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Sun J.,The 478 Hospital of Chinese PLA | Pan Z.-Q.,The 478 Hospital of Chinese PLA | Zhu Y.-L.,Chinese PLA General Hospital | Zhou T.-H.,Chinese PLA General Hospital
Chinese Journal of Tissue Engineering Research | Year: 2014

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 AAVMax™ 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 successfully 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 successfully 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. Source

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