| Spinal cord injury (SCI) is very common in clinic.SCI is usually caused by traffic accident, crash, industrial accident, violence and athletic injury. the incidence rate of spinal cord injury (SCI) increase year by year. SCI is a major reason that lead to functional disability. A series of pathologic and biologic changes would be succeeded after primary injury in spinal cord, which would bring about neuronal death and tissue degeneration in vary extent leading to neurological dysfunction and even paralysis. Nerve neurotrophic factors being deficient in internal environments, neurons being apt to be death or apoptosis, neuronal axon growing slow, glial scar influence and so on, which make neurological function recovery very difficult. How to reduce spinal cord lesion from secondary injury in maximum extent ; How to make use of nerve neurotrophic factors in maximum extent. It is one of the main task in spine surgery that needs to be solved urgently.Following the developing studies of spinal cord injury, scientists find out that the functional recovery depends on the preservation of neurons and the regeneration of axon. external interventions such as cell transplantation and neurotrophic factors delivery overcoming the inhibitory environment at the injury site could stimulate some of the remaining intrinsic plasticity of neurons and, thus, to protect neurons and stimulate regeneration of axon. GDNF is known to be the most active neurotrophic factor for motoneurons, however, GDNFcan't cross the blood-brain barrier. SCs known for their beneficial effects on survival of several neuronal cells, neuroprotection and neurite outgrowth. Local delivery of GDNF can counteract pathological events and induce a regenerative response after SCI. So SCs maybe the better candidates for cell implantation, but the number of regenerating axons distal from the injury site was still limited partly because of the limited expression of neurotrophic factors and the differences in the capacity of various axonal populations to regenerate SCs implants.In present experiment, Firstlly we cultured and puried SCs in vitro, Secondly we make GDNF gene modify SCs, Then we make use of SCs which modified by the GDNF in neuron cell and spinal cord injury experiment. we observe SCs which modified by the GDNF protective function and the mechanism on SCI. It is anticipated that rationale therapeutic strategies would been find to cure spinal cord injury.Part one Eukaryotic vector construction of rat gIiaI cell-line derived neurotrophic factor Method: To extract GDNF-RNA from rat tissues, and then go through RT-PCR, transcribe RNA into cDNA, cDNA was multipled considerably, finally it was identified; To link GDNF gene with pEGFP-N1 vector and construct recombinant plasmid, and then to be identified.Results: pEGFP-N1/GDNF plasmid was constructed successfully, agar-gel electrophoresis and sequence analysis indicate the gene we obtained is identical to the gene in Gene Bank.Part two Culture, purification and identification study of Schwann Cells Method: The bilateral sciatic nerves of the 4-5day SD rats were ligated and predegenerated. After 7th post-operation day. The nerve fascicle were extracted.Two enzymes were used to digest the nerve.G-418 was added in the culture mediumfor 4 days. Schwann cells was tested cell counting and force of Schwann cells. The Schwann cells was identicated under immunocytochemistry staining test and record the purity.Results: Approximally 3.84x106 cells were harvested with95.6%survival rate and a purity of Schwann cells over 94%by this method. Conclusion This is method yields large amount of viable Schwann cells with high purity and survival rate.Part three Building engineered SCs-GDNF and its biological assay Methods: we transfected GDNF into SCs with liposome, and built SCs which modified by the GDNF by using G418 culture medium to select positive clones. Finally, we assayed GDNF activation and quantity by Immunocytochemistry , Elisa and Western blot.Results: SCs which modified by the GDNF was constructed successfully.The bright green fluorescence could be observed with fluorescence microscopy in pEGFP-N1 transfected group and pEGFP-N1/ GDNF transfected group,but normal group could not observed. ELISA and Western blot assay demonstrated high GDNF protein secretion in SCs which modified by the GDNF conditioned medium, SCs which modified by the GDNF demonstrated higher GDNF protein than the others(P<0.05).Part four The affection of the SCs which modified by the GDNF on the expression of Bcl-2 and Bax in apoptosis of spinal nerve cell induced by radiationMethods: spinal nerve cells were performed serial subcultivation in vitro, and entered into experiment during the exponential phase of growth.After the 2Gy radiation induced apoptosis of spinal nerve cells,The experiment was devided into 5 groups , After apoptosis of spinal nerve cells and SCs are cultured in Transwell system. percentage of the cell apoptosis assayed by FCW, the expression of Bcl-2 and Bax was obversed by Immunocytochemistry.Results: The FCW result showed the percentage rate of apoptosis is 53.24±9.54,31.93±1.99,32.33±2.09和20.11±0.98,E group is superior to B, C and D group(p<0.05),C and D group is superior to B group(p<0.05 ) .Immunity histochemistry assay found Bcl-2 and Bax positive cells is brown. At the same time, The protein expression of Bcl-2 in E group is the most than the other(p<0.05). The protein expression of Bax is increased in B group and decreased in E group.Part five Effect of SCs modified with GDNF on spinal cord injuryMethods: rats were devided into 5 groups randomly, spinal cord injury model were made firstly, then SCs modified with GDNF was injected into injury site, HE, immunohistochemistry and RT-PCR were examined and protection effect of SCs modified with GDNF on spinal cord was observed.Results: HE indicate Spinal cord injury in the first day, a large tissue injury become inflammatory response increased; in the seventh day, in the C, D and E group, spinal cord injury and the surrounding area, the inflammatory cell infiltration reduced, and some spinal cord tissue injury were cystlike changed. in the fifteenth day, softening lesions occurring at the large kitchen, and bleeding lesions still visible; Inflammatory cells disappear .White matter edema reduced, we can see that gliocytes in the B group.In the E group, edema was reduced, inflammatory cell infiltration reduced, degeneration reduced, neuronal cells were rearranged.The E group is superior to C and D group in thenerve cell morphology and structure of restoring order. Immunocytochemistry showed In the first day. There is a small amount of Bcl-2 expression in the A group. the B, C, D and E group were Bcl-2 expression and cell deeper coloring.In the seventh day the B group continued high Bcl-2 expression, in the E group Bcl-2 positive cells increased, the number of positive cells more than the first day corresponding dose group.In the fifteenth day, expression of Bcl-2 is decreased in the B group other group still demonstrate this phenomenon.In the E group Bcl-2 positive cells ins more than the other group.In the first day. A group shows no expression of Bax. the B, C, D and E group were Bax expression and cell deeper coloring.In the seventh day the B group continued high Bax positive cells, in the E group Bax positive cells decreased, the number of positive cells is less than other group. Bax reduce the number of positive cells to the 15th day after, still show this phenomenon.RT-PCR assay got the similar immunocytochemistry's results.In this study, SCs modified with GDNF could afford significant neuroprotection against apoptosis of spinal nerve cells induced by radiation via upregulation of bcl-2, downregulation of bax , so the apoptosis was inhibited . SCs modified with GDNF transplantation had therapeutic effect on SCI rats, which could up-regulate Bcl-2 protein and downregulate Bax protein in on SCI rats.Using the technology of cell culture, molecular clone, immunocyto-chemistry, RT PCR and Western blot, we research the neuroprotection on spinal nerve cellsof SCs modified with GDNF and the therapic effect on SCI rats of SCs modified with GDNF transplantation. On cellular and molecular level, our study provided some experimental evidence on treating SCI using SCs modified with GDNF. |
PDF Full Text Request