LIF对小鼠视网膜光感受器视锥细胞的神经保护作用及其机制研究
发布时间：2018-08-07 10:17
【摘要】：目的建立小鼠视网膜光损伤模型,探讨白血病抑制因子(leuke-mia inhibitory factor,LIF)对小鼠视网膜光感受器视锥细胞光损伤的影响。 方法采用50001ux白色冷光源对BALB/c小鼠进行6h(下午6：00到凌晨12：00)持续照射,建立小鼠视网膜光损伤模型。30只小鼠分为五组：①正常对照组10只、玻璃体腔注药组10只(②右眼为玻璃体腔LIF注药组；③左眼为玻璃体腔PBS注药组)和光损伤+玻璃体腔注药组10只(④右眼为光损伤+玻璃体腔LIF注药组；⑤左眼为光损伤+玻璃体腔PBS注药组)。光照前2d给予玻璃体腔注药,右眼玻璃体腔LIF(0.4μg/μl,1μ1)注药,左眼玻璃体腔PBS(0.01M,1μ1)平衡缓冲液注射(作为自身对照)。(1)光照后7天,视网膜电流图(electroretinogram, ERG)检查分别采用白光、绿光及蓝光三种不同光刺激对视网膜光感受器视锥细胞的功能进行检测；(2)ERG检查后,运用免疫荧光组织化学通过M视锥细胞和S视锥细胞特异性标志蛋白M-opsin和S-opsin对视网膜光感受器视锥细胞的数量和形态进行检测。 结果(1)在白光、绿光及蓝光刺激下,与正常对照组ERGb波振幅相比,玻璃体腔LIF注药组和玻璃体腔PBS注药组均无明显变化,其差异无统计学意义(p0.05)；光损伤+玻璃体腔LIF注药组和光损伤+玻璃体腔PBS注药组均明显下降,其差异有统计学意义(p0.05)。同时,与光损伤+玻璃体腔LIF注药组ERGb波振幅相比,光损伤+玻璃体腔PBS注射组明显下降,其差异有统计学意义(p0.05)。(2)与正常对照组M-opsin和S-opsin的数量和形态相比,玻璃体腔LIF注药组和玻璃体腔PBS注药组均无明显变化；光损伤+玻璃体腔LIF注药组和光损伤+玻璃体腔PBS注药组M-opsin和S-opsin的数量均明显下降,并且光损伤+玻璃体腔PBS注药组M-opsin和S-opsin的形态均明显受损。 结论50001ux白色冷光源对BALB/c小鼠进行6h持续照射可对视网膜光感受器视锥细胞的功能和形态产生严重损伤。玻璃体腔注射LIF对视网膜光感受器视锥细胞光损伤具有保护作用。 目的研究过氧化氢(hydrogen peroxide, H2O2)对鼠视网膜光感受器视锥细胞661W细胞株细胞活性的影响,并探讨在氧化损伤条件下信号传导子与转录激活因子3(signal transducer and activator of transcription3, STAT3)信号通道、细胞外信号调节激酶1/2(extracellular signal-regulated kinase1and2, ERK1/2)信号通道和蛋白激酶B(protein kinase B, PKB (AKT))信号通道对661W细胞株细胞活性的影响。 方法体外培养鼠视网膜光感受器视锥细胞661W细胞株。(1)不同浓度H202(0,0.25,0.50,0.75,1mM)干预12h,采用MTT方法检测661w细胞株细胞活性；(2)运用不同浓度H202(0,0.005,0.01,0.05,0.5,1mM)干预661W细胞15min与1mM H202干预不同时间点(0,5,10,15,30min),采用Western blot方法检测p-Tyr705-STAT3、STAT3、p-ERK1/2、ERK1/2、p-ser473-AKT和AKT蛋白质的表达；(3)运用50μM STAT3特异性抑制剂S3I201、50μM MEK1(ERK1/2直接上游)特异性抑制剂PD98059和20μMAKT抑制剂分别干预661W细胞1h,采用Western blot方法检测细胞信号通路抑制剂对STAT3、ERK1/2和AKT信号通路的抑制作用。50μM S3I201、50μM PD98059或者20μM AKT抑制剂预处理1h后1mM H202干预12h,采用MTT方法检测信号通路抑制剂对661W细胞株细胞活性的影响。 结果(1)不同浓度H202作用12h后,661W细胞株的细胞活性呈浓度依赖性下降,其差异有统计学意义(p0.05)；(2)H202诱导661W细胞STAT3、ERK1/2和AKT磷酸化,其差异有统计学意义(P0.05)；(3)S3I201、PD98059和AKT信号通道抑制剂预处理后,H202诱导的661W细胞STAT3、ERK1/2或AKT磷酸化分别受抑制,其差异有统计学意义(p0.05)。用S3I201、PD98059和AKT信号通道抑制剂分别抑制STAT3、ERK1/2或AKT信号通路后,661W细胞株细胞活性均明显下降,其差异有统计学意义(p0.05)。 结论H202可激活661W细胞STAT3、ERK1/2和AKT信号通路。氧化损伤条件下,STAT3、ERK1/2和AKT信号通路的激活是661W细胞存活所必需的。 目的研究氧化损伤条件下,LIF对661W细胞活性的影响及其作用机制。 方法体外培养鼠视网膜光感受器视锥细胞661W细胞株。(1)5ng/mlLIF预处理661W细胞1h后1mM H202干预12h,采用MTT方法检测661W细胞株细胞活性；(2)5ng/mlLIF预处理661W细胞1h后1mM H202干预15min,采用Western blot方法检测661W细胞p-Tyr705-STAT、STAT3、p-ERK1/2、ERK1/2、p-Ser473-AKT和AKT蛋白质的表达。5ng/mlLIF干预661W细胞1h,采用免疫荧光组织化学方法检测661W细胞p-Tyr705-STAT、p-ERK1/2和p-Ser473-AKT蛋白质的表达和定位；(3)50μMSTAT3特异性抑制剂S3I201预处理661W细胞1h后5ng/mlLIF预处理1h,然后1mmH202干预12h,采用MTT方法检测661W细胞株细胞活性；(4)50μMS3I201预处理661W细胞1h后1mM H202干预15min,采用Western blot方法检测细胞周期蛋白D1(cyclinD1)和细胞周期蛋白E(cyclinE)蛋白质的表达。 结果(1)氧化损伤条件下,LIF可降低H202诱导的661W细胞死亡,其差异有统计学意义(p0.05)。(2)正常和氧化损伤条件下,LIF均只激活STAT3信号通道,其差异有统计学意义(p0.05)；不激活ERK1/2和AKT信号通道,其差异无统计学意义(p0.05)(3)氧化损伤条件下,S3I201减弱LIF的保护作用,其差异有统计学意义(p0.05)。(4)氧化损伤条件下,阻断STAT3信号通道减少STAT3靶基因cyclinD1和cyclin E蛋白的表达,其差异均有统计学意义(p0.05)。 结论我们的研究表明LIF作为661W细胞一个新的存活因子,氧化损伤条件下通过激活STAT3信号通道及其靶基因cyclin D1和cyclin E发挥细胞保护作用。
[Abstract]:Objective to establish a mouse retinal light damage model and to explore the effect of leuke-mia inhibitory factor (LIF) on the optical damage of retinal photoreceptor conical cells in mice.
Methods the BALB/c mice were irradiated continuously with 50001ux white cold light source (from 6:00 p.m. to 12:00 am), and the mouse retinal light damage model was set up in five groups: (1) the normal control group was 10, the glass cavity injection group 10 (the right eye was the vitreous body LIF injection group; the left eye was the glass cavity PBS injection group) and the light loss. 10 rats were treated with injections and vitreous cavity injection group ((4) the right eye was light injury + LIF injection group of vitreous cavity, the left eye was light injury + PBS injection of glass body cavity. 2D was injected into the vitreous cavity before illumination, LIF (0.4 mu g/, 1 mu 1) in the right eye, PBS (0.01M, 1 mu 1) in the left eye (as self control). (1) 7 after illumination. The retinal electroretinogram (electroretinogram, ERG) examination used three different light stimuli, white light, green light and blue light, to detect the function of retinal photoreceptor cone cells. (2) after ERG examination, immunofluorescent histochemistry was used to pass M cone cells and S cone cell specific marker protein M-opsin and S-opsin to retina. The number and morphology of photoreceptor cone cells were detected.
Results (1) under the light of white light, green light and blue light, there was no significant change in the LIF injection group and the PBS injection group in the vitreous cavity compared with the normal control group ERGb wave amplitude, and the difference was not statistically significant (P0.05). The difference was statistically significant between the light injury + the glass cavity LIF injection group and the light damage + Bose body cavity PBS injection group. Significance (P0.05). At the same time, compared with the amplitude of ERGb wave in the light injury + LIF injection group, the light injury and the PBS injection group of the vitreous cavity were significantly decreased, and the difference was statistically significant (P0.05). (2) there was no significant change in the amount and form of M-opsin and S-opsin in the normal control group and the morphology of the vitreous cavity LIF injection group and the vitreous cavity PBS injection group. The number of M-opsin and S-opsin in the LIF injection group and the light injury + PBS injection group were significantly decreased, and the morphology of M-opsin and S-opsin in the light injury + PBS injection group of the vitreous body was obviously damaged.
Conclusion the continuous irradiation of 50001ux white cold light on BALB/c mice can seriously damage the function and morphology of retinal photoreceptor conical cells. LIF injection of vitreous cavity can protect the optical damage of retinal photoreceptor cone cells.
Objective to study the effect of hydrogen peroxide (H2O2) on the cell activity of 661W cell line of retina photoreceptor conical cells, and to explore the signal transduction pathway of signal conductors and transcription activator 3 (signal transducer and activator of Transcription3, STAT3) under oxidative damage, and the extracellular signal regulating kinase 1/2. The effect of signal channel of acellular signal-regulated kinase1and2, ERK1/2) and protein kinase B (protein kinase B, PKB (AKT)) signal channel on the cell activity of 661W cell line.
Methods the rat retinal photoreceptor 661W cell lines were cultured in vitro. (1) different concentrations of H202 (0,0.25,0.50,0.75,1mM) were used to interfere with 12h, and MTT method was used to detect the cell activity of 661W cell lines. (2) the intervention of 661W cell 15min and 1mM H202 at different time points using H202 (0,0.005,0.01,0.05,0.5,1mM) concentration were used. The stern blot method was used to detect the expression of p-Tyr705-STAT3, STAT3, p-ERK1/2, ERK1/2, p-ser473-AKT and AKT proteins; (3) the inhibition of cell signaling pathway was detected by using the 50 micron STAT3 specific inhibitor S3I201,50 micron M inhibitors and 20 micron inhibitors. Inhibitory effects of agents on STAT3, ERK1/2 and AKT signaling pathways:.50, M S3I201,50, S3I201,50, M PD98059 or 20 micron AKT inhibitor pretreatment 1H 1mM, the effect of signaling pathway inhibitors on cell activity was detected by means of signaling pathway inhibitors.
Results (1) after 12h of different concentrations of H202, the cell activity of 661W cells decreased in a concentration dependent manner, and the difference was statistically significant (P0.05). (2) H202 induced 661W cells STAT3, ERK1/2 and AKT phosphorylation, and the difference was statistically significant (P0.05); (3) S3I201, PD98059 and signaling pathway inhibitors were pretreated. The phosphorylation of T3, ERK1/2 or AKT was inhibited respectively, and the difference was statistically significant (P0.05). The activity of 661W cell lines decreased significantly after the inhibition of STAT3, ERK1/2 or AKT signaling pathways with S3I201, PD98059 and AKT signal channel inhibitors, and the difference was statistically significant (P0.05).
Conclusion H202 activates the STAT3, ERK1/2 and AKT signaling pathways of 661W cells. The activation of STAT3, ERK1/2 and AKT signaling pathways is essential for the survival of 661W cells under oxidative damage.
Objective to study the effect of LIF on the activity of 661W cells under oxidative stress and its mechanism.
Methods the rat retinal photoreceptor 661W cell line was cultured in vitro. (1) after 5ng/mlLIF pretreated 661W cell 1H, 1mM H202 intervened 12h, and MTT method was used to detect the cell activity of 661W cell lines. The expression and localization of 661W cell p-Tyr705-STAT, p-ERK1/2 and p-Ser473-AKT proteins were detected by the expression of 1/2, p-Ser473-AKT and AKT protein.5ng/mlLIF, and the expression and localization of 661W cell p-Tyr705-STAT, p-ERK1/2 and p-Ser473-AKT proteins were detected by immunofluorescence histochemical method. The cell activity of 661W cell line was detected by MTT method; (4) 1mM H202 intervened 15min after 661W cell 1H was pretreated with 1H, and the expression of cyclin D1 (cyclinD1) and cyclin protein was detected by Western blot.
Results (1) under the condition of oxidative damage, LIF could reduce the death of 661W cells induced by H202, and the difference was statistically significant (P0.05). (2) all LIF only activated STAT3 channel under normal and oxidative damage conditions, and the difference was statistically significant (P0.05); no ERK1/2 and AKT signal channels were activated (P0.05) (P0.05) (3) oxidative damage strips The protective effect of LIF was weakened by S3I201, and the difference was statistically significant (P0.05). (4) the expression of cyclinD1 and cyclin E protein of STAT3 target gene was reduced by blocking the STAT3 signal channel, and the difference was statistically significant (P0.05).
Conclusion our study shows that LIF is a new survival factor of 661W cells. Under oxidative damage, the protective effect of STAT3 signal channel and its target gene cyclin D1 and cyclin E is activated by oxidative damage.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R774.1
本文编号：2169713
[Abstract]:Objective to establish a mouse retinal light damage model and to explore the effect of leuke-mia inhibitory factor (LIF) on the optical damage of retinal photoreceptor conical cells in mice.
Methods the BALB/c mice were irradiated continuously with 50001ux white cold light source (from 6:00 p.m. to 12:00 am), and the mouse retinal light damage model was set up in five groups: (1) the normal control group was 10, the glass cavity injection group 10 (the right eye was the vitreous body LIF injection group; the left eye was the glass cavity PBS injection group) and the light loss. 10 rats were treated with injections and vitreous cavity injection group ((4) the right eye was light injury + LIF injection group of vitreous cavity, the left eye was light injury + PBS injection of glass body cavity. 2D was injected into the vitreous cavity before illumination, LIF (0.4 mu g/, 1 mu 1) in the right eye, PBS (0.01M, 1 mu 1) in the left eye (as self control). (1) 7 after illumination. The retinal electroretinogram (electroretinogram, ERG) examination used three different light stimuli, white light, green light and blue light, to detect the function of retinal photoreceptor cone cells. (2) after ERG examination, immunofluorescent histochemistry was used to pass M cone cells and S cone cell specific marker protein M-opsin and S-opsin to retina. The number and morphology of photoreceptor cone cells were detected.
Results (1) under the light of white light, green light and blue light, there was no significant change in the LIF injection group and the PBS injection group in the vitreous cavity compared with the normal control group ERGb wave amplitude, and the difference was not statistically significant (P0.05). The difference was statistically significant between the light injury + the glass cavity LIF injection group and the light damage + Bose body cavity PBS injection group. Significance (P0.05). At the same time, compared with the amplitude of ERGb wave in the light injury + LIF injection group, the light injury and the PBS injection group of the vitreous cavity were significantly decreased, and the difference was statistically significant (P0.05). (2) there was no significant change in the amount and form of M-opsin and S-opsin in the normal control group and the morphology of the vitreous cavity LIF injection group and the vitreous cavity PBS injection group. The number of M-opsin and S-opsin in the LIF injection group and the light injury + PBS injection group were significantly decreased, and the morphology of M-opsin and S-opsin in the light injury + PBS injection group of the vitreous body was obviously damaged.
Conclusion the continuous irradiation of 50001ux white cold light on BALB/c mice can seriously damage the function and morphology of retinal photoreceptor conical cells. LIF injection of vitreous cavity can protect the optical damage of retinal photoreceptor cone cells.
Objective to study the effect of hydrogen peroxide (H2O2) on the cell activity of 661W cell line of retina photoreceptor conical cells, and to explore the signal transduction pathway of signal conductors and transcription activator 3 (signal transducer and activator of Transcription3, STAT3) under oxidative damage, and the extracellular signal regulating kinase 1/2. The effect of signal channel of acellular signal-regulated kinase1and2, ERK1/2) and protein kinase B (protein kinase B, PKB (AKT)) signal channel on the cell activity of 661W cell line.
Methods the rat retinal photoreceptor 661W cell lines were cultured in vitro. (1) different concentrations of H202 (0,0.25,0.50,0.75,1mM) were used to interfere with 12h, and MTT method was used to detect the cell activity of 661W cell lines. (2) the intervention of 661W cell 15min and 1mM H202 at different time points using H202 (0,0.005,0.01,0.05,0.5,1mM) concentration were used. The stern blot method was used to detect the expression of p-Tyr705-STAT3, STAT3, p-ERK1/2, ERK1/2, p-ser473-AKT and AKT proteins; (3) the inhibition of cell signaling pathway was detected by using the 50 micron STAT3 specific inhibitor S3I201,50 micron M inhibitors and 20 micron inhibitors. Inhibitory effects of agents on STAT3, ERK1/2 and AKT signaling pathways:.50, M S3I201,50, S3I201,50, M PD98059 or 20 micron AKT inhibitor pretreatment 1H 1mM, the effect of signaling pathway inhibitors on cell activity was detected by means of signaling pathway inhibitors.
Results (1) after 12h of different concentrations of H202, the cell activity of 661W cells decreased in a concentration dependent manner, and the difference was statistically significant (P0.05). (2) H202 induced 661W cells STAT3, ERK1/2 and AKT phosphorylation, and the difference was statistically significant (P0.05); (3) S3I201, PD98059 and signaling pathway inhibitors were pretreated. The phosphorylation of T3, ERK1/2 or AKT was inhibited respectively, and the difference was statistically significant (P0.05). The activity of 661W cell lines decreased significantly after the inhibition of STAT3, ERK1/2 or AKT signaling pathways with S3I201, PD98059 and AKT signal channel inhibitors, and the difference was statistically significant (P0.05).
Conclusion H202 activates the STAT3, ERK1/2 and AKT signaling pathways of 661W cells. The activation of STAT3, ERK1/2 and AKT signaling pathways is essential for the survival of 661W cells under oxidative damage.
Objective to study the effect of LIF on the activity of 661W cells under oxidative stress and its mechanism.
Methods the rat retinal photoreceptor 661W cell line was cultured in vitro. (1) after 5ng/mlLIF pretreated 661W cell 1H, 1mM H202 intervened 12h, and MTT method was used to detect the cell activity of 661W cell lines. The expression and localization of 661W cell p-Tyr705-STAT, p-ERK1/2 and p-Ser473-AKT proteins were detected by the expression of 1/2, p-Ser473-AKT and AKT protein.5ng/mlLIF, and the expression and localization of 661W cell p-Tyr705-STAT, p-ERK1/2 and p-Ser473-AKT proteins were detected by immunofluorescence histochemical method. The cell activity of 661W cell line was detected by MTT method; (4) 1mM H202 intervened 15min after 661W cell 1H was pretreated with 1H, and the expression of cyclin D1 (cyclinD1) and cyclin protein was detected by Western blot.
Results (1) under the condition of oxidative damage, LIF could reduce the death of 661W cells induced by H202, and the difference was statistically significant (P0.05). (2) all LIF only activated STAT3 channel under normal and oxidative damage conditions, and the difference was statistically significant (P0.05); no ERK1/2 and AKT signal channels were activated (P0.05) (P0.05) (3) oxidative damage strips The protective effect of LIF was weakened by S3I201, and the difference was statistically significant (P0.05). (4) the expression of cyclinD1 and cyclin E protein of STAT3 target gene was reduced by blocking the STAT3 signal channel, and the difference was statistically significant (P0.05).
Conclusion our study shows that LIF is a new survival factor of 661W cells. Under oxidative damage, the protective effect of STAT3 signal channel and its target gene cyclin D1 and cyclin E is activated by oxidative damage.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R774.1
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