LIF對(duì)小鼠視網(wǎng)膜光感受器視錐細(xì)胞的神經(jīng)保護(hù)作用及其機(jī)制研究
發(fā)布時(shí)間：2018-08-07 10:17
【摘要】：目的建立小鼠視網(wǎng)膜光損傷模型,探討白血病抑制因子(leuke-mia inhibitory factor,LIF)對(duì)小鼠視網(wǎng)膜光感受器視錐細(xì)胞光損傷的影響。 方法采用50001ux白色冷光源對(duì)BALB/c小鼠進(jìn)行6h(下午6：00到凌晨12：00)持續(xù)照射,建立小鼠視網(wǎng)膜光損傷模型。30只小鼠分為五組：①正常對(duì)照組10只、玻璃體腔注藥組10只(②右眼為玻璃體腔LIF注藥組；③左眼為玻璃體腔PBS注藥組)和光損傷+玻璃體腔注藥組10只(④右眼為光損傷+玻璃體腔LIF注藥組；⑤左眼為光損傷+玻璃體腔PBS注藥組)。光照前2d給予玻璃體腔注藥,右眼玻璃體腔LIF(0.4μg/μl,1μ1)注藥,左眼玻璃體腔PBS(0.01M,1μ1)平衡緩沖液注射(作為自身對(duì)照)。(1)光照后7天,視網(wǎng)膜電流圖(electroretinogram, ERG)檢查分別采用白光、綠光及藍(lán)光三種不同光刺激對(duì)視網(wǎng)膜光感受器視錐細(xì)胞的功能進(jìn)行檢測(cè)；(2)ERG檢查后,運(yùn)用免疫熒光組織化學(xué)通過(guò)M視錐細(xì)胞和S視錐細(xì)胞特異性標(biāo)志蛋白M-opsin和S-opsin對(duì)視網(wǎng)膜光感受器視錐細(xì)胞的數(shù)量和形態(tài)進(jìn)行檢測(cè)。 結(jié)果(1)在白光、綠光及藍(lán)光刺激下,與正常對(duì)照組ERGb波振幅相比,玻璃體腔LIF注藥組和玻璃體腔PBS注藥組均無(wú)明顯變化,其差異無(wú)統(tǒng)計(jì)學(xué)意義(p0.05)；光損傷+玻璃體腔LIF注藥組和光損傷+玻璃體腔PBS注藥組均明顯下降,其差異有統(tǒng)計(jì)學(xué)意義(p0.05)。同時(shí),與光損傷+玻璃體腔LIF注藥組ERGb波振幅相比,光損傷+玻璃體腔PBS注射組明顯下降,其差異有統(tǒng)計(jì)學(xué)意義(p0.05)。(2)與正常對(duì)照組M-opsin和S-opsin的數(shù)量和形態(tài)相比,玻璃體腔LIF注藥組和玻璃體腔PBS注藥組均無(wú)明顯變化；光損傷+玻璃體腔LIF注藥組和光損傷+玻璃體腔PBS注藥組M-opsin和S-opsin的數(shù)量均明顯下降,并且光損傷+玻璃體腔PBS注藥組M-opsin和S-opsin的形態(tài)均明顯受損。 結(jié)論50001ux白色冷光源對(duì)BALB/c小鼠進(jìn)行6h持續(xù)照射可對(duì)視網(wǎng)膜光感受器視錐細(xì)胞的功能和形態(tài)產(chǎn)生嚴(yán)重?fù)p傷。玻璃體腔注射LIF對(duì)視網(wǎng)膜光感受器視錐細(xì)胞光損傷具有保護(hù)作用。 目的研究過(guò)氧化氫(hydrogen peroxide, H2O2)對(duì)鼠視網(wǎng)膜光感受器視錐細(xì)胞661W細(xì)胞株細(xì)胞活性的影響,并探討在氧化損傷條件下信號(hào)傳導(dǎo)子與轉(zhuǎn)錄激活因子3(signal transducer and activator of transcription3, STAT3)信號(hào)通道、細(xì)胞外信號(hào)調(diào)節(jié)激酶1/2(extracellular signal-regulated kinase1and2, ERK1/2)信號(hào)通道和蛋白激酶B(protein kinase B, PKB (AKT))信號(hào)通道對(duì)661W細(xì)胞株細(xì)胞活性的影響。 方法體外培養(yǎng)鼠視網(wǎng)膜光感受器視錐細(xì)胞661W細(xì)胞株。(1)不同濃度H202(0,0.25,0.50,0.75,1mM)干預(yù)12h,采用MTT方法檢測(cè)661w細(xì)胞株細(xì)胞活性；(2)運(yùn)用不同濃度H202(0,0.005,0.01,0.05,0.5,1mM)干預(yù)661W細(xì)胞15min與1mM H202干預(yù)不同時(shí)間點(diǎn)(0,5,10,15,30min),采用Western blot方法檢測(cè)p-Tyr705-STAT3、STAT3、p-ERK1/2、ERK1/2、p-ser473-AKT和AKT蛋白質(zhì)的表達(dá)；(3)運(yùn)用50μM STAT3特異性抑制劑S3I201、50μM MEK1(ERK1/2直接上游)特異性抑制劑PD98059和20μMAKT抑制劑分別干預(yù)661W細(xì)胞1h,采用Western blot方法檢測(cè)細(xì)胞信號(hào)通路抑制劑對(duì)STAT3、ERK1/2和AKT信號(hào)通路的抑制作用。50μM S3I201、50μM PD98059或者20μM AKT抑制劑預(yù)處理1h后1mM H202干預(yù)12h,采用MTT方法檢測(cè)信號(hào)通路抑制劑對(duì)661W細(xì)胞株細(xì)胞活性的影響。 結(jié)果(1)不同濃度H202作用12h后,661W細(xì)胞株的細(xì)胞活性呈濃度依賴性下降,其差異有統(tǒng)計(jì)學(xué)意義(p0.05)；(2)H202誘導(dǎo)661W細(xì)胞STAT3、ERK1/2和AKT磷酸化,其差異有統(tǒng)計(jì)學(xué)意義(P0.05)；(3)S3I201、PD98059和AKT信號(hào)通道抑制劑預(yù)處理后,H202誘導(dǎo)的661W細(xì)胞STAT3、ERK1/2或AKT磷酸化分別受抑制,其差異有統(tǒng)計(jì)學(xué)意義(p0.05)。用S3I201、PD98059和AKT信號(hào)通道抑制劑分別抑制STAT3、ERK1/2或AKT信號(hào)通路后,661W細(xì)胞株細(xì)胞活性均明顯下降,其差異有統(tǒng)計(jì)學(xué)意義(p0.05)。 結(jié)論H202可激活661W細(xì)胞STAT3、ERK1/2和AKT信號(hào)通路。氧化損傷條件下,STAT3、ERK1/2和AKT信號(hào)通路的激活是661W細(xì)胞存活所必需的。 目的研究氧化損傷條件下,LIF對(duì)661W細(xì)胞活性的影響及其作用機(jī)制。 方法體外培養(yǎng)鼠視網(wǎng)膜光感受器視錐細(xì)胞661W細(xì)胞株。(1)5ng/mlLIF預(yù)處理661W細(xì)胞1h后1mM H202干預(yù)12h,采用MTT方法檢測(cè)661W細(xì)胞株細(xì)胞活性；(2)5ng/mlLIF預(yù)處理661W細(xì)胞1h后1mM H202干預(yù)15min,采用Western blot方法檢測(cè)661W細(xì)胞p-Tyr705-STAT、STAT3、p-ERK1/2、ERK1/2、p-Ser473-AKT和AKT蛋白質(zhì)的表達(dá)。5ng/mlLIF干預(yù)661W細(xì)胞1h,采用免疫熒光組織化學(xué)方法檢測(cè)661W細(xì)胞p-Tyr705-STAT、p-ERK1/2和p-Ser473-AKT蛋白質(zhì)的表達(dá)和定位；(3)50μMSTAT3特異性抑制劑S3I201預(yù)處理661W細(xì)胞1h后5ng/mlLIF預(yù)處理1h,然后1mmH202干預(yù)12h,采用MTT方法檢測(cè)661W細(xì)胞株細(xì)胞活性；(4)50μMS3I201預(yù)處理661W細(xì)胞1h后1mM H202干預(yù)15min,采用Western blot方法檢測(cè)細(xì)胞周期蛋白D1(cyclinD1)和細(xì)胞周期蛋白E(cyclinE)蛋白質(zhì)的表達(dá)。 結(jié)果(1)氧化損傷條件下,LIF可降低H202誘導(dǎo)的661W細(xì)胞死亡,其差異有統(tǒng)計(jì)學(xué)意義(p0.05)。(2)正常和氧化損傷條件下,LIF均只激活STAT3信號(hào)通道,其差異有統(tǒng)計(jì)學(xué)意義(p0.05)；不激活ERK1/2和AKT信號(hào)通道,其差異無(wú)統(tǒng)計(jì)學(xué)意義(p0.05)(3)氧化損傷條件下,S3I201減弱LIF的保護(hù)作用,其差異有統(tǒng)計(jì)學(xué)意義(p0.05)。(4)氧化損傷條件下,阻斷STAT3信號(hào)通道減少STAT3靶基因cyclinD1和cyclin E蛋白的表達(dá),其差異均有統(tǒng)計(jì)學(xué)意義(p0.05)。 結(jié)論我們的研究表明LIF作為661W細(xì)胞一個(gè)新的存活因子,氧化損傷條件下通過(guò)激活STAT3信號(hào)通道及其靶基因cyclin D1和cyclin E發(fā)揮細(xì)胞保護(hù)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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R774.1
本文編號(hào)：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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R774.1
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相關(guān)期刊論文 前9條
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