Effect on function of human nucleus pulposus(HNP) cells transfected by the recombinant adeno-associated virus vector-mediated human telomerase reverse transcriptase gene

English Keywords:Nucleus pulposus cells  Adeno-associated virus  Human Telomerase reverse transcriptase gene  Gene therapy

English Abstract:

  【Abstract】 Objectives: To investigate the effects on the function of HNP cells transfected by the rAAV-hTERT. Methods: The cultured homogeneous HNP cells were obtained and released by mechanical dissection and enzyme digestion.　The second generation of HNP cells cultured in monolayer culture was transfected by rAAV-hTERT. rAAV-EGFP was firstly used as mark gene to detect the efficiency of the transduction at MOI 103, 104, 105 vector genomes/cell(v.g/cell) by flow cytometry. Three groups were designed for the experiment: (1)group 1: HNP cells transfected by rAAV-hTERT; (2)control 1: HNP cells transfected by AAV; (3) control 2:　HNP cells as noviral transduction group. The expression of the hTERT gene was determined by RT-PCR and Western-blot. Cellular matrix transcript/translated levels were determined by real-time quantitative PCR/Elisa, respectively. Results: The expression of the rAAV-EGFP was 73.9% for MOI(105 v.g/cell) group which was much higher than the MOI(103, 104 v.g/cell) groups at 7 days after transfection.　The expression of the rAAV-hTERT was successfully detected at 7, 60, 90, 120 days after transfection in rAAV-hTERT group, while not present in two controls. The expressional levels of collagen 2 and aggrecan of rAAV-hTERT group were much higher than two controls in 120 days after transfection(P<0.05), while the two controls showed no difference from the beginning to the end(P>0.05). Conclusions: rAAV-hTERT can infect the HNP cells and also express in the transfected cells. The transfection of rAAV-hTERT can improve the cell potency to produce collagen 2 and aggrecan.