- 作者：Yong Wen ; Jing Lan ; Haiyun Huang ; Meijiao Yu ; Jun Cui ; Jin Liang ; Baoqi Jiang ; Xin Xu
- 关键词：Human periodontal ligament stem cells ; Lentiviral vector ; Enhanced green fluorescent protein ; Osteogenic capability ; Periodontal tissue engineering
- 刊名：Archives of Oral Biology
- 出版年：2012
- 出版时间：September, 2012
- 年：2012
- 卷：57
- 期：9
- 页码：1241-1250
- 全文大小：2072 K
Objectives
To establish human periodontal ligament stem cells (hPDLSC) with high and stable expression of enhanced green fluorescent protein (eGFP) and to obtain an ideal vector expression system that suitable for gene therapy in periodontal tissue engineering.Methods
hPDLSCs were transfected with eGFP for 48 h via different MOI (25, 50, 100, 200 and 400) by lentiviral vector, the transfection efficiency was evaluated by fluorescent microscopy and flow cytometry, and transfected hPDLSCs proliferation was evaluated by MTT. Pluripotent, differentiation capacity and ALP expression status were determined further. Osteoblast-associated genes expressions for osteogenesis were evaluated by quantitative-PCR. In addition, rat molar periodontal fenestration defect model was used for evaluating periodontal tissue engineering.
Results
The transfection efficiency after 48 h were 44.7 % , 60.9 % , 71.7 % , 85.8 % , and 86.9 % respectively. There was no significant effect of transfection (at different MOI levels of 25, 50, 100, and 200) on the proliferation of hPDLSCs (designated as eGFP-hPDLSCs) compared with hPDLSCs (P > 0.05). However, proliferation of eGFP hPDLSCs at MOI 400 became slower (P < 0.05). Both eGFP hPDLSCs and hPDLSCs were able to differentiate into osteocytes and adipocytes under certain conditioned media. At 7 days, expression levels of COL-1, RUNX2 in hPDLSCS were higher than those in eGFP hPDLSCs (P < 0.05); expression levels of ALP and OPN in eGFP hPDLSCs were similar to those in hPDLSCs (P > 0.05). Newly regenerated bone formation was observed in the defect model used.
Conclusions
Among the transfection conditions, 48 h transfection at MOI 200 is optimal for labelling hPDLSCs with eGFP in a lentiviral vector. There is no change in capability of the eGFP hPDLSCs osteogenesis. The lentiviral vector with eGFP is an appropriate expression vector system and hPDLSCs are ideal seeding cells for gene therapy in periodontal tissue engineering.