巨噬细胞集落刺激因子对氧诱导视网膜病变中小胶质细胞和血管化进程作用研究及机制探讨

发布时间：2018-05-27 09:40

本文选题：氧诱导视网膜病变 + 再血管化　；参考：《复旦大学》2013年博士论文

【摘要】：第一部分：探讨OIR模型视网膜小胶质细胞和血管变化的关系试验选用出生后7天(P7)小鼠,与同笼母鼠一起置入氧浓度为75±3%的密闭氧箱,5天后返回正常氧环境,制作OIR模型。分别取P12、P15、P17、P21小鼠视网膜,采用视网膜铺片及切片免疫组化观察血管改变。结果显示：与正常视网膜血管相比,OIR模型P12时可见视网膜中央仅第一级大血管存在,第二级、第三级血管及毛细血管网消失,大片无灌注区形成,周边部有血管网分布。P17时在视网膜周边部和中央部无灌注区交界处大量新生血管簇形成,中央部大血管迂曲。P21时视网膜中央部基本血管化。视网膜垂直切片可见新生血管芽突破内界膜,长入玻璃体。视网膜铺片及切片结果提示OIR造模成功。成功建立OIR模型后,本研究进一步观察了OIR模型中视网膜小胶质细胞动态变化及其与视网膜血管的关系。采用免疫荧光染色铺片和切片观察OIR模型血管化过程中血管和小胶质细胞的关系,应用RT-PCR及western blot检测方法对小胶质细胞的特异性标记物CDllb的mRNA和蛋白表达进行定量分析,提示小胶质细胞增减变化。结果显示：正常视网膜小胶质细胞主要分布在视网膜内层,胞体较小,突起细长伸展,呈静息态,和血管关系密切。OIR模型中,小胶质细胞胞体变大,突起变短粗,在血管化区域和新生血管区分布明显多于无血管区。RT-PCR检测提示P15时,OIR视网膜小胶质细胞特异性标记物CD11bmRNA显著低于正常组,随时间增加,P21时超过正常水平。Western blot定量检测CD11b蛋白结果与RT-PCR结果一致。体外低氧培养小胶质细胞提示OIR模型中视网膜血管闭塞后的改变引起了小胶质细胞改变。共聚焦图片显示小胶质细胞聚集在新生血管芽上面及前方,包绕出芽的内皮细胞尖端,提示小胶质细胞吞噬基质为新生血管形成开辟空间,在内皮细胞出芽形成血管腔的过程中起一定作用。本部分研究表明OIR模型中视网膜小胶质细胞被激活,开始时减少,随着时间增加,小胶质细胞增加。在视网膜无灌注区血管化过程中,小胶质细胞与新生血管芽密切相关,提示其在视网膜无灌注区血管化过程中发挥重要作用。第二部分：M-CSF对OIR模型中小胶质细胞作用研究 OIR造模后,采用免疫组化观察OIR模型视网膜M-CSF及CSF-1R分布变化,并且应用RT-PCR及western blot检测OIR模型视网膜M-CSF及CSF-1R mRNA和蛋白动态表达变化。结果提示OIR模型M-CSF和CSF-1R主要分布于视网膜内层,与正常视网膜组织对比,未见显著变化。正常小鼠和OIR模型小鼠视网膜部分小胶质细胞均可高表达CSF-1R。将OIR造模成功的小鼠随机分为两组,注药组腹腔注射M-CSF (40μg/kg),对照组注射等量注射用水。分别观察小鼠行为、记录各个时间点小鼠体重,视网膜铺片免疫组化计数小胶质细胞数目,应用RT-PCR及western blot检测OIR模型视网膜小胶质细胞标记性蛋白CD11b的mRNA和蛋白丰度动态表达变化。结果显示：注药组体重P15时高于对照组小鼠10%,P17和P21时同对照组比较无明显统计学差异,两组小鼠行为未见明显异常,主要组织器官大体解剖观察亦未见明显异常改变。视网膜铺片免疫组化显示P15、P17、P21时注药组小胶质细胞高于对照组71%、46%、31%,RT-PCR结果表明注药组CDllb的mRNA表达分布高于对照组2.61、2.24、1.05倍,Western blot检测CD11b蛋白表达与其mRNA趋势一致。本试验结果表明,OIR模型小鼠腹腔注射M-CSF可促进视网膜小胶质细胞增加,对小鼠无明显毒副反应产生。第三部分：M-CSF对OIR模型视网膜血管化进程的影响将OIR造模成功的小鼠随机分为两组,注药组腹腔注射M-CSF (40μg/kg),对照组注射等量注射用水,分别取P15、P17两个时间点视网膜铺片,FITC-GS染色、照相、拼图后采用Image Pro Plus软件分别描记视网膜边界、无灌注区边界、新生血管簇面积,分别计算每一张视网膜拼图无灌注区面积比、新生血管簇面积比、血管化面积比,并做统计比较。Western blot检测视网膜血管屏障蛋白ZO-1和Occludin动态变化,MMP-9动态变化,以及VEGF动态变化。结果表明系统性应用M-CSF可促进OIR视网膜无灌注区血管化进程,加速新生血管簇退化,增加视网膜血管屏障蛋白ZO-1表达,减少MMP-9表达,减少VEGF表达。为了进一步研究是否因为M-CSF引起的小胶质细胞增加而促进OIR视网膜无灌注区血管化进程,其中部分系统性M-CSF注药组小鼠接受腹腔和玻璃体腔注射氯膦酸二钠脂质体减少系统性巨噬细胞及视网膜小胶质细胞,视网膜铺片免疫组化观察视网膜小胶质细胞和血管变化,分别计算P15和P17时视网膜无灌注面积比,进行统计。体外试验通过小胶质细胞和人脐静脉内皮细胞(HUVEC)共培养,采用跨膜电阻仪测量HUVEC上下的跨内皮电阻抗(TER)。结果显示：氯膦酸二钠脂质体减少了M-CSF注药组视网膜小胶质细胞,血管发育障碍,血管较细,血管网稀疏。氯膦酸二钠脂质体处理的OIR视网膜无灌注区面积增加。体外小胶质细胞和HUVEC共培养试验结果提示,小胶质细胞增加可促进HUVEC细胞间TER增加。本试验结果表明,系统性应用M-CSF可促进OIR视网膜无灌注区血管化进程及血管屏障成熟,还可促进新生血管簇退化,减少VEGF表达。去除视网膜内小胶质细胞及其来源时,OIR视网膜内小胶质细胞减少,血管发育不良；小胶质细胞增加可促进血管内皮屏障功能形成。研究提示系统性应用M-CSF引起视网膜小胶质细胞增加,可促进缺血性视网膜病变无灌注区血管化进程。
[Abstract]:The first part : To investigate the relationship between retinal microglial cells and vascular changes in OIR model

The results showed that the central part of the retina was only the first order big blood vessel , the second stage , the third stage blood vessel and the capillary network disappeared , and the peripheral part had the distribution of vascular network .

After successfully establishing the OIR model , this study further observed the dynamic changes of retinal microglial cells in OIR model and their relationship with retinal vessels . The expression of CDllb mRNA and protein in microglial cells was quantitatively analyzed by RT - PCR and western blot .

The results showed that normal retinal microglial cells were mainly distributed in the inner layer of the retina , the cells were small , the protrusions were elongated and stretched , and the distribution of CD11bmRNA was significantly lower in the OIR model than in the normal group .

In this part , the retinal microglial cells in the OIR model were activated and decreased at the beginning . As the time increased , the microglial cells increased . During the vascularization of the retina without perfusion , the microglial cells were closely related to the neovascular shoots , suggesting that they play an important role in the vascularization of the retina without perfusion .

The Effect of M - CSF on Microglia in OIR Model

The expression of M - CSF and CSF - 1R in the retina of OIR model was observed by immunohistochemistry . The results suggested that the OIR model M - CSF and CSF - 1R were mainly distributed in the inner layer of the retina and compared with normal retinal tissues .

The mice were randomly divided into two groups : M - CSF ( 40 渭g / kg ) was injected intraperitoneally in the injection group , and the control group injected the same amount of water for injection . The mouse behavior was observed , the weight of mouse body weight was recorded at each time point , the number of microglial cells was counted by immunohistochemistry . RT - PCR and western blot were used to detect the changes of mRNA and protein abundance in the retinal microglial cell marker protein of OIR model .

The results showed that the expression of CDllb mRNA in the injection group was higher than that of the control group ( 71 % , 46 % , 31 % ) when compared with the control group ( 71 % , 46 % , 31 % ) when P17 and P21 were higher than that in the control group .

The results showed that the intraperitoneal injection of M - CSF by OIR model could promote the increase of retinal glial cells and no obvious toxic side effect to mice .

The third part : Effect of M - CSF on retinal vascularization in OIR model

The mice were randomly divided into two groups : M - CSF ( 40 渭g / kg ) was injected intraperitoneally in the injection group and the control group injected with the same amount of water for injection . The changes of the area ratio , the area ratio and the area ratio of the retinal vascular barrier protein ZO - 1 , and the area of the neovascularization were respectively calculated by Western blot . The results showed that the systemic application of M - CSF could accelerate the vascularization of the retinal blood vessel barrier protein ZO - 1 and the vascular endothelial cell , and to reduce the expression of MMP - 9 and reduce the expression of VEGF .

In order to further study whether the microglial cells induced by M - CSF were increased to promote the vascularization of the OIR retina , some systemic M - CSF injection group mice received intraperitoneal and vitreous cavity injection of diclofenac sodium liposome to reduce systemic macrophages and retinal microglial cells . The retinal microglial cells and vascular changes were observed by immunohistochemistry . The ratio of retinal free area ratio was calculated by immunohistochemistry . In vitro experiments were carried out by co - culture of microglial cells and human umbilical vein endothelial cells ( HUVEC ) , and the trans - endothelium electrical impedance ( TER ) of HUVEC was measured by a transmembrane resistance meter .

The results showed that disodium clodronate decreased the area of retinal microglial cells , vascular development disturbance , vessel thinner , and sparse vascular network in M - CSF injection group , and the area of OIR retinal non - irrigated area treated by diclofenac sodium liposome was increased .

The results showed that the systemic application of M - CSF could promote the vascularization and vascular barrier maturation of OIR retinal non - irrigated area , and also promote the degeneration of neovascularization and reduce the expression of VEGF . In the removal of microglial cells in the retina and their origin , the microglial cells in the retina of OIR decreased and the vascular dysplasia was poor ;
Conclusion Systemic application of M - CSF results in the increase of retinal glial cells , which can promote the vascularization of ischemic retinopathy .
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R774.1

【共引文献】