MFG-E8与视网膜色素变性过程中小胶质细胞吞噬行为的研究

发布时间：2018-01-18 16:11

本文关键词：MFG-E8与视网膜色素变性过程中小胶质细胞吞噬行为的研究　出处：《西南大学》2011年硕士论文　论文类型：学位论文

【摘要】：视网膜色素变性(retinitis pigmentosa, RP)是一组以进行性感光细胞功能丧失为特征的遗传性视网膜变性疾病,是遗传性视觉损害和盲目的最常见原因。皇家外科学院(Royal College of Surgeon rat, RCS)大鼠是研究视网膜色素变性的经典动物模型,其视网膜色素上皮细胞(retinal pigment epithelial cell, RPE)不能发挥其吞噬功能,吞噬功能障碍将导致周期性脱落的视杆外节膜盘(orduoetresmgnet, ROS)不能及时被吞噬,大量堆积于视网膜下腔,从而引起视觉功能障碍,导致自身生理功能丧失,加重RP的发展。视网膜小胶质细胞在视网膜发育和变性过程中起“清道夫”的作用,清除凋亡细胞。因此,加强小胶质细胞识别、吞噬凋亡的视杆细胞,可以减轻凋亡细胞对临近组织的毒性作用,维持视网膜微环境的稳定,从而有效清除堆积在视网膜下腔的ROS,起到保护健康的视锥细胞,保护残存中心视力的作用。乳脂球上皮生长因子-8(milk fat globule epidermal growth factor 8, MFG-E8)最早发现由淋巴器官的吞噬细胞分泌,是介导吞噬细胞发挥吞噬功能的关键因子,通过CrkⅡ-DOCK180-Rac1信号通路发挥作用。在RCS大鼠视网膜色素变性过程中,视网膜小胶质细胞发挥吞噬功能,是否也有MFG-E8参与,是否也通过该通路发挥作用,这一涉及神经免疫调节的基础性问题还未有研究者涉足。如果小胶质细胞吞噬功能的调节确实有MFG-E8的参与,那么外源性加入MFG-E8有可能加强其吞噬功能,进而保护残存的感光细胞,对治疗RP也具有积极意义。本研究的目的：研究视网膜小胶质细胞在RCS大鼠视网膜色素变性过程中的活化规律：MFG-E8在RCS大鼠视网膜色素变性过程中的表达规律；MFG-E8与视网膜小胶质细胞的关系；CDllb、MFG-E8及相关细胞因子的mRNA在视网膜发育和变性过程中的表达变化规律。方法1)鼠龄为14、35、60、90 d的RCS大鼠,采用免疫荧光染色技术标记视网膜小胶质细胞(标志物CDllb)。2)鼠龄为14、35、60、90 d的RCS大鼠,采用免疫荧光染色技术标记MFG-E8。3)鼠龄为14、35、60、90 d的RCS大鼠,采用免疫荧光染色双标技术标记MFG-E8和视网膜小胶质细胞(标志物CDllb)。4)选取鼠龄为0、3、7、14、30、45、60、90d的RCS大鼠,荧光实时定量聚合酶链反应(real-time PCR)检测CDllb、MFG-E8、TNF-α、IL-1β、MCP-1、αv及β5的mRNA的表达变化。结果1)免疫荧光染色显示,鼠龄为14d的RCS大鼠CDllb阳性细胞分布于视网膜的内层,主要为视网膜神经节细胞层(ganglion cells layer, GCL)和内丛状层(Inner plexiform layer, IPL),在内核层(Inner nuclear layer, INL)可见少量的CDllb阳性细胞。鼠龄为35d的RCS大鼠CDllb阳性细胞主要分布在视网膜内丛状层,少量细胞迁移至外核层(Outer plexiform layer,OPL)。鼠龄为60 d的RCS大鼠CDllb阳性细胞迁移至外核层,在外核层明显活化。鼠龄为90 d的RCS大鼠CDllb阳性细胞分布于外核层之外,阳性细胞数量较60d少。2)免疫荧光染色显示,鼠龄为14d的RCS大鼠MFG-E8阳性细胞主要分布于视网膜神经节细胞层(ganglion cells layer, GCL)。鼠龄为35d的RCS大鼠MFG-E8阳性细胞主要分布在视网膜内核层(Inner plexiform layer, IPL),少量细胞迁移至外核层(Outer plexiform layer,OPL)。鼠龄为60 d的RCS大鼠MFG-E8阳性细胞迁移至外核层,在外核层明显活化。鼠龄为90 d的RCS大鼠MFG-E8阳性细胞分布于外核层之外,与60d相比,阳性细胞数量较少。3)免疫荧光染色显示,MFG-E8免疫阳性细胞在RCS大鼠14、35、60、90d均与CDllb染色部位相同,且阳性细胞逐渐从内核层迁移至外核层。4)Real-time PCR检测结果显示,正常大鼠和RCS大鼠神经层视网膜胚后发育过程中(P0-14),MFG-E8 mRNA的表达水平在出生早期较低,然后逐渐增加.P7-P14的mRNA表达最为强烈,与P0比较,差异具有统计学意义(P0.05)。正常大鼠在神经层视网膜发育成熟后,MFG-E8 mRNA的表达水平与P0比较,差异不具有统计学意义(P0.05)。而RCS大鼠在神经层视网膜变性过程中(P14-P90),其mRNA表达逐渐增加；P30、P45与PO比较,差异具有统计学意义(P0.05)；P60 mRNA的表达达到高峰,与P0比较,差异具有统计学意义(P0.05)；P90与P0比较,差异具有统计学意义(P0.05)。整合素αv和β5与MFG-E8的mRNA表达变化一致。5)Real-time PCR检测结果显示,正常大鼠和RCS大鼠神经层视网膜胚后发育过程中(P0-P14),CD11bmRNA的表达水平在出生早期较低,然后逐渐增加。P7-P14的mRNA表达最为强烈,与PO比较,差异具有统计学意义(P0.05)。正常大鼠在神经层视网膜发育成熟(P14)后,MFG-E8 mRNA的表达水平与P0比较,差异不具有统计学意义(P0.05)。而RCS大鼠在神经层视网膜变性过程中(P14-P90),其mRNA表达逐渐增加.P30、P45与P0比较,差异具有统计学意义(P0.05)。P60 mRNA的表达达到高峰,与P0比较,差异具有统计学意义(P0.05)；P90与P0比较,差异具有统计学意义(P0.05)。细胞炎性因子TNF-α(肿瘤坏死因子)、IL-1β(白介素1-β)及细胞趋化因子MCP-1,其nRNA表达变化均与CD11bmRNA表达变化一致。结论1)RCS大鼠视网膜色素变性过程中：CD11b标记的视网膜小胶质细胞明显活化,并向外核层迁移；阳性细胞数量先增多后减少,P60达到高峰；视网膜小胶质细胞的活化可能在视网膜变性过程中发挥重要作用。2)RCS大鼠视网膜色素变性过程中：MFG-E8标记的阳性细胞数量先增多后减少,P60达到高峰；肯定了MFG-E8在视网膜中的表达。3)RCS大鼠视网膜色素变性的各个阶段,CD11b标记的视网膜小胶质细胞与MFG-E8标记的阳性细胞相同,确定MFG-E8表达于神经层视网膜小胶质细胞；MFG-E8可能在视网膜小胶质细胞吞噬过程中发挥重要作用。4)在RCS大鼠视网膜色素发育和变性过程中,整合素αv、β5和MFG-E8与CD11b(小胶质细胞的特异标记物)的mRNA表达变化规律表现出一致性,提示：MFG-E8介导神经层视网膜小胶质细胞的吞噬作用,且可能通过CrkⅡ-DOCK180-Rac1信号通路吞噬凋亡细胞。5)炎性相关因子TNF-α、IL-1β、MCP-1的mRNA表达变化与CDllb(小胶质细胞的特异标记物)表达变化规律一致,提示在RCS大鼠视网膜色素变性过程中,小胶质细胞介导炎症反应。
[Abstract]:Retinitis pigmentosa (retinitis pigmentosa RP) is a group for photosensitive cell function loss for hereditary retinal degeneration disease characteristics, is the most common genetic cause of visual impairment and blindness. The Royal College of Surgeons (Royal College of Surgeon rat, RCS) is a classical animal model of rat retinal degeneration. The retinal pigment epithelial cells (retinal pigment epithelial cell, RPE) can not play its phagocytic function, phagocytic dysfunction will result in periodic shedding of rod outer segment membrane disc (orduoetresmgnet, ROS) can not be swallowed up, a lot of accumulation in the subretinal space, causing visual impairment, resulting in the loss of their physiological function, development of exacerbations of RP retinal microglia plays a scavenger role in retinal development and degeneration during removal of apoptotic cells. Because of this, to strengthen the microglia The recognition and phagocytosis of rod cell apoptosis, toxicity can reduce the apoptosis of adjacent tissues, maintain retinal micro environment, so as to effectively eliminate accumulation in the subretinal space of ROS, to protect the health of cone cells, the protective effect of residual central vision. Milk fat globule epidermal growth factor -8 (milk fat globule epidermal growth factor 8, MFG-E8) was first discovered by the phagocytosis of lymphoid organs is mediated by cellular secretion and phagocytic cells play a key factor in phagocytosis, play a role through the Crk II -DOCK180-Rac1 signaling pathway in retinal pigment degeneration of RCS rats during retinal microglia play a phagocytic function, whether MFG-E8 participate in it through this pathway, which involves the basic issues of neuroimmunoregulation have not been involved. If the researchers adjusted the phagocytic function of microglia is M FG-E8 participation, then exogenous MFG-E8 may strengthen its phagocytic function, and then protect the surviving photoreceptor cells, and it is also of positive significance for the treatment of RP.
The purpose of this study: the research of retinal microglia in retinal pigment degeneration in RCS rats: activation of MFG-E8 expression pattern in the process of retinal pigment degeneration in RCS rats; the relationship between MFG-E8 and retinal microglia; CDllb, MFG-E8 and related cytokine mRNA in retinal development and degeneration in the process expression changes. Methods 1) rats aged rats with RCS 14,35,60,90 D, using immunofluorescence labeling of retinal microglia (.2) marker CDllb) rats aged rats with RCS 14,35,60,90 D, using immunofluorescence staining labeled MFG-E8.3) rats aged rats with RCS 14,35,60,90 D, by immune immunofluorescence double labeling technique and MFG-E8 marker of retinal microglia (.4) marker CDllb) selected rats aged rats with RCS 0,3,7,14,30,45,60,90d, fluorescence quantitative real-time polymerase chain reaction (real-time, PC The detection of CDllb, MFG-E8, R) TNF- alpha, IL-1 beta, MCP-1, expression of mRNA and alpha v beta 5. Results 1) immunofluorescence staining showed that the inner rat age RCS rats CDllb 14d positive cells distributed in the retina, mainly for retinal ganglion cell layer (ganglion cells layer, GCL) and the inner plexiform layer (Inner plexiform layer, IPL) in the core layer (Inner nuclear, layer, INL) showed a few of CDllb positive cells. Rats aged rats with RCS CDllb 35d positive cells were mainly distributed in the inner plexiform layer, cells migrate to the outer nuclear layer (Outer plexiform layer, OPL). 60 d rats aged RCS rats, CDllb positive cells migrate to the outer nuclear layer, outer nuclear layer was activated in rats. Age 90 d RCS rats CDllb positive cells distributed in the outer nuclear layer, the number of positive cells was less than 60d.2) immunofluorescence staining showed that rats aged RCS rats MFG-E8 14d positive cells were mainly divided into Distributed in retinal ganglion cell layer (ganglion cells layer, GCL). Rats aged rats with RCS MFG-E8 35d positive cells were mainly distributed in the inner nuclear layer of retina (Inner plexiform layer, IPL), a small number of cells migrate to the outer nuclear layer (Outer plexiform layer, OPL). Rats aged 60 d RCS rats MFG-E8 the positive cells migrate to the outer nuclear layer, outer nuclear layer was activated in rats. Age 90 d RCS rats MFG-E8 positive cells distributed in the outer nuclear layer, compared with 60d, the number of positive cells less.3) immunofluorescence staining showed that MFG-E8 immunoreactive cells in 14,35,60,90d RCS rats with CDllb staining were the same, and the positive cells gradually from the core layer to migrate to the outer nuclear layer).4 Real-time PCR showed normal and RCS rats retinal nerve layer postembryonic development process (P0-14), the expression level of MFG-E8 mRNA was born in the early low, and gradually increase the.P7-P 14 the expression of mRNA was the strongest, compared with P0, the difference was statistically significant (P0.05). Normal rats in mature retinal nerve layer, compared to expression of P0 and MFG-E8 mRNA, the difference was not statistically significant (P0.05). RCS rats in the nerve layer in the retinal degeneration (P14-P90). The expression of mRNA gradually increased; P30, P45 and PO, the difference was statistically significant (P0.05); the expression of P60 mRNA reached the peak, compared with P0, the difference was statistically significant (P0.05); P90 and P0, the difference was statistically significant (P0.05). Integrin alpha v beta 5 and the expression of mRNA and MFG-E8.5 Real-time PCR) consistent test results showed that the normal and RCS rats retinal nerve layer postembryonic development process (P0-P14), the expression level of CD11bmRNA was lower in early, and gradually increase the.P7-P14 expression of mRNA was the strongest, compared with PO, the difference There was statistical significance (P0.05). The rats in the normal maturation of retinal nerve fiber layer (P14), compare the expression level of P0 and mRNA MFG-E8, the difference was not statistically significant (P0.05). RCS rats in the nerve layer in the retinal degeneration (P14-P90), the expression of mRNA increased.P30, P45 and P0, the difference was statistically significant (P0.05) expression of.P60 mRNA reached the peak, compared with P0, the difference was statistically significant (P0.05); P90 and P0, the difference was statistically significant (P0.05). Cell inflammatory factor TNF- alpha (TNF), IL-1 beta (interleukin 1- beta) and cell chemokine MCP-1, nRNA expression changes were consistent with the expression of CD11bmRNA. Conclusion: 1) RCS retinal degeneration in rats: CD11b labeled retinal microglia was activated, and outer nuclear layer migration; the number of positive cells increased first and then decreased, and reached the peak at P60; Retinal microglia activation may play an important role in.2 retinal degeneration process) RCS retinal degeneration in rats: MFG-E8 labeled positive cells increased first and then decreased, and reached the peak at P60; the expression of.3 confirmed that MFG-E8 in the retina of RCS rats) in various stages of retinal degeneration, CD11b positive cells labeled retinal microglial cells labeled with MFG-E8, to determine the expression of MFG-E8 in neural retina microglia; MFG-E8 in retinal microglia phagocytosis play an important role in the process of.4 RCS) in rat retinal pigment degeneration and developmental process, integrin alpha v beta 5 and MFG-E8 and CD11b (specific marker microglia) prompted the expression pattern of mRNA showed consistency: phagocytosis mediated by MFG-E8 neural retina microglia, and possibly by Crk II -DOCK180-Rac1 signal pathway of phagocytosis of apoptotic cells.5) inflammatory related factor TNF- alpha, IL-1 beta, MCP-1 mRNA expression and CDllb (specific marker of microglia) expression changes, suggesting that in retinal degeneration of RCS rats in the process of microglia mediated inflammation.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R774.1

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