| BackgroundsAs the development of modern society, the use of electricity in industry and our day life become more and more frequently. The electrical injuries caused by abnormal touch are very common seen. Human body a kind of the electric conduction can be injured by accidently contact with the electric current which can cause heat when it goes through the tissue. So in clinical someone also call this kind of injury: electrical burn. The first part of skin contacts the electric current can be the entrance of the current and as the current going in a certain direction, it will exit from another part of body. However, once the current enter or exit from the body, the contact area of tissue will be damaged by electro-discharge,and this may cause more damage than electrical burn. Also we can find that the exit area of wound is often appeared to be more serious. There are multiple ways of surgeries to cover the wounds now, but none of them can solve the problems of vascular thrombosis,muscle necrosis, and nerve degeneration caused by electrical burn effectively. Peripheral nerve injury is in a high rate because the contact is always happening in our limbs. In clinical, suitable therapy is still not been found to solve the problems caused by peripheral nerve injuries and the prognosis are always disappointed. Further researches on recovery of peripheral nerve injury are required eagerly.Glial cell line-derived neurotrophic factor(GDNF) is the most bioactive target neurotrophic factor that ever been found which can decrease the death rate, improve the livability and promote the growth and differentiation of neurons. Adipose-derived stem cells(ADSCs) is the focus of lots of areas of research. There is a very cheerful prospect on peripheral nerve repair for its powerful potential on differentiation to multiple types of cells and its great paracrine of kinds of growth factors to improve the recovery of nerve injury. In this research, we intended to use the lentivirus which over expressing GDNF protein to transfect the ADSCs and ADSCs were injected to the surface of sciatic nerve of SD rats injured by electrical stimulation to observe the improvement of the motor function of the injured limbs.Methods1.ADSCs were isolate from bilateral inguinal adipose tissue of 5 SD rats by collagenase digestion and purefied by the different adhension method. The ADSCs from 3rd passage were induced to adipocytes and osteocytes and the surface marker of the ADSCs were detected by flow cytometry.2.Lentivirus were established to transfect the ADSCs in a appropriate titer.3. 33 SD rats were used in this part of trail. Sciatic nerve electrical injury model were established by electrical stimulation device in 220 V voltage and the injury degree was checked by pathological slice with 3 rats. Other 30 SD rats were divided into five groups randomly(6 rats in each):normal group, NS control group, ADSCs group, GDNF group and GDNF-ADSCs group. None treated were used in normal group and feat them as the others. In other four group, physiological saline, ADSCs cell suspension(cell density is1×107/m L), GDNF protein solution and GDNF-ADSCs cell suspension(cell density is1×107/m L)(100μL for all of above) were injected to the surface of the injury nerves soon after the injury. Wire hang score and hind limb stride were detected from the 1st week to the 8th week. Collect the sciatic nerves at the end of the last week, and observe the histological changes compared to the normal group.4.Other 120 SD rats were divided into five group randomly(24 for each group) and treated as the same way before and sacrificed in 4 weeks time, collected the sciatic nerves from the rats to analysis the expression level of GDNF protein in nerves by Western-blot test.Results Lentivirus with up-regulated expression of GDNF was constructed successfully, the infection rate of ADSCs is 95%. Wire hang average score figure showed a better trend in GDNF-ADSCs group. According to statistical result, at 8th week time, the wire hang score in GDNF-ADSCs group was significantly better than other three treated groups(μ value were 6.02, 2.54, 2.67, respectively(p < 0.05 or p < 0.01). There was no significant difference between other group pairs. In hind limb stride test analysis, all post-injury groups were decreased compared to normal group at every time point in the whole test(p<0.05). At the 3rd, 5th, 7th week, the hind limb stride test value in ADSCs group and GDNF group were significantly higher than the NS group( p<0.05). At the 8th week,value in GDNF group was significantly higher than NS group( p<0.05). From the 4th week to the 8th week, the value in GDNF-ADSCs group were significantly higher than that in the NS group, ADSCs group and GDNF group(p<0.05). According to the results of the pathological slices of the sciatic nerve cross cut from the test rats, the statistical analysis of the number of myelinated nerve fibers were significantly higher, the average diameter of axons were significantly smaller and the myelin thickness were significantly higher in ADSCs group, GDNF group and GDNF-ADSCs group than that in normal group(p < 0.05). The number of myelinated nerve fibers were significantly decreased in NS group(p < 0.05). The number of myelinated nerve fibers and myelin thickness were significantly higher in GDNF-ADSCs group than that in ADSCs group and GDNF group(p < 0.05). At 4 week time, GDNF protein expression was significantly higher in post-injury groups and that in normal group(p<0.05) and that in GDNF-ADSCs group was significantly higher than NS group, ADSCs group and GDNF group(p<0.05).Conclusion1.The transfection rate of ADSCs by lentivirus was stable, high and well. There was no negative effection in growth and differentiation on the ADSCs.2. ADSCs modified by GDNF protein had significant improvement in motor function recovery after sciatic nerve electrical injury in rats.3. Gene of ADSCs were improved effectively by the lentivirus transfection. The result of GDNF protein level in sciatic nerve in 4 weeks time showed that expression level of GDNF has positive effection in function recovery. |
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