The Experimental Study On Rabbit Nucleus Pulpous Cells Induce The Differentiation Of Rat Adipose-Derived Stem Cells By Co-cultured

Objective:To isolate, culture and identification of rat adipose-derived stem cells and rabbit intervertebral disc cells, and establish an immortalized adipose-derived stem cell line. Co-culture rat adipose-derived stem cells and rabbit intervertebral disc cells by Transwell, and study the induction of intervertebral disc cells to the adipose-derived stem cells, and to construct a kind of seed cell for tissue engineering.Methods:The inguinal fat pad adipose tissue from SD rats was digested by collagenaseâ… and the primary adipose stem cells were isolated from the adipose tissue. The proliferation of cells was observed by MTT method. Plasmid pCMVSV40T/PUR was transfected into adipose-derived stem cells by Lipofectamine TM 2000, and the resistant clones were selected by puromycin, and then the resistant cells were the immortalized adipose-derived stem cells. Primary nucleus pulposus cells were also gotten from Rabbit intervertebral discs. Cells were confirmed by immunocytochemical methods and RT-PCR. The immortalized Adipose-derived stem cells and nucleus pulposus cells were co-cultured with the ratio of 1:1 in Transwell system. After been co-cultured for 15 days, the two groups cells were collected and detected by immunocytochemistry and RT-PCR.Results:The adipose-derived stem cells were named immortalized adipose-derived stem cells after been transfected with Plasmid pCMVSV40T/PUR. The immunocyto-chemistry study and RT-PCR confirmed that the cells which we got were nucleus pulposus cells. After been co-cultured for 15 days, the immortalized adipose-derived stem cells began to express collagenâ…¡and aggrecan, which were found by immuneohistochemistry and RT-PCR.Conclusion:Using microscopy and immunological purification technique, we can successfully isolate adipose-derived stem cells and nucleus pulposus cells. After been transfected by plasmid pCMVSV40T/PUR, the adipose-derived stem cells can been immortalized. In the non-contact co-culture environment of transwell, nucleus pulposus cells can induce in vitro immortalized adipose-derived mesenchymal stem cells derived into a kind of immortalized nucleus pulposus cell-like cells. So, it can make use of undifferentiated adipose-derived mesenchymal stem cells to treat degenerative disc disease.