Abstract
Introduction
Adipose-derived stem cells (ADSCs) possess the potential of multi-differentiation which could differentiate into neuronal cells and Schwann cells under certain conditions. If we inoculate the ADSCs modified by Brain derived neurophic factor(BDNF) and Neurotrophin-3(NT-3)double genes on the silk fibroin/chitosan (SFCS), and transplant it into 2 mm pre-made spinal cord transection animal model, we can take advantage of the characteristic of ADSCs which could express BDNF and NT-3 stably after being double gene modified and the advantage of tissue engineering, we look forward to providing a new treatment method for spinal cord injury (SCI), which thereby could reduce SCI morbidity and mortality rate, so as to build up a solid theoretical foundation for the clinical treatment of SCI.
Material and Methods
According to the preliminary design, animal experiment is divided into seven groups: Group A: SFCS/ADSCs/Lenti-BDNF-GFP-NT-3-RFP, namely, BDNF and NT-3 double genes co-transfect ADSCs combining SFCS group; Group B: SFCS/ADSCs/Lenti-BDNF-GFP, namely, BDNF single gene transfect ADSCs combining SFCS group; Group C: SFCS/ADSCs/ Lenti-NT-3-RFP, namely, NT-3 single gene transfect ADSCs combining SFCS group; Group D: SFCS/ADSCs/Lenti-eGFP, namely, empty vector transfect ADSCs combining SFCS group; Group E:SFCS, namely, single SFCS group; Group F:only cut off spinal cord and without treatment; Group G: normal control group, just open the vertebral plate without any treatment. Group A, B, C, D and E transplant the scaffold into 2 mm spinal cord transection defect rat model. Group F cuts off spinal cord and makes 2 mm defect, Group G is normal group. Do a BBB scoring at 1 w, 4 w, 8 w and 12 w after surgery to analyze functional recovery, execute the rats 12 w later, take out the scaffolds, test them by HE staining, immunehistochemical staining (NSE, GAP-43, Caspase-3, GFAP), LFB staining, RT-PCR and Western blot, the purpose of which is to observe the effect of tissue engineering on the recovery of SCI.
Results
Group A (BDNF and NT-3 double genes modified ADSCs combining the use of SFCS group) has a better repair effect on rats spinal cord transection injury treatment, which reveals higher BBB scores, increased GAP-43 expression, more positive NSE cells, decreased expression of GFAP and Caspase-3. At the same time denser regenerating nerve fiber could move through the scaffold, and the most important thing is the repair effect of Group A is better than single gene modified Group B and Group C, which is better than Group D (empty vector transfect ADSCs combining SFCS group), which is better than Group E (single use SFCS), which is better than Group F (just cut off spinal cord and without treatment).
Conclusion
Under the guidance of tissue engineering method, ADSCs modified by BDNF and NT-3 double genes combining the use of SFCS can significantly reduce the inflammatory response after injury, inhibit scar hyperplasia and cell apoptosis and promote nerve fibers regeneration and the differentiation of ADSCs into neurons in vivo, so as to improve the repair effect of rat spinal cord transection injuries, which provides a new method for the treatment of SCI.