罂粟碱抑制视网膜小胶质细胞活化、保护神经节细胞的实验研究
发布时间：2018-09-04 12:35
【摘要】：青光眼是当今世界上第一大不可逆的致盲性眼病,典型特征是视网膜神经节细胞的进行性死亡和视野缺损。尽管多种原因与青光眼性神经节细胞损伤有关,但视网膜小胶质细胞的过度活化扮演着重要角色:(1)青光眼早期,视网膜小胶质细胞即出现增殖并迁徙至视网膜内层,由分支状为阿米巴样吞噬细胞;(2)活化的小胶质细胞释放的TNF-α、IL-1β等炎症介质与视神经损伤程度一致,抑制炎症介质释放可以减轻神经节细胞损伤;(3)活化后小胶质细胞异常的吞噬功能是造成视网膜神经节细胞二次损伤的重要原因。大量研究表明,抑制小胶质细胞过度活化能够有效减轻视网膜神经节细胞损伤,促进视觉功能恢复。c AMP与小胶质细胞活化和中枢神经元存活密切相关。(1)活化c AMP/PKA信号通路能够通过抑制NF-κB活性减轻小胶质细胞的炎症因子释放和吞噬功能,促进抗炎因子表达。(2)上调胞浆c AMP含量还能通过激活c AMP/PKA/CREB信号通路提高中枢神经元对抗应激损伤的能力,抑制神经元凋亡。罂粟碱是一种磷酸二酯酶抑制剂,能够抑制c AMP降解,提高胞浆内c AMP含量。因此,我们推测罂粟碱能够抑制小胶质细胞过度活化,促进视网膜神经节细胞存活。本研究中,我们拟通过(1)使用BV2小胶质细胞系和原代视网膜小胶质细胞,研究罂粟碱在体外培养条件下对LPS活化的小胶质细胞形态学改变、炎症因子表达、抗炎因子表达、表型转换的影响及分子机制;(2)使用大鼠视神经横断伤模型,研究活体条件下罂粟碱对视网膜小胶质细胞形态、增殖、迁徙、炎症因子表达及视网膜神经节细胞生存的作用。第一部分:罂粟碱通过NF-κB通路抑制LPS诱导的BV2细胞炎症活化1材料与方法我们使用LPS诱导的BV2小胶质细胞活化模型,观察不同浓度的罂粟碱作用下,BV2小胶质细胞(1)形态学变化;(2)炎症因子的转录和释放;(3)表型转换。然后定量分析NF-κB通路的活化水平,探讨罂粟碱抑制小胶质细胞活化的分子机制。分组:PBS对照组、LPS组、不同浓度的罂粟碱组。2结果2.1罂粟碱剂量依赖性的抑制LPS诱导的小胶质细胞形态学变化。正常培养下,大约(5.6±1.2)%的BV2细胞呈阿米巴样。LPS刺激活化后,阿米巴样细胞比例显著升高(89.4±4.5)%,罂粟碱可以剂量依赖性的抑制小胶质细胞形态学变化(P0.01)。2.2罂粟碱剂量依赖性的抑制LPS诱导的小胶质细胞炎症因子转录和释放。正常培养下,小胶质细胞释放极微量的TNF-α(24.54±5.23)pg/ml和IL-1β(20.04±2.13)pg/ml等炎症因子,LPS上调了上述炎症因子的表达数倍[TNF-α:(687.83±19.78)pg/ml,IL-1β:(153.88±4.69)pg/ml],而罂粟碱能够剂量依赖性的抑制这种效果[10μg/ml罂粟碱作用下:TNF-α:(315.42±14.58)pg/ml,IL-1β:(35.12±5.26)pg/ml]。2.3罂粟碱促进BV2细胞表型转换。正常培养下BV2细胞表达极低水平的M1标记(COX-2和i NOS)和M2b标记(IL-1ra和SOCS3),LPS能够显著上调M1型标记和M2b型标记的表达(P0.001),而2μg/ml和10μg/ml罂粟碱能剂量依赖性的逆转上述效果并促进M2a型标记(Arg 1和CD206)的表达(P0.05)。2.4罂粟碱通过NF-κB通路抑制LPS诱导的小胶质细胞活化。正常情况下,BV2小胶质细胞IKK磷酸化水平较低,P65主要分布于胞浆。LPS作用后,小胶质细胞IKK的磷酸化水平明显上调,P65出现显著的核转移。免疫印迹显示:罂粟碱能够剂量依赖性的抑制上述效果(P0.05)。第二部分:罂粟碱通过c AMP/PKA/NF-κB通路抑制LPS诱导的大鼠原代小胶质细胞活化1材料与方法分离、纯化大鼠视网膜小胶质细胞,建立LPS诱导的小胶质细胞活化模型。观察不同浓度的罂粟碱作用下原代视网膜小胶质细胞的形态学变化、炎症因子/抗炎因子的表达以及表型转换,观察(1)不同剂量的罂粟碱作用下,c AMP/PKA、NF-κB通路的活化水平;(2)抑制c AMP/PKA的关键酶后NF-κB的活化状态变化。进一步研究c AMP/PKA、NF-κB在罂粟碱抑制LPS诱导的小胶质细胞炎症中的关系。分组:PBS对照组、LPS组、罂粟碱组(0.4μg/ml组,2μg/ml组和10μg/ml组)。2结果2.1震摇法联合差速贴壁法获得纯度为97.99%的原代大鼠视网膜小胶质细胞。2.2罂粟碱抑制原代小胶质细胞炎症因子转录和释放:与阴性对照组相比,LPS刺激上调了(8.00±1.41)倍TNF-α转录和(14.48±2.08)倍IL-1β转录,罂粟碱剂量依赖性的抑制了这种效果(P0.01)。与转录水平类似,预置罂粟碱能够剂量依赖性的减少TNF-α和IL-1β的释放(P0.01)。2.3罂粟碱不逆转LPS引起的原代小胶质细胞形态学变化。静息状态下,(10.17±2.01)%的原代小胶质细胞呈阿米巴样,LPS显著增加了阿米巴样小胶质细胞的比例(92.03±5.64)%。与BV2细胞明显不同,不同剂量的罂粟碱罂粟碱组(0.4μg/ml组,2μg/ml组和10μg/ml组)并不能逆转小胶质细胞的形态改变(与LPS组比较,P=0.737,P=0.290,P=0.290)。2.4罂粟碱促进原代小胶质细胞抗炎因子(IL-10)的表达。静息状态下,原代小胶质细胞IL-10表达水平很低(20.09±3.30)pg/ml。LPS刺激显著提高了小胶质细胞IL-10的表达(77.83±9.31)pg/ml,而预置罂粟碱能够更进一步提高IL-10的表达(0.4μg/ml组除外)[2μg/ml罂粟碱:(77.83±9.31)pg/ml,与LPS组相比,P=0.020;10μg/ml罂粟碱:(77.83±9.31)pg/ml,与LPS组相比,P0.001]。2.5罂粟碱促进LPS活化的原代小胶质细胞表型转换。2μg/ml和10μg/ml的罂粟碱能够抑制原代小胶质细胞M1型标记(COX-2和i NOS)(P0.01),上调M2a型标记Arg1(P0.05)和CD206的表达。2.6罂粟碱抑制视网膜小胶质细胞活化受c AMP/PKA/CREB和NF-κB信号通路调控。2.6.1罂粟碱活化小胶质细胞c AMP/PKA/CREB通路。静息状态下,小胶质细胞c AMP的含量和CREB的磷酸化水平较低。10μg/ml的罂粟碱显著上调了小胶质细胞c AMP的含量(P0.001)和CREB的磷酸化水平(P0.001),但LPS部分抑制了这种效果(与LPS组相比,P0.01)。2.6.2罂粟碱抑制原代小胶质细胞NF-κB信号通路的激活:与BV2细胞类似,LPS刺激后原代小胶质细胞核P65显著上调约8倍,但该作用可以被10μg/ml的罂粟碱阻断约65%。预置10μg/ml罂粟碱也能抑制LPS诱导的小胶质细胞IKK磷酸化约55.7%。2.6.3 NF-κB受c AMP/PKA通路调控:200μM的rp-isomer或5μM的H-89能够显著阻断罂粟碱对视网膜小胶质细胞TNF-α、IL-1β等炎症因子释放(P0.001)及P65核转移(64.47%-69.30%)的抑制。第三部分:罂粟碱抑制视神经横断伤模型大鼠小胶质细胞活化和神经节细胞死亡1材料与方法制作大鼠视神经横断伤模型,玻璃体腔内注射不同浓度的罂粟碱。7天后处死大鼠,观察:小胶质细胞的数量、形态及迁徙、炎症因子的释放;视网膜神经节细胞的数量、c AMP/PKA/CREB信号通路的活化情况。分组:罂粟碱组(50μg/ml,200μg/ml,500μg/ml)和PBS对照组。2结果2.1罂粟碱抑制视网膜小胶质细胞的增殖、迁徙及形态学改变。视神经横断伤后,小胶质细胞大量增殖并由内、外从状层迁徙至神经节细胞层,变成阿米巴样吞噬细胞。罂粟碱显著减少,但不能完全阻断视网膜神经节细胞层阿米巴样小胶质细胞的迁徙和增殖([对照组(15.60±2.30)/切片V.S 500μg/ml罂粟碱组(4.80±1.30)/切片)。500μg/ml的罂粟碱还能减少吞噬视网膜神经节细胞的小胶质细胞数量([对照组(250.20±9.20)/mm2V.S 500μg/ml的罂粟碱组(149.60±14.33)/mm2)。2.2罂粟碱抑制视网膜TNF-α和IL-1β的表达。视神经横断伤后1周,TNF-α的转录上调了(3.86±1.27)倍,玻璃体腔内注射罂粟碱显著抑制了TNF-α的转录的转录(P0.05)。罂粟碱对IL-1β具有类似的抑制效果(P0.05)。2.3罂粟碱抑制视网膜神经节细胞的死亡2.3.1全视网膜铺片RGC密度:视神经横断伤后7天,视网膜周边部、中间部、中央部及平均RGC密度分别为1223.77±139.10/mm2,1393.62±112.59/mm2,1607.00±76.89/mm2,1408.13±192.90/mm2。玻璃体腔注射罂粟碱能够显著提高全视网膜各个区域RGC的密度。2.3.2视网膜切片RGC计数:与全视网膜铺片类似,玻璃体腔注射罂粟碱也显著提高了视网膜切片上Brn-3a阳性的神经节细胞数量(PBS对照组:(8.02±1.07)细胞/切片V.S 500μg/ml罂粟碱组:(12.82±1.71)细胞/切片)。2.4罂粟碱能够提高视网膜神经节细胞CREB的磷酸化水平。视神经横断伤后1周,PBS对照组各层视网膜几乎无p-CREB阳性的细胞。玻璃体腔内注射不同浓度(50μg/ml-500μg/ml)的罂粟碱均能显著提高视网膜神经节细胞和内核层神经元的CREB磷酸化水平。
[Abstract]:Glaucoma is the world's largest irreversible blinding ophthalmopathy, characterized by progressive retinal ganglion cell death and visual field defect. Although many causes are associated with glaucomatous ganglion cell injury, the excessive activation of retinal microglia plays an important role: (1) early glaucoma, retinal microglia Cells proliferate and migrate to the inner layer of the retina, and are branched into amoebic phagocytes. (2) Activated microglia release TNF-a, IL-1 beta and other inflammatory mediators consistent with the degree of optic nerve injury, inhibition of the release of inflammatory mediators can reduce the damage of ganglion cells; (3) After activation, the abnormal phagocytosis of microglia is caused. A large number of studies have shown that inhibition of microglia over-activation can effectively reduce retinal ganglion cell injury and promote visual function recovery. c-AMP is closely related to microglia activation and central neuron survival. (1) activation of c-AMP/PKA signaling pathway can inhibit NF-kappa B by inhibiting NF-kappa B. Papaverine is a phosphodiesterase inhibitor that can inhibit the degradation of C AMP. We hypothesized that papaverine could inhibit the over-activation of microglia and promote the survival of retinal ganglion cells. In this study, we intend to study the morphology of LPS-activated microglia in vitro by using BV2 microglia and primary retinal microglia. The effects of papaverine on the morphology, proliferation, migration, expression of inflammatory factors and survival of retinal ganglion cells in vivo were studied using rat optic nerve transection model. Pathway Inhibits LPS-induced BV2 Cell Inflammatory Activation 1 Material and Methods We used LPS-induced BV2 microglia activation model to observe the morphological changes of BV2 microglia (1), (2) transcription and release of inflammatory factors, (3) phenotypic transformation, and then quantitatively analyze the activation level of NF-kappa B pathway to explore the effect of papaverine on BV2 microglia. Results 2.1 Papaverine inhibited LPS-induced microglial morphological changes in a dose-dependent manner. Under normal culture, about (5.6 (1.2)) percent of BV2 cells showed amoebic morphology. After LPS stimulation, the proportion of amoebic cells was significant. Papaverine inhibited LPS-induced microglial inflammatory cytokine transcription and release in a dose-dependent manner (P 0.01). Under normal culture, microglial cells released minimal amounts of TNF-alpha (24.54+5.23) pg/ml and IL-1 beta (20.04+2.13) pg/ml. Factor and LPS up-regulated the expression of these inflammatory factors several times [TNF-a: (687.83 [19.78] pg/ml, IL-1 beta: (153.88 [4.69] pg/ml], and papaverine could inhibit this effect in a dose-dependent manner [10 ug/ml papaverine: TNF-a: (315.42 [14.58] pg/ml), IL-1 beta: (35.12 [5.26] pg/ml]. LPS could significantly up-regulate the expression of M1 and M2b markers (P 0.001) in BV2 cells at very low levels (COX-2 and I NOS) and M2b markers (IL-1ra and SOCS3), while 2 ug/ml and 10 ug/ml papaverine could reverse the above effect and promote the expression of M2a markers (Arg-1 and CD206) in a dose-dependent manner (P 0.05). Pathway inhibited LPS-induced microglial activation. Normally, the IKK phosphorylation level of BV2 microglia was low, and P65 was mainly distributed in the cytoplasm. After LPS treatment, the IKK phosphorylation level of microglia was significantly up-regulated, and P65 showed significant nuclear metastasis. Immunoblotting showed that papaverine could inhibit the above effects in a dose-dependent manner (P 0.05). Part two: Papaverine inhibits LPS-induced microglia activation in rats through the c-AMP/PKA/NF-kappa B pathway. Material and method were isolated and purified. LPS-induced microglia activation model was established. Morphological changes and inflammation of primary retinal microglia were observed under different concentrations of papaverine. To observe the expression of factor/anti-inflammatory factor and phenotypic transition, we observed (1) the activation level of c-AMP/PKA and NF-kappa B pathway under different doses of papaverine; (2) the activation state of NF-kappa B after inhibiting the key enzymes of c-AMP/PKA. Results 2.1 Shaking method combined with differential adherence method was used to obtain 97.99% purity of primary rat retinal microglia. 2.2 Papaverine inhibited the transcription and release of inflammatory factors in primary microglia: Compared with negative control group, LPS stimulation increased (8.00 (+ 1.41) times TNF-alpha The effect was inhibited by papaverine in a dose-dependent manner (P 0.01). Similar to the transcriptional level, preset papaverine reduced the release of TNF-a and IL-1 beta in a dose-dependent manner (P 0.01). 2.3 papaverine did not reverse the morphological changes of primary microglia induced by LPS. In resting state, (10.17+2.01)% of the primary microglia were inhibited by papaverine. Different from BV2 cells, different doses of papaverine (0.4 ug/ml group, 2 ug/ml group and 10 ug/ml group) could not reverse the morphological changes of microglia (compared with LPS group, P = 0.737, P = 0.290, P = 0.290).2.4 poppy. LPS stimulation significantly increased the expression of IL-10 in primary microglia (77.83+9.31) pg/ml, but papaverine pretreatment could further increase the expression of IL-10 (except 0.4 ug/ml group) [2 ug/ml papaverine]. Papaverine (77.83 [9.31] pg / ml, P = 0.020; 10 UG / ml papaverine: (77.83 [9.31] pg / ml, compared with LPS group, P 0.001]. 2.5 papaverine promoted LPS-activated primary microglia phenotypic transition. 2 UG / ml and 10 UG / ml papaverine inhibited primary microglia M1-type markers (COX-2 and I NOS) (P 0.01), up-regulated M2a-type markers Arg1 (P 0.0.01). 5) and CD206 expression.2.6 Papaverine inhibits retinal microglia activation regulated by C AMP/PKA/CREB and NF-kappa B signaling pathways. MP content (P 0.001) and CREB phosphorylation (P 0.001), but LPS partially inhibited this effect (compared with LPS group, P 0.01). Inhibition of IKK phosphorylation in LPS-induced microglia was also inhibited by 10 ug/ml papaverine. 2.6.3 NF-kappa B was regulated by the c-AMP/PKA pathway. 200 UG rp-isomer or 5 UG H-89 significantly blocked papaverine release of inflammatory factors such as TNF-a, IL-1 beta (P 0.001) and P65 nuclear metastasis (64.47% -69.30%). Part 3: Papaverine inhibited the activation of microglia and the death of ganglion cells in the rat model of optic nerve transection 1 Material and Methods The rat model of optic nerve transection was made by intravitreal injection of different concentrations of papaverine. Result 2.1 Papaverine inhibited the proliferation, migration and morphological changes of retinal microglia. After optic nerve transection, microglia proliferated and migrated from the inner and outer layers. Papaverine significantly decreased, but could not completely block the migration and proliferation of amebic microglia in the retinal ganglion cell layer ([control group (15.60 [2.30) / slice V.S 500 UG / ml papaverine group (4.80 [1.30) / slice)]. 500 UG / ml papaverine also reduced the phagocytosis of retinal nerves. The number of microglia in ganglion cells ([control group (250.20 Papaverine had a similar inhibitory effect on IL-1 beta (P 0.05). 2.3 Papaverine inhibited retinal ganglion cell death 2.3.1 RGC density: 7 days after optic nerve transection, the density of RGC in peripheral, middle, central and average retinal areas was 1223.77 (+139.10/mm2), 1393.62 (+112.59/mm2), 1607.00 (+76.89/mm2), 1408.8, respectively. Intravitreal papaverine injection significantly increased the RGC density in various regions of the whole retina. 2.3.2 RGC counts in retinal slices: Similar to panretinal slices, intravitreal papaverine injection significantly increased the number of Brn-3a-positive ganglion cells in retinal slices (PBS control group: (8.02 + 1.07) cells / slices V.S 50 0 UG / ml papaverine group: (12.82 (- 1.71) cells / slices).2.4 papaverine can increase the phosphorylation of CREB in retinal ganglion cells. CREB phosphorylation level in ganglion cells and inner nuclear neurons.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R775
，
本文编号：2222117
[Abstract]:Glaucoma is the world's largest irreversible blinding ophthalmopathy, characterized by progressive retinal ganglion cell death and visual field defect. Although many causes are associated with glaucomatous ganglion cell injury, the excessive activation of retinal microglia plays an important role: (1) early glaucoma, retinal microglia Cells proliferate and migrate to the inner layer of the retina, and are branched into amoebic phagocytes. (2) Activated microglia release TNF-a, IL-1 beta and other inflammatory mediators consistent with the degree of optic nerve injury, inhibition of the release of inflammatory mediators can reduce the damage of ganglion cells; (3) After activation, the abnormal phagocytosis of microglia is caused. A large number of studies have shown that inhibition of microglia over-activation can effectively reduce retinal ganglion cell injury and promote visual function recovery. c-AMP is closely related to microglia activation and central neuron survival. (1) activation of c-AMP/PKA signaling pathway can inhibit NF-kappa B by inhibiting NF-kappa B. Papaverine is a phosphodiesterase inhibitor that can inhibit the degradation of C AMP. We hypothesized that papaverine could inhibit the over-activation of microglia and promote the survival of retinal ganglion cells. In this study, we intend to study the morphology of LPS-activated microglia in vitro by using BV2 microglia and primary retinal microglia. The effects of papaverine on the morphology, proliferation, migration, expression of inflammatory factors and survival of retinal ganglion cells in vivo were studied using rat optic nerve transection model. Pathway Inhibits LPS-induced BV2 Cell Inflammatory Activation 1 Material and Methods We used LPS-induced BV2 microglia activation model to observe the morphological changes of BV2 microglia (1), (2) transcription and release of inflammatory factors, (3) phenotypic transformation, and then quantitatively analyze the activation level of NF-kappa B pathway to explore the effect of papaverine on BV2 microglia. Results 2.1 Papaverine inhibited LPS-induced microglial morphological changes in a dose-dependent manner. Under normal culture, about (5.6 (1.2)) percent of BV2 cells showed amoebic morphology. After LPS stimulation, the proportion of amoebic cells was significant. Papaverine inhibited LPS-induced microglial inflammatory cytokine transcription and release in a dose-dependent manner (P 0.01). Under normal culture, microglial cells released minimal amounts of TNF-alpha (24.54+5.23) pg/ml and IL-1 beta (20.04+2.13) pg/ml. Factor and LPS up-regulated the expression of these inflammatory factors several times [TNF-a: (687.83 [19.78] pg/ml, IL-1 beta: (153.88 [4.69] pg/ml], and papaverine could inhibit this effect in a dose-dependent manner [10 ug/ml papaverine: TNF-a: (315.42 [14.58] pg/ml), IL-1 beta: (35.12 [5.26] pg/ml]. LPS could significantly up-regulate the expression of M1 and M2b markers (P 0.001) in BV2 cells at very low levels (COX-2 and I NOS) and M2b markers (IL-1ra and SOCS3), while 2 ug/ml and 10 ug/ml papaverine could reverse the above effect and promote the expression of M2a markers (Arg-1 and CD206) in a dose-dependent manner (P 0.05). Pathway inhibited LPS-induced microglial activation. Normally, the IKK phosphorylation level of BV2 microglia was low, and P65 was mainly distributed in the cytoplasm. After LPS treatment, the IKK phosphorylation level of microglia was significantly up-regulated, and P65 showed significant nuclear metastasis. Immunoblotting showed that papaverine could inhibit the above effects in a dose-dependent manner (P 0.05). Part two: Papaverine inhibits LPS-induced microglia activation in rats through the c-AMP/PKA/NF-kappa B pathway. Material and method were isolated and purified. LPS-induced microglia activation model was established. Morphological changes and inflammation of primary retinal microglia were observed under different concentrations of papaverine. To observe the expression of factor/anti-inflammatory factor and phenotypic transition, we observed (1) the activation level of c-AMP/PKA and NF-kappa B pathway under different doses of papaverine; (2) the activation state of NF-kappa B after inhibiting the key enzymes of c-AMP/PKA. Results 2.1 Shaking method combined with differential adherence method was used to obtain 97.99% purity of primary rat retinal microglia. 2.2 Papaverine inhibited the transcription and release of inflammatory factors in primary microglia: Compared with negative control group, LPS stimulation increased (8.00 (+ 1.41) times TNF-alpha The effect was inhibited by papaverine in a dose-dependent manner (P 0.01). Similar to the transcriptional level, preset papaverine reduced the release of TNF-a and IL-1 beta in a dose-dependent manner (P 0.01). 2.3 papaverine did not reverse the morphological changes of primary microglia induced by LPS. In resting state, (10.17+2.01)% of the primary microglia were inhibited by papaverine. Different from BV2 cells, different doses of papaverine (0.4 ug/ml group, 2 ug/ml group and 10 ug/ml group) could not reverse the morphological changes of microglia (compared with LPS group, P = 0.737, P = 0.290, P = 0.290).2.4 poppy. LPS stimulation significantly increased the expression of IL-10 in primary microglia (77.83+9.31) pg/ml, but papaverine pretreatment could further increase the expression of IL-10 (except 0.4 ug/ml group) [2 ug/ml papaverine]. Papaverine (77.83 [9.31] pg / ml, P = 0.020; 10 UG / ml papaverine: (77.83 [9.31] pg / ml, compared with LPS group, P 0.001]. 2.5 papaverine promoted LPS-activated primary microglia phenotypic transition. 2 UG / ml and 10 UG / ml papaverine inhibited primary microglia M1-type markers (COX-2 and I NOS) (P 0.01), up-regulated M2a-type markers Arg1 (P 0.0.01). 5) and CD206 expression.2.6 Papaverine inhibits retinal microglia activation regulated by C AMP/PKA/CREB and NF-kappa B signaling pathways. MP content (P 0.001) and CREB phosphorylation (P 0.001), but LPS partially inhibited this effect (compared with LPS group, P 0.01). Inhibition of IKK phosphorylation in LPS-induced microglia was also inhibited by 10 ug/ml papaverine. 2.6.3 NF-kappa B was regulated by the c-AMP/PKA pathway. 200 UG rp-isomer or 5 UG H-89 significantly blocked papaverine release of inflammatory factors such as TNF-a, IL-1 beta (P 0.001) and P65 nuclear metastasis (64.47% -69.30%). Part 3: Papaverine inhibited the activation of microglia and the death of ganglion cells in the rat model of optic nerve transection 1 Material and Methods The rat model of optic nerve transection was made by intravitreal injection of different concentrations of papaverine. Result 2.1 Papaverine inhibited the proliferation, migration and morphological changes of retinal microglia. After optic nerve transection, microglia proliferated and migrated from the inner and outer layers. Papaverine significantly decreased, but could not completely block the migration and proliferation of amebic microglia in the retinal ganglion cell layer ([control group (15.60 [2.30) / slice V.S 500 UG / ml papaverine group (4.80 [1.30) / slice)]. 500 UG / ml papaverine also reduced the phagocytosis of retinal nerves. The number of microglia in ganglion cells ([control group (250.20 Papaverine had a similar inhibitory effect on IL-1 beta (P 0.05). 2.3 Papaverine inhibited retinal ganglion cell death 2.3.1 RGC density: 7 days after optic nerve transection, the density of RGC in peripheral, middle, central and average retinal areas was 1223.77 (+139.10/mm2), 1393.62 (+112.59/mm2), 1607.00 (+76.89/mm2), 1408.8, respectively. Intravitreal papaverine injection significantly increased the RGC density in various regions of the whole retina. 2.3.2 RGC counts in retinal slices: Similar to panretinal slices, intravitreal papaverine injection significantly increased the number of Brn-3a-positive ganglion cells in retinal slices (PBS control group: (8.02 + 1.07) cells / slices V.S 50 0 UG / ml papaverine group: (12.82 (- 1.71) cells / slices).2.4 papaverine can increase the phosphorylation of CREB in retinal ganglion cells. CREB phosphorylation level in ganglion cells and inner nuclear neurons.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R775
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本文编号：2222117
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