| Objective: Our previous study has found that TPM4was changed after spinal cord transection (SCT) in SD rats. In this study, we detected the expression of TPM4on the cellular localization at the rostral of transected spinal cord, then explore the role of TPM4in neural plasticity following SCT.Methods: First of all, thirty two adult SD rats were randomly divided into the Normal controlled group and the Operation group. And the Operation group was subdivided into the3day,14day and28day post operation (dpo) with SCT.Each group was comprised with8SD rats (n=8). Q-PCR and Western Blot were employed to detect the expression level of mRNA and protein; immunofluorescence (IF) was used to localize TPM4in the spinal cord. TPM4overexpression and interference recombinant HIV lentivirus were synthesized to study the functional recovery in spinal cord SD rats. Thereafter, another32SD rats were randomly subdivided into shame,TPM4Control group, SCT+TPM4ORF group and TPM4SH group Each group was comprised with8SD rats (n=8). Then TPM4overexpression and interference recombinant HIV lentivirus were microinjected into the rostral of the transection spinal cord. BBB scores were employed to detect the posterior limb’s motor function of SCT rats. Western Blot was employed to detect the expression level of TPM4protein, and confirm the effect of TPM4ORF and TPM4SH.IF was used to determine the localization of TPM4in the spinal cord. And the TPM4ORF and TPM4SH recombinant HIV lentivirus were transfected to the primary cultured spinal cord neurons, respectively. Then images were taken before and at2and4days after transfection. Lastly the number, cell body area and neurite length were measured, respectively.Results:1.①Q-PCR showed a gradually increase on the level of TPM4mRNA expression was found at3,14and28dpo,compared to the normal group; and TPM4mRNA expression exhibits an significant increase at14dpo compared to the3dpo group(P=0.036<0.05),28dpo compared to the3dpo group (P=0.003<0.05); but not28dpo compared to the14dpo group,(P=0.326>0.05)②Western Blot showed a gradually increase level of TPM4protein at3,14and28dpo, compared to the normal group; and at14dpo compared to the3dpo group(P=0.006<0.05);but not at28dpo compared to the14dpo (P=1>0.05)③IF showed that TPM4expressed in both neurons and glial cells in the spinal cord tissue, but neurons are main cell types.2.①We successfully packaged TPM4ORF and TPM4SH HIV lentivirus. The recombinant TPM4ORF and TPM4SH HIV lentivirus were micro-injected in the rostral of the transection spinal cord, respectively. Both TPM4ORF and TPM4SH group showed a bit of higher increase of BBB scores compared to the TPM4Control group. There was no significant statistic difference at7d,14d and28dpo in the TPM4ORF compared to the TPM4Control group. Compared to the TPM4Control group, the TPM4SH group showed a significant increase in the BBB scores at14d (P=0.007<0.05),21d (P=0.000<0.05) and28dpo (P=0.000<0.05).②Western Blot result showed TPM4ORF group had a deep strip when compared to the TPM4Control group; and TPM4SH group had a light strip when compared to the TPM4Control group.③IF showed that TPM4expressed in neurons.3. Compared to TPM4Control group, both the TPM4ORF and TPM4SH group showed no significant changes of neurons number, cell body area and the axon length at2d after transfection (P>0.05).However, at4d after the transfection, there was a significantly increase in axon length in TPM4SH group compared to the TPM4Control group (P=0.019<0.05), while that is no significant statistic difference in terms of the neuronal number and the cell body area. Moreover,the cell body area and neuronal number were significant increase in TPM4ORF group compared to the TPM4Control group at4d after transfection (P<0.05) but the axon length showed no significant changes within the two groups(P>0.05).Conclusion:The recombinant lentivirus technique was successfully employed to detect the role of TPM4in neural plasticity following SCT, which indicated that TPM4may be involved in the repair of spinal cord injury.TPM4SH could stimulate the motor function recovery following SCT in SD rats. And the mechnism may involve in neurite out growth. This study provided a molecular evidence for clinical treatment of spinal cord injury in the future based on TPM4. |
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