LIF对小鼠眼视网膜缺血—再灌注损伤中视网膜神经节细胞保护作用的实验研究
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摘要
目的:观察白血病抑制因子(Leukemia inhibitory factor, LIF)在小鼠眼视网膜神经节细胞(retinal ganglion cells,RGCs)中的表达,探讨其对小鼠眼视网膜缺血-再灌注(retinal ischemia reperfusion,RIR)损伤中视网膜神经节细胞的保护作用,为临床治疗视网膜缺血-再灌注损伤类疾病尤其是青光眼提供一种有效的新方法。
方法:将54只正常无眼疾昆明小鼠(108只眼)随机分为正常对照组(18只小鼠,36只眼)、视网膜缺血-再灌注组(RIR组,18只小鼠,36只眼)、LIF治疗组(18只小鼠,36只眼)。每组根据再灌注的时间不同分为4h、8h、12h、18h、24h、48h 6个小组(每小组3只小鼠,6只眼)。采用小鼠眼前房灌注至高眼压的方法制作实验性视网膜缺血-再灌注模型,治疗组于造模完成后立即向小鼠玻璃体腔内注射0.5ul/g LIF。分别于再灌注后4h、8h、12h、18h、24h、48h摘除眼球,固定、石蜡包埋、切片,进行HE染色及免疫荧光染色。普通光学显微镜下观察视网膜形态变化,应用医学图像分析系统对视网膜神经节细胞进行量化,并进行统计学分析。荧光显微镜下观察LIF的表达。
结果:
1.光镜观察: RIR组组织学变化早期为视网膜水肿(尤以视网膜神经纤维层及内丛状层为明显)。晚期视网膜内层萎缩、变薄,视网膜神经节细胞数目减少。治疗组视网膜神经节细胞明显多于RIR组。
2.荧光显微镜观察:LIF表达于各组视网膜神经节细胞上。
3.细胞计数结果:治疗组4h与RIR组4h、正常对照组4h视网膜神经节细胞数目相比较差异无显著性(P>0.05),治疗组8h、12h、18h、24h、48h比RIR组8h、12h、18h、24h、48h视网膜神经节细胞数目明显增多,差异有显著性(P<0.05)。但比正常对照组8h、12h、18h、24h、48h数目为少,相比差异有显著性(P<0.05)。
结论:
1.病理学改变显示视网膜缺血-再灌注模型建模成功。
2.LIF在小鼠正常眼及视网膜缺血-再灌注后视网膜神经节细胞上均有表达。
3. LIF对小鼠眼视网膜缺血-再灌注后视网膜神经节细胞具有保护作用。
Objective: To observe the expression of Leukemia Inhibitory Factor on mouse retinal gonglion cell and investigate its neuroprotective effect on mouse retinal gonglion cell after ischemic-reperfusion injury.To supply a effected and new method for treating retinal ischemic-reperfusion injury especially glaucoma.
Methods: Healthy fifty-four kunming mice(one hundred and eight eyes)were randomly divided into normal control group(eighteen mice,thirty-six eyes), retinal ischemia reperfusion group(RIR group,eighteen mice,thirty-six eyes),and LIF treatment group(eighteen mice,thirty-six eyes) . These three groups were subdivided into group 4 ,8,12 ,18, 24 and 48 hours after reperfusion respectively(every group three mice,six eyes).Transiently elevating the pressure of the anterior chamber with perfusion of normal saline to induce experimental retina ischemia reperfusion injury modle. .After madding mouse models, 0.5ul/g LIF was injected into vitreous cavity of the treatment group mice after ischemia/reperfusion immediately. The eyes were enucleated after reperfusion for 4,8,12,18,24,48 hours.,then fixed, embedded and sectioned.made the sample HE staining and immunofluorescence staining.The morphological change of retina was studied by light microscopy.The number of retinal Gonglion Cell was measured with compute picture analytic system.and statistical conclusions were obtained. The expression of LIF on retinal Gonglion Cell was observed with fluorescence microscope.
Result:
1. Light microscope: In the ischemia reperfusion group, we can found edema of retinal (especially in the neuron fibrary layer and inner plexiform layer) in the early stage and the retinal atrophy, degeneration and necrosis were found in the later period. We can found that the RGCs was much less than the normal control group in the ischemia reperfusion group. In the treatment group, the RGCs was much more than the ischemia reperfusion group.
2. Fluorescence microscope: LIF expressed on retinal gonglion cell in every griup.
3. The number of RGCs :Compared with the control group and the retinal ischemia reperfusion group, the number of RGCs had no significant differences (P>0.05) at 4th hour after reperfusion .The number of RGCs is much more (P<0.05)in the LIF treatment group than the ischemia reperfusion group at 4th,8th,12th ,18th, 24th and 48th hour after reperfusion ,but is less (P<0.05)than the control group at 4th,8th,12th ,18th, 24th and 48th hour after reperfusion .
Conclusion:
1. Pathological changes displayed: the retinal ischemia reperfusion model was made successfully.
2. As to mouse, there was the expression of LIF on retinal gonglion cell .
3. LIF could provide neuroprotective effect for mouse retinal Gonglion Cell after ischemic-reperfusion induced injury
方法:将54只正常无眼疾昆明小鼠(108只眼)随机分为正常对照组(18只小鼠,36只眼)、视网膜缺血-再灌注组(RIR组,18只小鼠,36只眼)、LIF治疗组(18只小鼠,36只眼)。每组根据再灌注的时间不同分为4h、8h、12h、18h、24h、48h 6个小组(每小组3只小鼠,6只眼)。采用小鼠眼前房灌注至高眼压的方法制作实验性视网膜缺血-再灌注模型,治疗组于造模完成后立即向小鼠玻璃体腔内注射0.5ul/g LIF。分别于再灌注后4h、8h、12h、18h、24h、48h摘除眼球,固定、石蜡包埋、切片,进行HE染色及免疫荧光染色。普通光学显微镜下观察视网膜形态变化,应用医学图像分析系统对视网膜神经节细胞进行量化,并进行统计学分析。荧光显微镜下观察LIF的表达。
结果:
1.光镜观察: RIR组组织学变化早期为视网膜水肿(尤以视网膜神经纤维层及内丛状层为明显)。晚期视网膜内层萎缩、变薄,视网膜神经节细胞数目减少。治疗组视网膜神经节细胞明显多于RIR组。
2.荧光显微镜观察:LIF表达于各组视网膜神经节细胞上。
3.细胞计数结果:治疗组4h与RIR组4h、正常对照组4h视网膜神经节细胞数目相比较差异无显著性(P>0.05),治疗组8h、12h、18h、24h、48h比RIR组8h、12h、18h、24h、48h视网膜神经节细胞数目明显增多,差异有显著性(P<0.05)。但比正常对照组8h、12h、18h、24h、48h数目为少,相比差异有显著性(P<0.05)。
结论:
1.病理学改变显示视网膜缺血-再灌注模型建模成功。
2.LIF在小鼠正常眼及视网膜缺血-再灌注后视网膜神经节细胞上均有表达。
3. LIF对小鼠眼视网膜缺血-再灌注后视网膜神经节细胞具有保护作用。
Objective: To observe the expression of Leukemia Inhibitory Factor on mouse retinal gonglion cell and investigate its neuroprotective effect on mouse retinal gonglion cell after ischemic-reperfusion injury.To supply a effected and new method for treating retinal ischemic-reperfusion injury especially glaucoma.
Methods: Healthy fifty-four kunming mice(one hundred and eight eyes)were randomly divided into normal control group(eighteen mice,thirty-six eyes), retinal ischemia reperfusion group(RIR group,eighteen mice,thirty-six eyes),and LIF treatment group(eighteen mice,thirty-six eyes) . These three groups were subdivided into group 4 ,8,12 ,18, 24 and 48 hours after reperfusion respectively(every group three mice,six eyes).Transiently elevating the pressure of the anterior chamber with perfusion of normal saline to induce experimental retina ischemia reperfusion injury modle. .After madding mouse models, 0.5ul/g LIF was injected into vitreous cavity of the treatment group mice after ischemia/reperfusion immediately. The eyes were enucleated after reperfusion for 4,8,12,18,24,48 hours.,then fixed, embedded and sectioned.made the sample HE staining and immunofluorescence staining.The morphological change of retina was studied by light microscopy.The number of retinal Gonglion Cell was measured with compute picture analytic system.and statistical conclusions were obtained. The expression of LIF on retinal Gonglion Cell was observed with fluorescence microscope.
Result:
1. Light microscope: In the ischemia reperfusion group, we can found edema of retinal (especially in the neuron fibrary layer and inner plexiform layer) in the early stage and the retinal atrophy, degeneration and necrosis were found in the later period. We can found that the RGCs was much less than the normal control group in the ischemia reperfusion group. In the treatment group, the RGCs was much more than the ischemia reperfusion group.
2. Fluorescence microscope: LIF expressed on retinal gonglion cell in every griup.
3. The number of RGCs :Compared with the control group and the retinal ischemia reperfusion group, the number of RGCs had no significant differences (P>0.05) at 4th hour after reperfusion .The number of RGCs is much more (P<0.05)in the LIF treatment group than the ischemia reperfusion group at 4th,8th,12th ,18th, 24th and 48th hour after reperfusion ,but is less (P<0.05)than the control group at 4th,8th,12th ,18th, 24th and 48th hour after reperfusion .
Conclusion:
1. Pathological changes displayed: the retinal ischemia reperfusion model was made successfully.
2. As to mouse, there was the expression of LIF on retinal gonglion cell .
3. LIF could provide neuroprotective effect for mouse retinal Gonglion Cell after ischemic-reperfusion induced injury
引文
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