金納多和蘿卜硫素對小鼠視網(wǎng)膜神經(jīng)細胞凋亡的保護作用與機制研究
發(fā)布時間：2018-07-25 12:16
【摘要】：目的：光照可引起視網(wǎng)膜細胞凋亡,致視網(wǎng)膜外核層損傷。本實驗通過對視網(wǎng)膜光損傷小鼠進行金納多(GBE)和蘿卜硫素(SF)心腔注射,探討GBE和SF對光照引起的視網(wǎng)膜細胞凋亡是否具有預防保護作用及其機制。 方法： 1.建立小鼠光損傷模型：Bal b/c小鼠暗室飼養(yǎng)24小時,次日置于光照箱內(nèi),分別于照射24h、48h和72h處死,取眼球制作石蠟切片。HE染色進行視網(wǎng)膜形態(tài)學觀察；進行外核層厚度測量和外核層層數(shù)統(tǒng)計,分析不同光照長度對視細胞造成的損傷;DNA梯度電泳及TUNEL法檢測視細胞凋亡。 2. Bal b/c小鼠隨機分為6組即正常對照組,實驗對照組(生理鹽水),GBE組(GBE-Ⅰ組100 ul/二次；GBE-Ⅱ組為50μl/一次,濃度為3.5mg/ml),SF組(SF-Ⅰ組為60μl/二次；SF-Ⅱ組為30μl/一次,濃度為50mg/ml),每組5只,心臟注射給藥(替代靜脈注射),除正常對照組之外的5組小鼠均在自制光照箱內(nèi)光照72h后斷頸處死,常規(guī)制備眼球石蠟切片,HE染色后形態(tài)學觀察并測量外核層厚度;RT-PCR檢測細胞色素C和Bak-1的表達；Caspase活性試劑盒檢測Caspase-3活性。使用SPSS13.0統(tǒng)計軟件,單因素方差分析法進行分析。 結(jié)果： 1.小鼠光損傷模型建立：HE染色形態(tài)學觀察發(fā)現(xiàn)正常組小鼠視網(wǎng)膜結(jié)構(gòu)層次清楚；光照各組外核層明顯變薄,視網(wǎng)膜視細胞內(nèi)、外節(jié)排列明顯紊亂,分界不清,出現(xiàn)不規(guī)則細胞核。凋亡檢測及定位：DNA電泳結(jié)果顯示,光照各組均出現(xiàn)DNA梯狀條帶,其中光照72h組DNA梯狀條帶最為顯著；TUNEL法檢測顯示細胞凋亡位于外核層。 2.GBE和SF對視網(wǎng)膜光損傷的預防保護作用：形態(tài)學觀察發(fā)現(xiàn),實驗對照組視網(wǎng)膜細胞排列稀疏,內(nèi)、外節(jié)排列明紊亂、腫脹、破碎,外核層明顯變�。唤o藥組及正常對照組的視網(wǎng)膜結(jié)構(gòu)均優(yōu)于實驗對照組。外核層厚度測量結(jié)果,正常對照組49.23±2.32μm,實驗對照組40.36±2.17μm; GBE-Ⅰ組、GBE-Ⅱ組外核層厚度分別為42.0770±0.8999μm和40.9411±0.706μm; SF-Ⅰ組、SF-Ⅱ組外核層厚度分別為48.2352±2.0447μm和46.8004±0.6014μm。對結(jié)果進行統(tǒng)計學分析表明,GBE-Ⅰ組、GBE-Ⅱ組、SF-Ⅰ組、SF-Ⅱ組與實驗對照組比較均有顯著差異(P0.001)。 3.GBE和SF對視網(wǎng)膜光損傷的預防保護機制：GBE-Ⅰ組、GBE-Ⅱ組、SF-Ⅰ組、SF-Ⅱ組與實驗對照組比較,RT-PCR檢測結(jié)果顯示細胞色素C和Bak-1的表達均不同程度下調(diào)。GBE-Ⅰ組、GBE-Ⅱ組、SF-Ⅰ組、SF-Ⅱ組mRNA水平表達與對照組比較有顯著性差異(P0.01,P0.05); Caspase-3活性檢測結(jié)果：GBE-Ⅰ組、GBE-Ⅱ組、SF-Ⅰ組、SF-Ⅱ組與實驗對照組比較,Caspase-3活性均降低。 結(jié)論： 1.可以采用定時光照建立小鼠視網(wǎng)膜光損傷模型； 2.GBE和SF對視網(wǎng)膜光損傷具有預防保護作用。 3.GBE和SF對視網(wǎng)膜光損傷的預防保護機制均與抑制視網(wǎng)膜細胞凋亡及相關(guān)蛋白表達下調(diào)有關(guān)。
[Abstract]:Objective: light can induce apoptosis of retina cells and damage the outer nuclear layer of retina. By injecting GBE and sulforaphin (SF) into the heart of mice with retinal light injury, this experiment is to explore whether GBE and SF have protective protective effect and mechanism on retinal cell apoptosis induced by light.
Method:
1. the mouse light damage model was established: the Bal b/c mice were kept in the dark room for 24 hours, and the next day was placed in the light box. They were irradiated with 24h, 48h and 72h respectively. The eyeball was taken to make the retinal morphology observation with.HE staining of paraffin section; the thickness of outer nuclear layer and the number of outer layer layers were counted, and the damage caused by different light length to visual cells was analyzed. DNA gradient electrophoresis and TUNEL assay were used to detect the apoptosis of the optic cells.
2. Bal b/c mice were randomly divided into 6 groups: normal control group, experimental control group (physiological saline), group GBE (group GBE- I, 100 ul/ two times, GBE- II group 50 mu l/, 3.5mg/ml), group SF (SF- I group 60 mu l/ two, SF- II group 30 micron, concentration for concentration), 5 in each group, with cardiac injection (instead of intravenous injection), except normal pair 5 groups of mice outside the group were killed in the self-made light box after light 72h, and the eyeball paraffin section was routinely prepared. After HE staining, morphological observation and measuring the thickness of outer nuclear layer; RT-PCR to detect the expression of cytochrome C and Bak-1; Caspase active kit to detect Caspase-3 activity. SPSS13.0 statistical software, single factor variance analysis method was used. Line analysis.
Result:
1. the model of light damage in mice was established: HE staining morphological observation found that the structure of retina in the normal group was clear; the outer nuclear layers were obviously thinner in each group, the outer segments of the retina were obviously disorganized, the boundary was disorderly, the irregular nuclei were unclear, the apoptosis detection and location were found. The results of DNA electrophoresis showed that the DNA ladder appeared in all groups of light. TUNEL assay showed that apoptosis was located in the outer nuclear layer.
The preventive and protective effects of 2.GBE and SF on retinal light damage: morphological observation showed that the retinal cells in the experimental control group were arranged sparsely, the outer segments were arranged in disorder, swelling, broken, and the outer core layer was thinner; the retina structure of the drug group and the normal control group were superior to those in the test control group. The outer nuclear thickness measurement results, the normal control group 49 .23 + 2.32 mu m, experimental control group 40.36 + 2.17 micron m, GBE- I group, GBE- II Group outer nucleus thickness was 42.0770 + 0.8999 mu m and 40.9411 + 0.706 mu m, SF- I group, SF- II Group outer core layer thickness of 48.2352 + 2.0447 mu m and 46.8004 + 0.6014 M. on the results of statistical analysis, GBE- I, group II, group I, group II and experiment There were significant differences between the control group and the control group (P0.001).
The prevention and protection mechanism of retinal light damage by 3.GBE and SF: group GBE- I, group GBE- II, group SF- I, group SF- II and experimental control group. The results of RT-PCR detection showed that the expression of cytochrome C and Bak-1 were all down regulated in the.GBE- I group, GBE- II group and SF- group. Caspase-3 activity test results: GBE-I group, GBE-II group, SF-I group, SF-II group compared with the experimental control group, Caspase-3 activity decreased.
Conclusion:
1. The model of retinal light damage in mice can be established by fixed-time irradiation.
2. GBE and SF have preventive and protective effects on retinal light damage.
3. The protective mechanism of GBE and SF on retinal light injury is related to inhibition of retinal cell apoptosis and down-regulation of related protein expression.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R774.1
本文編號：2143794
[Abstract]:Objective: light can induce apoptosis of retina cells and damage the outer nuclear layer of retina. By injecting GBE and sulforaphin (SF) into the heart of mice with retinal light injury, this experiment is to explore whether GBE and SF have protective protective effect and mechanism on retinal cell apoptosis induced by light.
Method:
1. the mouse light damage model was established: the Bal b/c mice were kept in the dark room for 24 hours, and the next day was placed in the light box. They were irradiated with 24h, 48h and 72h respectively. The eyeball was taken to make the retinal morphology observation with.HE staining of paraffin section; the thickness of outer nuclear layer and the number of outer layer layers were counted, and the damage caused by different light length to visual cells was analyzed. DNA gradient electrophoresis and TUNEL assay were used to detect the apoptosis of the optic cells.
2. Bal b/c mice were randomly divided into 6 groups: normal control group, experimental control group (physiological saline), group GBE (group GBE- I, 100 ul/ two times, GBE- II group 50 mu l/, 3.5mg/ml), group SF (SF- I group 60 mu l/ two, SF- II group 30 micron, concentration for concentration), 5 in each group, with cardiac injection (instead of intravenous injection), except normal pair 5 groups of mice outside the group were killed in the self-made light box after light 72h, and the eyeball paraffin section was routinely prepared. After HE staining, morphological observation and measuring the thickness of outer nuclear layer; RT-PCR to detect the expression of cytochrome C and Bak-1; Caspase active kit to detect Caspase-3 activity. SPSS13.0 statistical software, single factor variance analysis method was used. Line analysis.
Result:
1. the model of light damage in mice was established: HE staining morphological observation found that the structure of retina in the normal group was clear; the outer nuclear layers were obviously thinner in each group, the outer segments of the retina were obviously disorganized, the boundary was disorderly, the irregular nuclei were unclear, the apoptosis detection and location were found. The results of DNA electrophoresis showed that the DNA ladder appeared in all groups of light. TUNEL assay showed that apoptosis was located in the outer nuclear layer.
The preventive and protective effects of 2.GBE and SF on retinal light damage: morphological observation showed that the retinal cells in the experimental control group were arranged sparsely, the outer segments were arranged in disorder, swelling, broken, and the outer core layer was thinner; the retina structure of the drug group and the normal control group were superior to those in the test control group. The outer nuclear thickness measurement results, the normal control group 49 .23 + 2.32 mu m, experimental control group 40.36 + 2.17 micron m, GBE- I group, GBE- II Group outer nucleus thickness was 42.0770 + 0.8999 mu m and 40.9411 + 0.706 mu m, SF- I group, SF- II Group outer core layer thickness of 48.2352 + 2.0447 mu m and 46.8004 + 0.6014 M. on the results of statistical analysis, GBE- I, group II, group I, group II and experiment There were significant differences between the control group and the control group (P0.001).
The prevention and protection mechanism of retinal light damage by 3.GBE and SF: group GBE- I, group GBE- II, group SF- I, group SF- II and experimental control group. The results of RT-PCR detection showed that the expression of cytochrome C and Bak-1 were all down regulated in the.GBE- I group, GBE- II group and SF- group. Caspase-3 activity test results: GBE-I group, GBE-II group, SF-I group, SF-II group compared with the experimental control group, Caspase-3 activity decreased.
Conclusion:
1. The model of retinal light damage in mice can be established by fixed-time irradiation.
2. GBE and SF have preventive and protective effects on retinal light damage.
3. The protective mechanism of GBE and SF on retinal light injury is related to inhibition of retinal cell apoptosis and down-regulation of related protein expression.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R774.1
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