中华眼科杂志
中華眼科雜誌
중화안과잡지
Chinese Journal of Ophthalmology
2014年
6期
440-447
,共8页
华宁%刘宏伟%钱学翰%东莉洁%武晶晶%李筱荣
華寧%劉宏偉%錢學翰%東莉潔%武晶晶%李篠榮
화저%류굉위%전학한%동리길%무정정%리소영
视网膜病,早产儿%信号素3A%细胞凋亡%疾病模型,动物
視網膜病,早產兒%信號素3A%細胞凋亡%疾病模型,動物
시망막병,조산인%신호소3A%세포조망%질병모형,동물
Retinopathy of prematurity%Semaphorin-3A%Apoptosis%Disease models,animal
目的：探讨信号素3A（ SEMA3A）在大鼠氧诱导视网膜病变（ OIR）模型中的表达变化及其对视网膜组织损伤的影响。方法实验研究。采用随机数字表法将新生大鼠48只分为4组，每组12只（12眼）。正常对照组于正常空气环境下饲养，余3个组隔天交替吸入氧含量为50％和10％的氮氧混合气体，制备OIR模型。 OIR＋SEMA3A抑制组于出生后7 d给予单眼玻璃体腔注射兔抗大鼠SEMA3A多克隆抗体（40 mg/L，2μl）；OIR＋IgG组出生后7 d给予单眼玻璃体腔注射等量兔IgG （40 mg/L，2μl）；OIR组不进行玻璃体腔注射。各组大鼠出生后18 d取材，HE染色检测视网膜厚度、神经节细胞层（ RGCL）细胞数和突破内界膜内皮细胞数，TUNEL染色检测视网膜细胞凋亡并计算凋亡指数（ AI）；免疫印迹法检测视网膜SEMA3A和Caspase-3蛋白p17亚基表达；免疫荧光染色定位检测SEMA3A表达。各组大鼠视网膜厚度、RGCL细胞数、突破内界膜内皮细胞数，视网膜AI，SEMA3A和Caspase-3蛋白p17亚基相对表达量的比较采用单因素方差分析，进一步的两两比较采用LSD-t检验。结果 HE染色结果显示，正常对照组视网膜厚度为（117.07±8.13）μm，OIR组为（70.93±5.68）μm，OIR＋SEMA3A抑制组为（91.28±4.58）μm，OIR＋IgG组为（67.27±10.15）μm，差异有统计学意义（F＝13.222，P＝0.002）。正常对照组RGCL细胞数为42.7±3.6，OIR组为24.3±3.1， OIR＋SEMA3A抑制组为35.0±6.2，OIR＋IgG组为22.8±4.3，差异有统计学意义（ F＝22.537，P＝0.000）。正常对照组突破内界膜的内皮细胞数为1.0±0.3，OIR组为14.2±3.2，OIR＋SEMA3Aab组为9.6±1.1， OIR ＋IgG 组为10.8±1.6，差异有统计学意义（ F ＝14.478， P ＝0.002）。 OIR ＋SEMA3A抑制组视网膜厚度、RGCL细胞数均低于正常对照组（P＜0.01），但均较OIR组和OIR＋IgG组明显增高（P＜0.01）；OIR＋SEMA3A抑制组突破内界膜的内皮细胞数高于正常对照组（P＜0.01），但与OIR组和OIR＋IgG组差异无统计学意义（P＞0.05）。正常对照组视网膜AI值为27.67±2.51， OIR组为58.33±8.50，OIR＋SEMA3A抑制组为37.33±5.03，OIR＋IgG组为61.67±6.65，差异有统计学意义（F＝19.250，P＝0.001）。 OIR＋SEMA3A抑制组视网膜AI较OIR＋IgG组和OIR组降低（P＜0.01）。应用TatolLab 2.01软件对免疫印迹法检出的蛋白条带进行分析，正常对照组SEMA3A相对表达量为0.64±0.03，OIR组为0.97±0.05，OIR＋SEMA3A抑制组为0.41±0.02，OIR＋IgG组为1.03±0.15，差异有统计学意义（F＝38.59，P＝0.000）。 OIR组视网膜组织SEMA3A蛋白相对表达量较正常对照组明显增高（ P＜0.01）；OIR＋SEMA3Aab组视网膜组织SEMA3A蛋白相对表达量明显低于OIR组和OIR＋IgG组（P＜0.01）。免疫荧光染色结果显示，正常对照组SEMA3A主要分布于光感受器细胞层， OIR 组和 OIR ＋IgG 组 SEMA3A 荧光强度明显增加，各层均可见着染， OIR ＋SEMA3A抑制组其荧光强度明显减弱。免疫印迹法检测各组视网膜组织Caspase-3蛋白p17亚基相对表达量，正常对照组为0.00±0.00，OIR组为0.30±0.02，OIR＋SEMA3A抑制组为0.12±0.01， OIR＋IgG组为0.27±0.02，差异有统计学意义（ F＝304.619，P＜0.01）。其中OIR＋SEMA3A抑制组较OIR＋IgG组和OIR组明显降低（P＜0.01）。结论大鼠OIR模型视网膜组织中SEMA3A表达上调可能促进视网膜神经细胞凋亡。阻断SEMA3A可减轻视网膜组织的凋亡程度。（中华眼科杂志，2014，50：440-447）
目的：探討信號素3A（ SEMA3A）在大鼠氧誘導視網膜病變（ OIR）模型中的錶達變化及其對視網膜組織損傷的影響。方法實驗研究。採用隨機數字錶法將新生大鼠48隻分為4組，每組12隻（12眼）。正常對照組于正常空氣環境下飼養，餘3箇組隔天交替吸入氧含量為50％和10％的氮氧混閤氣體，製備OIR模型。 OIR＋SEMA3A抑製組于齣生後7 d給予單眼玻璃體腔註射兔抗大鼠SEMA3A多剋隆抗體（40 mg/L，2μl）；OIR＋IgG組齣生後7 d給予單眼玻璃體腔註射等量兔IgG （40 mg/L，2μl）；OIR組不進行玻璃體腔註射。各組大鼠齣生後18 d取材，HE染色檢測視網膜厚度、神經節細胞層（ RGCL）細胞數和突破內界膜內皮細胞數，TUNEL染色檢測視網膜細胞凋亡併計算凋亡指數（ AI）；免疫印跡法檢測視網膜SEMA3A和Caspase-3蛋白p17亞基錶達；免疫熒光染色定位檢測SEMA3A錶達。各組大鼠視網膜厚度、RGCL細胞數、突破內界膜內皮細胞數，視網膜AI，SEMA3A和Caspase-3蛋白p17亞基相對錶達量的比較採用單因素方差分析，進一步的兩兩比較採用LSD-t檢驗。結果 HE染色結果顯示，正常對照組視網膜厚度為（117.07±8.13）μm，OIR組為（70.93±5.68）μm，OIR＋SEMA3A抑製組為（91.28±4.58）μm，OIR＋IgG組為（67.27±10.15）μm，差異有統計學意義（F＝13.222，P＝0.002）。正常對照組RGCL細胞數為42.7±3.6，OIR組為24.3±3.1， OIR＋SEMA3A抑製組為35.0±6.2，OIR＋IgG組為22.8±4.3，差異有統計學意義（ F＝22.537，P＝0.000）。正常對照組突破內界膜的內皮細胞數為1.0±0.3，OIR組為14.2±3.2，OIR＋SEMA3Aab組為9.6±1.1， OIR ＋IgG 組為10.8±1.6，差異有統計學意義（ F ＝14.478， P ＝0.002）。 OIR ＋SEMA3A抑製組視網膜厚度、RGCL細胞數均低于正常對照組（P＜0.01），但均較OIR組和OIR＋IgG組明顯增高（P＜0.01）；OIR＋SEMA3A抑製組突破內界膜的內皮細胞數高于正常對照組（P＜0.01），但與OIR組和OIR＋IgG組差異無統計學意義（P＞0.05）。正常對照組視網膜AI值為27.67±2.51， OIR組為58.33±8.50，OIR＋SEMA3A抑製組為37.33±5.03，OIR＋IgG組為61.67±6.65，差異有統計學意義（F＝19.250，P＝0.001）。 OIR＋SEMA3A抑製組視網膜AI較OIR＋IgG組和OIR組降低（P＜0.01）。應用TatolLab 2.01軟件對免疫印跡法檢齣的蛋白條帶進行分析，正常對照組SEMA3A相對錶達量為0.64±0.03，OIR組為0.97±0.05，OIR＋SEMA3A抑製組為0.41±0.02，OIR＋IgG組為1.03±0.15，差異有統計學意義（F＝38.59，P＝0.000）。 OIR組視網膜組織SEMA3A蛋白相對錶達量較正常對照組明顯增高（ P＜0.01）；OIR＋SEMA3Aab組視網膜組織SEMA3A蛋白相對錶達量明顯低于OIR組和OIR＋IgG組（P＜0.01）。免疫熒光染色結果顯示，正常對照組SEMA3A主要分佈于光感受器細胞層， OIR 組和 OIR ＋IgG 組 SEMA3A 熒光彊度明顯增加，各層均可見著染， OIR ＋SEMA3A抑製組其熒光彊度明顯減弱。免疫印跡法檢測各組視網膜組織Caspase-3蛋白p17亞基相對錶達量，正常對照組為0.00±0.00，OIR組為0.30±0.02，OIR＋SEMA3A抑製組為0.12±0.01， OIR＋IgG組為0.27±0.02，差異有統計學意義（ F＝304.619，P＜0.01）。其中OIR＋SEMA3A抑製組較OIR＋IgG組和OIR組明顯降低（P＜0.01）。結論大鼠OIR模型視網膜組織中SEMA3A錶達上調可能促進視網膜神經細胞凋亡。阻斷SEMA3A可減輕視網膜組織的凋亡程度。（中華眼科雜誌，2014，50：440-447）
목적：탐토신호소3A（ SEMA3A）재대서양유도시망막병변（ OIR）모형중적표체변화급기대시망막조직손상적영향。방법실험연구。채용수궤수자표법장신생대서48지분위4조，매조12지（12안）。정상대조조우정상공기배경하사양，여3개조격천교체흡입양함량위50％화10％적담양혼합기체，제비OIR모형。 OIR＋SEMA3A억제조우출생후7 d급여단안파리체강주사토항대서SEMA3A다극륭항체（40 mg/L，2μl）；OIR＋IgG조출생후7 d급여단안파리체강주사등량토IgG （40 mg/L，2μl）；OIR조불진행파리체강주사。각조대서출생후18 d취재，HE염색검측시망막후도、신경절세포층（ RGCL）세포수화돌파내계막내피세포수，TUNEL염색검측시망막세포조망병계산조망지수（ AI）；면역인적법검측시망막SEMA3A화Caspase-3단백p17아기표체；면역형광염색정위검측SEMA3A표체。각조대서시망막후도、RGCL세포수、돌파내계막내피세포수，시망막AI，SEMA3A화Caspase-3단백p17아기상대표체량적비교채용단인소방차분석，진일보적량량비교채용LSD-t검험。결과 HE염색결과현시，정상대조조시망막후도위（117.07±8.13）μm，OIR조위（70.93±5.68）μm，OIR＋SEMA3A억제조위（91.28±4.58）μm，OIR＋IgG조위（67.27±10.15）μm，차이유통계학의의（F＝13.222，P＝0.002）。정상대조조RGCL세포수위42.7±3.6，OIR조위24.3±3.1， OIR＋SEMA3A억제조위35.0±6.2，OIR＋IgG조위22.8±4.3，차이유통계학의의（ F＝22.537，P＝0.000）。정상대조조돌파내계막적내피세포수위1.0±0.3，OIR조위14.2±3.2，OIR＋SEMA3Aab조위9.6±1.1， OIR ＋IgG 조위10.8±1.6，차이유통계학의의（ F ＝14.478， P ＝0.002）。 OIR ＋SEMA3A억제조시망막후도、RGCL세포수균저우정상대조조（P＜0.01），단균교OIR조화OIR＋IgG조명현증고（P＜0.01）；OIR＋SEMA3A억제조돌파내계막적내피세포수고우정상대조조（P＜0.01），단여OIR조화OIR＋IgG조차이무통계학의의（P＞0.05）。정상대조조시망막AI치위27.67±2.51， OIR조위58.33±8.50，OIR＋SEMA3A억제조위37.33±5.03，OIR＋IgG조위61.67±6.65，차이유통계학의의（F＝19.250，P＝0.001）。 OIR＋SEMA3A억제조시망막AI교OIR＋IgG조화OIR조강저（P＜0.01）。응용TatolLab 2.01연건대면역인적법검출적단백조대진행분석，정상대조조SEMA3A상대표체량위0.64±0.03，OIR조위0.97±0.05，OIR＋SEMA3A억제조위0.41±0.02，OIR＋IgG조위1.03±0.15，차이유통계학의의（F＝38.59，P＝0.000）。 OIR조시망막조직SEMA3A단백상대표체량교정상대조조명현증고（ P＜0.01）；OIR＋SEMA3Aab조시망막조직SEMA3A단백상대표체량명현저우OIR조화OIR＋IgG조（P＜0.01）。면역형광염색결과현시，정상대조조SEMA3A주요분포우광감수기세포층， OIR 조화 OIR ＋IgG 조 SEMA3A 형광강도명현증가，각층균가견착염， OIR ＋SEMA3A억제조기형광강도명현감약。면역인적법검측각조시망막조직Caspase-3단백p17아기상대표체량，정상대조조위0.00±0.00，OIR조위0.30±0.02，OIR＋SEMA3A억제조위0.12±0.01， OIR＋IgG조위0.27±0.02，차이유통계학의의（ F＝304.619，P＜0.01）。기중OIR＋SEMA3A억제조교OIR＋IgG조화OIR조명현강저（P＜0.01）。결론대서OIR모형시망막조직중SEMA3A표체상조가능촉진시망막신경세포조망。조단SEMA3A가감경시망막조직적조망정도。（중화안과잡지，2014，50：440-447）
Objective To observe the change of semaphorin 3A(SEMA3A)expression in the retina of oxygen induced retinopathy ( OIR ) in rats and to investigate its influence on retinal degeneration in OIR.Methods Experimental study.Forty-eight newborn pups were randomly classified into four groups and only one eye was examined in each pup.Thirty-six pups was induced into OIR model through aspirating 50%and 10% oxygen every 24 hours alternatively while the control group ( 12 pups ) was raised under normal atmosphere.OIR+SEMA3Aab group accepted intravitreous injection of rabbit anti-rat SEMA3A antibody ( Abcam Co.LTD, 40 mg/L, 2μl) while OIR+IgG group was injected the same amount of non-active rabbit IgG at postnatal day 7.And the OIR group had no injections.All the pups were executed at postnatal day 18.Histological changes of retinas were examined through HE stain while Immunoflurecence staining and TUNEL procedure were used to detect focal expression of SEMA 3A and apoptosis respectively.Total tissue protein was extracted and the expression of SEMA 3A and Caspase-3 p17subunit were examined by Western blot.The data including retinal thickness, cell counting of retinal ganglion cells layers (RGCL), endothelia outside inner limiting membrane, retinal apoptosis index (AI), the relative expression of SEMA3A and Caspase3 p17 subunit were analyzed by One-way ANOVA and followed LSD-t test to compare group differences.Results There were significant differences of retinal thickness , cell counting of RGCL and endothelia outside inner limiting membrane among each groups respectively ( F=13.222,F=22.537,F=14.478;P<0.01 ).The retinal thickness was ( 117.07 ±8.13 ) μm in normal control group , ( 70.93 ± 5.68) μm in OIR group, (91.28 ±4.58) μm in OIR+SEMA3Aab group, and (67.27 ±10.15) μm in OIR+IgG group;the cell counting of RGCL was 42.7 ±3.6 in normal control group , 24.3 ±3.1 in OIR group, 35.0 ±6.2 in OIR+SEMA3Aab group, and 22.8 ±4.3 in OIR+IgG group while the endothelia outside inner limiting membrane was 1.0 ±0.3 in normal control group , 14.2 ±3.2 in OIR group, 9.6 ± 1.1 in OIR +SEMA3Aab group, and 10.8 ±1.6 in OIR +IgG group.The retinal thickness and cell counting of RGCL in OIR+SEMA3Aab group were significantly lower than those in the normal control group (P<0.01), but were apparently higher than the data of OIR group and OIR +IgG group respectively(P<0.01).There were no more endothelia on the vitreal side of the inner limiting membrane in OIR +SEMA3Aab group than OIR group or OIR +IgG group(P>0.05)although these three groups had much more endothelia than it in the normal control group (P<0.01).The retinal AI detected by TUNEL staining was 27.67 ±2.51 in normal control,58.33 ±8.50 in OIR group,37.33 ±5.03 in OIR+SEMA3Aab group and 61.67 ±6.65 in OIR+IgG group.There was significant difference of the retinal AI among the four groups (F=19.250,P=0.001).Apoptotic cells were significantly reduced in OIR +SEMA3Aab group compared with OIR +IgG group or OIR group(P<0.01).The difference of SEMA3A protein expression among the groups detected by Western blot was significant (F=38.59,P=0.000).SEMA3A expression in OIR group was 0.97 ±0.05, which was significantly upregulated compared with the normal control group (0.64 ±0.03) ( P<0.01).And its expression was successfully neutralized in OIR +SEMA3Aab group(0.41 ±0.02)with comparison with OIR+IgG group(1.03 ±0.15)through Western blot(P<0.01).SEMA3A was detected apparently in the photoreceptors layer in the normal control while its fluorescence was stronger and much more scattered in the whole retina.However,anti-SEMA3Aab intravitreous injection successfully reduced its fluorescence compared with the IgG injection.The cleavage of Caspase-3 was not detected in the normal control while the relative expression of Caspase-3 p17 subunit was significantly different in the other three groups(F=304.619,P<0.01).OIR+SEMA3Aab group had much less Caspase-3 p17 subunit(0.12 ± 0.01)than OIR group(0.30 ±0.02)or OIR +IgG group(0.27 ±0.02)(P<0.01).Conclusions The over regulation of SEMA3A probably enhances the aggravation of apoptosis in OIR rats.Inhibition of SEMA3A is helpful to protect neuroretina.
