Dissertation > Excellent graduate degree dissertation topics show

The Effects of Neuroserpin on the Neurons and Microglias Undergoing Hypoxia/Reoxygenation

Author: YangXueLian
Tutor: DongQiang;WangLiang;RenHuiMing
School: Fudan University
Course: Neurology
Keywords: Neurogenic serine protease inhibitor Tissue-type plasminogen activator Hypoxia and reoxygenation model Microglia Conditioned medium
CLC: R741
Type: PhD thesis
Year: 2009
Downloads: 294
Quote: 1
Read: Download Dissertation

Abstract


The first part, Neuroserpin and tPA in the dynamic expression of neurons in different time periods of hypoxia and reoxygenation purpose: to study neurons under normal conditions of hypoxia and reoxygenation synthesis neurogenic serine protease inhibitor (Neuroserpin, the NSP) and tissue-type amount of plasminogen activator (Tissue plasminogen activator, tPA) and their dynamic changes during hypoxia and reoxygenation. Methods: In vitro primary cultured rat cortical neurons and identified. Hypoxia and reoxygenation (hypoxia / reoxygenation, H / R) model. Use double immunofluorescence staining, Western-blot assay neurons in the amount of the release of oxygen, glucose deprivation (Oxygen-glucose deprivation, OGD) 1.5h and different reoxygenation periods NSP under the same conditions using an ELISA assay release tPA content to observe the dynamic changes in their expression. Results: 1, hypoxia for 90 min, 6 h after reoxygenation, swelling of the cell bodies of neurons, MAP-2 immunofluorescence staining was beaded changed or broken part of the axon, live cell counting kit (Cell CountingKit-8 , CCK-8) The results showed that the survival rate of neurons decreased to normal training for about 50% of the state. 2, immunofluorescence staining was observed in normal cultured neurons visible expression of NSP; neurons after hypoxia-reoxygenation injury still visible expression of the NSP. Western blot results further: normal cultured neurons there are higher levels of NSP protein expression, OGD 1.5h protein expression after mild increase, peaked at 6 h of reoxygenation (before hypoxia levels 3. 29 times), the difference was statistically significant (P <0.05). After the NSP protein expression level of slow decline, the basic back before hypoxia reoxygenation 24h. 3, ELISA results showed that: normal neurons tPA content basic undetectable OGD1.5h, cells can express a small amount of tPA (P <0.05). With the increase in the time of reoxygenation, tPA, there is an increasing trend. The reoxygenation 6h tPA content hypoxia significantly improved (P <0.01), followed by the content gradually reduce reoxygenation 24h basically back to the level of OGD1.5h reoxygenation 0h. Conclusion: neurons during hypoxia and reoxygenation model. NSP and tPA expression in neurons during hypoxia and reoxygenation injury volume has significantly increased, and both basic dynamic changes. The second part, Neuroserpin purpose of a protective effect in neurons during hypoxia and reoxygenation injury: observation tPA different conditions of in vitro cultured neuronal injury, and explore the NSP tPA can alleviate neuronal damage. Methods: In vitro primary cultured rat cortical neurons and identified. Hypoxia and reoxygenation (hypoxia / reoxygenation, H / R) model. First detection of role of tPA neuronal damage. 1, different concentrations of tPA intervention in normal neurons at different times: random cells into normal group, of PBS intervention group, and tPA intervention group, respectively, to intervene in the neurons at different times (0.5h, 1h, 4h, 6h, 8h and 124h). 2, tPA before oxygen and glucose deprivation intervention neurons: cells were divided into normal group, H / R group (OGD1.5h reoxygenation 4h), PBS H / R group, tPA H / R group. That PBS and tPA the OGD1.5h intervention neurons 4 hours after reoxygenation 4h. 3, tPA after oxygen and glucose deprivation intervention neurons: cells were randomly divided into the H / R group (OGD1.5h reoxygenation group), H / R ± PBS group, H / R tPA group. PBS and tPA OGD1.5h added, and then reoxygenation 0.5h, 1h, 4h, 6h. tPA is divided into four different concentrations (final concentrations were 0.01,0.05,0.075,0.1 μg / μL). Were detected by CCK-8 cell viability in each group. PBS H / R followed by the detection of NSP neurons: 1, NSP intervention before oxygen and glucose deprivation neurons: the H / R group (OGD1.5h reoxygenation 4h) cells were randomly divided into control group (PBS intervention cells hours after OGD1.5h reoxygenation 4h), tPA H / R group (tPA intervention the 4h after OGD1.5h reoxygenation 4h), the NSP of tPA H / R group (NSP pretreatment normal neurons 0.5h plus tPA common culture 4h, then OGD1.5h of reoxygenation 4h). MAP-2 staining under a fluorescence microscope to directly observe the changes in neuronal morphology, CCK-8 assay cell viability, LDH method determination of the degree of cell damage and Tunel staining (DAB) Determination of apoptosis rate. 2, NSP intervention neurons after oxygen and glucose deprivation: the cells into the H / R group (OGD1.5h reoxygenation 4h), H / R PBS group (OGD1.5h instantly added the PBS after reoxygenation 4h), H / After the R tPA group instantly added tPA (OGD1.5h reoxygenation cultured 4h), H / R tPA the NSP group instantly added tPA (OGD1.5h intervention 0.5h, then add the NSP co-cultured 4h) / R NSP group (OGD1.5h instantly joined NSP reoxygenation culture 4h) detection indicators with the first part. Results: 1, tPA normal conditions in cultured neurons has a toxic effect, the survival rate trends change over time, and the intervention time role with the intervention in the drug concentration. 2, tPA before oxygen and glucose deprivation intervention The neurons will aggravate neuronal damage. 3, tPA after oxygen and glucose deprivation interfere with the neurons, the cell survival rate is still the trend of change over time, and the intervention time role with the intervention in the drug concentration. That tPA cells hypoxia reoxygenation injury will increase, and the with tPA increased concentration and reoxygenation time extension, this damage will increase. 4, NSP neurons can inhibit tPA intervention before or after oxygen and glucose deprivation. Conclusion: of tPA on cultured normal nerve yuan toxic effects, cell viability and tPA dose and duration of the intervention was a negative correlation. tPA can increase the dose and duration of OGD-complex sugar reoxygenation neuronal damage, cell viability remains with tPA intervention was negatively correlated. NSP under different conditions can reduce toxic injury tPA neurons play a role to protect neurons. The third part, the role of microglia Neuroserpin intervention neurons, Neuroserpin microglia hypoxia reoxygenation model purpose: To observe the tissue-type plasminogen activator (tissue plasminogen activator, tPA role) of the different conditions in vitro cultured microglia (Microglia, MG). Explore neurogenic serine protease inhibitor (Neuroserpin, NSP) tPA can change the impact of the MG. Methods: In vitro primary cultured rat cortical microglial cells and identified. Hypoxia and reoxygenation (hypoxia / reoxygenation, H / R) model. The first to use the ELISA kit microglia release tPA content in hypoxia reoxygenation state itself. Secondly role of Neuroserpin hypoxia and reoxygenation in microglia, were divided into three groups: the first group hypoxia reoxygenation intervention group: divided into normal group, and at different times of the PBS control group OGD3h reoxygenation group ( reoxygenation 1h, 3h, 5h, 7h and 24h); second-largest group for tPA intervention group: divided into the normal group, the PBS control group, different concentrations of tPA group (final concentration of 0.01,0.025,0.05 , 0.075 and 0.1μg/μl for intervention 24h); third largest group of NSP pretreatment group: divided into normal group, the PBS control group, tPA group (final concentration 0.05μg/μl of tPA intervention 24h) NSP tPA group (final concentration 0.025μg/μl NSP pretreatment 0.5h final concentration of tPA 0.05μg/μl intervention 24h) of OGD3h group, tPA OGD3h group (final concentration of 0.05μg/μl the tPA intervention the 24h after OGD3h), NSP tPA OGD3h group the final concentration 0.051μg/μl of tPA (NSP pretreatment 0.5h intervention 24h, OGD3h). Respectively using inverted phase contrast microscope morphological changes were observed directly, CCK-8 assay cell proliferation rate in each group, IL-1β and NO content was measured by ELISA kit. Results: MG H / R synthesis of tPA, OGD3h reoxygenation 3h, release. 2, H / R MG activation, increased release of inflammatory cytokines IL-1β and NO. 3, of tPA intervention MG cell activation and proliferation, IL-1β and NO in the increased release. 4, each NSP pretreatment group cell activation, increased proliferation and inflammatory cytokine release are not obvious the tPA or H / R intervention group. Conclusion: MG synthesis in the H / R tPA. Early stage H / R-induced activation of the MG. The tPA may further induce the proliferation and activation of the MG. H / R and tPA-induced release of IL-1β and NO. The NSP can alleviate the activation and proliferation of MG caused by hypoxia and reoxygenation and tPA intervention, and can reduce the release of of microglial cells IL-1β and NO. The microglia cell conditioned medium on neurons Objective: To investigate to NSP whether by affecting the microglial cells and protective effect of nerve yuan. Methods: In vitro primary cultured rat cortical neurons and microglia, and they were identified. Respectively hypoxia and reoxygenation (hypoxia / reoxygenation, H / R) model. Preparation microglia cell conditioned culture medium (conditioned medium, CM), divided into 1, without any intervention microglia conditioned medium (N-CM); 2, PBS intervention microglial cell conditioned medium ( PBS-CM); OGD3h reoxygenation 1h, 3h, 5h, 7h, 24h stimulate resulting conditioned medium (H / R-CM); 4, a final concentration of 0.025μg/μL of the NSP pretreatment 0.5h after the liquid the hypoxia reoxygenation stimulation resulting conditioned medium (NSP H / R-CM); join different final concentrations of tPA (0.01,0025,0.05,0.075,0.1 μg / μL) stimulus from conditions medium (TPA-CM); 6, the final concentration of 0.025μg/μL NSP pretreatment 0.5h after adding tPA stimulation conditions obtained culture liquid (NSP TPA-CM). Immunofluorescence staining of MAP-2 marked morphological changes observed neurons, CCK-8 assay neuronal survival, LDH release test for the determination of cytotoxic cells was determined KGI living cells / apoptotic cells / necrotic cell identification kit The rate of apoptosis and necrosis rate and Tunel kit (DAB staining) the determination of the rate of apoptosis. Results: the hypoxia reoxygenation group made microglia conditions is strong damage to the culture medium for neurons, mainly caused by cell necrosis. When OGD3h when this injury has been very obvious, and with the time extension of reoxygenation Injury increase (P <0.05). The tPA group made microglia conditioned medium liquid neurons injury induces apoptosis, the final concentration when 0.01μg/μL Injury lighter, but with tPA concentration increases, damage The role was significantly increased (P <0.05). The preconditioning group cell damage and various NSP NSP group of reduce (P <0.05). Conclusion: hypoxia reoxygenation or tPA intervention after preparation the microglial cell conditioned medium damage to neurons have NSP pretreatment can reduce these injuries. The fourth part Neuroserpin protective mechanism of Objective: To observe the the microglial activation signal transduction pathways, explore the possible mechanisms NSP tPA inhibition of microglia and produce neuroprotective effects. Methods: In vitro primary cultured rat cortical microglial cells and identified, the establishment of the OGD model. The cells were randomly divided into: normal group, tPA group (final concentration 0.05μg/μL, interfering with cell 24h) the OGD3h group, NSP tPA group (0.025μg/μL NSP pretreatment 0.5h 0.05μg / μL of tPA intervention the 24h) the NSP OGD3h group (final concentration after 0.025μg/μL NSP pretreatment 0.5h OGD3h). Immunofluorescence double staining was detected in each intervention group MAPK pathway phosphorylated protein expression, Western-blot method further changes in the expression of proteins detected in each group phosphorylated MAPK pathway. Immunofluorescence double staining method to detect the intervention group NF-κB nuclear translocation. Results: Immunofluorescence double staining prompt microglial cells in the normal state, there is a small amount of ERK1 / 2, and P38 pathway protein phosphorylation, tPA and after OGD intervention, phosphorylation protein expression was significantly increased. Further use of the result of Western blot analysis: the tPA and OGD intervention group ERK1 / 2 and P38 pathway phosphorylation levels than normal group were significantly increased ERK1 / 2 phosphorylation levels more significantly increased (P <0.01). NSP pretreatment group phosphorylation level is lower than the intervention group. Microglial cells under normal and intervention status phosphorylation of both JNK pathway expression of the higher amount of protein, and further use of the result of Western blot analysis of protein phosphorylation in: tPA and OGD intervention after mild increase, NSP pretreatment The group decreased expression. Immunofluorescence double staining Tip: to normal microglia NF-κB is mainly present in the cytoplasm, in tPA and OGD intervention after, NF-κB nuclear translocation phenomenon occurs mainly expressed in the nucleus. NSP pretreatment group nuclear translocation of NF-κB and significantly reduced. Conclusion: Ras / ERK, JNK / SAPK and P38 pathways for microglia activation after tPA and OGD intervention played a certain role, which ERK1 / 2 and P38 play a more important role. the the tPA and OGD intervention microglia, NF-κB nuclear translocation occurs, this pathway is activated. NSP pretreatment intervention groups, various signaling pathways are different degrees of inhibition. Conclusion 1, NSP and tPA expression in hypoxia-reoxygenation injury volume has significantly increased, and both basic dynamic changes. Of tPA normal neurons in vitro toxic effects, tPA can aggravate the the OGD-complex sugar reoxygenation neuronal damage. 4, NSP reduce tPA toxic injury to the neurons play a role to protect neurons. 5, microglial cells in the H / R themselves can be synthesized tPA. 6, hypoxia and reoxygenation early that induce the activation of microglia. The tPA may further induce the proliferation and activation of the MG. Hypoxia reoxygenation and tPA can cause microglia IL-1β and NO release. 7, NSP can reduce the activation and proliferation of MG caused by hypoxia and reoxygenation and tPA intervention, and can reduce the release of the MG of IL-1β and NO. 8, anoxic and re the oxygen and tPA intervention made microglia cell conditioned medium damage to neurons have NSP can reduce these injuries. 9, MAPK pathway for the activation of microglia tPA and after OGD intervention, the role of ERK1 / 2 and P38 play a more important role. the the tPA and OGD intervention microglia, NF-κB nuclear translocation occurs, this pathway is activated. NSP pretreatment intervention groups, various signaling pathways are different degrees of inhibition.

Related Dissertations

  1. The Variations of MAPK Signaling Pathway in Microglia and the Effect of Catalpol,R363
  2. The Inhibitory Effects of Corilagin on Irradiated BV-2 Cells Inflammatory Response in Vitro,R730.5
  3. Influence of Mild Hypothermia Treatment on Serum tPA/PAI-1 Activity in Patients with Acute Massive Cerebral Infarction,R743.3
  4. Inducible nitric oxide synthase involved in the pathological process of Alzheimer's disease model mice,R749.16
  5. Effects of DHA Supplementation During Pregnancy on the Lps-induced Brain Microglia Activation and IL-1β Expression of Rat Pups,R742.3
  6. The Stem Cell Antigen Phenotype of Glial Cells,R329
  7. Regulation of Phenotype by Dibutyryl-cAMP in Irradiation-stimulated Murine BV2 Microglial Cells,R329
  8. Effect of Nao Xintong Capsule Manual on the Apoptosis of Neuronsandthe Inflammatory Reaction of Microglias After the Injuryof Acute Cerebral Ischemia Reperfusion in Rats,R285.5
  9. The Effect of Rosiglitazone on Biological Characteristics of the HSC and Approach to the Mechanism,R96
  10. Effects of Activated Retinal Microglia on Blood-retinal Barrier,R774.1
  11. The Effect of NGF on P75NTR Expression and Apoptosis of HSC in Microenvironment of Hepatocellular Carcinoma,R735.7
  12. The Mechanism of LPS Induced Injury in PC12 Cells and the Protective Effect of EPA,R742.5
  13. Expression and Significance of ICAM-1 in Inflammation of Nervous System,R392
  14. A Preliminary Study on the Role of TNF-α in Rats during Early Inflammaion,R364.5
  15. A Preliminary Study on β-1,4-GalT-I in CNS Inflammation,R392
  16. Synthesis and biological activity of resveratrol derivatives and analogues,R284
  17. The Research of DPSCs and PDLSCs’Microenvirnment Construction by Cell Sheet Engineering Techniques,R780.2
  18. Effects of CyclosporinA on Expression of OX-42 and INOS in Cerebral Ischemia Reperfusion Injury in Rats and Research of Mylin Protection,R741
  19. Effects of Recombinant Human Erythropoietin on Microglia and Inflammatory Factors of Rat Model of Parkinson’s Disease,R742.5
  20. Study of Advanced Glycation Endproducts on Microglia Activation and Inflammatory Expression in Vivo and Vitro Experiments,R741.02
  21. Research on the Establishment of Model of Microglial Activation and Effects of Piper Kadsura Ohwi,R741.02

CLC: > Medicine, health > Neurology and psychiatry > Neurology
© 2012 www.DissertationTopic.Net  Mobile