視網(wǎng)膜膠質(zhì)細胞在大鼠視神經(jīng)夾持損傷后的作用研究
發(fā)布時間：2018-11-08 21:17
【摘要】：[目的]建立不同時間點大鼠視神經(jīng)夾持損傷模型,觀察大鼠視神經(jīng)損傷后視網(wǎng)膜炎癥因子和膠質(zhì)細胞的反應(yīng),探討視神經(jīng)損傷后視網(wǎng)膜炎癥因子和膠質(zhì)細胞在視神經(jīng)夾持損傷中的作用,為臨床治療外傷性視神經(jīng)損傷的相關(guān)疾病提供新的思路和方法。[方法]采用50 g力度的視神經(jīng)夾持鑷建立大鼠視神經(jīng)夾持損傷模型;將99只成年SPF級雌性SD大鼠隨機分成正常組(n=3)、手術(shù)組(n=48)和假手術(shù)組(n=48)。手術(shù)組做視神經(jīng)夾持損傷模型,假手術(shù)組僅分離暴露視神經(jīng),但不行夾持。手術(shù)組與假手術(shù)組按損傷后時間點分別在1d、3d、5 d、7 d進行取材,每個時間點共12只大鼠。Western blot實驗(n=4)分別觀察視網(wǎng)膜IL-1β、TNF-α在相應(yīng)時間點的蛋白表達水平;采用免疫熒光組織化學(xué)染色法(n=4)觀察大鼠視神經(jīng)損傷后不同時間點視神經(jīng)及視網(wǎng)膜上膠質(zhì)細胞的變化情況;并采用免疫熒光共定位(n=4)觀察視網(wǎng)膜TNF-α IL-1β與膠質(zhì)細胞的共表達情況。[結(jié)果]Western blot實驗結(jié)果顯示:視神經(jīng)夾持損傷后IL-1β、TNF-α的表達均發(fā)生變化。IL-1β在3d的表達量達峰值,與假手術(shù)組相比差異具有統(tǒng)計學(xué)意義(p0.05),其它時間點的差異較假手術(shù)組相比無統(tǒng)計學(xué)意義(p0.05);TNF-α在損傷后1d表達量明顯上升,3d達峰值,5-7d稍有下降但差異較假手術(shù)組相比均有統(tǒng)計學(xué)意義(均為p0.05)。視神經(jīng)上膠質(zhì)細胞免疫熒光單染結(jié)果顯示:視神經(jīng)夾持損傷后視神經(jīng)上的細胞結(jié)構(gòu)排列不整齊,分布不均勻,且夾持部位細胞缺失。視神經(jīng)夾持損傷后小膠質(zhì)細胞的綠色熒光信號表達較假手術(shù)組相比明顯增強,且分布的小膠質(zhì)細胞數(shù)量增加;星形膠質(zhì)細胞與假手術(shù)組相比,GFAP熒光信號表達也有所增強,軸突變長。視網(wǎng)膜上小膠質(zhì)細胞和星形膠質(zhì)細胞免疫熒光單染結(jié)果顯示:視神經(jīng)夾持損傷后膠質(zhì)細胞的形態(tài)、數(shù)量和分布發(fā)生變化,小膠質(zhì)細胞主要分布在視網(wǎng)膜神經(jīng)節(jié)細胞層和內(nèi)核層,損傷后小膠質(zhì)細胞1d突觸變短變粗,3d胞體變圓、突觸變短,二級分支減少或消失,5-7 d出現(xiàn)活化的阿米巴狀,且數(shù)量上,活化的小膠質(zhì)細胞數(shù)量明顯增多,差異與假手術(shù)組相比具有統(tǒng)計學(xué)意義(p0.05);而星形膠質(zhì)細胞主要分布在視網(wǎng)膜神經(jīng)節(jié)細胞層之外,在夾持損傷后被激活,表現(xiàn)為胞體變得肥大,軸突增粗,GFAP熒光表達增強。免疫熒光共定位顯示:視神經(jīng)損傷后小膠質(zhì)細胞出現(xiàn)活化現(xiàn)象,視網(wǎng)膜中TNF-α、IL-1β的表達明顯,且TNF-α、IL-1β主要分布于視網(wǎng)膜中的小膠質(zhì)細胞中。而由星形膠質(zhì)細胞分泌的IL-1β在3d表達明顯,但在視網(wǎng)膜星形膠質(zhì)細胞中未檢測到TNF-α的表達。[結(jié)論]1)視神經(jīng)夾持損傷后視網(wǎng)膜炎癥因子IL-1β和TNF-α表達上升;2)視神經(jīng)損傷后視神經(jīng)、視網(wǎng)膜小膠質(zhì)細胞和星形膠質(zhì)細胞均出現(xiàn)激活現(xiàn)象;3)小膠質(zhì)細胞和星形膠質(zhì)細胞活化后均可表達IL-1β,視網(wǎng)膜中的TNF-α可能主要來源于小膠質(zhì)細胞。
[Abstract]:[objective] to establish a rat model of optic nerve clamp injury at different time points and to observe the response of inflammatory factors and glial cells to retinal inflammation after optic nerve injury in rats. To explore the role of retinal inflammatory factors and glial cells in optic nerve clamping injury after optic nerve injury, and to provide new ideas and methods for clinical treatment of traumatic optic nerve injury related diseases. [methods] the optic nerve clamp injury model was established by 50 g forceps, 99 adult SPF female SD rats were randomly divided into normal group (n = 3), operation group (n = 48) and sham operation group (n = 48). The optic nerve clamp injury model was made in the operation group, but only the optic nerve was isolated in the sham operation group, but it could not be clamped. In the operation group and the sham operation group, the retinal IL-1 尾 was observed in 12 rats by. Western blot test (N4) at 1 day, 3 days, 5 days and 7 days after injury, respectively. The protein expression level of TNF- 偽 at the corresponding time points; The changes of optic nerve and retinal glial cells at different time points after optic nerve injury in rats were observed by immunofluorescence histochemical staining (nb4). The co-expression of TNF- 偽 IL-1 尾 and glial cells in retina was observed by immunofluorescence co-localization (nb4). [results] the results of Western blot experiment showed that the expression of IL-1 尾 and TNF- 偽 were all changed after optic nerve clamping injury. The expression of IL-1 尾 reached its peak value on the 3rd day, and the difference was statistically significant compared with the sham operation group (p0.05). There was no significant difference in other time points compared with sham operation group (p0.05). The expression of TNF- 偽 increased significantly at 1 day after injury, reached its peak at 3 days, and decreased slightly at 5 to 7 days, but the difference was statistically significant compared with that in sham operation group (all p0. 05). The results of immunofluorescence single staining of glial cells on the optic nerve showed that the structure of the optic nerve was irregular, the distribution was not even, and the cells were missing in the gripping position after the injury of optic nerve. The expression of green fluorescence signal in microglia after optic nerve clamp injury was significantly higher than that in sham operation group, and the number of distributed microglia was increased. The expression of GFAP fluorescence signal was also increased in astrocytes compared with sham operation group, and the axon became longer. The results of immunofluorescence single staining of microglia and astrocytes in the retina showed that the morphology, number and distribution of glial cells were changed after optic nerve clamping injury, and microglia were mainly distributed in the retinal ganglion cell layer and the nuclear layer. After injury, the synapses of the microglia became shorter and thicker at day 1, the cell body became round at day 3, the synapse became shorter, the secondary branches decreased or disappeared, and the activated amoeba appeared at 5-7 days, and the number of activated microglia increased obviously. The difference was statistically significant compared with sham operation group (p0.05). The astrocytes mainly distributed outside the retinal ganglion cell layer and were activated after clamping injury, which showed that the cell body became hypertrophic, axon thickened, and the GFAP fluorescence expression increased. Immunofluorescence co-localization showed that the microglia were activated after optic nerve injury, the expression of TNF- 偽 and IL-1 尾 in retina was obvious, and TNF- 偽 and IL-1 尾 were mainly distributed in microglia of retina. However, IL-1 尾 secreted by astrocytes was significantly expressed on day 3, but no expression of TNF- 偽 was detected in retinal astrocytes. [conclusion] 1) the expression of IL-1 尾 and TNF- 偽 increased after optic nerve clamp injury, 2) the activation of optic nerve, retinal microglia and astrocytes was observed after optic nerve injury. 3) IL-1 尾 was expressed in both microglia and astrocytes, and TNF- 偽 in retina probably originated from microglia.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R774.6
[Abstract]:[objective] to establish a rat model of optic nerve clamp injury at different time points and to observe the response of inflammatory factors and glial cells to retinal inflammation after optic nerve injury in rats. To explore the role of retinal inflammatory factors and glial cells in optic nerve clamping injury after optic nerve injury, and to provide new ideas and methods for clinical treatment of traumatic optic nerve injury related diseases. [methods] the optic nerve clamp injury model was established by 50 g forceps, 99 adult SPF female SD rats were randomly divided into normal group (n = 3), operation group (n = 48) and sham operation group (n = 48). The optic nerve clamp injury model was made in the operation group, but only the optic nerve was isolated in the sham operation group, but it could not be clamped. In the operation group and the sham operation group, the retinal IL-1 尾 was observed in 12 rats by. Western blot test (N4) at 1 day, 3 days, 5 days and 7 days after injury, respectively. The protein expression level of TNF- 偽 at the corresponding time points; The changes of optic nerve and retinal glial cells at different time points after optic nerve injury in rats were observed by immunofluorescence histochemical staining (nb4). The co-expression of TNF- 偽 IL-1 尾 and glial cells in retina was observed by immunofluorescence co-localization (nb4). [results] the results of Western blot experiment showed that the expression of IL-1 尾 and TNF- 偽 were all changed after optic nerve clamping injury. The expression of IL-1 尾 reached its peak value on the 3rd day, and the difference was statistically significant compared with the sham operation group (p0.05). There was no significant difference in other time points compared with sham operation group (p0.05). The expression of TNF- 偽 increased significantly at 1 day after injury, reached its peak at 3 days, and decreased slightly at 5 to 7 days, but the difference was statistically significant compared with that in sham operation group (all p0. 05). The results of immunofluorescence single staining of glial cells on the optic nerve showed that the structure of the optic nerve was irregular, the distribution was not even, and the cells were missing in the gripping position after the injury of optic nerve. The expression of green fluorescence signal in microglia after optic nerve clamp injury was significantly higher than that in sham operation group, and the number of distributed microglia was increased. The expression of GFAP fluorescence signal was also increased in astrocytes compared with sham operation group, and the axon became longer. The results of immunofluorescence single staining of microglia and astrocytes in the retina showed that the morphology, number and distribution of glial cells were changed after optic nerve clamping injury, and microglia were mainly distributed in the retinal ganglion cell layer and the nuclear layer. After injury, the synapses of the microglia became shorter and thicker at day 1, the cell body became round at day 3, the synapse became shorter, the secondary branches decreased or disappeared, and the activated amoeba appeared at 5-7 days, and the number of activated microglia increased obviously. The difference was statistically significant compared with sham operation group (p0.05). The astrocytes mainly distributed outside the retinal ganglion cell layer and were activated after clamping injury, which showed that the cell body became hypertrophic, axon thickened, and the GFAP fluorescence expression increased. Immunofluorescence co-localization showed that the microglia were activated after optic nerve injury, the expression of TNF- 偽 and IL-1 尾 in retina was obvious, and TNF- 偽 and IL-1 尾 were mainly distributed in microglia of retina. However, IL-1 尾 secreted by astrocytes was significantly expressed on day 3, but no expression of TNF- 偽 was detected in retinal astrocytes. [conclusion] 1) the expression of IL-1 尾 and TNF- 偽 increased after optic nerve clamp injury, 2) the activation of optic nerve, retinal microglia and astrocytes was observed after optic nerve injury. 3) IL-1 尾 was expressed in both microglia and astrocytes, and TNF- 偽 in retina probably originated from microglia.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R774.6
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