The Biological Effects Of Lentivirus GV115-Survivin Transfection On The Differentiation Of Intervertebral Disc Nucleus Pulposus Cells And The Construction Of GV115-Caspase3 SiRNA And Its Biological Effects

[Objective] To construct GV115-Caspase3 si RNA and study the biological effects of GV115-Survivin or GV115-Caspase3 si RNA on human dedifferentiated intervertebral disc nucleus pulposus(IDNP) cells.[Methods] The human NP cells were isolated and cultured using tissue culture technique.Lentivirus transfect rate to the earlier or later dedifferentiated IDNP cells was detected with PEGF. The expression and biological effects of Lentivirus-Survivin were detected and compared by the immunofluence, MTT,-Blot or Antonopulos methods. On the basis of RNAi design principle, four Caspase-3 si RNA sequence were designed.GV115-Caspase3 si RNA was constructed by gene synthesis and subclone technique. The GV115-Caspase3 si RNA was detected by PCR and DNA sequencing. After the lentivirus had been packaged, the 293 T cells were transfected by GV115-Caspase3 si RNA. The translation of Caspase-3 gene transfected by GV115-Caspase3 si RNA was detected using RT-PCR,and the most effective GV115-Caspase3 si RNA was screened. The IDNP cells were isolated and cultured using tissue culture technique. The GV115-Caspase3 si RNA transfect rate to later dedifferentiated IDNP cells was detected with PEGF. The GV115-Caspase3 si RNA expression and biological effects for later dedifferentiated IDNP cells were detected by the immunofluence, MTT,Western-Blot and Antonopulos methods.[Results] Lentivirus could transfect efficiently the earlier and later dedifferentiated IDNP cells, and promote Survivin protein to be expressed efficiently. Lentivirus-Survivin could enhance the earlier dedifferentiated IDNP cells viability and the synthesis of the glycosaminoglycan or collagen type II, but it inhibit those of the later dedifferentiated IDNP cells.The GV115-Caspase3 si RNA was proved to be right using PCR and DNA sequencing. The gene knocking rate of the most efficient GV115-Caspase3 si RNA was84.2%. GV115-Caspase3 si RNA could transfect efficiently the later dedifferentiated IDNP cells and inhibit the expression of Caspase-3. GV115-Caspase3 si RNA could enhance the activity of the later dedifferentiated IDNP cells and the synthesis of the glycosaminoglycan or collagen type II.[Conclusion] Lentivirusvirus-Survivin can be used to the earlier dedifferentiated IDNP cells, but it is not applicable to the later dedifferentiated IDNP cells; the GV115-Caspase3 si RNA was constructed successfully and can enhance the activity of the later dedifferentiated IDNP cells and promote the synthesis of extracellular matrix.