Effects Of Lentivirus-mediated RNAi P21Gene Silence On Biological Characteristics Of Antler Stem Cells

Background and purpose: Organ regeneration is quite important for organism tosurvival and reproduction. The development of regenerative medicine has brought aboutnew dawn for human organ regeneration. The study of regeneration mechanism mainlycomes from the lower vertebrates, and regenerative capacity is rare in mammals. Velvetantler is a subsidiary organ which could completely regenerate. Antler growth is very similarto the limb development, so it just provides us with a good chance to get to knowmammalian organ regeneration. Antler stem cells can proliferate rapidly without goingcancerous. Therefore, it is particularly important to explore the molecular mechanism ofantler regeneration. The recent study of MRL mouse provides a reference for our researchon deer antler regeneration at the genetic and molecular level. One study found that MRLmice has a unique cell cycle(G2/M accumulation), which is similar to many other modelorganism who can regenerate organs. At the same time, the study found that p21down-regulation mouse closes ear holes similar to that of MRL mice, providing a firm linkbetween cell cycle checkpoint control and tissue regeneration. However, we do not foundthe similar cell-cycle (G2/M accumulation) in regenerating antler cells (unpublished). Inorder to determine the relationship between cell cycle control of the pedicle periosteum cellsand p21gene, and whether p21gene plays an important role in the antler regenerativeprocess as it acts in MRL mice, we have done our study by RNA interference.Methods:1. The high score RNAi target sequences of p21gene aiming to sika deer wasselected based on our laboratory and literature reports, and all the candidate sequences wereblasted in NCBI database to remove the off target ones. Then an oligo deoxynucleotide ofshRNA (short hairpin RNA) was designed empirically and synthesized by a commercialcompany. The oligo DNA was ligated to pLVTHM (the lentivirus carrier plasmid) using T4DNA ligase. The constructed plasmid was subsequently confirmed by PCR reaction andsequencing.2. The constructed plasmid was transfected into293t cells with pMD2.G andpCMV-dr8.9plasmids after no-endotoxin extraction. The lentivirus containing the purposegene was obtained by collecting and enriching. The pedicle periosteum cells (PP cell) wasrecovered, and subsequently infected with the recombinant lentivirus, the positive cellswere sorted by flow cytometry. Real-time PCR determined the mRNA expression afterinfection in order to establish the interferential effect.3. The cell proliferation rate wasdetermined by MTT assay; The cell cycle and cell apotosis were determined by flow cytometry; The cell senescence was determined by β-galactosidase staining; DNA damagewas determined by comet assay.Result:1. Two shRNAs aiming at p21gene were successfully selected and wereverified by PCR and sequencing, and oligo DNA was synthesized by annealing response;2.The oligo DNA was successfully inserted into the vector plasmid pLVTHM, therecombinant plasmid was obtained;3. The recombinant lentivirus was obtained bytransfected293t cells;4. The stable genetic cell lines was obtained by infecting PP cells andsorting;5. The results of real-time PCR showed that the expression level of p21gene weresignificantly decreased;6. The detection of cell biology behavior showed that-In the earlyperiod of culture the cells behaved no significant change in proliferation rate, on thecontrary, in the later period of culture the cell multiplication rate was significantly higherthan the control group; No significant change in the cell cycle; The number of apoptoticcells increased significantly; The number of aging cells decreased significantly; The p21transfected cell showed evident DNA damage.Conclusion: The recombinant lentiviral vector aiming at p21gene was successfullystructured, and the stable cell line which expresses p21gene lowly were obtained, whichthus laid foundation for future study about the role of p21gene in deer antler regeneration;This study showed that the way of antler regeneration is different from the existing models,p21gene is not the key growth signs molecules for the rapid cell proliferation and cell cycle,however it was just associated with the stability of PP cell genome.