二氢睾酮对大鼠RGC-5细胞神经突起再生及RhoA和Neuritin表达的影响
发布时间：2019-06-04 15:14
【摘要】：背景：从解剖上看，视神经属于周围神经系统（PNS），但如果从其发育过程中所表现出来的特点，以及所具有的结构特征和功能特性来看，视神经与中枢神经系统（CNS）的神经更为相似，所以常被视作一种特殊分化的中枢神经。因此，与CNS的神经元类似，，RGCs损伤后的再生也是极为困难的，这是眼科临床上外伤性视神经疾病和青光眼等引起不可逆盲的主要原因。 近年来研究表明：CNS难以有效的再生，神经元自身的性质并不是最为主要的因素，损伤导致神经周围形成的具有强烈抑制作用的微环境是更为重要的原因。再生轴突往往由于这种微环境的存在，在出芽的初始阶段就发生了萎缩，最终导致了神经再生的失败。目前，微环境在神经再生研究领域的关注度正在快速增加。 雄激素是近年来研究得较多的一种性激素。除了具有促进生殖系统发育和维持第二性征的作用外，还具有脑保护功能。然而，关于雄激素是否对视神经具有保护作用，以及是否通过调节神经再生微环境来发挥作用报道不多。 目的：1.建立诱导分化后的大鼠RGC-5细胞神经突起损伤模型；2.观察二氢睾酮（DHT）对神经突起损伤后再生的作用；3.检测二氢睾酮（DHT）对损伤局部微环境中RhoA和Neuritin表达的影响。 方法：1.用Na2S2O4制备大鼠RGC-5细胞神经突起氧糖剥夺/复氧损伤模型；2.用不同浓度的DHT（1、10和100nmol/L）对损伤模型进行干预，MTT法检测细胞存活率，光学显微镜下观察细胞神经突起数量和长度，RT-PCR反应和Western blot法检测细胞RhoA和NeuritinmRNA和蛋白的表达水平。 结果：1.5mmol/L的Na2S2O4为制备RGC-5细胞神经突起氧糖剥夺/复氧损伤模型的最佳浓度。2.氧糖剥夺/复氧损伤后，模型组细胞存活率、神经突起数量和长度均较正常对照组减少（P＜0.05），DHT干预后，与模型组比较，10和100nmol/L组细胞存活率和神经突起数量增加（P＜0.05），而1nmol/L组细胞存活率和神经突起数量差异没有统计学意义（P0.05），1、10和100nmol/L DHT组神经突起长度较模型组增加（P＜0.05）。3.氧糖剥夺/复氧损伤后，模型组RhoA mRNA和蛋白表达量均较正常对照组明显增加（P＜0.05），而Neuritin mRNA及蛋白表达量均较正常对照组明显减少（P＜0.05），DHT干预后，1、10和100nmol/L DHT组RhoA mRNA和蛋白表达量均较模型组减少（P＜0.05），而Neuritin mRNA及蛋白表达量均较模型组增加（P＜0.05）。 结论：RhoA和Neuritin可能是氧糖剥夺/复氧损伤后影响大鼠RGC-5细胞神经突起再生的重要因子之一，DHT可能通过调节RhoA和Neuritin的表达，从而发挥神经保护作用。
[Abstract]:Background: from the anatomical point of view, the optic nerve belongs to the peripheral nervous system (PNS), but if you look at the characteristics of its development, as well as the structural and functional characteristics, The optic nerve is more similar to the nerve of the central nervous system (CNS), so it is often regarded as a specially differentiated central nervous system. Therefore, similar to the neurons of CNS, the regeneration after RGCs injury is extremely difficult, which is the main cause of irreversible blindness caused by traumatic optic nerve disease and glaucoma in ophthalmic clinic. In recent years, it has been shown that CNS is difficult to regenerate effectively, and the nature of neurons is not the most important factor, and the microenvironment around the nerve is more important because of the strong inhibitory microenvironment around the nerve. Because of the existence of this microenvironment, the regenerated axons atrophy at the initial stage of budding, which leads to the failure of nerve regeneration. At present, the attention of microenvironment in the field of nerve regeneration is increasing rapidly. Androgen is a kind of sex hormone which has been studied more and more in recent years. In addition to promoting the development of the reproductive system and maintaining the secondary sexual characteristics, it also has the function of brain protection. However, there are few reports about whether androgen has protective effect on optic nerve and whether androgen plays a role by regulating nerve regeneration microenvironment. Objective: 1. The neural process injury model of rat RGC-5 cells induced by differentiation was established. To observe the effect of dihydrotestosterone (DHT) on regeneration after nerve process injury. To detect the effect of dihydrotestosterone (DHT) on the expression of RhoA and Neuritin in local microenvironment. Methods: 1. The rat model of neuronal oxygen glucose deprivation / reoxidation injury in RGC-5 cells was established by Na2S2O4. Different concentrations of DHT (1, 10 and 100nmol/L) were used to intervene the injury model. The cell survival rate was measured by MTT assay, and the number and length of neuronal processes were observed under optical microscope. The expression of RhoA, NeuritinmRNA and protein were detected by RT-PCR reaction and Western blot assay. Results: the Na2S2O4 of 1.5mmol/L was the best concentration for the establishment of neuronal oxygen glucose deprivation / reoxidation injury model in RGC-5 cells. 2. After oxygen-glucose deprivation / reoxygenation injury, the cell survival rate, the number and length of nerve processes in the model group were lower than those in the normal control group (P < 0.05), DHT), compared with those in the model group. The cell survival rate and the number of nerve processes increased in 10 and 100nmol/L groups, but there was no significant difference in cell survival rate and neurite number in 1nmol/L group (P 0.05). 1the length of nerve processes in 10 and 100nmol/L DHT groups was higher than that in model group (P < 0.05). After oxygen glucose deprivation / reoxygenation injury, the expression of RhoA mRNA and protein in the model group was significantly higher than that in the normal control group (P < 0.05), while the expression of Neuritin mRNA and protein in the model group was significantly lower than that in the normal control group (P < 0.05), DHT). 1the expression of RhoA mRNA and protein in 10 and 100nmol/L DHT groups was lower than that in model group (P < 0.05), while the expression of Neuritin mRNA and protein in 10 and RhoA mRNA group was higher than that in model group (P < 0.05). Conclusion: RhoA and Neuritin may be one of the important factors affecting neuronal regeneration in rat RGC-5 cells after oxygen-glucose deprivation / reoxygenation injury. DHT may play a neuroprotective role by regulating the expression of RhoA and Neuritin.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R774.6
本文编号：2492809
[Abstract]:Background: from the anatomical point of view, the optic nerve belongs to the peripheral nervous system (PNS), but if you look at the characteristics of its development, as well as the structural and functional characteristics, The optic nerve is more similar to the nerve of the central nervous system (CNS), so it is often regarded as a specially differentiated central nervous system. Therefore, similar to the neurons of CNS, the regeneration after RGCs injury is extremely difficult, which is the main cause of irreversible blindness caused by traumatic optic nerve disease and glaucoma in ophthalmic clinic. In recent years, it has been shown that CNS is difficult to regenerate effectively, and the nature of neurons is not the most important factor, and the microenvironment around the nerve is more important because of the strong inhibitory microenvironment around the nerve. Because of the existence of this microenvironment, the regenerated axons atrophy at the initial stage of budding, which leads to the failure of nerve regeneration. At present, the attention of microenvironment in the field of nerve regeneration is increasing rapidly. Androgen is a kind of sex hormone which has been studied more and more in recent years. In addition to promoting the development of the reproductive system and maintaining the secondary sexual characteristics, it also has the function of brain protection. However, there are few reports about whether androgen has protective effect on optic nerve and whether androgen plays a role by regulating nerve regeneration microenvironment. Objective: 1. The neural process injury model of rat RGC-5 cells induced by differentiation was established. To observe the effect of dihydrotestosterone (DHT) on regeneration after nerve process injury. To detect the effect of dihydrotestosterone (DHT) on the expression of RhoA and Neuritin in local microenvironment. Methods: 1. The rat model of neuronal oxygen glucose deprivation / reoxidation injury in RGC-5 cells was established by Na2S2O4. Different concentrations of DHT (1, 10 and 100nmol/L) were used to intervene the injury model. The cell survival rate was measured by MTT assay, and the number and length of neuronal processes were observed under optical microscope. The expression of RhoA, NeuritinmRNA and protein were detected by RT-PCR reaction and Western blot assay. Results: the Na2S2O4 of 1.5mmol/L was the best concentration for the establishment of neuronal oxygen glucose deprivation / reoxidation injury model in RGC-5 cells. 2. After oxygen-glucose deprivation / reoxygenation injury, the cell survival rate, the number and length of nerve processes in the model group were lower than those in the normal control group (P < 0.05), DHT), compared with those in the model group. The cell survival rate and the number of nerve processes increased in 10 and 100nmol/L groups, but there was no significant difference in cell survival rate and neurite number in 1nmol/L group (P 0.05). 1the length of nerve processes in 10 and 100nmol/L DHT groups was higher than that in model group (P < 0.05). After oxygen glucose deprivation / reoxygenation injury, the expression of RhoA mRNA and protein in the model group was significantly higher than that in the normal control group (P < 0.05), while the expression of Neuritin mRNA and protein in the model group was significantly lower than that in the normal control group (P < 0.05), DHT). 1the expression of RhoA mRNA and protein in 10 and 100nmol/L DHT groups was lower than that in model group (P < 0.05), while the expression of Neuritin mRNA and protein in 10 and RhoA mRNA group was higher than that in model group (P < 0.05). Conclusion: RhoA and Neuritin may be one of the important factors affecting neuronal regeneration in rat RGC-5 cells after oxygen-glucose deprivation / reoxygenation injury. DHT may play a neuroprotective role by regulating the expression of RhoA and Neuritin.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R774.6
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本文编号：2492809
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