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BACKGROUND: Wnt signaling pathway plays an important regulative role in the embryonic development processes. Accordingly, it is of great significance to establish the cell model of Wnt signaling pathway so as to conduct study on it. OBJECTIVE: To establish Wnt signaling pathway cell model by transfecting L-M (TK-) cells with Wnt3a eukaryotic expression plasmid, and to investigate the effect of canonical Wnt signal pathway on the β-catenin subcellular distribution. METHODS: The eukaryotic expression plasmid pgk-Wnt3a-pcDNA3.0 after amplification was digested by restriction endonuclease first. Then it was transfected together with the control plasmid pgk-neo-pcDNA3.0 into L-M (TK-) cells via lipofection, after which the cell colony was screened by G418 for amplification. RT-PCR was used for detecting the expression products and the indirect immunofluorescence assay for observing the effect of Wnt3a on the β-catenin subcellular localization of L-M (TK-) cells. RESULTS AND CONCLUSION: The Wnt3a plasmid was verified by endonuclease digestion to have produced the expected plasmids after amplification. According to the RT-PCR detection to the 10 stably-transfected cell colonies achieved by 3 weeks of G418 screening, it was seen, on the L-Wnt3a cDNA, a strip of bright band of 320 bp in length, which showed that the products of amplification were exactly the expected fragments and that the Wnt3a plasmid was expressed on mRNA transcriptional level after being transfected with L-M (TK-) cells. In contrast, no expected band was found on the cDNA of L-M (TK-) calls transfecting the control plasmid. In addition, the immunofiuorescence assay detection showed that the protein expression of Wnt3a was found in the cytoplasm of the L-M(TK-) cells tranfecting Wnt3a plasmid, while for those transfecting the control plasmid, it was opposite. β-catenin, as showing by bright red fluorescence, was found to concentrate and enter into the nucleus of the L-M (TK-) cells transfecting Wnt3a plasmid, while for those transfecting the control plasmid, it was opposite. Cell model with continually activated Wnt signaling pathway is established. The stable expression of Wnt3a in L-M (TK-) cells transfected with pgk-Wnt3a-pcDNA3.0 is obtained. The expression of Wnt3a is able to promote the transfer of β-catenin from cytoplasms into nucleus in L-M (TK-) cells.
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Objective To analyses the magnetic resonance imaging(MRI)findings of different clinical patterns of neurosyphilis(NS).Methods Clinical records and MRI of 26 patients with NS were retrospectively studied.Results Abnormal MRI was found in 17 patients of 26 patients with NS.In 7 patients were with meningo-vascular syphilis,the MRI commonly showed multiple cerebral ischemia focus and cerebral infarction focus,very few similar to those of encephalitis;Six patients had general paresis,who presented cerebral MRI abnormalities of frontal and temporal atrophy,and few simultaneously with cerebral ischemia focus,granular apendymitis and hippocampus sclerosis;Three patients had syphilitic myelitis,their MRI showed mild tumefaction with multiple ischemic focus all the way through lower cervical spinal cord to lower thoracic spinal cord:One patient was with tabes dorsalis,whose cerebral MRI showed ischemic locus.Another 9 patients had normal MRI,of whom 4 patients with meningitis NS and 5 with tabes dorsalis.Conclusion The MRI of neurosyphilis has diverse presentations,and clinicians should pay much attention to it.
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<p><b>OBJECTIVE</b>To construct the recombinant plasmid containing human microdystrophin cDNA, and study the microdystrophin expression in vivo and in vitro.</p><p><b>METHODS</b>Microdystrophin cDNA was obtained from recombinant plasmid pBSK-MICRO digested with restrictive endonuclease Not I, the product was inserted into plasmid pVAX1, resulting in pAMICDYS. And then 3T3 cells were transfected with pAMICDYS. Forty-eight hours after transfection, the expression of the microdystrophin was detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. Finally, TA muscles of mdx mice were injected with the recombinant plasmid pAMICDYS through i.m. and the pathological change of TA was evaluated by histology, and the expression of microdystrophin in mdx TA was detected by immunohistochemical analysis.</p><p><b>RESULTS</b>The recombinant plasmid containing human microdystrophin cDNA was constructed successfully. The recombinant plasmid was proved to be able to express microdystrophin protein both in vivo and in vitro. Moreover, treatment of the TA of mdx mice with the recombinant plasmid could decrease the number of centrally nucleated myofibers.</p><p><b>CONCLUSION</b>Recombinant plasmid containing the microdystrophin gene was constructed successfully, and it could express microdystrophin protein both in vivo and in vitro. It provides basis for further study on microdystrophin as a target gene to treat Duchenne muscular dystrophy (DMD) by electrotransfer, i.v, arterial injection and combining with other exogenous gene to enhance microdystrophin expression.</p>
