The Establish Of Huamn Dental Follicle Cell Line By Using PiggyBac Transposon System

Objective:Dental follicle is connective tissue surrounding growing dental germ, and dental follicle will develop into periodontal tissues. Dental follicle stem cells are cells inside dental follicle and have been proved to be able to differentiate into multiple kinds of cells both in vitro and in vivo. But the supply of dental follicle cells is limited and the life span of the cells is also limited. Thus, how to gain enough biologically identical cells is a problem to be solved. Immortalization, as an effective way to establish cell lines, has been used widely in tissue engineering. But the old ways for immortalization is with low-efficiency and has safety concerns. PiggyBac transposon system is easy to perform and effective。 Thus we intend to use this piggyBac transposon system to reversibly immortalize human dental follicle cells and analyze their multiple differentiation potential.Standard mesenchymal stem cells hold limited differentiation capacity, and how to induce stem cells for specified differentiation is of great importance. Growth factors are important for specified differentiation.There are no researches about the effect of BMP9 on osteogenic and chondrogenic differentiation and PPARy2 on adipogenic differentiation of dental follicle cells. Thus we intend to analyze the effect of BMP9 and PPARy2 on dental follicle cells differentiation. Methods:1. Using limiting dilution to gain single clone dental follicle cells2. Obtaining immortalized dental follicle cells and deimmortalized dental follicle cells using piggyBac transposon system3. Cell proliferation assay using CCK8 and cell couting assay4. Cell immunofluorescence staining and flow cytometry for cell surface marker detection5. Telomeric Repeat Amplification Protocol (TRAP) and quartz crystal microbalance (QCM) to test the telomerase activity of DFCs,iDFCs and dDFC.6. To assess the multiple differentiation potential of DFCs,iDFCs and dDFC in vitro.7. To assess the effect of BMP9 on osteogenic and chondrogenic differentiation potential of DFCs,iDFCs and dDFC.8. To assess the effect of PPARy2 on adipogenic differentiation potential of DFCs, iDFCs and dDFC.9. To assess the multiple differentiation potential of DFCs,iDFCs and dDFC in vivo. Results:1.iDFCs have higher proliferation rate than primary DFCs,while dDFCs has similar proliferation rate to DFCs2.DFCs, iDFCs and dDFCs express the same cell surface marker.3.The telomerase activity of DFCs decreased with every passage while the telomerase activity of iDFCs are higher and didn’t change due to passages. The telomerase activity of dDFCs are similar with DFCs.4.DFCs, iDFCs and dDFCs are ALP and alizarin red positive and the osetogenic related genes and protein markers were expressed after osteogenic induction.5.DFCs, iDFCs and dDFCs are alcian blue positive and the chondrogenic related genes and protein markers were expressed after chondrogenic induction.6.DFCs, iDFCs and dDFCs are oil red positive and the adipogenic related genes and protein markers were expressed after adipogenic induction.7.BMP9 could enhance the osteogenic and chondrogenic differentiation potential of DFCs, iDFCs and dDFCs8.could enhance the adipogenic differentiation potential of DFCs, iDFCs and dDFCs9.DFCs, iDFCs and dDFCs could form bone-like tissue after induction in vivo.1. PiggyBac transposon system could establish human dental follicle cell lines2. BMP9 could enhance osteogenic and chondrogenic differentiation potential of DFCs,iDFCs and dDFC.3. PPARy2 could enhance adipogenic differentiation potential of DFCs,iDFCs and dDFC.