色素上皮衍生因子联合巨噬细胞条件培养液或晶状体损伤对视神经损伤的修复作用

发布时间：2018-06-07 01:07

本文选题：视网膜神经节细胞 + 巨噬细胞条件培养液　；参考：《第四军医大学》2010年博士论文

【摘要】： 研究背景: 视网膜神经节细胞(RGCs)将视觉信号传入大脑,但是神经损伤或疾病(如青光眼)总是会导致神经元的死亡和视功能的丧失。成年哺乳动物视神经损伤后RGCs的轴突很难再生。要修复受损的视神经必须保护神经元的存活和促进轴突的再生。色素上皮衍生因子(PEDF)是一种具有抗血管生成、神经营养和神经保护作用的蛋白,能够保护神经元免受多种病理性的损害。另外它还具有抗炎能力。当视神经损伤时晶状体同时损伤,许多RGCs可以存活,而且其轴突再生进入远端视神经。晶状体损伤可以诱导巨噬细胞活化,巨噬细胞能够分泌多种刺激RGCs轴突再生的细胞因子。巨噬细胞在这个过程中似乎起关键作用,因为单独玻璃体注射酵母多糖而没有晶状体损伤,也可以诱导RGCs轴突再生进入远端视神经,而酵母多糖可以刺激组织的单个核细胞。目的: 本研究探讨联合两种方法,即PEDF联合巨噬细胞条件培养液(MCM)或晶状体损伤是否能协同保护RGCs的存活和促进RGCs轴突的再生。方法: 1.细胞培养:培养出生24 h的SD仔鼠的RGC细胞以及SD大鼠腹腔巨噬细胞,观察它们的形态、进行免疫组化鉴定,用MTT法绘制它们的生存曲线,揭示它们的培养规律。 2.细胞实验:(1)巨噬细胞与酵母多糖共培养,观察培养液中血清对巨噬细胞吞噬酵母多糖颗粒的影响。(2)制备细胞爬片及MCM,观察联合应用PEDF和MCM对培养的RGC细胞的存活数和轴突再生长度的影响。 3.动物实验:(1)将荧光金注射到SD成年大鼠及出生2 w的SD幼鼠脑内,逆行性标记RGC细胞,观察RGCs的分布规律和细胞数目。(2)制备视神经损伤模型和晶状体损伤模型,观察联合应用玻璃体注射PEDF和晶状体损伤对视网膜RGC细胞的存活数、视神经轴突再生以及GAP-43 mRNA表达的影响。结果: 1.细胞培养:(1)成功培养了SD大鼠RGC细胞,GAP-43呈阳性,培养第4 d时,有71.4%细胞存活,而到第6 d时,大量细胞开始死亡,46.3%细胞存活,第8 d时,只有7.1%细胞存活。(2)成功培养了SD大鼠腹腔巨噬细胞,CD68呈阳性,可以存活2 w左右,在培养第8 d时,约有一半细胞死亡。 2.细胞实验:(1)巨噬细胞同酵母多糖共培养2 d,血清组细胞数多于对照组(P0.01)。(2)观察对RGCs存活数目的影响: MCM组在细胞培养第3 d时细胞数目为31.4±3.7(P0.05),PEDF联合MCM组为34.7±4.2(P0.01),而对照组为27.6±2.2,与对照组均有显著统计学差异。PEDF联合MCM组的细胞数大于MCM组,两者有显著统计学差异(P0.05)。对RGC轴突再生的影响:细胞培养第3 d时,MCM组细胞轴突长度为57.6±3.7μm,PEDF联合MCM组的细胞轴突长度为88.6±9.5μm,与对照组(41.8±5.4μm)均有显著统计学差异(P0.01)。PEDF联合MCM组的轴突长于MCM组(P0.01)。 3.动物实验:(1)成功制备荧光金逆标模型,RGCs密度为2219±113 RGC / mm2。(2)玻璃体注射PEDF联合晶状体损伤可以大幅提高视神经损伤后RGCs存活数目。与单独应用玻璃体注射PEDF或晶状体损伤相比,两者联合可以增加RGCs总存活数(1411±84 RGC/mm2),与前两者均有显著统计学差异(P 0.01)。单纯的晶状体损伤能够刺激远端的RGCs轴突再生,PEDF联合晶状体损伤能够强烈刺激远端视神经轴突再生,与晶状体损伤组有显著统计学差异(P0.01)。PEDF联合晶状体损伤在同一时间点可以大幅提高视神经损伤后GAP-43 mRNA的表达,与两者单独进行干预均有显著统计学差异(P 0.01)。结论: 1.血清能够刺激巨噬细胞的吞噬功能。 2.单独应用PEDF不能保护培养的RGC细胞存活以及促进轴突再生, MCM能保护RGC细胞存活以及促进轴突再生,与PEDF可以协同保护RGC细胞存活以及促进轴突再生。 3.玻璃体内注射PEDF和晶状体损伤都可以增加RGCs存活数目,两者可以协同使这一作用增加。晶状体损伤可以刺激轴突再生,联合PEDF可以增强刺激再生作用。晶状体损伤和PEDF都能提高GAP-43 mRNA的表达,两者联合使这一作用加强。创新点: 1.首次观察幼鼠荧光金逆行性标记RGCs。 2.首次探讨PEDF对培养RGCs轴突再生和视神经损伤修复的作用。 3.首次探讨联合PEDF和MCM或晶状体损伤对视神经夹伤后RGCs的存活和促进RGCs轴突再生的影响。
[Abstract]:Research background:
Retinal ganglion cells (RGCs) transmit visual signals into the brain, but nerve damage or disease (such as glaucoma) always leads to neuronal death and loss of visual function. The axon of RGCs is difficult to regenerate in adult mammalian optic nerve injury. To repair the damaged optic nerve must protect the survival of the neuron and promote the regeneration of the axon. Pigment epithelial derived factor (PEDF) is a protein with antiangiogenic, neurotrophic and neuroprotective effects that can protect neurons from a variety of pathological damage. In addition, it has the ability to resist inflammation. When the optic nerve is damaged, many RGCs can survive, and its axon regenerates into the distal optic nerve. It can induce macrophage activation and macrophages secrete a variety of cytokines that stimulate the regeneration of RGCs axons. Macrophages seem to play a key role in this process, because the single vitreous injection of Yeast Polysaccharides without lens damage can also induce the regeneration of RGCs axons into the distal optic nerve, and yeast polysaccharide can be induced. To stimulate the mononuclear cells of the tissue.
Objective:
The present study explored whether the combined two methods, namely PEDF combined macrophage conditioned medium (MCM) or lens injury, could synergistically protect the survival of RGCs and promote the regeneration of RGCs axons.
Method:
1. cell culture: RGC cells of 24 h born SD mice and peritoneal macrophages of SD rats were cultured. Their morphology was observed and identified by immunohistochemistry. Their survival curves were plotted by MTT, and their cultivation rules were revealed.
2. cell experiment: (1) the effect of macrophage and yeast polysaccharide co culture to observe the effect of serum on macrophage phagocytosis of yeast polysaccharide particles. (2) to prepare cell crawling tablets and MCM, to observe the effect of combined application of PEDF and MCM on the survival number of cultured RGC cells and the axon regeneration length.
3. animal experiments: (1) injection of fluorescent gold into the brain of SD adult rats and SD young rats born with 2 W, retrograde labeling of RGC cells to observe the distribution of RGCs and the number of cells. (2) to prepare the optic nerve injury model and lens injury model, to observe the survival of the retina RGC cells combined with vitreous injection of PEDF and lens injury, and to observe the deity of the retina of the retina RGC cells. The effect of the regeneration of the axon process and the expression of GAP-43 mRNA.
Result:
1. cell culture: (1) SD rat RGC cells were successfully cultured, GAP-43 was positive, and 71.4% cells survived at fourth D, while at sixth D, a large number of cells began to die, 46.3% cells survived, and eighth D, only 7.1% cells survived. (2) the peritoneal macrophages of SD rats were successfully cultured, CD68 was positive, and could survive 2 W, about eighth D, about 71.4% D. Half of the cells died.
2. cell experiment: (1) the macrophage and yeast polysaccharide co culture 2 D, the number of serum group more than the control group (P0.01). (2) the effect of observation on the number of RGCs survival: MCM group in cell culture third D cell number of 31.4 + 3.7 (P0.05), PEDF combined MCM group of 34.7 + 4.2 (P0.01), and the control group is 27.6 + 2.2, and the control group were significantly worse than the control group The number of cells in group MCM combined with.PEDF was larger than that of group MCM, and there was significant difference between the two groups (P0.05). The effect on the axon regeneration of RGC: when cell culture was third D, the cell axon length of the MCM group was 57.6 + 3.7 mu m, and the length of the cell axon in the PEDF combined MCM group was 88.6 + 9.5 micron m, and there were significant statistical differences between the control group and the control group (41.8 + 5.4). The axon of group M was longer than that of group MCM (P0.01).
3. animal experiments: (1) the successful preparation of the fluorescent gold reverse standard model, the RGCs density of 2219 + 113 RGC / mm2. (2) of vitreous injection of PEDF combined with lens injury can significantly increase the number of RGCs survival after optic nerve injury. Compared with the single use of vitreous injection of PEDF or lens injury, the combination of both can increase the total number of RGCs survival (1411 + 84 RGC/mm2), There were significant statistical differences between the two groups (P 0.01). Pure lens injury could stimulate the regeneration of RGCs axons in the distal end. PEDF combined with lens injury could strongly stimulate the regeneration of the distal optic axon. There was a significant statistical difference between the lens injury group and the lens injury group (P0.01).PEDF joint lens injury could be greatly improved at the same time point. The expression of GAP-43 mRNA after nerve injury was significantly different from that of intervention alone (P 0.01).
Conclusion:
1. the serum can stimulate the phagocytosis of macrophages.
2. PEDF alone can not protect the cultured RGC cells to survive and promote axon regeneration. MCM can protect the survival of RGC cells and promote axon regeneration, and PEDF can protect the survival of RGC cells and promote axon regeneration in collaboration with PEDF.
3. intravitreal injection of PEDF and lens injury can increase the number of RGCs survival. Both can increase the effect. Lens injury can stimulate axon regeneration, combined with PEDF can enhance stimulation of regeneration. Lens injury and PEDF can increase the expression of GAP-43 mRNA, both combined to strengthen the effect.
Innovation point:
1. the first observation of fluorescent gold retrograde RGCs. in young rats
2. to explore the effect of PEDF on axonal regeneration and repair of optic nerve injury in RGCs.
3. the effects of combined PEDF and MCM or lens injury on RGCs survival and RGCs axonal regeneration after optic nerve crush were first discussed.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R774.1

【参考文献】