The Research Of The Relations Between Apoptosis And Fas/Daxx/Ask1/p38/nNOS Pathway In SMA Cell Model

The Research of The relations between Apoptosis and Fas/Daxx/Ask1/p38/nNOS Pathway in SMA Cell ModelSpinal muscular atrophy(SMA)is an autosomal recessive disease in humans that is characterized by degeneration of the motor neurons of the spinal cord anterior horn,and SMN gene is the virulence gene of SMA. SMN gene has two copies called SMN1 and SMN2,of which products are fl-SMN protein andΔ7-SMN protein.The dysfunction of SMN1 gene is the cause of SMA,and the quantity of fl-SMN protein determinate its clinical phenotype.The reduction of fl-SMN protein may induce the apoptosis of motor neurons.Motor neuron of SMA showed signs of apoptosis which mechanisms are unknown.Recently a specific motor neuron-related apoptosis pathway(Fas/Daxx/Ask1/p38/nNOS)were discovered,which is different from the classical pathway(Fas/FADD/ caspase8).No evidence was found in cells other than motoneurons. Motoneurons from transgenic mice overexpressing ALS-linked SOD1 mutants displayed increased susceptibility to activation of this pathway: they were more sensitive to Fas-triggered cell death but not to trophic deprivation or excitotoxic stimulation.If the motor neurons which lack of SMN protein are sensitive to this pathway,we assume that we can cure SMA by blocking the apoptosis pathway.In the specific neuron-related apoptosis pathway(Fas/Daxx/Ask1/ p38/nNOS),Daxx is a key factor.It play an important role in the transcriptional regulation and apoptosis by transfer from the nucleus to cytoplasm.In this research,the expression of SMN1 gene was inhibited in MSCs by RNAi,and then MSCs were differentiated to neuron-like cells which were called SMA cell model.To explore the apoptosis regulation of Daxx protein on SMA model cell,the subcellular localization of Daxx and the susceptibility of SMA model cell to the apoptosis pathway(Fas/ Daxx/Ask1/p38/nNOS).Chapter 1 The Establishment of SMA Experiment Cell Model by RNAiobjective:To construct the plasmid containing short hairpin RNA (shRNA)of SMN1 expression vector and to investigate the effect of shRNA to the expression of SMN1 gene in human mesenchymal stem cells(hMSCs).To establish SMA experiment cell model by blocking the expression of SMN1 gene with shRNA.Methods:The hMSCs come from the patients without SMA.Short chain oligonucleotide was designed according to the SMN1 mRNA sequence provided by Genscript,and then DNA segment was gained through annealing after chemosynthesis,and then was cloned to pRNATU6.2/Lenti vector.The recombinant SMN1 shRNA expression vector was evaluated by using enzyme cutting.At last,the constructed SMN1 expression vector was transfected into MSCs by lentivirus,and its effect on SMN1 expression was observed by RT-PCR and Western-blot.The pRNAT-SMN-1 vector which could inhibit the expression of SMN1 furtherest was choosed according to the experiment,and it was transfected into mesenchymal stem cells by lentivirus.The clones of transfected cells were selected in G418 medium,and they were differentiated to neuron-like cells.Immunocytochemical analysis was used to check the expression of NSE,MAP2 and GFAP on the neuronlike cells,and the expression of f1-SMN gene and protein were observed by RT-PCR and Western-blot analysis.Results:Successful construction was identified by enzyme cutting and the constructed plasmid was called pRNAT-SMN1.Expression of SMN1 mRNA and protein can be effectively inhibited by pRNAT-SMNI-1 and pRNAT-SMN1-3,of which the ratio of interference was 66.08%,65.15%on the level of mRNA,58.20%,56.30%on the level of protein.Compared with control groups,the differences showed the statistical significance(P＜0.05).After differentiation,NSE,MAP2 protein was detected on neuron-like cells and GFAP protein expression negative.After induction,the expression of f1-SMN,A7-SMN mRNA and protein of SMA model group was upregulated(P＜0.05),but the expression in pRNAT-SMN-1 group is less than that in control group (P＜0.05),and the differences have the statistical significations.The expression ofΔ7-SMN mRNA between the groups have no statistical difference(P＞0.05). Conclusion:The vector constructed by SMN1 shRNA can effectively block the expression of SMN1 mRNA and their protein in mesenchymal stem cells.The neuron like-cells,which were blocked the expression of SMN mRNA and protein,can be regarded as SMA experiment cell model.Chapter 2 The Susceptibility of SMA Model Cell to the Apoptosis Pathway(Fas /Daxx/Ask1/p38/nNOS)and Apoptosis Regulation of Daxx Protein on SMA Model CellSection 1 The Spontaneous Apoptosis in SMA Model Cell and the Expression and Subcellular Locatization of Daxx Protein Before and After the Intervention of Valproic AcidObjective:To investigate the spontaneous apoptosis in SMA experiment model cell and to study the expression and subcellular locatization of Daxx Protein before and after the intervention of valproic acid.To explore the apoptosis regulation role of Daxx on SMA model cell.Methods:By using SMA experiment model cell and normal neuron like cell as research target,and cells were divided into SMA model group, normal control group and VPA intervention group.①Cell proliferation was observed by drawing cellular growth curve;②The activity of cells was detected using the method of MTT spectrography;③Using FITC-Annexin V and PI double staining,apoptosis was detected by flow cytometry;④Immunofluorescence assay the subcellular localization of Daxx protein;⑤The position of fl-SMN protein and Daxx protein was detected by confocal microscopy;⑥The expression of fl-SMN,Daxx mRNA and protein was detected by RT-PCR and Western-blot.Results:①Cellular growth curve:Two negative proliferations were showed in all groups.During the phase of the second negative proliferations(at the 4th,5th and 6th day),the difference of cells numbers was showed between different time point(P＜0.05);the negative proliferation of cells in SMA model cell group was more obvious than normal NLCs group(P＜0.05),and the negative proliferation of cells was decreased after the intervention by VPA(P＜0.05);②MTT spectrography:Two downtrends of cellular activity was observed in all groups,and the difference of their MTT value had statistic significance (P＜0.05)at the 3rd,4th,5th,6th and 7th day in the second downtrend.In addition,the cellular activity of SMA model cell group was lower than normal NLCs(P＜0.05),but no difference after the intervention by VPA (P＞0.05);③Flow cytometry:The ratio of apoptosis in SMA experiment model group is higher than in normal control group(P＜0.05);After VPA intervention,the apoptosis of SMA model cell was significantly decreased(P＜0.05);④The expression of Daxx mRNA and protein in SMA model cell group were more than normal control group(P＜0.05). With the addition of VPA concentration,the expression of fl-SMN mRNA and protein in SMA model cell group tend to upregulate,and quantity-effect correlation was elucidated.The expression of Daxx mRNA and protein tend to downregulate with the concentration of VPA from 1μmol/ml to 7μmol/ml,and quantity-effect correlation was elucidated;⑤Immunofluorescence tests showed that Daxx protein mainly located in the nucleus in normal neuron-like cells and the SMN protein localized in the cytoplasm and nucleus;Daxx protein mainly located in the cytoplasm in SMA model cell group and SMN protein expression decreased;After the intervention of VPA,Daxx protein mainly localized in the nucleus and the SMN protein expression increased; Confocal microscopy showed that fl-Daxx SMN protein and Daxx protein interact in nuclei.Condnsion:①Daxx play a pro-apoptotic role in the SMA model cell;②Daxx protein and fl-SMN protein interact in the nuclei.③Valproic acid can increase the expression of fl-SMN transcription and protein by inhibiting expression of Daxx.Section 2 The Sensitivity of SMA Model Cells to Fas/Daxx/Ask1/ p38/nNOS Apoptosis PathwayObjective:To study the sensitivity of neurous which lack of SMN protein to Fas/Daxx/Ask1/p38/nNOS apoptosis pathway.Methods:By using SMA experiment model cell and normal neuron like cell as research target.①Anti-Fas antibody was added to SMA model cells and normal neuron-like cells at final concentrations between 0.01 and 100 ng/ml.Cell survival was measured using the MTT assay after 24,48 and 72 h.The ratio of apoptosis cells was investigated by Flow cytometry;②100 ng/ml anti-Fas antibody was added to the two cell groups,The expression of nNOS,Daxx,p-p38 protein was detectd by Western-blot;③SMA model cells were preincubated with or without nNOS inhibitor,p38 inhibitor and then treated with anti-Fas antibody. The ratio of apoptosis cells was investigated by Flow cytometry.Results:①With the addition of anti-Fas antibody concentration and with the extension of time,the activity of SMA model cell decreased significantly(P＜0.05),normal neuron-like cells decreased was not significant(P＞0.05).With the addition of anti-Fas antibody Concentration and with the extension of time,flow cytometry analysis showed that the apoptosis ratio of SMA model group cell was significantly increased (P＜0.05)and the increasing of normal neuron-like cells was not significant(P＞0.05);②In SMA model cells with 100 ng/ml anti-Fas antibody intervention,Daxx,p-p38 and nNOS protein expression levels were significantly higher than those without the intervention group (P＜0.05).In normal neuron-like cells with 100 ng/ ml anti-Fas antibody intervention,Daxx,p-p38 and nNOS protein expression did not change significantly(P＞0.05);③P38 inhibitor SB203580 and nNOS inhibitor 7 NI not only protect the apoptosis of the SMA model cells induced by anti-Fas antibody(P＜0.05),but also protect the spontaneous apoptosis of SMA model cells(P＜0.05).Conclusion:①Deficiencies of SMN protein may activate Fas/Daxx/Ask1/p38/nNOS apoptosis pathway;②Daxx may be cause the apoptosis of SMA cell by translocation from cytoplasm to nucleus and by Fas/Daxx/Ask1/p38/nNOS pathway.