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[Objective] To compare the biological characteristics of neuron-like cells from rat mesenchymal stem cells (MSC) induced by β-mercaptoethanol (BME) and basic fibroblast growth factor (bFGF) respectively. [Method] BME and bFGF were added to rat MSC respectively for 9 h and 5 days. The neuron-like cells from MSC with the neuron specific protein NF200 were identified using immunofluorescence. The gene expression of NF were analyzed using RT-PCR and the protein expression of NF200 were analyzed using Western blotting. [Results] MSC induced by BME appeared morphology change in an hour and the neurite can be seen at six hour. Some cell bodies became lighter and died. MSC induced by bFGF had neurite in the third day, and appeared network structure in the fifth day, then cells died in the seventh day. At the same time, The result of immunofluorescence showed the NF200 expression of neuon-like cells induced by two ways were beth positive, but MSC induced by bFGF were stronger than those induced by BME. The results of RT-PCR and western blotting were same as that of immunofluorescence. [Conclusion] Both bFGF and BME can induce rat MSC to neuron-like cells, but the neuon-like cells induced by bFGF were more like real neurons in morphology and expression of neuron specific protein and mRNA.
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Pdx-1, an important transcription factor highlighting in the early pancreatic development,islet functions and pancreatic disorders, needs to be more investigated in zebrafish, and siRNA is still seldom applied in zebrafish embryo-related research.Our aim was to explore the role of pdx-1 in pan-creatic development of zebrafish embryos by using siRNA approach. Microinjection, reverse tran-scriptase-PCR (RT-PCR), in situ hybridization and immunofluorescent staining were used in this re-search, and the morphology of the islet in normal zebrafish embryos, and in those treated with the siRNA specific to pdx-1 (siPDX-1) or siGFP was observed and compared. The expression of pdx-1 was detected in the stages of 1-cell, 2-cell, 4-cell, 8-cell, 16-cell, 16-hour by RT-PCT. The in situ hy-bridization and immunofluorescent staining results showed that siPDX-I disturbed the formation of the islet in zebrafish embryos. Pdx-1 played multiple roles in maintaining the phenotype of the islet during embryogenesis in zebrafish.
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Objective To investigate if the transcription factor Brn 3a can induce committedly ES cells into neurons. Methods pJ5 Brn 3a recombinant expression factors containing objective gene Brn 3a were introduced into mouse ES D3 cells by liposome mediated transfection. The Brn 3a transfected ES cells were cultured to undergo differentiation into neurons. Finally immunohistochemical technique was implied to detect the expression of Brn 3a before and after transfection and some specific marked antigens of mature neural cell and glia cell were detected after the ES cells differentiation into neuron like cells. Results 1 Before transfection ES cells do not express Brn 3a and the Brn 3a immunoreaction is negative. After transfection of Brn 3a gene 24?h the ES cells could be detected to have Brn 3a and the immunoreaction is positive.2 When this Brn 3a transfected ES cells were cultured whithin 1 week over 70% ES cells could differentiate into neuron like cells bearing obvious neurites. These neuron like cells express NF H+L 、 NSE、SY and do not express GFAP.Conclusion Brn 3a transcription factor can successfully induce ES cells to differentiate into neuron like cells. [
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AIM: To analyze neuron-related gene expression before and after mesenchymal stem cells (MSCs) differentiating into neuron-like cells. METHODS: MSCs were induced to neuron-like cells with ?-mercaptoethanol. Before inducement and at 8 h after inducement, the total RNA was extracted, then the expression of microtubule-associated protein-2 (MAP-2), growth-associated protein-43 (GAP-43), NSE, nestin and neurofilament (NF) mRNA were detected with RT-PCR. RESULTS: NSE mRNA expressed before and after inducement, MAP-2, GAP-43, nestin and NF mRNA only expressed after inducement. CONCLUSION: The differentiation of MSCs into neuron-like cells may be related to MAP-2, GAP-43, nestin and NF expression. [
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0.05). CONCLUSION: NPY could downregulate the expression of LDL-R in hVSMCs and the downregulation was blocked by benextramine.
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Objective To investigate the whole differentiating process of myogenic satellite cells in vitro,and the expression of N-ACh receptors.Methods Skeletal muscle satellite cells were isolated by trypsin digestion in vitro and purified by preferential attachment method.The cells were observed morphologically and identified by ?-sarcomeric actin,desmin and skeletal myosin antibodies.Contraction of myotubes was observed after adding ACh in the culture medium, N-AChRs in myotube were also be identified by bungarotoxin.Results Cultured satellite cells in vitro differentiated into myoblasts and fused to form myotubes later.These cells were positive by ?-sarcomeric actin, skeletal myosin and desmin antibodies' staining.Adding ACh directly in the medium stimulated the contraction of myotubes.No obvious expression of ACh receptors was observed on the surface of satellite cells.Cluster of receptors aggregated on myotubes from neonatal rats' satellite cells cultured in differentiation media.Conclusion The myotubes differentiated from myogenic satellite cells have the function of contraction.N-ACh receptors were synthesized gradually during the process of differentiation.
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Objective To study the effects of directional inducement of factors produced by injured sciatic nerve on the regenerating axons of adult frog in vivo. Methods Conditioned medium with fraction of 30kD to 100kD was obtained from sciatic nerve of adult frog and pumped out and formed a gradient of concentration toward the end of central nerve stump of cutaneous pectoris nerve with different distances and angles by miniosmotic pumps.After 28\|35 days,the chest tissue was dissected and stained with immunocytochemistry to show the regenerated motor\|axons and the directional value is analyzed. Results The regenerated motor axons grew obviously towards the tip of the cannula with high concentration of CM.The directional value is 0^84 whereas the value is 0^04 with random direction.The average directional value between the two groups is significant and is not influenced by the distances and angles of the cannula to the nerve stump. Conclusion Damaged peripheral nerve can produce diffusible active factors with molecular weight between 30kD to 100kD,which induce the directional outgrowth of regenerating motor axons from sectioned nerve stump of adult frogs in vivo. [
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Objective To study the relationship between the inhibitory effects that myelin membrane proteins exerted on nerve regeneration and the changes of intracellular cAMP levels in neurons. Methods To observe the effects of myelin membrane proteins extracted from central nervous system(CNS) by density centrifugation on the outgrowth of neurite of cerebella granule cells in culture and detect the changes of intracellular cAMP levels of the neurons with radioimmunoassay. Results 1^Myelin membrane proteins of CNS inhibited neurite outgrowth of cultured cerebella granule cells. 2^The cAMP level in neurons decreased in 5 minutes after contacting myelin membrane proteins and reached to the lowest level in 12 hours of contacting. Conclusion The inhibitory effects of myelin membrane proteins on the outgrowth of neurites may be related to the inhibitory factors which cause the decrease of cAMP level in neuron through the passway of signal transduction pathway. [
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Objective In previous experiment we proved that rat marrow stromal cells(MSCs) could be induced to differentiate to neuron\|like cells by ? mercaptoethanol and all\|trans\|retinoic acid(ATRA), but the neuron\|like cells showed apoptosis sooner after the differentiation.In this study, we used several sets of combinations of anti\|apoptotic reagents and inductive reagents to induce the differentiation of MSCs,to prevent the differentiated cells from apoptosis and to elongate the lifetime of the neuron\|like cells which might be use in clinic. Methods The MSCs were isolated primarily from bone marrow of adult rat,and purified by passage culture.The 5th passage of MSCs was induced by single or combinative induction,which included RA,Melatonin(10 -9 mol/L), bFGF,bFGF+RA,Melatonin+RA,Melatonin+bFGF.At 1?h,5?h and 24?h of induction, the ratio of neuron\|like cells and the numbers of survived neuron\|like cells among 6 groups were compared.In addition,immunocytochemical stainings with anti\|MAP\|2 and GFAP were also performed on cells of 5?h of induction. Results Neuron\|like cells were all MAP\|2 positive,but GFAP negative.The ratios of neuron\|like cells in MSCs at 1?h after induced by six combinations of reagents were 0 24,0 28,0,0,0 44,0 64;at 5?h were 0 56,0 28,0,0,0 76,0 83 and at 24?h were 0 10,0 27,0,0 14,0 35,0 59 respectively.The ratios of survived neuron\|like cells of differentiated cells at 24?h after induction by RA,Melatonin,Melatonin+RA and Melatonin+bFGF were 0 08,0 37,0 05,0 51 respectively.Conclusions\ 1.bFGF can't induce MSCs into neuron\|like cells independently.Moreover,it suppressed the differentiation effects of RA,while enhancing the effects of MT.2.MT can induce MSCs differentiating into neuron\|like cells and has the anti\|apoptotic effects on the induced neuron\|like cells.3.When MT and bFGF were added together,not only the ratio of neuron\|like cell in MSCs was increased, the lifetime of neuron\|like cells was also elongated.