Objective To investigate the expression pattern of hypoxia inducible factor-1?(HIF-1?) in fetal vertebra development of the mouse. Methods The development of mouse fetal vertebra was observed dynamically,and the expression of HIF-1? mRNA at various stages was also detected by reverse transcription-polymerase chain reaction. Results The cartilaginous spine column began to form at E13.5.The primary ossification center was observed at E15.5,then the osteogenesis expanded and extended to both sides.HIF-1? mRNA began to express at E13.5,and more significantly at E14.5(P

Objective To explore the expression of SP-A in surface active substance of sputum pulmonary of dysfunctional venti-latory weaning response patients and to study changes of expression of SP-A and oxygenation index ,arterial partial pressure of oxy-gen .Methods The mechanical ventilatory patients were divide into dysfunctional ventilatory weaning response group (group A) and easily weaning group(group B) ,then collected sputum from ventilators at different times :on the first day ,two and seven days after using ventilators .Used ELISA to test level of SP-A and oxygenation index ,arterial partial pressure of oxygen in sputum of group A and group B .Results For group A ,SP-A content oxygenation index and arterial partial pressure of oxygen were significantly less than those for group B(P<0 .05);after two days ,for group A ,SP-A content in sputum was significantly less than that on the first day ,and SP-A content in sputum was significantly less than that on the second day (P<0 .05);two days later ,oxygenation index and arterial partial pressure of oxygen were significantly lmore than those on the first day (P<0 .05) and there was no statistical difference in oxygenation index and arterial partial pressure of oxygen between the seventh day and the second day (P>0 .05) .Con-clusion Dysfunctional ventilatory weaning response and lung injury related to ventilator can be diagnosed early through the test of SP-A content in sputum ,which provides a brand-new way and method of treatment in dysfunctional ventilatory weaning response and lung injury related to ventilator .

OBJECTIVETo investigate the effects of RCCS on cell seeding onto 3-D scaffold and cell-scaffold composite culture in vitro.

METHODSRabbit skin fibroblasts of passage 2 were seeded at 2 x 10(6) cell per cm(3) onto/into polyglycolic acid (PGA) foams by static seeding (dropping a cell suspension onto foams) or dynamic seeding (rotating PGA foams and a cell suspension in RCCS). Attachment of cells in foams was observed by cell-counting after trypsin digestion. The effects of culture condition were next studied by culturing cell-PGA complexes in RCCS versus static culture condition. Distribution and proliferation of cells in foams were investigated with MTT, stereomicroscope and scan electron microscope.

RESULTSNumbers of cells adhering to polymers increased gradually during an initial period of 24 hours. Eight, 12 and 24 hours after seeding, the rates of adhering cells were significantly higher in the dynamic seeding group than in the static seeding group (46.70% + 2.16% vs. 31.50% +/- 3.54%; 56.36% +/- 3.18% vs. 34.28% +/- 3.16%; 66.32% +/- 4.60% vs. 37.38% +/- 4.66%; P < 0.01). The dynamic culture method as compared to the static method resulted in new tissues with a higher cellularity and more uniform cell distribution during a 3 period of weeks.

CONCLUSIONSRCCS has advantages of promoting cell attachment, uniform migration and proliferation in polymer scaffolds and can be used for construction of 3-D cell-polymer tissues in vitro.

Animals ; Cell Adhesion ; Cell Culture Techniques ; methods ; Cell Division ; Cell Movement ; Fibroblasts ; cytology ; Polyglycolic Acid ; pharmacology ; Rabbits ; Time Factors

OBJECTIVETo observe dynamically the development of fetal long bone and detect the expression and distribution of HIF-1alpha,to investigate the expression pattern and possible effects of hypoxia inducible factor-1alpha (HIF-1alpha) in fetal long bone development of mouse.

METHODSE12.5, E13.5, E14.5, E15.5, E16.5 and E17.5 pregnant C57BL6 mice were sacrificed. After sacrifice, the embryos were delivered by caesarean section. The development of fetal long bone was dynamically observed by stereoscopic microscope, and the distributional expression of HIF-1alpha protein was detected by using method of immunohistochemistry. The expression of HIF-1alpha mRNA and osteoblast marker gene at various stage were also detected by using methods of reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSThe cartilaginous long bone began to form and joints outline arised at E13.5, then the primary ossification center was observed at E14.5, showing opaque ossification under stereoscopic microscope,and then the osteogenesis expanded and extended to both sides. Immunohistochemistry demonstrated lots of HIF-1alpha protein positive chondrcytes in the center of primary ossification at E14.5, then they decreased dramatically. HIF-1alpha mRNA expressed at high level from E13.5 to E15.5, and then decreased to low level.

CONCLUSIONFetal long bone development pattern appeared to be endochondral osteogenisis process, existing hypoxia microenviroment may increase HIF-1alpha mRNA expression and thus initiate the cascade of endochondral osteogenisis.

Animals ; Bone Development ; Female ; Hypoxia-Inducible Factor 1, alpha Subunit ; analysis ; genetics ; physiology ; Immunohistochemistry ; Male ; Mice ; RNA, Messenger ; analysis

In this paper, based on the special influence of supramolecular chemistry on the basic theory of Chinese medicines ( CM) , the authors further analyzed the history of meridian tropism and natural origins of CM organisms and explained CM ingredients and the universal regularity of the automatic action of the supramolecular "imprinting templates" hole channel structure. After entering human bodies, CMs, as the aggregation of supramolecular "imprinting templates" , automatically seek supramolecular subjects that are matched with their "imprinting templates" in human meridians and organs for the purpose of self-recognition, self-organization, self-assembly and self-replication, so as to generate specific efficacy in meridians and organs, which is reflected as the meridian tropism phenomena at macro level. This regularity can be studied by in vitro and in vivo experimental studies. In vitro methods are mostly supra molecular structure analysis and kinetic and thermodynamic parameter calculation; Whereas in vivo methods are dominated by the analysis on object component distribution, chromatopharmacodynamic parameters and network chromatopharmacodynamic parameters; Particularly, the acupoint-medicine method can simplify to study the supramolecular subject-object relations. Consequently, CM's'meridian tropism reveals the universal regularity for interactions of macromolecular and micromolecular "imprinting templates" of subjects and objects in natural organisms. As the first barrier for the material base of the CM theory and breakthrough in the modernization of the basic CM theory, meridian tropism plays an important role in studies on basic theories of the basic CM theory.
Acupuncture Points ; Drug Therapy ; Drugs, Chinese Herbal ; chemistry ; Humans ; Medicine, Chinese Traditional ; Meridians

Objective To explore the expression and regulatory mechanism of hypoxia inducible factor-1?(HIF-1?)during fracture healing. Methods Mouse models of tibia fracture healing were established,and callus samples were collected 1,3,7,14,21 and 28 days after fracture.The development of callus and new bone formation were evaluated with roentgenology,Micro-CT and tetracycline double labeling method,and the expression of HIF-1?,vascular endothelial growth factor(VEGF),Runx2 and ALP in callus were detected with RT-PCR,Western blotting and immunohistochemistry.The relationship between HIF-1? and fracture healing was analysed. Results The expression of HIF-1? was detected in cells in the fracture sites as well as in evolved osteoblasts,chondrocytes and osteocytes in early callus under hypoxia.The highest expression rate of HIF-1? achieved on the 7th day after fracture,lasted for about 7 days,then decreased gradually,and returned to intact level on the 28th day after fracture.The expression tendency of VEGF resembled that of HIF-1?.Bone formation activity was more active in early callus,and the callus volume peaked on the 14th day after fracture and decreased gradually.The mineralization of callus mainly took place in the late healing period(14th to 28th day after fracture).Conclusion Cells involved in fracture healing are hypoxia-responsive cells,which express HIF-1?.HIF-1? can regulate cell state and function,and can promote angiogenesis so as to play a crucial role in fracture healing.

The paper aimed to elucidate the specific impact of supramolecular chemistry on the Chinese medicine theories (CMT) in their modernization, after had summarized up the research status of supramolecular chemistry and analyzed the possible supramolecular forms of Chinese medicine (CM), as well as considered the problems in modernization of CM theories. On comparison of the classical chemistry that delt with chemical bonds among atoms, the supramolecular chemistry was rather concerned with varietes of weak noncovalent bonds intermolecules, and reflected the macro-apparent chemical properties of each molecules, and was the most appropriate chemical theories to explain the CMT and microcosmic materials. The molecules in the human body and Chinese material medica (CMM) formed supramolecules by way of self-assembly, self-organization, self-recognition and self-replication, with themselves or with complexation, composition, chelation, inclusion, neutralization etc. Meridian and Zang-fu viscera in CMT might be a space channel structure continuously consisted of unique molecules cavity that was imprinted with the supramolecularly template inside and outside of cells, through which the molecules in CMM interacted with the meridian and Zang-fu viscera. When small molecules in human body imprinted with macromolecules in meridian and Zang-fu viscera, in other words, they migrated along within imprinting channels of meridian and Zang-fu viscera on behavior of "Qi chromatography" impulsed by the heart beat, finally showed up on macroscopic the anisotropy of tissue and organ, as described namely as visceral manifestation in Chinese medical science. When small molecules in CMM interacted with imprinting channel on meridian and Zang-fu viscera, the natural properties and efficacy regularities of CMM was reflected on macroscopic. Therefore, the special representation forms of basic CMT is based on the macroscopic expression of "Qi chromatography" abided by imprinting effect regularities, and on whether the imprinted template of small molecules matched with cavity template of macromolecules in meridian and Zang-fu viscera, only is the adequate representation of supramolecular chemistry for them. The CMM materials is the mixture including single molecules and supramolecules. The compatibility for CM prescriptions can significantly change the function rules. Therefore in the study of basic CMT, we should pay special attention to the laws of supramolecular chemistry. It is the most essential differences of the CMT from the modern medicine which established by the laws of single molecular theories.
Chemistry, Pharmaceutical ; Drug Therapy ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Humans ; Meridians ; Nanotechnology

The paper, based on the previous publication as special impact of Chinese medicine theories on supramolcular chemistry, aims to analyze the natural origination for the Chinese medicine and to explain the special impact of "Qi chromatography" reaction on "imprinting templates" in supramolcular host of human being with Chinese medicine, in order to reveal the CM's properties of "medical element" with "imprinting templates" autonomisation generally took place in natural supramolecules, and also to discover that the CM's pharmacology are satisfied with its own approaches different form western pharmacology. It was decided, for CM's pharmacology guided by CM's theories, to "Qi chromatography" relations between the CM's ingredient groups and the meridian zang-fu viscera. The supramolcular chemistry played an all-through role in procession of making macro-regularities and special presentation on behavior of "Qi chromatography" impulse owning to the matching action of all kinds of ingredients on the meridian zang-fu viscera with similar "imprinting templates". The CM's pharmacology were guided by CM's theories, owing to its interpretation of supramolecular chemistry. The pharmacology was achieved to construct up completely on base of classical chemical single molecular bonds whereas the CM's pharmacology be configured to big building by way of "imprinting templates" as multi-weak bonds among "supramolecular society". CM's pharmacology was supramolcular pharmacology dealt with "molecular society" on the base of western pharmacology, and employed to double research approaches both math-physical quantitative representation on macroscope and qualitative analyses in microscope.
Animals ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Humans ; Meridians ; Molecular Imprinting ; Qi

OBJECTIVETo study osteoblastic phenotype expression of New Zealand rabbit skin fibroblasts transfected with mouse core binding factor a1/osteoblast specific transplanting factor-2 gene (Cbfa1/Osf2).

METHODSCbfa1/Osf2 gene, engineered into eukaryotic expression vector pSG5, was introduced into New Zealand rabbit skin fibroblasts with catholyte liposomes-Lipofectamine 2000. Meanwhile, those transfected pSG5 and un-transfected were set the control groups. The expression of Cbfa1 gene, osteocalcin (OCN) gene, alkaline phosphatase (ALP) gene and pre-peptide 2 alpha gene of collagen type I were detected by RT-PCR assay. Cbfa1 protein was detected by Western-Blot assay, in-cell ALP activity by p-nitrophenyl phosphate (PNPP) assay and OCN content in the supernatant by radio-immunity method. The ossification nodules was detected by Alizarin-Red staining and scanning electron microscope.

RESULTSCbfa 1mRNA and Cbfa1 protein were expressed in New Zealand rabbit skin fibroblasts transfected with pSG5-Cbfa1/Osf2 from the first day to the fifth day, but they were not detected in the control groups. In the pSG5-Cbfa1/Osf2 transfected group, the expression of ALP gene and OCN gene were respectively induced from the third day and the forth day, pre-peptide 2 alpha gene of collagen type I was enhanced from the third day. From the sixth day, ALP activity greatly increased, OCN strongly secreted, and they were maintained at a high level for about 4 weeks, and the difference was significant compared with the control group (P < 0.05). On the forty-second day, ossification nodules were found on the surface of pSG5-Cbfa1/Osf2 gene transfected cells.

CONCLUSIONSNew Zealand rabbit skin fibroblasts transfected with pSG5-Cbfa1/Osf2 can express osteogenesis-related genes and proteins, and form ossification nodules on their surface.

Alkaline Phosphatase ; biosynthesis ; genetics ; Animals ; Cell Adhesion Molecules ; biosynthesis ; genetics ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit ; biosynthesis ; genetics ; Fibroblasts ; cytology ; physiology ; Gene Expression ; Genetic Vectors ; Mice ; Osteocalcin ; biosynthesis ; genetics ; Osteogenesis ; genetics ; physiology ; Rabbits ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVETo study the feasibility of osteogenic phenotype expression by human skin fibroblasts induced in polyglycolic acid (PGA) foams and the effect of tumor necrosis factor-alpha (TNF-alpha) on the expression of bone morphogenetic protein (BMP) receptors.

METHODSThe fibroblasts were isolated, purified from human skin. (1) Fibroblasts were seeded onto PGA foams. The cell-PGA complexes were cultured in RCCS for 6 weeks, in the media of TNF-alpha (50 U/ml) and BMP-2 (0.1 microg/ml). 1 d, 3 and 6 weeks later, cells and extracellular matrix were investigated by electron microscopic and histochemistry observation respectively. Secretion of osteogenic markers were analyzed by biochemical methods. (2) Fibroblasts were seeded on the glass fragments or culture flasks and treated with TNF-alpha (50 U/ml) in different usage (one-time, all-time). The RT-PCR method and the immunohistochemistry fluorescence staining were used to examine the influence of TNF-alpha on the mRNA expression and the protein expression of the type I BMP receptors at 2, 4, 6, 8 d after treatment.

RESULTSFibroblasts seeded on the PGA foams formed 3-dimensional matrix 3 weeks after seeding, which was demonstrated as osteo-tissue by tetracycline labeling and ARS staining. Cells secreted much more bone-specific alkaline phosphatase (B-AKP) and osteocalcin (OCN) into supernatant than the cells that were cultured in the media without TNF-a and BMP2. Eight days after all-time usage, the TNF-alpha (50 U/ml) increased the expression of the mRNA and protein of the type IB BMP receptor.

CONCLUSIONSFibroblasts on 3-D cell-foam structures can express osteoblastic phenotype under certain inducing conditions. The numerous fibroblasts in body would be a promising resource for cell seeds candidate of tissue- engineered bone. TNF-alpha provides the essential condition for BMP2's target effect on fibroblasts, and combined use of TNF-alpha and BMP2 is one of the regulating factors.

Bone Morphogenetic Protein 2 ; Bone Morphogenetic Protein Receptors, Type I ; biosynthesis ; genetics ; Bone Morphogenetic Protein Receptors, Type II ; biosynthesis ; genetics ; Bone Morphogenetic Proteins ; pharmacology ; Cell Culture Techniques ; methods ; Cell Differentiation ; drug effects ; Cells, Cultured ; Drug Synergism ; Fibroblasts ; cytology ; drug effects ; metabolism ; Humans ; Phenotype ; Polyglycolic Acid ; Transforming Growth Factor beta ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology