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.

Objective To evaluate the characteristics of remodeling of Achillis tendon under different training methods, including the functional and structural changes in its insertion, and to extend these experimental findings to improve strategies of prevention of military training injuries. Methods With a special equipment to provoke running and jumping, adult rabbits were trained under different loads and the length of time, then the Achillis tendons were harvested. The tendons and their attachment were examined with light and scanning electron microscopy, as well as analyzed biomechanically. Results The findings showed that the structure of tendon became weak at the fourth week of excessive exercise, as evidenced by changes in micro-structures. Conclusion Excessive load is the major cause of enthesiopathy of the Achillis tendon, and training in cycles may have positive effects in prevention of the pathology.

Objective To discuss clinical significance on early diagnosis of brain injury in premature infants with multiple sequence joint inspection of magnetic resonance imaging (MRI).Methods The brain MRI findings of 160 premature infants treated by Neonatal Intensive Care Unit were analyzed retrospectively.Results In 160 premature infants,brain injury occurred in 76 cases,the incidence of brain injury was 47.5％.Ischemic lesions were seen more in brain injury in premature infants,cerebral white matter injury was the most common,especially periventricular leukomalacia.Ischemic brain injury performed patchy or large sheet increased signal intensity on T1-weighted images(T1 WI),decreased signal intensity on T2-weighted images (T2WI) and obviously increased signal intensity on diffusion weighted imaging (DWI) in half egg circle center and around the lateral ventricle.Periventricular leukomalacia performed patchy decreased signal intensity on T1WI,increased signal intensity on T2WI and decreased signal intensity on DWI.Periventricular-intraventricular hemorrhage was seen more in hemorrhagic lesions.Hemorrhage stove was performed different signal because of different bleeding time.MRI performance in acute phase was iso-signal or slightly decreased signal intensity on T1WI,increased signal intensity on T2WI,increased signal intensity on T1WI,slightly decreased signal intensity on T2WI in early subacute,increased signal intensity on T1 WI and T2WI in late subacute and obviously decreased signal intensity on magnetic sensitive weighted imaging.The detection rate of ischemic lesions by DWI was higher than the conventional MRI,and DWI could show cerebral white matter damage of premature infants much earlier than the conventional MRI.The detection rate of hemorrhage stove by susceptibility weighted imagingc (SWI) was higher than the conventional MRI (x2 =23.78,P ＜ 0.05),and SWI could show hemorrhagic lesions much earlier than conventional MRI (x2 =27.02,P ＜ 0.05).Conclusions MRI,especially combined multiple sequence checking,could provide accurate imaging evidence for the early diagnosis of brain injury in premature infants.

BACKGROUND:Radiotherapy alone is not suitable for tumor-caused vertebral fractures and neurological dysfunction. In recent years, 125I radiation particles have been widely used in a variety of primary or secondary tumors and achieved good results. Percutaneous kyphoplasty can restore vertebral height efficiently, remodel spinal stability, and relieve pain.
OBJECTIVE:To evaluate safety and effectiveness of percutaneous kyphoplasty combined with 125I in patients with metastatic spinal tumors.
METHODS:A retrospective study was conducted to review 30 cases of metastatic spinal tumors undergoing percutaneous kyphoplasty combined with 125I from March 2011 to July 2012. Symptoms, signs, and imaging findings were col ected and analyzed. Al the patients had a refractoriness back pain. CT scan showed osteolytic changes in the vertebrae. The visual analogue scales, WHO standards for pain relief and Owestry disability index were recorded to analyze the clinical symptoms outcome and recovery of neurological function, and the change of height in abnormal vertebrae was measured. The fol ow-up time was 1 day, 1 month and 6 months postoperatively. RESULTS AND CONCLUSION:Operations in al the 30 patients were done successful y. Al patients got a conspicuous pain relief in 24 hours after operation, and nospinal injury or compression was found. There were significant differences in scores of visual analogue scales, pain levels, Owestry disability index, and the height of vertebral bodies before and after operation (P<0.05). During postoperative fol ow-up of 1 and 6 months, scores of visual analogue scales, pain levels, Owestry disability index, and the height of vertebral bodies showed no difference from those at 24 hours postoperatively (P>0.05). Bone cement leakage occurred in the anterior longitudinal ligament (n=2) and intervertebral space (n=2), and no serious complications occurred. Percutaneous kyphoplasty combined with 125I is a safe and effective way to treat metastatic spinal tumors, which can quickly ease the pain caused by spinal tumor, recover the abnormal vertebral height, reduce complications and improve life quality of patients.

AIM: To investigate the expression of matrix metalloproteinases (MMPs), membrane type MMPs (MT-MMPs) and their inhibitors (TIMPs) in human primary cultured prostatic cells and malignant prostate cell lines.METHODS: Reverse transcription-polymerase chain reaction-based measurements of the mRNA levels of MMP-2, MMP-7, MMP-9, MT1-MMP, MT3-MMP, TIMP-1 and TIMP-2 in relation to the house-keeping gene glyceraldehydes phosphate dehydrogenase were performed in cancerous and non-cancerous prostatic tissue samples, in primary cell cultures of epithelial cells, in both fibroblasts and smooth-muscle cells as stromal cells, and in the human malignant prostatic cell lines DU-145, LNCaP and PC-3.RESULTS: MMP-2 was mainly expressed in stromal cells. MMP-7 and MMP-9 showed their highest values in epithelial cells. MT1-MMP, MT3-MMP, TIMP-1 and TIMP-2 were found both in stromal and in epithelial cells, but there were some differences between the expression in fibroblasts and smooth-muscle cells. Different expression was also observed between the cells deriving from the primary cell cultures, the benign cell line BPH-1, and the malignant cell lines LNCaP, DU-145, and PC-3.CONCLUSION: These results concerning different expression of MMPs and TIMPs in cells from prostatic tissue suggest that a better insight into changes observed in prostatic tissue needs studies on cells cultured from the tissue.

Objective To study magnetic resonance imaging(MRI) features of dysembryoplastic neuroepithelial tumor(DNT) and to improve accurate diagnosis of DNT.Methods The MRI appearance and clinical features of 10 patients with DNT confirmed by surgery and pathology were analyzed retrospectively.Results In 10 cases,9 tumors located in supratentorial hemisphere cortex,3 tumors located in the temporal lobe,5 in the frontal lobe,1 in the parietal lobe,and 2 of them encroached the adjacent white matter.In 9 tumors located in supratentorial hemisphere cortex,8 cases had decreased signal intensity on T1-weighted MR images,1 case iso-decreased mixed signal intensity on T1-weighted MR images,and 9 cases increased signal intensity on T2-weighted images,9 cases slightly increased signal intensity on fluid attenuated inversion recovery weighted images.The manifestation of tumors was cystic or cystic partially oriented and was seen separate section intratumoral in some cases.Three cases appeared as hyperintense ring sign and internal septation,2 cases appeared as a triangle in shape,3 cases appeared as gyms-like shape,and 1 case as round shape,similar to cyst.Nine tumors had no significant mass effect and peritumoral edema.Enhanced MR imaging showed only 1 case with slight and heterogeneous enhancement,the rest 6 cases showed non enhancement.One case located in cerebellar hemisphere,and appeared cystic-solid mass,the solid part had decreased signal intensity on T1-weighted MR images,and increased signal intensity on T2-weighted images,the cystic part had decreased signal intensity on T1-weighted MR images,and increased signal intensity on T2-weighted images.On enhanced MR imaging,the wall-node obviously contrast enhancement,cyst wall slightly contrast enhancement,cystic part non enhancement.The tumor had peritumoral edema and mass effect.Ten cases had no hemorrhage and calcification.Conclusion The MRI appearance of DNT is characteristic and is helpful for the preoperative diagnosis of DNT.

OBJECTIVE To investigate the prevalence of ?-lactamases in nosocomial infections of Enterobacter cloacae,study the different genotyping of ?-lactamases,and analyze its independence in drug resistance. METHODS AmpC ?-lactamases and ESBLs were detected by three-dimensional extract tests.PCR was used to determine the genotype of ?-lactamases and their resistance to 13 kinds of antibiotics was detected by Kirby-Bauer method. RESULTS The sensitivity test showed that they were resistant to the most antibiotics.Among the 80 ESBLs strains,29 strains carried ESBLs gene,3 strains carried two different gene.Such as:TEM existed in 6,SHV-Ⅰ existed in 2,CTX-M-2 existed in 5,CTX-M-3 existed in 8 and CTX-M-9 existed in 11 strains.Twenty-six strains carried ampC.The resistant rate of SSBLs-producing E.cloacae to 12 antibiotics except merapenem was 66.7-100.0%.SSBLs strains of E.cloacae were higher than AmpC ?-lactamases in resistance to levofloxacin,cefepime,aztronem and trimethoprim sulpfamethoxazole with significant difference(P

Objective: To investigate the molecular mechanism of down-regulation of monocarboxylic acid transporter 1 (MCT1) on the proliferation inhibition of glioma cell. Methods: siMCT1, siMCT4 and negative control siRNA were transfected into glioma cell lines including U-251 and U-87. The proliferation activities of U-251 and U-87 cells were detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay and clonogenic assay. Glucose consumption and lactic acid efflux of U-251 and U-87 cells were determined by spectrophotometry.Western blot was used to detect the expressions of MCT1, MCT4, human glucose transporter 1 (GLUT1), GLUT4, tuberous sclerosis associated protein (TSC2), p-TSC2, 4E binding protein 1 (4EBP1), p-4EBP1, ribosomal S6 protein kinase (S6) and p-S6 protein in U-251 and U-87 cells. Results: Compared with negative control group, siMCT1 and siMCT4 significantly inhibited the expressions of MCT1 and MCT4 protein in U-251 and U-87 cells (both P<0.05). However, only knockdown of MCT1, the proliferation activities of U-251 and U-87 cells significantly decreased (P<0.05). The clone formation rates of U-251 and U-87 cells decreased to (55.20±3.27)% and (68.33±4.58) %, respectively (P<0.05). The glucose consumption of U-251 and U-87 cells in the negative control group at 72 hours were (82.65±6.66) pmol/L and (63.33±5.27) pmol/L, respectively, significantly higher than (31.70±3.17) pmol/L and (26.41±3.19) pmol/L of the siMCT1 transfected group (P<0.05). The extracellular lactate flow of U-251 and U-87 cells in negative control group at 72 h were (155.49±8.15) mmol/L and (135.37±8.21) mmol/L, respectively, significantly higher than (42.69±4.66) mmol/L and (38.91±4.83) mmol/L of the siMCT1 transfected group (P<0.05). Western blot analysis showed that knockdown of MCT1 significantly decreased the protein levels of GLUT1 p-TSC2, p-4EBP1 and p-S6 in U-251 and U-87 cells. Conclusions Downregulation of MCT1 expression can inhibit the proliferation of glioma cells. Deletion of MCT1 inhibits the glycolysis and metabolism of glioma cells through regulating the mTOR signaling pathway.

Six atrazine-degrading strains, Pseudomonas spp. AD1, AD2, AD6, Agrobacterium sp. AD4, Xanthomonas AD5, and Erwinia sp. AD7, were isolated from industrial wastewater. These strains are able to grow on atrazine as sole nitrogen source. Strain AD1 is able to degrade atrazine of 0. 3g/L in minimal medium at a percentage of 99.9% in 72 hours. PCR products that are homologous to the atrazine chlorohydrolase gene atzA)from Pseudomonas sp. strain ADP were obtained by PCR method using total DNA of the strains AD1 ,AD4,AD5,AD6,and AD7 as templates.