Study Of Neural Cell Apoptosis After Spinal Cord Injury And RNAi To Downregulated Caspase-3 Gene Expression

The treatment of Spinal Cord Injury is a very difficult problem .Now the basic research of spinal cord injury mainly lies in preventing secondary injury. To prevent neuron and glial cell death in the early spinal cord injury is an important therapeutic strategies. After the spinal cord injury, the nerve cell and the organization have necrosis and apoptosis. Apoptosis is a complex pathophysiological process, and the regulative mechanism to be extremely complex. Mainly has the proteinase cascade which the Caspase family members lies between leads to respond completes. It can be induced by promoting factor such as increased excitatory amino acid level, increased intracellular calcium ions level, and the presence of free radials or nitric oxide, and cell-secreted cytokines. These factors stimulate membrane receptor such as TNFR1, FAS/Apo1, DR3,DR4,DR5 et al and generate death signals. These signals can act via a caspase activation pathway that activated caspase cleave substrates important to cell preservation such as DNA maintenance enzyme inducing cell apoptosis. Caspase-3 is playing the key role, Caspase-3 can cut and activate Caspase-6, Caspase-7, Caspase-9. Caspase-3 itself receives Caspase-8,-9,-10 cut and activate.RNA interference (RNAi) is one block gene expression method which the recent years. It is by double strand RNA ( dsRNA) induces sequence gene silence, Lies between leads the sequence specificity mRNA degeneration. It is in the eukaryote one kind of extremely conservative mechanism. The RNAi exists naturally in biology and so on plant, fungus, fruit fly. The RNA disturbance technology has the specificity and the high efficiency, and it is a effective tool of the gene function research and the protein group's study. In medicine target gene screening, disease and so on prevention and treatment tumor, viral infection , it has provided the mentality.This research designed and synthesized siRNA to Caspase-3 gene. Studies the effect to block the Caspase-3 gene expression. Using RNAi effect to inhibit Caspase-3 expression and activeness. In order to lay the foundation of the research which the cell apoptosis, and discusses Caspase-3 the function and the influence which apoptosis in the neuron cell, and Caspase-3 and Fas the factor relations. To expectate for the inhibitory apoptosis of nerve cell, treats the spinal cord injury to provide one kind of new strategy. The study is composed of four parts:1. The Neuronal cell apoptosis and the expression of Caspase-3, Fas after spinal cord injury in rats, and the Caspase-3 , Fas correlation with apoptosis.2. The Experimental study in treatments of rats at nerve function improve and expression of apoptosis after spinal cord injury with sodium P-aescin.3. To investigate the effect of Caspase-3 inhibitor Z-DEVD-FMK on neural cell apoptosis and nerve function improve after spinal cord injury in rats.4. To investigate the inhibition effects of RNAi on Caspase-3 expression, designed Si-Caspase-3-3 and synthesized siRNAs against Caspase-3 gene, so as to provide a research tool for gene therapy on spinal cord injury.Part 1. Neural Cell Apoptosis and Significance Expression of Caspase-3, Fas after Spinal Cord Injury in Rats[Object]: To study the express of neural cell apoptosis and the changes of Caspase-3, Fas after spinal cord injury in rats.[Methods]: With improved Allen's method to make rats acute SCI at the level of T_9,10 in moderate degree. The study is divided into six groups including control group, injured 5 groups. The segments of injured spinal cord were cut for morphological studies at 6, 24, 48 hours and 7, 15 days after injury, including HE staining, Hoechst 33258 staining, and TLTNEL methods. The expression of caspase-3 was detected by immunohistochemical staining and RT-PCR. [Results]: After injury, TUNEL-positive celts were found in the compression region after 6h and mostly located in the gray matter. TUNEL-positive cells were present both in the gray matter and white matter with a maximum presence at injured 3d, Its decreased from 3d to 7d and positive cells mostly in the white matter. Fas increased at 6h and peaked at 3d .Caspase-3 mRNA increase at 6h and peaked at 48h after trauma, its decreased at 7d. The protein expression of caspase-3 by immunohistochemical staining is similar to the TUNEL time-course date.[Conclusion]: There were neuronal apoptosis after spinal cord injury, Caspase-3 mRNA and protein expressions are enhanced in combination with neuronal apoptosis. The expression of Caspase-3 have some certainty correlation with Fas' expression. Part 2. The Experimental Study in Treatments of Rats Spinal Cord Injury by Sodium β-Aescin[Objective]: To observe the manifold change of the spinal cord injured SD rat treatedwith Sodium β-Aescin and study the protection of β-Aescin to the nerve.[Methods]: The twenty-two SD rats were divided into three groups including controlgroup, injured group, cured group . The spinal cords of injured group were middle-degreeinjured with the improved Allen's method. It was observed that the change of nervefunctions . After executing the treated mice at twelfth day, the samples were examinedwith HE dyeing, immunohistochemical technique (bcl-2, bax, fas), Caspase-3 andTUNEL dyeing. Compare with differences and effect of every group.[Results]: The grade of nerve functions was improved distinctly in cured group. Thenumber of the positive cells for fas and bax in injured group was much more than one incured group (P<0.05) . The number of the positive cells for bcl-2 in injured group wasmuch less than one in cured group.[Conclusions]: Apoptosis was one important mode of neuron death after spinal cordinjury. Sodium β-Aescin could advance distinctly the comeback of the nerve function andprotect the nerve tissue. Part 3. Effect of Caspase-3 Inhibitor Z-DEVD-FMK on Cell Apoptosis after Spinal Cord Injury in Rats[Objective]: To investigate the effect of Caspase-3 inhibitor Z-DEVD-FMK on neural cell apoptosis and nerve function improve after spinal cord injury in rats.[Methods]: The models of acute spinal cord injury in 36 Sprauge-Dawley (SD) rats were made with the improved Allen's method to get middle-degree injured. The rats were divided into two groups including control group(injury without treatment), cured group(injury with Z-DEVD-FMK cured). After executing the treated rats at 6h,3d,8d, the samples were examined with HE dyeing, immunohistochemistry technique Fas and TUNEL dyeing and the expression of Caspase-3 by immunohistochemistry. The neuro functional of spinal cord was measured by BBB score and slope plate experiment.[Results]: The apoptosis cells positive for TUNEL and Caspase-3 expression were detectable in both two groups. In the cured group, the number of apoptotic cells and the express of Caspase-3 were decreased and the neurofunctional of the spinal cord improved as compared with those in the control group.[Conclusion]: Caspase-3 inhibitor Z-DEVD-FMK can reduce the numbers of apoptotic cells and promote the nerve function recovery after spinal cord injury. The two groups have distinctive difference at p<0.05.Part 4. Inhibition effects of RNAi on Caspase-3 gene expression[Objective] :To investigate the inhibition effects of RNAi on Caspase-3 expression, so as to provide a research tool for gene therapy on spinal cord injury.[Methods]: According to the gene sequence and the secondary structure characteristics of Caspase-3, designed three siRNA (Si-Caspase-3-1, Si-Caspase-3-2, Si-Caspase-3-3) with 21 basic group, synthesized siRNA against Caspase-3 gene. With the liposome conduct, transferred the synthesized siRNA into neuroglia cells. After the cells crept the slices, dye them with fluor Hoechst33258; the apoptotic cells were stained with TUNEL, controlling with the group of normal neuroglia cells, the Caspase-3 protease activity were detected with hemi-quantitative RT-PCR and Western blot. [Result]: The synthesized siRNAs could inhibit the express of gene Caspase-3. ForSi-Caspase-3-3, the expression inhibition rate of Caspase-3 mRNA was 86.32% inneuroglia cell. [Conclusion]: The synthesized siRNAs inhibit the expression of Caspase-3 gene whichprovide a tool for the research of the inhibition effect of RNAi on Caspase-3 activityand expression , and also for the study of the action of Caspase-3 in the process of cellapoptosis...