BACKGROUND: Small conductance,Ca2+-activated potassium(SK)channel,presents in various cell types and plays a crucial role in action potential profile.However,coupling and modulation of calcium and associated molecules to SK2 channel remains unclear.OBJECTIVE: To construct the recombinants of pGBAT7 and target fragments of SK2 gene,so as to observe the coupling and regulation of SK2 channel gene to calcium and other molecules.METHODS: Three pairs of primers of the target fragments of SK2 gene were designed and synthesized based on the full-length sequences of SK2.After being identified,they were individually sub-cloned into the yeast expressive plasmid pGBKT7 to construct pGBKT7-SK2 vectors.The recombinant pGBKT7-SK2 vectors were transformed into yeast AH109 by electroporation,and their activation was tested.The recombinants were extracted from yeast AH109 and verified by electrophoresis and sequencing.RESULTS AND CONCLUSION: The target fragments of SK2 gene by PCR were 411,546 and 729 bp,respectively.Three sub-clones of pGBKT7-SK2 were successfully constructed.Electrophoresis and sequencing showed that the constructed sub-clones of pGBKT7-SK2 met the expected requirements.The recombinant pGBKT7-SK2 vectors transformed into the yeast could be activated.The successful construction of the sub-clones of SK2 gene provides an important material basis for further study in the SK2 channel and function-associated molecules.

Objective To develop a method to prepare human articular cartilage derived microcarrier for both rapid propagating chondrocytes and being used as scaffold to support chondrogenesis. Methods Human articular cartilage was crushed into small pieces by muller after lyophilization, and sorted through two different meshes to collect only those specimens measuring 150-200 microns. Then, in turn, the specimens were subjected to 0.25% trypsin at 37 ℃ for 24 hours and 1% Triton X-100 for 72 hours, respectively. The specimens were observed by inverted phase contrast microscopy, and assessed by staining with haematoxylin-eosin, safranin-O (for GAG), as well as by the immunohistochemistry of aggrecan, collagen type Ⅱ. The microcarriers were seeded with human chondrocytes after being irradiated by 60Co. Results Using inverted phasecontrast microscope, the freezing-dry cartilage particles were observed as yellow, different shapes, and their surfaces were uneven, and with many pits. After treating with trypsin and Triton X-100, the microcarriers showed light yellow, without cartilage morphology. The microcarriers became flocculous or like a hairbrush, and the area of contacting surface significant increased. After culture with cartilage cell for 2 hours, lots of spherical chondrocytes adhered to the microcarriers. HE stain of section confirmed that the celluar constituents of the specimens were removed, the specimens stained weakly positive for GAG, negatively for aggrecan, and positively for collagen type Ⅱ, respectively. Conclusion The detergent and trypsin can remove the cellular constituents and knock out the aggrecan from human articular cartilage while maintaining collagen type Ⅱ and GAG, and made the cartilage pieces flocculous or hairbrush-like. The chondrocytes can be well maintained in human articular cartilage derived microcarriers. Human articular cartilage derived microcarriers were prepared successfullly.

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.