An Experimental Study On The Treatment Of Sciatic Nerve Injury By Transplantation Of Genetically Modified NSCs Combined With Tissue-engineering Material

The regeneration and repair of nervous system is the focus and difficulty of neural research. Nowadays, the effect of functional restoration on injury nerve is not much satisfied, although the surgical technology in nerve repair has gotten a great achievement. Studies have showed that the restoration of injured nerve need three factors with their cooperation, they are neurotrophic factors (NTFs), substrates and neural cells.Glial cell line-derived neurotrophic factor (GDNF) is known to have important functions in neuronal suvival, differentiation and plasticity. In addition to its roles as survival-promoting factors, GDNF reportedly can enhance dopaminergic neurons and motor neurons. Chitosan is a widely used tissue-engineering material which has histocompatibility and ability of biological degradation. It's important that chitosan can restraint cicatricle. We have found that chitosan has the capacity of protecting motor neurons from death induced by peripheral nerve injury, and of enhancing recovery of nervous function in our former study. Moreover, recent attention has focused on neural stem cells (NSCs). NSCs is an undifferentiated cells which is characterized self-renewal and multiple differentiation potentials in the central nervous system (CNS). Studies have show that NSCs can proliferate greatly in vitro and differentiate into types of nervous cells: neurons, astrocytes and oligodendrocytes, in the CNS under certain inductive conditions. NSCs have become a suitable vehicle for gene transfection.In the present study, methods including cell culture, gene transfection, immunocytochemistry etc, were used in the isolation and culture of rat NSCs, to observed the growth and differentiation of NSCs cultured with Schwann's cells (SCs) and chitosan, and preparation of GDNF genetically modified NSCs. In addition to this, by the model of transected sciatic nerve linked by chitosan-tubulization in the adult rats, either NSCs or GDNF-NSCs were injected into the chitosan chamber, and compared with SAL injected. The regeneration and function recovery of injured nerve were examined at some daysintervals after operation, using immunocytochemistry, flow cytometry, hydrogen-peroxide (HRP) retrograde trace, measurement of sciatic function index (SFI), and patch clamp. The results were as follows:1. The NSCs were isolated from cerebral cortex of fetal rats. NSCs floated in the serum free media as spherical. NSCs can proliferate in vitro and differentiate multi-potemtially induced by 10% fetal bovine serum (FBS). NSCs represented a positive reaction to Anti-Nestin-IgG, and the differentiated cells represented the positive reaction to either Anti-GFAP-IgG or Anti-MAP2-IgG.2. It was observed that chitosan membrane had not changed the growth status of NSCs when they were cultured on it. NSCs kept their characteristics, and also proliferated in vitro. That shows chitosan has good histocompatibility with NSCs.3. NSCs differentiated into neurons and glial cells when they were cultured on chitosan membrane and induced by 10% FBS. But both the differentiated neurons and glial cells were much more than control group which had no chitosan membrane. That's to say chitosan is more suitable for differentiation of NSCs.4. The differentiation phenomenon was observed when NSCs co-cultured with SCs in the serum free media. The number of differentiated cells was biger than control group which induced by FBS only. Furthermore, the percentage of Anti-MAP2 positive cells among differentiated cells was much higher than control group. The flow cytometry data also showed the same conclusion. These results indicated that SCs enhanced the NSCs differentiated into neurons.5. We constructed a retrovirus recombinant pLXSN-GDNF, and identified by PCR. The recombinant retrovirus containing GDNF gene was applied to transfect the isolated NSCs, followed by selection by G418 and the identification by both ICC and RT-PCR. All the results suggested that the GDNF genetically modified NSCs were constructed successfully.6. Treatment of transplant NSCs or GDNF-NSCs combined with chitosan-tubulization decreased the death and promoted the survival of neurons induced by sciatic nerve transection. It also enhanced the regeneration of axons and myelin sheath; promoted the recovery of axoplasm transportation and SFI. Furthermore, the effect of GDNF-NSCs was better than NSCs transplantation only.7. Whole-cell patch clamp showed that passive membrane electrophysiologicalcharacters of motor neurons in the anterior horn of spinal cord did not change markedly after sciatic nerve injury. But the threshold and time course of action potential (AP) changed significantly at 7 days after sciatic nerve injury, but these changes in NSCs and GDNF-NSCs group were fewer than SAL group. And the effect of GDNF-NSCs was better than both NSCs and SAL transplantation.In shortly, because of expression of GDNF continually by GDNF genetically modified NSCs, inhibition of cicatricle by chitosan and supplement of neural cells by differentiated cells from NSCs, the treatment by transplantation of genetically modified NSCs combined with tissue-engineering material has achieved a good effect. And this method has good application prospect.