The Research On The Neuron Protection Of SMN Protein By SMA Cell Model

Spinal muscular atrophy (SMA) is an autosomal recessive disease which affects motor neurons of spinal cord, and SMN gene is the virulence gene of SMA. SMN gene has two copies called SMN1 andSMN2, of which products are fl-SMN protein and△7-SMN protein. The dysfunction of SMN1 gene is the cause of SMA, and the quantity of fl-SMN protein determinate its clinical phenotype. We assumed that fl-SMN protein is related with the apoptosis process of motor neurons, that is to say, the reduction of fl-SMN protein may induce the apoptosis of motor neurons.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 effect of fl-SMN protein toαmotor neuron, the apoptosis of SMA cell model were investigated by a series of molecular biological methods.Chapter 1 The Effect Of shRNA to the Expression of SMN1 Gene in Human Mesenchymal Stem Cellsobjective: 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).Methods: The hMSCs come from the patients without SMA. Short chain oligonucleotide was designed according to the SMN1 mRNA sequence provided by Genebank, and then DNA segment was gained through annealing after chemosynthesis, and then was cloned to pRNAT-U6.1/Neo 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 Lipofectomine TM 2000, and its effect on SMN1 expression was observed by RT-PCR, Western-Blot, and flow cytometry.Results: Successful construction was identified by enzyme cutting and the constructed plasmid was called pshRNA-SMN1. Expression of SMN1 mRNA and protein can be effectively inhibited by pshRNA-SMN1-1 and pshRNA-SMN1-4, of which the ratio of interference was 64.87%,61.45% on the level of mRNA, 60.25%,54.05% on the level of protein and 96.00%,94.29% indicated by flow cytometry analysis. Compared with control groups, the differences showed the statistical significance (P＜0.05).Conclusion: The vector constructed by SMN1 shRNA can block the expression of SMN1 mRNA and their protein in mesenchymal stem cells. Chapter 2The Establishment of SMA Experiment Cell Model by RNAiObjective: To establish SMA experiment cell model by blocking the expression of SMN1 gene with shRNA.Methods: The pshRNA-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 Lipofectomine TM 2000. 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, NF on the neuron like cells, and the expression of fl-SMN gene and protein were observed by RT-PCR and Western-Blot analysis.Results: After differentiation, NSE, NF protein was detected on neuron like cells. After induction, the expression of fl-SMN,△7-SMN mRNA and protein of SMA model group was upregulated (P＜0.05), but the expression in PshRNA-SMN-1 group is less than that in control group (P＜0.05), and the differences have the statistical significations. The expression of△7-SMN mRNA between the groups have no statistical difference (P＞0.05).Conclusion: The neuron like cells, which recombinant SMN1 shRNA expression vector was transfected into, can be regarded as SMA experiment cell model. Chapter 3The Spontaneous Apoptosis in SMA Experiment Model Cell and the Effect of SMN ProteinObjective: To investigate the spontaneous apoptosis in SMA experiment model cell and the effect of SMN protein.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;③The ratio of apoptosis cells was investigated by Tunel fluorescent staining;④Using PE-Annexin V and 7-AAD double staining, apoptosis or necrosis was detected by flow cytometry;⑤The expression of Caspase-3 mRNA was detected by RT-PCR;⑥After intervention by different dose of VPA, the expression of Caspase-3 mRNA and fl-SMN mRNA was detected by RT-PCR;⑦The expression of Caspase-3 protein and fl-SMN protein was detected by Western-Blot.⑧The position of fl-SMN protein and Caspsase-3 protein was detected by confocal microscopy.Results:①Cellular growth curve: Two negative proliferations were showed in all groups. During the phase of the second negative proliferations (at the 4^th, 5^th and 6^th day), the difference of cells numbers was showed between different time point (principle effect of time: P＜0.05); the negative proliferation of cells in SMA model cell group was more obvious than normal NLC group (principle effect of RNAi: P＜0.05), and the negative proliferation of cells was decreased after the intervention by VPA (principle effect of 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 3^rd, 4^th, 5^th, 6^th and 7^th day in the second downtrend. In addition, the cellular activity of SMA model cell group was lower than normal NLC (P＜0.05), but no difference after the intervention by VPA (P＞0.05).③Tunel fluorescent staining: The ratio of apoptosis cells was (40.82±3.66)% in SMA experiment model groups, (31.69±2.53)% after VPA intervention, (22.98±2.04)% in normal control groups, (26.62±2.62)% after VPA intervention, and the difference in all groups had statistic significance (P<0.05).④Flow cytometry: The ratio of apoptosis was 41.39% in SMA experiment model group, 36.88% after VPA intervention group, 32.93% in normal control group, 34.67% after VPA intervention group and the difference in all groups had statistic significance (P＜0.05).⑤RT-PCR: The expression of Caspase-3 mRNA in SMA model cell group (0.85583±0.05241) was more than normal control group (0.14975±0.03093) (P＜0.05). After intervention by VPA, the expression of Caspase-3 mRNA in SMA model cell group tend to downregulate (0.61311±0.05093) (P＜0.05), but the expression in normal NLC group tend to upregulalte (0.21796±0.03354) (P＜0.05).⑥With 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 (R=0.9424, P=2.047e-31; R=0.9424, P=5.682e-30). The expression of Caspase-3 mRNA and protein tend to downregulate with the concentration of VPA from 1μmol/ml to 5μmol/ml, and quantity-effect correlation was elucidated (R= 0.959, P= 4.510e-21; R=0.942, P=8.651e-19). When the concentration of VPA exceed 5μmol/ml, the expression of Caspase-3 mRNA came back to unregulated, and hook-like curve was elucidated.⑦Confocal microscopy: The direct interaction between fl-SMN protein and Caspase-3 protein was not identified.Conclusion:①The process of apoptosis of SMA experiment model cell is earlier than normal neuron like cell, which may relate with the reduction of fl-SMN protein and the increase of Caspase-3 protein.②VPA can decrease the apoptosis of SMA experiment model cell at the certain dose range by increaseing the expression of fl-SMN protein an reducing the expression of Caspase-3 protein, which suggested SMN protein have the effect of neuron protection, but it is not the result of the direct interaction between fl-SMN protein and Caspase-3 protein.