Transplantation of human telomerase reverse transcriptase gene-transfected Schwann cells for repairing spinal cord injury

Abstract

Transfection of the human telomerase reverse transcriptase (hTERT) gene has been shown to increase cell proliferation and enhance tissue repair. In the present study, hTERT was transfected into rat Schwann cells. A rat model of acute spinal cord injury was established by the modified free-falling method. Retrovirus PLXSN was injected at the site of spinal cord injury as a vector to mediate hTERT gene-transfected Schwann cells (1 × 10(10)/L; 10 μL) or Schwann cells (1 × 10(10)/L; 10 μL) without hTERT gene transfection. Between 1 and 4 weeks after model establishment, motor function of the lower limb improved in the hTERT-transfected group compared with the group with non-transfected Schwann cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and reverse transcription-polymerase chain reaction results revealed that the number of apoptotic cells, and gene expression of aquaporin 4/9 and matrix metalloproteinase 9/2 decreased at the site of injury in both groups; however, the effect improved in the hTERT-transfected group compared with the Schwann cells without hTERT transfection group. Hematoxylin and eosin staining, PKH26 fluorescent labeling, and electrophysiological testing demonstrated that compared with the non-transfected group, spinal cord cavity and motor and sensory evoked potential latencies were reduced, while the number of PKH26-positive cells and the motor and sensory evoked potential amplitude increased at the site of injury in the hTERT-transfected group. These findings suggest that transplantation of hTERT gene-transfected Schwann cells repairs the structure and function of the injured spinal cord.

Keywords: Schwann cells; cells; modification; motor function; nerve regeneration; neural regeneration; proliferation; reverse transcriptase; spinal cord injury; telomerase; transplantation.

Figure 1

Identification of Schwann cells 4 days after the primary culture. (A) Inverted microscope image (40× magnification) of the morphology of primary cultured Schwann cells; cells are spindle in shape (arrow). Immunofluorescence staining showing (B) nestin-positive Schwann cells (arrow) where bodies and processes of Schwann cells are labelled green (40× magnification) and (C) PKH-26-labeled Schwann cells (stained red and indicated by arrow; 100× magnification).

Figure 2

Protein expression of hTERT in Schwann cells 48 hours after transfection (western blot assay). Protein expression for hTERT is noticeably higher in the hTERT-transfected group compared with the EV group and in the group without hTERT transfection of Schwann cells. hTERT: Human telomerase reverse transcriptase; GAPDH: glyceraldehyde phosphate dehydrogenase; EV: empty virus transfection; I: EV group; II: Schwann cells without hTERT transfection group; III: hTERT transfection group.

Figure 3

Effect of the transplantation of hTERT gene-transfected Schwann cells at the site of injury on cell proliferation. Between 2 and 5 days from the time of culturing, the proliferation of Schwann cells was significantly (P < 0.05) higher in the hTERT-transfected group compared with the Schwann cells without hTERT transfection group, which was markedly (P < 0.05) higher than the SCI group (n = 6; paired comparison; one-way analysis of variance with the least significant difference test). hTERT: Human telomerase reverse transcriptase; EV: empty virus transfection; d: day(s); SCs group: Schwann cells without hTERT transfection.

Figure 4

Effect of the transplantation of hTERT-transfected Schwann cells at the site of injury on cell apoptosis (TUNEL assay, fluorescence microscope). Specific brown particles were seen in the nuclei of apoptotic nerve cells (arrow; 200× magnification). Compared with the SCI group, the apoptotic rate was significantly (P < 0.05) less in both the Schwann cells without hTERT transfection group and the hTER-transfected group, with the latter group exhibiting the lowest rate (P < 0.05) (n = 5; paired comparison; one-way analysis of variance with the least significant difference test). hTERT: Human telomerase reverse transcriptase; SCI: spinal cord injury; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; SCs group: Schwann cells without hTERT transfection.

Figure 5

Effect of the transplantation of hTERT-transfected Schwann cells at the site of injury on mRNA expression of AQP4/9 and MMP9/2. AQP4/9 and MMP9/2 mRNA expression was lower in the SCs group compared with the SCI group. AQP4/9 and MMP9/2 mRNA expression (/GAPDH optical density) is the lowest in the hTERT-transfected group followed by the SCs group, and the highest in the SCI group (n = 6; one-way analysis of variance followed by the least significant difference test; P < 0.05). AQP: Aquaporin; GAPDH: glyceraldehyde phosphate dehydrogenase; hTERT: human telomerase reverse transcriptase; MMP: matrix metalloproteinases; SCI: spinal cord injury; SCs group: Schwann cells without hTERT transfection group.

Figure 6

Morphology of nerve cells (arrows) in the rat spinal cord after transplantation with hTERT-transfected Schwann cells. Hematoxylin and eosin staining reveals spinal cord loss and cavitation 4 weeks after injury. The cavities are small in the SCs group, and no cavities are found in the hTERT-transfected group (upper panels; 40× magnification). Immunofluorescence staining shows that the number of PKH-26-positive cells is the highest in the hTERT-transfected group followed by the SCs, and the lowest in the SCI group (lower panels; 200× magnification). hTERT: Human telomerase reverse transcriptase; SCI: spinal cord injury. SCs group: Schwann cells without hTERT transfection group.