Study On The Proliferation Of Human Dental Pulp Stem Cells Transfected By Recombinant Adenovirus Mediated Epidermal Growth Factor In Vitro

Objective:To observe the effect of recombinant adenovirus mediated epidermal growth factor (rAd-EGF) transfecting the human dental pulp stem cells (hDPSCs) on its growth, proliferation and apoptosis by culturing human dental pulp stem cells in vitro, and to provide an experimental evidence for the role of EGF on hDPSCs and a possible new idea and method of dental defects and regeneration.Methods:1The hDPSCs routinely collected in exponential phase of growth were made into cells suspension with density of1×105/ml, and inoculated into6-well culture plates with suspension volume of2ml in each well. According to the experimental purpose, The cells were divided into three groups, including control group (the hDPSCs medium only), the negative control group (hDPSCs transfected by rAd-EGFP) and experimental group (hDPSCs transfected by rAd-EGF). When the cells grew and adhered to the wall24hours later, the rAd-EGF and rAd-EGFP were added into specified well to transfect hDPSCs. After72h, used reverse transcription polymerase reaction(RT-PCR) to analyze the mRNA expression of EGF in hDPSCs transfected by rAd-EGF.2The cells in exponential phase of growth cultured in vitro were made into cell suspension with density of4.0×103, then inoculated into96-well culture plates, with suspension volume of200μl each cell. According to the experimental purpose, the cells were divided into three groups, the control group, negative control group and experimental group. After cell adherence, MTT was used to assess the cell line proliferation in vitro after transfected by rAd-EGF at the same time point on the1st、3rd、5th、7th、9th、11th Day.3. The cells in exponential phase of growth which were made into cell suspension, with3.0×106cells per well, were inoculated into6-well culture plates. After cell attachment, the recombinant rAd-EGF and rAd-EGFP were added into specified well to transfect hDPSCs. After48h, used flow cytometry Annexin V-FITC/PI to detect the effect of the apoptosis of hDPSCs after transfected by rAd-EGF Results:The EGF mRNA expression of hDPSCs in the experimental group were significantly up-regulated compared with the negative control group and blank control group (P<0.05). The proliferation of hDPSCs in experimental group proliferation was markedly increased compared with the negative control group and the blank group in vitro, during the first7days, it increased significantly(P<0.05), while diminished after7days, there was no significant difference (P>0.05). The results of flow cytometry the Annexin V-FITC/PI method appeared in the same role time, compared with the negative control group and the blank group, the apoptotic index of hDPSCs in the experimental in vitro was significantly lower (P<0.05). Conclusions:The rAd-EGF could transfect into hDPSCs successfully, and the EGF mRNA expression of hDPSCs was increased, so in the short term role, The rAd-EGF transfected into hDPSCs could promote the proliferation and inhibit apoptosis.