【作者】 陈芝清；

【导师】 姚克；

【作者基本信息】 浙江大学 ， 眼科学， 2008， 博士

【摘要】 视网膜缺血再灌注损伤是眼科常见的病理过程以及致盲的主要原因。已有众多研究证实视网膜缺血再灌注损伤导致细胞凋亡且伴有视网膜细胞内Ca²⁺浓度的升高。钙蛋白酶（calpains）是一种全身广泛存在的中性限制性钙蛋白水解酶。在病理状态下,异常增高的Ca²⁺浓度可激活calpains,引起疾病的发生。E-64d是一种具有细胞膜高通透性的、与calpains的Ca²⁺结合位点高度选择性结合的calpains抑制剂,已被证实对神经系统有较好的抗蛋白水解作用。本研究从形态学水平、细胞学水平、蛋白质水平和分子水平四个层次研究了急性高眼压诱导的视网膜缺血再灌注后视网膜神经上皮水肿和坏死程度、细胞凋亡、calpains的蛋白表达和mRNA的改变,以及calpains抑制剂E-64d对上述改变的抑制作用,探讨了以急性高眼压动物模型研究视网膜缺血再灌注发病机制的可行性,calpains在其发生机制中的作用及机理,以及E-64d对视网膜缺血再灌注的预防作用。第一部分急性高眼压性视网膜缺血再灌注损伤模型的建立目的:本研究将急性高眼压诱发大鼠视网膜缺血再灌注损伤作为实验模型,来研究急性高眼压对视网膜的水肿、细胞形态学改变以及视网膜神经元细胞凋亡的影响,探讨将急性高眼压诱发的缺血再灌注损伤作为动物模型研究其发病机制的可行性。方法:采用清洁级200～300g SD大鼠。将连接生理盐水输液管的20号留置针刺入大鼠前房,然后升高升高输液瓶形成109.7 mmHg的眼压,持续1h后降低输液瓶高度至动物水平,拔出前房灌注针头,可见眼结膜充血,恢复血液供应,缺血再灌注后1、3、6、24、72h处死动物,摘除眼球。标本常规10%中性多聚甲醛固定,石蜡包埋,切片,HE染色后置光镜下观察。Bax和m-calpain蛋白表达测定采用免疫组化方法。结果:SD大鼠视网膜缺血再灌注后1～24h,光镜可见视网膜明显水肿,神经元细胞胞体及线粒体肿胀,细胞膜崩解,核染色体不规则分布,细胞坏死。细胞坏死最早见于再灌注1h后,72小时视网膜变薄。在细胞坏死的同时可见凋亡,凋亡相关蛋白Bax在急性高眼压导致缺血的视网膜各层细胞的表达上调,呈强阳性反应。随着再灌注作用时间的延长,钙蛋白酶m-calpain阳性细胞百分比逐渐增多。结论:本研究证实了急性急性高眼压确可诱发视网膜神经元细胞坏死、凋亡,视网膜萎缩变薄,在细胞坏死的同时可见凋亡,凋亡相关蛋白Bax以及钙蛋白酶m-calpain表达上升,将急性急性高眼压性模型作为动物模型研究视网膜缺血再灌注损伤的发病机制中有一定可行性。第二部分:急性高眼压诱导的视网膜缺血再灌注损伤中的凋亡及钙蛋白酶表达目的:探讨caspase-3、m-calpain、calpastatin蛋白相关的细胞凋亡、以及钙蛋白酶在急性高眼压诱导的视网膜缺血再灌注损伤中的发生机制中的作用。方法:用急性高眼压诱发大鼠发生视网膜缺血再灌注损伤,用western-blot的方法研究m-calpain蛋白、caspase-3的表达,用RT-PCR法测定calpains、calpastatin的mRNA的改变,并与对照组进行比较。结果:SD大鼠在视网膜缺血再灌注前均有m-calpain和caspase-3蛋白的基础表达。缺血再灌注后24 h m-calpain和caspase-3的表达均较正常组上升,视网膜缺血再灌注后m-calpain/calpastatin的比值在6h后增加,24h达到最高峰,72h后开始回落。结论:急性高眼压诱导大鼠视网膜缺血再灌注损伤中的细胞凋亡,m-calpain和caspase-3的表达上升,缺血损伤导致calpain/calpastatin的比例上升。第三部分:钙蛋白酶抑制剂E-64d对急性高眼压诱导的视网膜缺血再灌注损伤预防作用目的:探讨E-64d（半胱氨酸蛋白酶抑制剂）在大鼠视网膜缺血再灌注损伤（RIRI）中对calpain和caspase-3表达的调节作用。方法:将SD大鼠随机分为视网膜缺血再灌注组,E-64d治疗组和正常组。通过前房穿刺加压法制成视网膜缺血再灌注损伤（RIRI）动物模型,缺血再灌注分为1、3、6、24、72小时5个时间段。采用Western-blot以及RT-PCR方法测定在大鼠视网膜缺血再灌注损伤的calpian蛋白和mRNA表达情况以及caspase-3的蛋白表达改变。结果:缺血再灌注后24小时m-calpain、caspase-3蛋白的表达均较正常组上升,而玻璃体腔注射E-64d组m-calpain、caspase-3蛋白表达与正常组相似,比缺血再灌注组下降。视网膜缺血再灌注后m-calpain/calpastatin mRNA的比值在6小时后上升,24小时达到最高峰,72小时后开始回落,其中缺血再灌注组6小时、24小时、72小时与正常组比较P值均小于0.05,其差异有显著统计学意义;E-64d组与视网膜缺血再灌注24小时组比较,P值小于0.05,其差异也有显著统计学意义。但是三组的m-calpain和calpastatin mRNA转录强度差异均无显著的统计学意义（P＞0.05）。结论:E-64d作用后较缺血再灌注同一时间段的视网膜细胞形态明显好转,水肿减轻,视网膜各层细胞m-calpain蛋白阳性率明显下降。E-64d能降低视网膜缺血再灌注时m-calpain、caspase-3蛋白的表达,调控m-calpain/calpastatin的mRNA比值。可能对视网膜的缺血再灌注损伤有保护作用。更多还原

【Abstract】 Retinal ischemia is a common clinical entity which remains a common cause of visual impairment and blindness in the world because of the lack of effective treatment. Ischemia/reperfusion injury （IRI） of the retina is one of the deleterious pathophysiological processes leading to visual impairment. It is well studied that apoptosis and calcium concentration elevates in IRI. The family of calpains is one kind of neutral restrictive proteolytic enzyme which distributes diffusely. Under pathological condition, abnormal elevated calcium can activate calpains and induce diseases. E-64d is a specific inhibitor of calpains with high permeability and high combinative ability. It is known that E-64d can protect neural system from proteolytic enzyme.The retinal thickness and morphologic changes was detected by histology. The protein expression of m-calpain （a calpain isoform） in the retina was accessed by immunohistochemistry and Western blot assay. The mRNA of m-calpain as well as calpastatin （an endogenous protein inhibitor of calpain） in the retina was accessed by RT-PCR, and the ratio of m-calpain/calpastatin was then calculated. To evaluate the effect of E-64d on the expression calpain, the drug （5ul of 100uM） was injected intravitreously immediately after IRI. Our study showed that there were retinal edematous changes, particularly in the inner plexiform layer after IRI. Calpain is involved in the retina injury of rats induced by ischemia/reperfusion. Calpain inhibitor E-64d decreased the protein expression of calpain, as well as the ratio increase of calpain/calpastatin mRNA. E-64d also inhibited retinal damage induced by IRI, suggesting the possible role of E-64d in the protection of retina apoptosis induced by IRI..Role of Calpains in the Mechanism of High intraocular pressure （IOP）-induced Ischemia/Reperfusion Injury in Rat RetinaIntroductionRetinal ischemia is a common clinical entity which remains a common cause of visual impairment and blindness in the world because of the lack of effective treatment. Ischemia/reperfusion injury （IRI） of the retina is one of the deleterious pathophysiological processes leading to visual impairment. Understanding the pathophysiological effect of IRI on the retina is important for the development of therapeutic strategies for protecting the retinal neurons. Studies have suggested that IRI initiates apoptosis in the retinal neuro-epithelium.The cascade of neuronal apoptosis, as reported in other tissues, is evidenced by the increased expression of bcl-2 family genes such as bax （pro-apoptosis） and bcl-2 （anti-apoptosis）, with an increased bax/bcl-2 ratio favoring apoptosis. The final ’execution’ of apoptosis is mediated by the activation of cysteine proteases, including caspases and calpains. Calpains, which mainly consist of two isoforms named m-calpain(activated in mili-molar Ca²⁺) and u-calpain (activated in micro-molar Ca²⁺), which are regulated by the endogenous inhibitor, calpastatin. The activity of calpastatin is considered to play a critical role in preventing calpain-mediated catabolism in tissues. With an increased calpain to calpastatin ratio, calpastatin is digested by calpain and loses its inhibitory activity. Since calpain activity may mediate cell death and damage in the IRI, it is of considerable interest to explore the benefit of a therapeutic approach that involves calpain inhibitors in neurodegenerative diseases. It has already been shown that E-64d, the first reported cell permeable calpain inhibitor, is capable of providing potent neuro-protective effect by inhibiting calpain-related neuron apoptosis in the injury of spinal cord. However, the involvement of calpains, and further, the effect of E-64d, is still unclear in the retina neuronal apoptosis induced by IRI.High intraocular pressure （IOP）-induced ocular ischemia is a commonly used model for retinal ischemia research and has been described in a number of animal species, such as the rat and rabbit. High IOP induces global ischemia with obstruction of both the retinal and uveal circulation. This particular model presents pathological features which are almost identical to that seen after central retinal artery obstruction （CRAO）. In such a model, we investigated the effect of E-64d on the expression of m-calpain and calpastatin in rat retina subjected to IRI. 1. Establishment of the Animal model of Ischemia/Reperfusion Injury in the Rat RetinaPurpose:To study the possibility of using high intraocular pressure （IOP）-induced Ischemia/Reperfusion Injury as the animal model.Methods:Sprague-Daley rats weighing 200-300g were purchased from the Shanghai laboratory animal center, the Chinese Academy of Science. Rats were maintained under standard lighting conditions with a 12:12h light/dark cycle at a room temperature between 21～24°C. A slit-lamp microscopic examination was performed on each rat to exclude any ocular defect. The rats were then anesthetized with 10% chloral hydrate （400 mg/kg） intraperitoneally. A 20-gauge needle connected with normal saline tubing was introduced into the anterior chamber of the right eye. A high IOP （109.7 mmHg） was achieved and maintained by lifting the infusion bottle to a height of 150 cm for 1 hour. The high IOP was attenuated by taking out the needle from the anterior chamber. The rat was then sacrificed the eyeball enucleated at 1、3、6、24 or 72 hours after the procedure. The eyeballs were fixed with paraformaldehyde and embedded in paraffin. The retinas were cut into 5μm thick sections and were processed for hematoxylin and eosin staining as well as immunohistochemistry.Results:There was an obvious increase in retinal thickness after IRI, particularly in the inner plexiform layer. Edematous changes were also seen in cells in the ganglion cell layer （GCL）. Many pyknotic cells were found in the inner nuclear layer （INL） and outer nuclear layer （ONL） at 6h and 24h. By the end of the experiment, 72h after the IRI, cells in the retinal layers did exhibit obvious degenerative changes. Immunostaining of m-calpain as well as bax were detected slightly or not at all in GCL and INL. The staining became more intense from 1 to 24h, and was largely confined to the cells of the GCL and INL. There was most intense staining in cells located in the outer regions of the INL. The staining in the INL appeared to mark a structure lining the outer boundary of this cellular layer and the outer plexiform layer.Conclusions:High intraocular pressure （IOP） can induce Ischemia/Reperfusion Injury in the rat retina. The changes of retinal morphology were similar with retinal ischemia. It is a recommendable way to use high intraocular pressure （IOP）-induced animal as the model of Ischemia/Reperfusion Injury. 2.Role of Calpains in the Mechanism of High intraocular pressure （IOP）-induced Ischemia/Reperfusion Injury in Rat RetinaPurpose:To investigate the role of apoptosis and Calpains expression in the high intraocular pressure （IOP）-induced Ischemia/Reperfusion Injury in Rat Retina. Methods: Animal model retina IRI was set up by increasing the intraocular pressure （110 mmHg） of a rat eye for 1 h. The retinal thickness and morphologic changes was detected by histology. The protein expression of m-calpain （a calpain isoform） in the retina was accessed by immunohistochemistry and Western blot assay. The mRNA of m-calpain as well as calpastatin （an endogenous protein inhibitor of calpain） in the retina was accessed by RT-PCR, and the ratio of m-calpain/calpastatin was then calculated.Results:There were retinal edematous changes, particularly in the inner plexiform layer after IRI. The protein expression of m-calpain in the retina was increased 24h after IRI. The mRNA expression of m-calpain and calpastatin was also increased 24h and 3h after IRI, respectively. The mRNA ratio of mcalpain/calpastatin was increased at the 6h, 24h and 72h after IRI （P < 0.05）.Conclusions:There were apoptosis and increase of m-calpain protein expression, as well as the mRNA ratio increase of m-calpain/calpastatin in the rat retina of IRI. 3. Inhibition of Calpain Expression by E-64d in the Rat Retina Subjected to Ischemia/Reperfusion InjuryPurpose:To investigate the effect of E-64d, a selective inhibitor of calpain, on the expression of calpain and calpastatin in rat retina of ischemia/reperfusion injury （IRI）.Methods:Animal model of retina IRI was set up by increase the intraocular pressure （110 mmHg） of a rat eye for 1 h. The retinal thickness and morphologic changes was detected by histology. The protein expression of m-calpain （a calpain isoform） in the retina was accessed by immunohistochemistry and Western blot assay. The mRNA of m-calpain as well as calpastatin （an endogenous protein inhibitor of calpain） in the retina was accessed by RT-PCR, and the ratio of m-calpain/calpastatin was then calculated. To evaluate the effect of E-64d on the expression calpain, the drug （5 ul of 100 uM） was injected intravitreously immediately after IRI.Results:There was retinal edematous changes, particularly in the inner plexiform layer after IRI. The protein expression of m-calpain in the retina was increased 24h after IRI, an effect that was inhibited by E-64d （P < 0.05）. The mRNA expression of m-calpain and calpastatin was also increased 24h and 3h after IRI, respectively. Neither m-calpain nor calpastatin mRNA expression was influenced by E-64d （P > 0.05）. The mRNA ratio of mcalpain/calpastatin was increased at the 6h, 24h and 72h after IRI and only at 24h the increase of the ratio was inhibited by E-64d （P < 0.05）. Conclusions:In the rat retina of IRI, E-64d inhibits the increase of m-calpain protein expression, as well as the mRNA ratio increase of m-calpain/calpastatin. E-64d also inhibited retinal damage induced by IRI, suggesting the possible role of E-64d in the protection of retina apoptosis induced by IRI更多还原