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The teaching contents of graduate medical developmental biology are rich and abstruse, which makes it insufficient to reach the goal of cultivating higher-quality graduate students by traditional teaching methods. In current years, we have constructed and practiced the modular teaching model for the course of graduate medical developmental biology. By dividing the teaching contents into models, including basic development principle, model organisms, hotspot of medicinedevelopmentalbiologyresearch and main technologies and methods, we aim to meet all needs of students, enhance the practicality of our course, improve students' innovation abilities and comprehensive quality, and efficiently increase the quality of our education.
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With the rapid development of biotechnology, we can change the trait of organism using transgenetic technology. In recent years, there are growing interests in the establishment of sperm mediated gene transfer (SMGT) technology as an effective and convenient method to produce transgenic animals. SMGT technology is a transgenetic method, which is easy in operation and does little harm to the cell compared with the other transgenetic methods. In this review, we expound the background, development, mechanism, operation and application of SMGT.
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Animals , Male , Animals, Genetically Modified , Gene Transfer Techniques , Genetic Engineering , Methods , Sperm Transport , Physiology , Spermatozoa , Metabolism , PhysiologyABSTRACT
BACKGROUND: NOV protein encoded by nephroblastoma overexpression gene(NOV) is IGF(insulin-like growth factor) -binding protein. What is its impact on human neural stem cell(hNSC) proliferation and differentiation?OBJECTIVE: To investigate the impacts of NOV protein on hNSCs proliferation and differentiation.DESIGN: A single factor analysis of variance experimental study using cells as subjectsSETTING: Department of histology and embryology, and department of neurobiology in a military medical university.MATERIALS: Study was conducted in the Department of Histology and Embryology of the Third Military Medical University of Chinese PLA. Subjects were hNSCs cultured from 10 to 14 weeks human embryo cerebral cortex.INTERVENTIONS: COS-7 cells were transfected by NOV gene recombined plasmid. COS-7 cell and COS-7 cell modified by NOV gene conditioned culture media(COS-CM and NOV-CM) were collected and reacted with the cultured HNSCs.MAIN OUTCOME MEASURES: hNSCs proliferation was detected by 3H-TdR scintillation analysis, and hNSCs differentiation was detected by immunocytochemistry and flow cytometer(FCM).RESULTS: Both COS-CM and NOV-CM could significant promote the intake of 3H-TdR by HNSCs, of which the 1/minute of NOV-CM group was significantly higher than that of COS-CM group(P ＜ 0.05), which indicated that NOV-CM contained component that could facilitate hNSCs proliferation, and moreover, there was certain dose-effect relationship in NOV-CM' s facilitation of cellular proliferation. The results of immunocytochemistry and FCM revealed that there were more NF-200 positive cells in NOV-CM group, while many glial fibrillary acidic protein positive cells could be seen in COS-CM group.CONCLUSION: NOV protein might have facilitative effects on hNSCs proliferation and differentiation into neurons.
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Objective To investigate the effect of estrogen on the pain score,c-Fos and substance P expressions in the dorsal horn of the spinal cord in the mice following formalin stimulation.Methods Fifteen C57/BL6 mice were randomized to 3 groups: control group(intact mice without estrogen treatment),OVX+V group(ovariectomized mice given vehicle) and OVX+E group(ovariectomized mice with subcutaneous injection of 2 ?g/d 17?-estradiol for 10 d).Pain score was used to assay the role of estrogen in affecting pain threshold in the mice following formalin injected into the right hind paw,and expressions of c-Fos and substance P in the dorsal horn of spinal cord(L3 to L5) in 2 h after injection of formalin was tested with immunohistochemisty to evaluate neuron activity and pain afferent fibers.Results Pain score was increased in ovariectomized mice following formalin stimulation,which was inhibited by estrogen especially in the early stage of secondary phase.The number of c-Fos-like immunoreactivity neuron(FLIN,P
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Objective To obtain eukaryotic expression vectors containing coding region of nephroblastoma overexpression gene (NOV) and detect its expression in COS-7 cells. Methods A 1 165-bp cDNA fragment was amplified from the total RNA of normal rat brain tissue by RT-PCR and cloned into eukaryotic expression vector pcDNA3.1/Myc-His(+)/lacZ. The cloned insert was identified by double digestion of the recombinant plasmid with restriction enzymes HindⅢ and BamHⅠ. The recombinant plasmid was transfected into COS-7 cells with liposome. The expression of NOV gene was detected by Western blotting and immunocytochemistry. Results Eukaryotic expression vectors containing 1 165 -bp coding region of NOV gene was constructed. COS-7 cells transfected with the recombinant plasmid expressed high level of NOV protein in cytoplasm. Conclusion That eukaryotic expression vectors containing coding region of NOV gene was constructed can provide a strong molecular tool for the studies of effect of NOV gene.
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Objective To observe the effects of fetal bovine serum(FBS) on differentiation of human neural stem cells (NSCs). Methods The effects of FBS with different concentrations on differentiation of human fetal NSCs were observed by cell culture, immunocytochemistry and flow cytometry. Results Human fetal NSCs could be induced to differentiate mainly three types of nerve system cells(neuron, astrocyte and oligodentrocyte). There were 80%～90% astrocytes of differentiated cells from human fetal NSCs with the concentration of 15% FBS induced. Conclusion Concentration dependent FBS in culture medium may have effect on the ratio of neurons to glial cells differentiated from human NSCs in vitro .
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Objective To observe the changes of the gene expressions at spinal cord injury site of rat after transplantation of BDNF genetically modified neural stem cells(NSCs) so as to provide basic data for the repair of spinal cord injury. Methods The Wistar rats were randomly divided into 4 groups: control group, operation group, NSCs transplantation group, BDNF NSCs transplantation group. Four time points(7 day, 1 month, 2 month, 3 month) were divided for each group. The expressions of ? galactosidase and BDNF, GFAP, NF 200 at the site of spinal cord injury were observed by cell transplantation, X gal histochemistry, immunocytochemistry, in situ hybridization, etc. Results After transplantation of BDNF genetically modified NSCs, some X gal positive cells were found at the sections of spinal cord injury. The expressions of BDNF were strong, especially at 1 week and 1 month post transplantation in transplantation group. The GFAP and NF 200 positive cells were also found at each time point in each group. Conclusion BDNF genetically modified NSCs can survive at the site of spinal cord injury and can strongly express BDNF, suggesting that BDNF genetically modified NSCs can be used as the material for the repair of spinal cord injury.
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Objective To investigate the nerve recanalization and the motor function of hind legs after transplantation of BDNF genetically modified neural stem cells(NSCs) at spinal cord injury site in rat. Methods After L4 spinal cord transection of rat, BDNF genetically modified NSCs were transplanted immediately. Retrograde HRP tracing through sciatic nerve were practiced at 1 week, 1 month, 2 month, 3 month after transplantation of BDNF genetically modified NSCs. The morphological changes were observed at section of spinal cord and the motor functions of both hind legs of rat were detected. Results The morphology of the injured spinal cord sections turned better. Retrograde HRP tracing through sciatic nerve showed some HRP positive neurons and nerve fibers at the site of near rostral end of the nearly injured part at one month after transplantation and increased with the time going by. Motor function of hind legs of rats recovered significantly in all transplantation groups. Conclusion BDNF genetically modified NSCs have repairing effect on spinal cord injury in rat.