【作者】 赵东波；

【导师】 崔树森；

【作者基本信息】 吉林大学 ， 外科学， 2010， 博士

【摘要】 神经缺损同手内在肌功能恢复、臂丛神经根性撕脱伤一样是至今尚未有效解决的难题。自体神经移植仍是目前临床治疗的首选方法,也是其他研究周围神经缺损治疗方法的“金标准”。移植神经存在经过缺血性改变,易形成瘢痕,再生神经纤维需经过两个吻合口,易使再生神经纤维错长或被阻断在远端吻合口等缺点。除了继发性损害的不断加重,移植的神经同样面临着中枢神经元的再生修复,然而关于移植神经远端吻合口对神经元再生影响的研究,国内外报导较少。本实验在国内外研究的基础上,利用大鼠坐骨神经移植模型,以GAP-43、bcl-2和bax作为观察指标,通过免疫组化、RT-PCR、western-blot以及TUNEL技术,研究二次处理移植神经远端吻合口对感觉和运动神经元再生的影响。实验结果显示:移植神经远端吻合口二次处理后,实验组GAP-43在相应脊髓和背根神经节中再次出现表达高峰,与对照组有明显差异;实验组bcl-2和bax的比值在相应脊髓和背根神经节中的表达再次达到低值,与TUNEL检测相一致,但与对照组相比无统计学意义。本实验可以得出如下结论:通过二次处理移植神经远端吻合口,轴突的延长和重建可被重新诱导,给神经元提供了二次再生的能力;二次处理虽然引起了神经元的再次凋亡,但是影响较小,形态学上观察没有统计学意义。更多还原

【Abstract】 Peripheral nerve regeneration has been a great concern of medical science studies.The first question encountered is the source of nerve transplant donor. Nerve conduit and variant nerve can’t be widely used in the clinic due to a variety of difficulties.The autograft nerve transplant takes the priority in the clinic treatment,“the golden standard”measuring other peripheral nerve treatments as well.Regeneration and scar reparation are the only two means of repairing after the body tissues get injured, which coexist after the peripheral nerve injury.Ischemic in the process of nerve transplant would bring about a scar, and desmoplasia will hamper the regenerative nerve fibers to pass through the second stomas because these fibers have two stomas to pass. The apoptosis of the neuron body and secondary lesion will be following. With the development of base medicine, people get a deeper knowledge of genes and molecule.Now we find that as a result of weaker stimuli, less factors, and restraint from the external conditions, nerve regeneration declines and enters into a relative“platform stage”after undergoing a peak of repair. Whether we can find a way to protect neuron body and overcome the defect in the autograft nerve transplant, rendering new stimulus and re-activating nerve growing, will substantially propel the studies on nerve regeneration. That’s exactly what we are working at.The adult wistar rats were divided into the pseudo-operation group (exposure only), the control group (excision of 2cm and suture) and the experimental group(excision of 2cm and retreatment after 8 weeks). Based on the animal model of sciatic nerve transplant, the corresponding dorsal root ganglia and spinal cord (L4-L6) were taken at different time after the operation.The growth-associated protein GAP-43 is a presynaptic membrane phosphoprotein that is expressed at high levels with the growth of axone. The protein was considered related with the nerve growth, axone regeneration and synapse reconnection strongly. Immunohistochemistry, Western Blot and RT-PCR were adopted to evaluate the different expressions of GAP-43 in dorsal root ganglia and spinal cord in the different period of time.As a new type of tumor family, bcl-2 can inhibit the cellule apoptosis and prolong the activity of cellule. The survival of nerve depends on the ration of bcl-2 and bax. Immunohistochemistry was used to detect the different expressions of bcl-2, bax and their ratio to study nerve variation rules at the protein level and the molecule mechanisms of nerve cellule apoptiosis.TUNEL is a widely-adopted method of cellule apoptiosis studies, by which we ascertain whether the stoma retreatment could lead to another apoptosis of the corresponding spinal segment motor neuron or not.Results of the study1 The detection of the GAP-431)spinal cord motor neurons: (1) the pseudo-operation group: the results show that GAP-43 in the pseudo-operation group maintains a certain level in spinal cord but only has a small amount of expressions, and shows no statistical difference over time. (2)the control group : The amount of GAP-43 in the control group increases significantly on the first week, peaks in the second week, keeps high in the fourth to the sixth weeks, and in the eighth to the sixteenth weeks it descends and stays invariable. (3)The experiment group: The amount of GAP-43 in the experiment group is the same with that in the control group in the third day to the eighth week. GAP-43 increases again from the third day after the eighth week to the twelfth week, and peaks in the tenth week again. It descends in the fourteenth to the sixteenth weeks over time.2)dorsal root ganglion:(1) the pseudo-operation group: the results show that GAP-43 in the pseudo-operation group maintains a certain level in spinal cord but only has a small amount of expressions, and shows no statistical difference over time. (2) the control group : GAP-43 expression has increased in the first week and reached peak in the second week after sciatic nerve transplant. It keeps high in the fourth to the sixth weeks, falls in the eighth week, and shows no statistical difference over time. (3) the experiment group: The expression of GAP-43 in the experiment group is the same with that in the control group in the third day to the eighth week, increases again from the third day after the eighth week to the twelfth week, comes to the second peak in the tenth week after the first operation, and finally falls over time in the fourteenth to the sixteenth week.2 The detection of bcl-2 and bax1) spinal cord motor neurons: chromagen was buffy and located in cytoplasm,excluding nucleus. (1)the pseudo-operation group: the immunoreactive cells stained are not found.(2) the control group : the immunoreactive cells stained shown in neuron and glioncyte on the third day after the sciatic nerve transplant, are located in cytoplasm, excluding nucleus. The expression of bcl-2 and bax increases gradually, reaches the peak in the second week, then descends little by little. (3) the experiment group: The result is the same with the control group in the third day to the eighth week. The expression increases again on the third day after the eighth week, peaks in the tenth week again, and then descends.(4) bcl-2/ bax in the control group: The ration of bcl-2/ bax falls down from the third day, comes to the lowest point in the second week, then increases little by little and stays invariable from the eighth week.(5) bcl-2/ bax in the experiment group: The ration of bcl-2/ bax is the same with the control group in the third day to the eighth week. It descends again in the third day after the eighth week, peaks in the tenth week again, and then increases. The Student’s test results show no statistical significance over time.2) dorsal root ganglion:chromagen was buffy and located in cytoplasm ,excluding nucleus.(1) pseudo-operation group: the immunoreactive cells stained are not found.(2) the control group : the immunoreactive cells stained shown in neuron and glioncyte on the third day after the sciatic nerve transplant, are located in cytoplasm ,excluding nucleus . The expression of bcl-2 and bax increases gradually, reaches the peak in the second week, and then descends little by little. (3) the experiment group: The expression is the same with the control group on the third day to the eighth week. After that, it increases, peaks in the tenth week again, and then descends.(4) bcl-2/ bax in the control group: The ration of bcl-2/ bax falls down from the third day, comes to the lowest point in the second week, and then increases little by little.(5) bcl-2/ bax in the experiment group: The ration of bcl-2/ bax is the same with the control group onn the third day to the eighth week. After it, it descends, peaks in the tenth week again, then increases. The Student’s test results show no statistical significance over time compared with the control group.3 The detection of TUNEL 1) the control group: The electropositive cells are microamount on the third day, and becomes dense in the second week, when the number density of apopotosis comes to the peak, and then descends obviously from the third day after the eighth week and the tenth week.2) the experiment group: it is the same with the control group on the third day and in the second week, but it becomes more in the tenth week than on the third day after the eighth week. There is no statistical significance in the apopotosis cell over time.Conclusion:In the rat model of sciatic nerve transplant, after surgical removal and renovation of the distal stoma of transplant nerves, elongation and reconstruction of an axon can be induced again, and then transcription and translation of growth-associated protein gene are stimulated, thus GAP-43 arrives at the second peak. The expression of bcl-2 and bax coincides with apoptosis following the nerve regeneration. The change of bax/bcl-2 ratio determines the neurocytes to survive or die following the nerve regeneration. The surgical removal and renovation of the distal stoma induce the neurocytes apoptosis, but have little effect. There is no statistical significance on morphology observation. This method can not only stimulate the regeneration of the neuron body but also release the scar restraint for nerve regeneration, which displays the reparation unity of neural body, neurite and microenvironment, and thus offers a new scientific idea and technique for the reinforcement of nerve transplant.更多还原