【作者】 曾勇；

【导师】 冯忠堂； 王廷华；

【作者基本信息】 昆明医学院 ， 神经外科， 2009， 博士

【摘要】 目的：探索视神经损伤大鼠视神经移植后,再生的视网膜神经节细胞的基因表达变化以及激肽释放酶结合蛋白(KBP)对视网膜神经节细胞的保护作用。方法：采用对照研究的方法,将39只Listed Hooded大鼠分为①正常对照组,②神经截断组,③截断+移植组,即神经截断后外周神经移植到神经截断端实验组。采用经典的外周神经缝接模型,用免疫组化技术检测GFAP、GAP-43、NF-KB、HPS-27、视紫红质蛋白等在研究组、对照组以及实验组不同时期表达的变化。通过采用RT-PCR、Real-time PCR技术检测大鼠视网膜中12个基因的mRNA浓度的变化,使用ANOVA进行统计比较,用LSD检测进行Post-Hoc检测。另外34只SD大鼠分为①实验组,②对照组,③正常组。实验组、对照组采用直接挤压法建立不完全损伤模型,采用免疫组化技术用以观察视网膜各层结构和视网膜厚度,并对各时点视网膜神经节细胞进行计数,并用JRuler软件进行分析,行Western-blot实验检测GAP-43蛋白的表达量,以Band Scan 5.0图像分析软件进行分析,旨在明确KBP是否有视网膜神经节细胞保护和促进其轴突再生作用。结果：①视网膜切片显示了神经截断组GFAP在5和14天有明显的表达上调；与截断组的视网膜相比较,在移植后5,14和21天,截断+移植组的GFAP表达下调；而对照组GFAP在GCL染色呈阴性反应。在5天时,坐骨神经段移植后GAP-43在GCL出现明显表达上调,并且GAP-43免疫反应性在移植后的14天有强烈的上调,但在21天后,只有少量的GAP-43在GCL表达。在再生的视网膜中NF-κb迁移到核仁样结构内。HPS-27蛋白质似乎在视神经切断后的5天在RGCs中重新分布。在视神经截断后的14天,此时视网膜RGCs已耗竭,在神经节细胞层中几乎检测不到HSP-27。在截断+移植组的视网膜中,HSP-27在术后持续表达21天。②RT-PCR检测结果中,这些基因未发现明显变化。③Real-time PCR结果,5天后,视网膜中的GFAP表达在截断组和截断+移植组与对照组相比有显著的增加(P<0.01)。然而,GFAP表达在任一时点的截断+移植组和截断组没有显著差异。在5天后,截断+移植组比对照组的视网膜中GAP-43的表达有显著增加(146±11%,P<0.05),然而在截断组中没有观察到同样的增加(108±31%,P<0.05)。在其后的时点,GAP-43的表达在两个实验组都比对照组有显著降低(P<0.05)。截断+移植组大鼠的降低程度的显著性较小,与第5天相比,差异没有统计学意义(14天：64±12% vs.39±4%；21天：46±6% vs.42±1%,)。所有时点,两实验组比对照组视网膜Thy-1的表达都有显著的减少(P<0.01)。Thy-1的表达在截断+移植和截断组大鼠中没有显著差异。截断+移植组与截断组视网膜的ET-1相比,随时间而出现明显的表达上调(14天：108±24% vs.148±21%)。但是所有时点两组组别之间的视网膜ET-1表达的差异没有统计学意义。ETB在各组之间所有时间点,视网膜中的ETB受体的表达没有统计学差异。④SD大鼠视神经不完全损伤后,通过HE染色,我们观察了视网膜各层结构变化,通过双盲法计数RGCs,和视网膜厚度测量,经统计学分析,组间差异具有统计学意义。⑤对神经再生的标记蛋白GAP-43进行Western-blot分析,经统计学处理,组间GAP-43表达差异具有统计学意义。结论：(1)正常大鼠GFAP分布于视网膜神经节细胞层(GCL)和神经纤维层(NFL)的星形胶质细胞中；神经截断大鼠视网膜GFAP呈高表达,提示Muller细胞和星形胶质细胞可能受到抑制；神经截断后外周神经移植大鼠视网膜GFAP表达水平降低,提示神经截断的部分视网膜神经节细胞(RGCs)能够产生再生反应。(2)正常大鼠和神经截断大鼠视网膜无GAP-43表达,而神经截断后外周神经移植大鼠视网膜GAP-43表达上调,提示GAP-43表达可能与神经截断的RGCs再生有关。(3)正常大鼠视网膜中NF-κB定位于细胞质内,神经截断后外周神经移植大鼠视网膜NF-κB易位到细胞核,提示NF-κB活化参与了神经截断的RGCs再生过程。(4)正常大鼠视网膜中RGCs中有HSP-27表达,神经截断大鼠视网膜HSP-27无表达,神经截断后外周神经移植大鼠视网膜HSP27持续表达,提示作为应激蛋白,HSP-27可能对神经截断的RGCs具有保护作用。(5)正常大鼠、神经截断大鼠和神经截断后外周神经移植大鼠视网膜视紫红质的表达无明显变化,提示紫红质与神经截断的RGCs再生无明显关系。(6)神经截断大鼠和神经截断后外周神经移植大鼠视网膜GFAP mRNA表达增加,提示作为神经营养因子,GFAP增加可为截断的神经细胞提供一个适宜再生的微环境,可能在神经截断的RGCs的再生中发挥作用。(7)神经截断后外周神经移植大鼠视网膜GAP-43 mRNA的表达增加,提示GAP-43作为神经元生长相关蛋白,可能参与神经截断的RGCs的再生。(8)神经截断大鼠和神经截断后外周神经移植大鼠视网膜Thy-1 mRNA表达降低,可能作为评价神经截断的RGCs再生能力的一个参考指标。(9)神经截断后外周神经移植大鼠视网膜ET-1 mRNA表达明显增加,提示作为神经因子,能够维持神经和血管完整性,可能与促进神经截断的RGCs再生有关。(10)正常大鼠、神经截断大鼠和神经截断后外周神经移植大鼠视网膜ETB mRNA表达无明显变化,提示ETB与神经截断的RGCs再生无明显关系。(11)视神经不完全损伤后玻璃体腔注入PBS溶液,大鼠视网膜视神经节细胞核染色深浅不一,核碎裂,有的细胞核呈空泡状改变,各层次间结构尚存,视神经节细胞数量减少明显,细胞极性极度紊乱,内丛状层变薄,甚至消失,提示PBS对视神经损伤无保护作用。(12)视神经不完全损伤后玻璃体腔注入KBP蛋白溶液,大鼠视网膜各层次结构尚清晰,视神经节细胞细胞呈单层排列,细胞核染色均匀,视神经节细胞数量随时间的增加而减少,出现轻度的内丛状层变薄,细胞极性轻度紊乱,提示KBP蛋白对视神经损伤具有一定的保护作用。(13)视神经不完全损伤后玻璃体腔注入KBP蛋白溶液,大鼠视网膜视神经节细胞细胞平均数量、视网膜平均厚度和视神经GAP-43蛋白表达均高于视神经不完全损伤后玻璃体腔注入PBS溶液大鼠,提示提示KBP蛋白对视神经损伤具有一定的保护作用。(14)玻璃体腔注入KBP蛋白溶液可为临床治疗视神经损伤提供科学的理论和实验依据。更多还原

【Abstract】 Objective To explore the changes of gene expression in regeneration of retina gangling cells after neural transplantation and protection of Kallikrein-Binding Protein(KBP) to Retinal Ganglion Cells (RGCs)Methods 39 Listed Hooded rats were researched by the approach of case control, by spliting which into normal control group, neural amputation group and experiment group on rats of which neural amputations were performed and then peripheral nerves were transplanted. Classic peripheral nerve spliced models were adopted, and immunohistochemical technique was performed to detect changes of expression of GFAP, GAP-43, NF-Kb, HPS-27 and rhodopsin protein during different periods in control and experimental group. RT-PCR, Real-time PCR techniques were adopted to detect mRNA concentration changes of 13 genes in Rats’ RGCs. NOVA was adopted to compare, and LSD test was used to Post-Hoc test to explore changes of gene expression in regenerating of RGCs after Optic nerve injury. In addition,34 SD rats, splited in to the experimental group, control group and normal group. In the rat model of optic nerve injury by direct pressurization method, immunohistochemical technique was adopted to observe the structure of retinal, measure the thick of retina and count the number of survival RGCs, and data were analyzed by JRuler Software. The rat optic nerves were collected to extraction protein for Western blot analysis to determining expression of GAP-43 and data was analyzed by Software of Band Scan 5.0 to identify whether KBP has protective function to retinal ganglion cells and promote regeneration of axon.Results (1) Amphiblestrodes cut sheets displayed GFAP increased visibly on the 5th and 14th day after neural amputation. Compared with injured amphiblestrodes, on the 5th,14th and 21st day after neural transplantation, GFAP expressed in a low level in transplanted and anageneticed nerves.Tinction in control group and neural amputation group showed negative immune reaction on GCL. On the 5th day after a piece of ischiadic nerve was transplanted, GAP-43 espression inceased visibly on GCL, and GAP-43 immunoreactivity increaseed greatly on the 14th day after transplantion, and after 21 days, only a little GAP-43 expressed on GCL. In anagenetic amphiblestrodes, NF-κb transferred into nucleoloid structures. HPS-27 seemed to redistribute in RGC on the 5th day after optic neural amputation. On the 14th day after optic neural amputation, amphiblestrode RGCs had exhausted, and little HPS-27 could be detected on GCL. In nerve amputated+transplanted group, HSP-27 expressed persistently for 21 days. (2)No marked change was found in these gene’s RT-PCR test results. (3) Real-time PCR results indicated 5 days later, amphiblestrode GFAP increased significantly in amputation group, experiment group and control group (p<0.01). But GFAP expressiong was not showed significantly differences at all times. 5 days later, amphiblestrode GAP-43 in nerve amputated+transplanted group increased much more siginificantly than that in control group (146±11%, P<0.05), however, same increase was not found in amputation group(108±31%, P<0.05). At time points after that, GAP-43 expression in both two experiment groups was decreased much more significantly than control group (P<0.05).The degree of decrease in nerve amputated+transplanted group was much lighter, and differences were not statistically significant, compared with the 5th day (14days:64±12% vs.39±4%; 21 days:46±6% vs.42±1%). At all times, amphiblestrode Thy-1 expression in both two experiment groups decreased significantly (P<0.01). There was no marked qualitative difference about Thy-1 expression in both nerve amputated+transplanted group and amputation group. Results:Amphiblestroid ET-1 expression of nerve amputated+ transplanted group increased more significantly than amputation group as time went on(14 days:108±24% vs 148±21%). Instead, differences of amphiblestroid ET-1 expression between the two groups were not statistically significant at all times P.There was significant difference between all groups about amphiblestroid ETB receptors’ expression at all timesP. (4)The optic nerve injury rats with HE staining, we observed the structural changes in the all levels of the retina. The double-blind RGCs counting and retinal thickness measurement showed the statistical significance differences between the groups. (5)We measured the expression of the nerve regeneration marker protein GAP-43 by Western-blot. The statistical signify can were showed between the inter-group. Conclusions (1)GFAP is distributed in astrocytes of GCL and NFL of retinal ganglial cells (RGCs) in normal rats; up-regulated expression of GFAP in nerve amputated indicated Muller cells and astrocytes may be inhibited; GFAP’s low expression of in nerve amputated+transplanted group indicated RGCs may regenerate. (2)There is no GAP-43 expression in normal and amputated rats’retina, however, GAP-43’s up-regulated expression indicated expression of GAP-43 may related to regeneration of RGCs. (3)NF-κB is located in cytoplasm in normal rats’retina, and NF-κB shifted into karyon after nerve amputeated. This indicated activation of NF-κB participates regeneration of RGCs. (4)HSP-27 may expressed in RGCs of normal rats but not in nerve amputated group. HSP-27 expressed persistently in nerve amputated+ transplanted group suggested HSP-27 may have protective effect on RGCs as a reactive protein. (5)Erythropsin didn’t express in the three groups and suggested no significant relation between erythropsin and regeneration of RGCs. (6)GFAP mRNA up-regulation expressed in both in nerve amputated+transplanted group and amputated group, showing increased GFAP may provide an eligible microcircumstance for nerve regeneration as a neurotrophic factor. (7)GAP-43 mRNA increased in expression in RGCs of nerve amputated+transplanted group, showing GAP-43 may participate regeneration of RGCs. (8)Thy-1 mRNA decreased in expression in RGCs of nerve amputated+transplanted group, which can be used as a index to assess regeneration capacity of RGCs. (9)ET-1 mRNA increased significantly in expression in retina of nerve amputated+transplanted group. As a neurotropic factor, it can maintain integrality of nerve and vessels and promote regeneration of RGCs. (10)No significant change of ETB mRNA expression in the three groups showed no relationship between ETB and regeneration of RGCs. (11)PBS solution was pured into vitreous chamber after incomplete injury of optic nerves, and nucleolus dye of rats’ RGCs was deep mixed. Nuclear fragmentation, vesicular nucleus, reduced RGCs, disorganized cell polarity and thinner inner plexiform layer indicated PBS can’t protect injured nerves. (12)KBP protein solution was pured into vitreous chamber after incomplete injury of optic nerves, and every layers was clear in rats’ retina. RGCs was arrayed in a monolayer, nucleolus were level dyeing, number of RGCs was reduced, inner plexiform layer was slight thinner and cell polarity was disorganized slightly. These suggested KBP protein may protect injured nerves. (13)KBP protein solution was pured into vitreous chamber after incomplete injury of optic nerves, and average quantity of RGCs, average thickness of retina and GAP-43 expression were higher than rats that were pured into PBS solution in their vitreous chambers. This suggested KBP protein may protect injured nerves. (14)KBP protein solution was pured into vitreous chamber, which can provide theoretical and practical foundations to clinical treatment of optic nerve injury.更多还原