Construction and expression of adeno-associated virus type-2 vector carrying human telomerase reverse transcriptase in human nucleus pulposus cells / 中国组织工程研究

ABSTRACT

BACKGROUND:In animal experiments, transplantation of autologous nucleus pulposus cellscan effectively repair the intervertebral disk degeneration. However, nucleus pulposus cells have a poor ability of proliferation in vitro, which limits its application as seed cells in treatment of intervertebral disk disease. OBJECTIVE:To construct recombinant adeno-associated virus type-2 vector carrying human telomerase reverse transcriptase and observe the human telomerase reverse transcriptase mRNA expression in human nucleus pulposus cells in vitro. METHODS:After the plasmid pSNAV2.0-pRSV-hTERT was constructed and identified, recombinant adeno-associated virus type-2 vector carrying human telomerase reverse transcriptase were constructed, amplified and purified by AAVMaxTM package and purification system. The optimal multiplicity of infection for human nucleus pulposus cells was detected by recombinant adeno-associated virus type-2 vector carrying enhanced green fluorescent protein. According the optimal multiplicity of infection (5 × 104 v·g/cell), three different multiplicity of infection (1×104, 5×104, 1×105 v·g/cell) of recombinant adeno-associated virus type-2 vector carrying human telomerase reverse transcriptase were determined to transfect the first passage human nucleus pulposus cells in vitro. In control group, the cells were transfected with adeno-associated virus type-2 vector without human telomerase reverse transcriptase. At 1, 2, 4 weeks after transfection, mRNA expression of human telomerase reverse transcriptase in human nucleus pulposus cells were semi-quantitatively detected by RT-PCR. RESULTS AND CONCLUSION:The recombinant adeno-associated virus type-2 vector carrying human telomerase reverse transcriptase was successful y constructed, and the titer of the obtained vector was more than 2×1011 v·g/mL. The optimal multiplicity of infection was 5×104 v·g/cell. The mRNA expression of human telomerase reverse transcriptase in human nucleus pulposus cells could be detected in different multiplicity of infection (1×104, 5×104, 1×105 v·g/cell). At 2 weeks post-transfection, mRNA expression of human nucleus pulposus cells was the highest (P<0.05), as detected by semi-quantitative RT-PCR. Moreover, the stable and high mRNA expression of human telomerase reverse transcriptase could be detected at 4 weeks post-transfection. In control group, no human telomerase reverse transcriptase mRNA expression was found. The recombinant adeno-associated virus type-2 vector carrying human telomerase reverse transcriptase can be successful y constructed, and can mediate a stable mRNA expression of human telomerase reverse transcriptase in human nucleus pulposus cells. Our findings provide a novel strategy of enhancing the properties of nucleus pulposus cells.