[Recombinant hFOXA2 and hPDX1 lentivirus induced dental pulp stem cells from deciduous teeth reprogramming for insulin-producing cells].
OBJECTIVE
The purpose of this study was to culture and identify dental pulp stem cells(DPSCs) from deciduous teeth in vitro and construct the recombinant hFOXA2 and hPDX1 lentivirus vectors and transfect the DPSCs to induce insulin-producing cells (IPCs).


METHODS
DPSCs were separated and cultured by enzyme digest method, and purified by limited dilution method. Flow cytometry was used to determine the surface marker expression of the DPSCs, and the ability of multiple differentiations was determined by specific staining. hFOXA2 and hPDX1 genes were amplified by PCR, and the recombinant hFOXA2 and hPDX1 lentivirus vectors were reconstructed and transfected into 293T cells by lipofectamine2000 for virus packaging. The viral infection efficiency and titer were determined through fluorescence cell count. The recombinant virus was used to infect the DPSCs cells via multiplicity of infection (MOI) and induce the DPSCs reprogramming for IPCs. Immunofluorescence staining was used to measure the expression of proinsulin, FOXA2 and PDX1. ELISA method was used to detect the insulin secretion. The data was analyzed Using SPSS13.0 software package.


RESULTS
DPSCs were isolated and cultured successfully. Cell surface highly expressed STRO-1 (98.01%), CDl46 (98.51%), CD34 (99.54%) and CD45 (24.08%). The multi-lineage differentiation capacity into osteoblasts, chondrocytes, and adipose was achieved. The recombinant hFOXA2 and hPDX1 lentivirus vectors were successfully constructed. Double enzyme digestion and sequencing appraisal showed that the sequence was fully consistent with GenBank retrieval. Virus packing efficiency was (96.15±0.17) % and (95.49±0.21) % respectively, and the infection titer was about 1.80±108 GTU/mL. The best MOI of the virus was 20. After inducing the cells to express proinsulin, FOXA2 and PDX1, insulin secretion volume was about 1.92 μmol/L. Compared with the uninduced group and control group, insulin secretion increased significantly (P<0.01).


CONCLUSIONS
The recombinant transcription factor virus can activate cell reprogramming mechanism, form insulin-producing cells, and can be used for gene therapy of diabetes seed cells. Supported by Science and Technology Research Program of Shaanxi Province (2009K17-06) and Science and Technology Innovation as a Whole Plan Resources Leading Industry Key Technology (Chain) Project of Shaanxi Province (2011KTCL03-24).
Medical Research Centre, Stomatological Hospital of Xi'an Jiaotong University. Xi'an 710004, Shaanxi Province, China.E-mail:921676599@qq.com.
Jian-feng
JF
Shi
Chun-hui
CH
Zhu
Jin
J
Liu
Jun-yi
JY
Sun
Guo-zhou
GZ
Rao
Ang
A
Li
D002454:Cell Differentiation; D002478:Cells, Cultured; D003782:Dental Pulp; D005822:Genetic Vectors; D051542:Hepatocyte Nuclear Factor 3-beta; D018398:Homeodomain Proteins; D006801:Humans; D007328:Insulin; D016086:Lentivirus; D059630:Mesenchymal Stem Cells; D010006:Osteoblasts; D013234:Stem Cells; D014094:Tooth, Deciduous; D015534:Trans-Activators; D014162:Transfection
C496502:FOXA2 protein, human; D018398:Homeodomain Proteins; D007328:Insulin; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D051542:Hepatocyte Nuclear Factor 3-beta