【作者】 陈翾；

【导师】 唐罗生；

【作者基本信息】 中南大学 ， 眼科学， 2009， 博士

【摘要】 第一部分大鼠视网膜缺血再灌注损伤模型的建立及细胞凋亡的检测目的:采用前房高压灌注方法建立RIR损伤模型,并检测视网膜细胞凋亡的情况。方法:72只SD大鼠随机分为对照组和RIR损伤组。RIR组分别包括6个亚组即再灌注后3h、6h、12h、24h、72h、和7d,每亚组6只,采用前房高压灌注法(110mmHg×60min)建立大鼠RIR损伤模型。应用尼氏染色观察视网膜组织形态学改变,应用核酸末端转移酶介导的dUTP末端标记法(TUNEL)原位检测视网膜凋亡细胞。结果:1、在大鼠前房高压灌注建模过程中,可以清楚观察到模型动物发生视网膜缺血-血液复流征象。2、RIR后视网膜组织形态学改变:尼氏染色结果显示RIR后6h、12h内丛状层出现水肿及空泡形成,24h可见核固缩;RIR后7d视网膜内丛状层则明显变薄;与正常对照组比较有统计学意义(P＜0.01)。3、视网膜细胞凋亡情况:对照组视网膜在各时间点偶见TUNEL阳性细胞;与对照组比较:RIR组于再灌注后6h、12h、24h和、72h视网膜TUNEL阳性细胞显著性增多(P＜0.01),72h时则明显减少;而7d时,TUNEL阳性细胞极少见。TUNEL阳性细胞主要位于视网膜神经节细胞层和内核层,外核层均未见TUNEL阳性细胞。结论:采用升高眼压(110mmHg×60min)的方法能成功建立伴视网膜细胞凋亡的RIR损伤模型。第二部分PAP1基因在大鼠视网膜RIR中表达目的:了解PAP1基因在大鼠视网膜的表达情况及RIR损伤后PAP1基因表达的变化方法:120只SD大鼠随机分为对照组和RIR组,分别包括6个亚组即再灌注后3h、6h、24h、72h及7d组,每组10只。RT-PCR方法及原位杂交方法检测PAP1基因在正常视网膜中的表达及RIR后表达的变化。结果:1、PAP1基因大鼠视网膜组织中有表达;其表达部位主要在视网膜节细胞层和内核层。2、与正常对照组相比,RIR组PAP1-mRNA的表达在RIR损伤后3h表达增加(P＜0.01);RIR损伤后6h、12h、24h其表达明显减少,至24h达最低值(P＜0.01);RIR损伤后72h、7d组其表达增加(P＜0.01)。3、原位杂交法观察显示:与对照组比较,PAP1基因在RIR损伤后3h表达增加(P＜0.01);RIR后6h、12h、24h其表达明显减少,;RIR.后72h、7d其表达又开始增加(P＜0.01);其表达部位主要在视网膜神经节细胞层和内核层。结论:1、PAP1基因在大鼠视网膜组织中有表达。2、RIR损伤后PAP1基因的表达有变化。第三部分RIR损伤后PAP1基因表达与细胞凋亡目的:探讨RIR损伤后PAP1基因表达的变化与所致细胞凋亡的关系,以期进一步了解RIR损伤致细胞凋亡的可能机制。方法:采用前房高压灌注(110mmHg×60min)建立大鼠RIR损伤模型。1.48只SD大鼠随机分为对照组和RIR组,分别包括6个亚组即再灌注后3h、6h、12h、24h、72h和7d每亚组4只,用TUNEL法原位检测视网膜细胞凋亡情况。2.60只SD大鼠随机分为对照组和RIR组,RIR组同样分为6个亚组即再灌注后3h、6h、12h、24h、72h、7d,每亚组5只,用半定量RT-PCR方法检测PAP1-mRNA的表达水平。结果:1、与对照组相比较:RIR损伤后3h,视网膜TUNEL阳性细胞无明显变化(P＞0.05),RIR损伤后6h、12h、24h、72h视网膜TUNEL阳性细胞明显增加(P＜0.01)。RIR后7d时TUNEL阳性细胞极少见(P＞0.05)。2、与对照组相比较:PAP1-mRNA的表达在RIR后3h有所增加(P＜0.01)而RIR损伤6h、12h、24h则明显减少;RIR损伤后72h、7d时又开始增加(P＜0.01)。3、PAP1基因表达与细胞凋亡之间呈负相关(r=-0.679,P＜0.01)。结论:PAP1基因在RIR损伤致细胞凋亡过程中可能具有保护性作用。更多还原

【Abstract】 PART I The rat model of retinal ischemia-reperfusion injury can be established and the apoptosis of the retinal cells was detected.[Objective]To establish a rat model of retinal ischemia-reperfusion (RIR) imjury by transiently elevated intraocular pressure (IOP) and to detect the apoptosis of the retinal cells.[Methods]72 sprague-Dawley (SD) rats were divided randomly into control group and RIR group .Each group Was consisted of 6 subgroups (3h、6h、12h、24h、72h and 7d after RIR; n=6).Retinal ischemia was induced of a needle into the anterior chamber connected to a saline column.Elevated IOP at 110 mmHg was maintained for 60 minutes.Morphological changes of the model’ s retinas were study using nissel staining. The apoptosis of the retinal cells were evaluated dynamically by in situ terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling (TUNEL).[Results]1. The sign of retinal ischemia and reperfusion were clearly investigated in the model of RIR.2.The morphological changes of the rats retinas:The edema and vaculoatoin in inner plexiform layer (IPL)presented at 6h and 12h after RIR; karyopycnosis presented at 24h after RIR; The thickness of retinal IPL in RIR group at 7d was significantly thinner than that of control group (P< 0.01).3.The apoptosis of the retinal cells: The TUNEL-postive cells presented mainly in the retinal ganglion cell layers and the inner nuclear layers, and few postive cells in external nuclear layers.Few TUNEL postive cells were observed in the normal retina; Compared with the control group, the mumber of TUNEL postive cells in RIR group was significantly increased at the time points of 6h to 72h after reperfusion, respectively (P<0.01).The number of TUNEL postive cells reached a maximum at 24h and followed by a decreased at 72h after reperfusion.[Conclusions]The model of retinal ischemia-reperfusion with apoptosis of the retinal cells can be successfully established by elevated intraocular pressure at 110 mmHg and maintained for 60 minutes. PART II The expression of PAPl gene in rat retinaAfter RIR[Objective]To observe the expression of PAP1 gene in rat retina and the change of PAP1 gene expression after RIR.[Methods]120 Sprague-Dawley (SD) rats were divided randomly into cotrol group and RIR group. Each group was consisted of 6 subgroups (3h、6h、12h、24h、72h、and 7d after RIR n=10) Retinal ischemia was induced by transiently elevated Intraocucar pressure through the insertion of a needle into the anterior chamber connected to a saline column。Elevated the intraocular perssure at 110 mmHg was maintained for 60 minutes.The transcription levels and the changes of the expression of PAP1 gene in the retina after RIR were dynamically detected by semiquantitative reverse transcriptase-polymerase chain (RT-PCR) and in situ hybridization.[Results]1. There is the expression of PAP1 gene in the retina in the control group.The expression of PAP1 gene was detected in the GCL and INL of the retina sections.2.Compared with the control group,the transcription levels of PAP1-mRNA significantly increased as early as 3h after RIR(P < 0.01)and then significantly decreased at 6h、12h、24h(P<0.01).But, the expression amount of PAP1-mRNA signifcantly increased at 72h> 7d after RIR(P<0.01).3.The expression levels of PAP1 gene was detected in situ hybridization technique .Compared with the control group,the expression anount of PAP1 gene significantly increased at 3h(P<0.01),and then decreased at 6h、12h and 24h(P<0.01).The expression amount of PAP1 gene significantly increased at 72h、7d after RIR(P<0.01),the expression of PAP1 gene was detected in GCL and INL of the retina sections .[Conclusion]The expression of PAP1 gene was detected in the retina.There is the changes of the PAP1 gene expression amount in the retina after RIR. PART III PAP1 gene expression and the apoptosis in ratRetinal cells after RIR[Objective]To investigate the possible mechanism by exploring the relationship between the changes of PAP1 gene expression and the apoptosis in rat retinal cells after RIR.[Metheds]The model of RIR was established by elevated intraocular pressure at 110mmHg and maintained for 60 minutes.48 SD rats were randomly divided into control group and RIR group.Each group was consisted of 6 subgroups (i.e. 3h、6h、12h、24h、72h and 7d after RIR, n=4) , The apoptosis of retinal cells were evaluated dynamically by in situ terminal deoxynucleotidyl transferase mediated biotin -deoxyuridine triphosphate nick-end labeling (TUNEL).[Results]1 .Compared with the control group,the number of TUNEL postive cells in RIR group was significantly increased at the time points of 6h to 72h after reperfusion,respectively(P < 0.01).The number of TUNEL postive cells was not significantly higher than the control group at the time points of 3h and 7d after reperfusion(P>0.05).2.Compared with the control group ,the expression amount of PAP1-mRNA in RIR group was significantly increased at the time points of 3h(P<0.01);and the expression amount of PAP1-mRNA in RIR group was significantly decreased at the time points of 6h 12h and 24h (P< 0.01)and then increased significatly at the time points of 72h and 7d (P< 0.01).3. There was negative correlation between the expression of PAP1 gene and the apoptosis in retinal cells (r= - 0.679, P<0.01) .[Conclusion] PAP1 gene may be involved in the mechanisms of retinal ischemia-reperfusion injury and cell apoptosis. PAP1 gene maybe protects the retina.更多还原