sheep).METHODS: Under general anesthesia and aseptic condition, 10-15 mL of sheep marrow was extracted; MSCs were separated and cultured before combined with porous β-tricalcium phosphate china for tissue engineering bone construction. Rats in each group were cut off 21mm long metatarsus in the middle section of metatarsus bonestem. Β-tricalcium phosphate china and autogenous MSC compound was implanted into the sheep of the complex implantation group; β-tricalcium phosphate china was implanted into the simple implantation group; and the bone defects in the blank control group remained untouched. Then the incision was sutured.X-ray filming was carried out right after the operation, as well as 1, 3,and 6 months after the operation for radiological appraisal (scored for1 if bone union formed in one surface of bone defect, but scored 0 if no boneunion formed in any surface of bone defect, and scored 4 if bone union formed in front, back, lateral surfaces and the center of bone defect), Xray radiation-resisting density was analyzed to compare the results of bone defect repair.MAIN OUTCOME MEASURES: The postoperative general condition and general observation, as well as the results of the radiological analysis of the bone defects of all sheep.RESULTS: Totally 20 sheep were brought into this xperiment and all entered the stage of result analysis. ① The postoperative general condition:Sheep regained consciousness 2-6 hours after the operation without incision infection and loosing of internal fixation. Their spirit gradually was back to normal 1 week after the operation, at which time the injured legs could touch ground but were incapable of bearing load, and the affected legs could bear load 2 weeks after the operation, walked slightly lamely 3 weeks after the operation, and even moved freely without limp 4 weeks after the operation. ②The general condition 6 months after the operation: In pure implantation group, the surface white hyaline cartilage-like tissues were gradually calcified, with both ends connected with the host bone by bone bridge, but china granules could still be easily observed; while no implantation substance could be observed in compound implantation group,with the boundary between implantation substance and host bone vanished,and bone defect became basically the same as host bone. However there was no bone tissue formed in bone defect at various postoperative time points in the blank control group. ③ Radiological analysis of the bone defects at various postoperative time points: The radiological rating score was obviously higher in complex implantation group at the time poin ts of 3, 6 months after the operation compared with the pure implantation group [(2.3±0.3), (1.8±0.5); (3.3±0.5), (2.6±0.6), P ＜ 0.05]. ④ Radiological analysis of bone callus thickness and the relative value of radiation-resisting density at various postoperative time points: The bone callus thickness in the complex implantation group was obviously lower than that of the pure implantation group at the postoperative time point of 6 months (4.62 vs 7.64, P ＜ 0.05), with relative value of radiation-resisting density obviously higher than that of the pure implantation group (70.4±1.5 vs 61.18±1.2, P ＜ 0.05).CONCLUSION: Radiological appraisal and bone defect density measurement can well reflect the dynamical repairing process of bone defects; the implantation of porous β-tricalcium phosphate china and autogenous BMMSCs compound into sheep can enhance the repair of large sectional bone defect.

BACKGROUND:Sirius red is a strong acid anionic dye. Being not-easyto-fade and specific, sirius red becomes the best dye for collagen staining.Collagen is a major component of extracellular matrix and has some specific physiological functions. Through synthesis and reconstruction of collagen, bone fracture repair will be accomplished.OBJECTIVE: Picric acid-Sirius red stained slides were observed under a polarized light microscopy for evaluation the dynamic changes in the ratio of different collagen types and their distributions in bone fracture healing.DESIGN: It was a controlled observation.SETTING: It was conducted in the Department of Orthopedics, Renmin Hospital, Wuhan University; Department of Traumatic Orthopaedics, Tianjin Hospital; Department of Traumatic Orthopaedics, Jishuitan Hospital,Medical Department, Peking University; Tissue Engineering Center of Institute of Basic Medical Sciences, Academy of Military Medical Sciences of Chinese PLAMATERIALS: It was conducted at Tissue Engineering Center of Institute of Basic Medical Sciences, Academy of Military Medical Sciences of Chinese PLA from March 2002 to September 2003. Three healthy adult Chinese sheep, male and in weight from 25 to 35 g, were selected.METHODS: All the animals were anesthesized and sterilized; a transverse osteotomy of the trunk of metatarsus was performed; and the end of fracture was fixed with a six-hole Medoff sliding plate. At the post-operative month 1, 3 and 6, samples were taken from bone fractures. After decalcification with EDTA, they were stained with Picric acid-sirius red, and the types and distribution of collagens were observed under a polarized light microscopy.MAIN OUTCOME MEASURES: Types and distributions of collagens in bone lesion in different period of bone healing were investigated.RESULTS: Three sheep used in this study entered the statistical analysis.①Morphological features of various collagens under a polarized light microscopy postoperatively: Type Ⅰ collagen packed tightly, with a strong refraction and yellow, orange or red thick fibres. Type Ⅱ collagen formed a loose reticulation with fibres exhibiting different colour and a weak refraction. Thin fibres of type Ⅲ collagen with weak refraction and green colour formed a loose reticulation. ②Quantitative studies on various collagens under a polarized light microscopy postoperatively: At postoperative month 1,red or orange fibres (type Ⅰ collagen) were rarely seen in bone fracture,while green fibres (typical of type Ⅲ collagen) were dominant with a disorder pack. At postoperative month 3, red or orange fibres increased significantly and the ratio of type Ⅲ collagen reduced. The collagen fibres assembled regularly. At postoperative month 6, thick yellow-red collagen became dominant and thin green type Ⅲ collagen decreased dramatically and arranged in an obvious oblique, spiral and crossed orientations.CONCLUSION: Picric acid-sirius red stain combined with polarized light microscopy technique is not only capable of identifing type Ⅰ and type Ⅲ collagens in bone fraction, but also can reflect the morphological features,distribution and the ratio of these two type collagens. This approach has the virtues of easiness in operation, strong specificity and high sensitivity.

Bioethics, one of the focus subjects in 21 century,attracts the attention in the worldwide.Originated from western developed countries,it has been branded with distinct western civilization,which is disadvantageous to the development of the subject.Some suggestions are put forward as following: 1.Communication between the East and the West should be promoted in order to form a solid multi-cultural base for bioethics. For instance, in America ethics bears the imprint of religious conventionality,while in China politics is infiltrated into traditional ethical ideas. Both of them are not perfect. So they cannot form the base of bioethics. 2.Research on the "base line" of the bioethics should be emphasized in order to provide freer academic atmosphere. Transplanting the western bioethical ideas to the developing countries will never work. As for the bioethical"base line",different countries should establish feasible ethical codes according to their own actual situations. 3.Scientists should be encouraged to adopt various technical ways to explore the problems in the field of bioethics.Such methods as tissue-engineering,antologous stem-cell replanting and cell diffrenttation inducing may bring about earlier breakthrough than fetus stem-cell.Equal attention,encouragement and support should be given to bioethics.

Objective To explore the feasibility of the construction and formation of cartilage with immortalized chondrocytes by tissue engineering technology in vitro . Methods Human telomerase reverse transcriptase(hTERT) gene was introduced into rabbit mandibular condylar chondrocytes by eukaryotic vector. After screening with G418, the positive clones were amplified for culture. Normal or immortalized chondrocyte loaded cytoskeleton ? tricalcium phosphate (? TCP) complexes were incubated in vitro for 1～2 d and then implanted into subcutaneous tissue of nude mouse. The complexes of immortalized chondrocyte ? TCP and chondrocyte ? TCP or ? TCP alone were established as the experimental group and control groups respectively. The specimens were harvested within 3 and 6 months after surgical procedure for histological and immunohistochemical observation. Results In experimental groups and control group 1, the complexes packed with cartilage like tissue were found, but there was only a little fiber like tissue formed in control group 2. Immunohistochemistry revealed strong positive staining with safranine O, toluidine blue and collagen type II and obvious formation of cartilage. There was significant difference between the experimental group and the control groups( P

Objective To explore the method and technique to gain adequate seed cells for cartilage tissue engineering Methods hTERT gene was introduced into rabbit mandibular condylar chondrocytes by eukaryotic vector Rapidly proliferated immortalized chondrocytes in positive clones in micro carrier rotary cell culture system (RCCS) were screened and selected The growth of immortalized chondrocytes and the metabolic rate were observed Collagen type Ⅱ expression of immortalized chondrocytes of the experimental groups was observed The immunohistochemical results were compared with those of the control groups Results The immortalized chondrocytes in experimental groups could grow rapidly with a high metabolic rate and shorter population doubling time (PD) ( P