中华眼底病杂志
中華眼底病雜誌
중화안저병잡지
CHINESE JOURNAL OF OCULAR FUNDUS DISEASES
2012年
5期
501-506
,共6页
刘兵%郭大东%毕宏生%郭媛媛%陈宁虹%马晓华
劉兵%郭大東%畢宏生%郭媛媛%陳寧虹%馬曉華
류병%곽대동%필굉생%곽원원%진저홍%마효화
视网膜神经节细胞/病理学%硫辛酸/治疗应用%细胞,培养的%动物实验
視網膜神經節細胞/病理學%硫辛痠/治療應用%細胞,培養的%動物實驗
시망막신경절세포/병이학%류신산/치료응용%세포,배양적%동물실험
Retinal ganglion cells/ pathology%Thioctic acid/therapeutic use%Cells,cultured%Animal experimentation
目的 观察α-硫辛酸对高压条件下培养大鼠视网膜神经节细胞(RGC)的保护作用.方法 将RGC分为正常对照组、阴性对照组、单纯加压组、加压联合不同浓度α-硫辛酸干预组.正常对照组和单纯加压组不做任何处理;阴性对照组仅接受200 μmol/Lα-硫辛酸预处理;加压联合不同浓度的α-硫辛酸干预组分别用50、100、200 μmol/L等3种不同浓度的α-硫辛酸对其进行干预.α-硫辛酸预处理1h后,单纯加压组和各干预组置于50 mm Hg(1 mm Hg=0.133 kPa)下培养24 h;正常对照组、阴性对照组置于常压下培养24 h.分别用噻唑蓝比色法检测各组细胞的活性;4,6-联脒2-苯基吲哚(DAPI)染色观察各组细胞核形态改变;实时聚合酶链反应(Real-time PCR)及蛋白免疫印迹法(Western blot)检测并比较各组细胞中锰超氧化物歧化酶(MnSOD) mRNA和蛋白表达水平的变化.结果 单纯加压组细胞活性为(65.6±3.4)％,50、100、200 μmol/L3种浓度的α-硫辛酸干预组细胞活性分别为(75.1±3.3)％、(81.8±2.9)％、(87.9±3.1)％,均较单纯加压组显著增高(t=5.108、10.007、12.513,P＜0.05).DAPI染色显示,加压后RGC-5细胞核可见空泡、染色质高度凝聚、边缘化等典型的凋亡特征,而α-硫辛酸干预组仅有部分细胞核染色质表现出浓缩现象.Real-time PCR及Western blot检测结果显示,加压培养后细胞内MnSODmRNA和蛋白相对表达量较正常对照组显著降低(t=22.045、26.979,P＜0.01);与单纯加压组相比,α-硫辛酸干预组细胞内MnSOD mRNA和蛋白相对表达量均显著增高,差异有统计学意义(t=9.171、12.267、23.567、7.723、12.009、28.198,P＜0.05);不同浓度的α-硫辛酸干预组之间,随α-硫辛酸浓度增加,MnSODmRNA和蛋白相对表达量呈升高趋势,且差异有统计学意义(F=134.273、194.597,P＜0.01).结论 应用α-硫辛酸可以显著提高高压条件下大鼠RGC的细胞活性及细胞内MnSOD的表达水平,增强细胞抗氧化损伤能力.
目的 觀察α-硫辛痠對高壓條件下培養大鼠視網膜神經節細胞(RGC)的保護作用.方法 將RGC分為正常對照組、陰性對照組、單純加壓組、加壓聯閤不同濃度α-硫辛痠榦預組.正常對照組和單純加壓組不做任何處理;陰性對照組僅接受200 μmol/Lα-硫辛痠預處理;加壓聯閤不同濃度的α-硫辛痠榦預組分彆用50、100、200 μmol/L等3種不同濃度的α-硫辛痠對其進行榦預.α-硫辛痠預處理1h後,單純加壓組和各榦預組置于50 mm Hg(1 mm Hg=0.133 kPa)下培養24 h;正常對照組、陰性對照組置于常壓下培養24 h.分彆用噻唑藍比色法檢測各組細胞的活性;4,6-聯脒2-苯基吲哚(DAPI)染色觀察各組細胞覈形態改變;實時聚閤酶鏈反應(Real-time PCR)及蛋白免疫印跡法(Western blot)檢測併比較各組細胞中錳超氧化物歧化酶(MnSOD) mRNA和蛋白錶達水平的變化.結果 單純加壓組細胞活性為(65.6±3.4)％,50、100、200 μmol/L3種濃度的α-硫辛痠榦預組細胞活性分彆為(75.1±3.3)％、(81.8±2.9)％、(87.9±3.1)％,均較單純加壓組顯著增高(t=5.108、10.007、12.513,P＜0.05).DAPI染色顯示,加壓後RGC-5細胞覈可見空泡、染色質高度凝聚、邊緣化等典型的凋亡特徵,而α-硫辛痠榦預組僅有部分細胞覈染色質錶現齣濃縮現象.Real-time PCR及Western blot檢測結果顯示,加壓培養後細胞內MnSODmRNA和蛋白相對錶達量較正常對照組顯著降低(t=22.045、26.979,P＜0.01);與單純加壓組相比,α-硫辛痠榦預組細胞內MnSOD mRNA和蛋白相對錶達量均顯著增高,差異有統計學意義(t=9.171、12.267、23.567、7.723、12.009、28.198,P＜0.05);不同濃度的α-硫辛痠榦預組之間,隨α-硫辛痠濃度增加,MnSODmRNA和蛋白相對錶達量呈升高趨勢,且差異有統計學意義(F=134.273、194.597,P＜0.01).結論 應用α-硫辛痠可以顯著提高高壓條件下大鼠RGC的細胞活性及細胞內MnSOD的錶達水平,增彊細胞抗氧化損傷能力.
목적 관찰α-류신산대고압조건하배양대서시망막신경절세포(RGC)적보호작용.방법 장RGC분위정상대조조、음성대조조、단순가압조、가압연합불동농도α-류신산간예조.정상대조조화단순가압조불주임하처리;음성대조조부접수200 μmol/Lα-류신산예처리;가압연합불동농도적α-류신산간예조분별용50、100、200 μmol/L등3충불동농도적α-류신산대기진행간예.α-류신산예처리1h후,단순가압조화각간예조치우50 mm Hg(1 mm Hg=0.133 kPa)하배양24 h;정상대조조、음성대조조치우상압하배양24 h.분별용새서람비색법검측각조세포적활성;4,6-련미2-분기신타(DAPI)염색관찰각조세포핵형태개변;실시취합매련반응(Real-time PCR)급단백면역인적법(Western blot)검측병비교각조세포중맹초양화물기화매(MnSOD) mRNA화단백표체수평적변화.결과 단순가압조세포활성위(65.6±3.4)％,50、100、200 μmol/L3충농도적α-류신산간예조세포활성분별위(75.1±3.3)％、(81.8±2.9)％、(87.9±3.1)％,균교단순가압조현저증고(t=5.108、10.007、12.513,P＜0.05).DAPI염색현시,가압후RGC-5세포핵가견공포、염색질고도응취、변연화등전형적조망특정,이α-류신산간예조부유부분세포핵염색질표현출농축현상.Real-time PCR급Western blot검측결과현시,가압배양후세포내MnSODmRNA화단백상대표체량교정상대조조현저강저(t=22.045、26.979,P＜0.01);여단순가압조상비,α-류신산간예조세포내MnSOD mRNA화단백상대표체량균현저증고,차이유통계학의의(t=9.171、12.267、23.567、7.723、12.009、28.198,P＜0.05);불동농도적α-류신산간예조지간,수α-류신산농도증가,MnSODmRNA화단백상대표체량정승고추세,차차이유통계학의의(F=134.273、194.597,P＜0.01).결론 응용α-류신산가이현저제고고압조건하대서RGC적세포활성급세포내MnSOD적표체수평,증강세포항양화손상능력.
Objective To observe the neuroprotective effect of α-lipoic acid (ALA) on cultured retinal ganglion cells (RGC-5) under elevated pressure in vitro.Methods Cultured RGC-5 cells were divided randomly into 4 groups,including normal control group (group A),negative control group (group B),elevated pressure group (group C) and elevated pressure + ALA group (group D).The cells of group A and C were not intervened with ALA.The cells of group B were treated with 200 μmol/L ALA.The cells of group D were treated with different concentrations of ALA (50,100,200 μmol/L) for one hour.Then cells of group C and D were exerted to 50 mm Hg (1 mm Hg=0.133 kPa) for 24 hours,while the cells of group A and B were exerted to normal pressures for 24 hours.The cell viability was measured using the methyl thiazolyl tetrazolium (MTT) assay and apoptosis was evaluated using 4',6 diamidino-2-phenylindole (DAPI) staining.Expression of MnSOD was determined by real-time polymerase chain reaction (RT-PCR)and Western blot,respectively.Results The cell viability of group B was (65.6 ± 3.4) ％,which lower than that in group D of three concentrations of ALA[ (75.1± 3.3)％,(81.8 ± 2.9 ) ％,(87.9 ± 3.1 ) ％ ],the differences were significantly (t =5.108,10.007,12.513; P＜0.05).DAPI staining revealed that characteristic apoptotic changes,such as chromatin condensation,convoluted nuclei with cavitations,fragmentation of the nucleus,and apoptotic bodies appeared in RGC-5 cells after 24 ours pressure.There was almost no evidence of apoptosis in group D.RT-PCR and Western blot analysis revealed that the expression of MnSOD mRNA and protein were weakly expressed in group C compared with control A (t=22.045,26.979; P＜0.01).Compared group C with group D,the level of MnSOD mRNA and protein in group D increased significantly (t=9.171,12.267,23.567,7.723,12.009,28.198;P＜0.05).In addition,the presence of ALA was found to inhibit hydrostatic pressure induced damage of RGC-5 cells in a dose-dependent manner (F=134.273,194.597;P＜0.01).Conclusion ALA can effectively improve the expression of MnSOD in RGC-5 cells under the condition of elevated pressure,enhance the ability of RGC-5 cells against oxidative damage.
