中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2015年
1期
27-32
,共6页
贺玲%郑晓龙%张杰%张善峰%余晓红%田芳
賀玲%鄭曉龍%張傑%張善峰%餘曉紅%田芳
하령%정효룡%장걸%장선봉%여효홍%전방
视网膜%新生血管%熊果酸%环氧合酶%血管内皮生长因子%基质金属蛋白酶%C57BL/6J小鼠
視網膜%新生血管%熊果痠%環氧閤酶%血管內皮生長因子%基質金屬蛋白酶%C57BL/6J小鼠
시망막%신생혈관%웅과산%배양합매%혈관내피생장인자%기질금속단백매%C57BL/6J소서
Retina%Neovascularization%Ursolic acid%Cyclooxygenase%Vascular endothelial growth factor%Matrix metalloproteinase%Mouse,C57BL/6J
背景 视网膜新生血管疾病严重影响视功能,目前虽然有较多的抗新生血管药物,但多针对单一的干预靶点,疗效有限.研究证实,熊果酸具有多种生物学效应,其中包括抗血管新生作用,但其对眼科血管疾病的疗效尚不清楚. 目的 观察熊果酸对小鼠氧诱导视网膜新生血管形成的抑制作用. 方法 采用随机数字表法将7日龄清洁级C57BL/6J小鼠60只随机分为6个组,即空白对照组、PBS对照组、阳性对照组和低、中、高剂量熊果酸组.空白对照组小鼠在正常环境中喂养,其他各组小鼠与哺乳的母鼠置于体积分数(75±2)％的高氧环境中连续饲养5d,小鼠12日龄时将模型小鼠及其哺乳母鼠返回正常空气环境中,以诱导视网膜新生血管产生.造模成功后按照分组不同分别于小鼠玻璃体腔注射无菌PBS 3μl、曲安奈德注射液(1 ml∶40 mg)3μl或1.5、3.0、6.0μg熊果酸各3μl.小鼠17日龄时过量麻醉法处死,摘除双侧眼球制备视网膜组织切片.采用组织病理学方法检查各组小鼠视网膜血管新生情况,计数突破视网膜内界膜的新生血管内皮细胞核数目;采用逆转录PCR(RT-PCR)法分别检测和比较各组小鼠视网膜组织中血管内皮生长因子(VEGF) mRNA、环氧合酶-2(COX-2)mRNA及基质金属蛋白酶-2(MMP-2)mRNA的相对表达量. 结果 视网膜切片后苏木精-伊红染色显示,PBS对照组小鼠视网膜中突破内界膜的新生血管内皮细胞核数为(18.65±3.24)个/视野,明显高于空白对照组的(0.78±0.11)个/视野,差异有统计学意义(t=2.24,P＜0.05);中剂量熊果酸组、高剂量熊果酸组小鼠视网膜突破内界膜的新生血管内皮细胞核数分别为(13.32±1.87)个/视野和(8.93±1.09)个/视野,明显少于PBS对照组和低剂量熊果酸组的(18.65±3.24)个/视野和(15.44±2.02)个/视野,差异均有统计学意义(均P＜0.05),而高剂量熊果酸组小鼠视网膜突破内界膜的新生血管内皮细胞核数与阳性对照组的(9.14±1.13)个/视野接近,差异无统计学意义(t=1.17,P＞0.05).RT-PCR检测表明,PBS对照组小鼠视网膜组织中COX-2 mRNA、VEGF mRNA和MMP-2 mRNA相对表达量均明显高于空白对照组,差异均有统计学意义(t=13.45、12.49、14.32,均P＜0.05),且高剂量熊果酸组小鼠视网膜组织中COX-2mRNA、VEGF mRNA和MMP-2 mRNA相对表达量均明显低于PBS对照组和低剂量熊果酸组,差异均有统计学意义(均P＜0.05),而高剂量熊果酸组与阳性对照组间的差异均无统计学意义(均P＞0.05).结论 熊果酸以剂量依赖的方式下调氧诱导的缺血小鼠视网膜组织中VEGF、COX-2及MMP-2的表达,从而抑制视网膜新生血管的产生.
揹景 視網膜新生血管疾病嚴重影響視功能,目前雖然有較多的抗新生血管藥物,但多針對單一的榦預靶點,療效有限.研究證實,熊果痠具有多種生物學效應,其中包括抗血管新生作用,但其對眼科血管疾病的療效尚不清楚. 目的 觀察熊果痠對小鼠氧誘導視網膜新生血管形成的抑製作用. 方法 採用隨機數字錶法將7日齡清潔級C57BL/6J小鼠60隻隨機分為6箇組,即空白對照組、PBS對照組、暘性對照組和低、中、高劑量熊果痠組.空白對照組小鼠在正常環境中餵養,其他各組小鼠與哺乳的母鼠置于體積分數(75±2)％的高氧環境中連續飼養5d,小鼠12日齡時將模型小鼠及其哺乳母鼠返迴正常空氣環境中,以誘導視網膜新生血管產生.造模成功後按照分組不同分彆于小鼠玻璃體腔註射無菌PBS 3μl、麯安奈德註射液(1 ml∶40 mg)3μl或1.5、3.0、6.0μg熊果痠各3μl.小鼠17日齡時過量痳醉法處死,摘除雙側眼毬製備視網膜組織切片.採用組織病理學方法檢查各組小鼠視網膜血管新生情況,計數突破視網膜內界膜的新生血管內皮細胞覈數目;採用逆轉錄PCR(RT-PCR)法分彆檢測和比較各組小鼠視網膜組織中血管內皮生長因子(VEGF) mRNA、環氧閤酶-2(COX-2)mRNA及基質金屬蛋白酶-2(MMP-2)mRNA的相對錶達量. 結果 視網膜切片後囌木精-伊紅染色顯示,PBS對照組小鼠視網膜中突破內界膜的新生血管內皮細胞覈數為(18.65±3.24)箇/視野,明顯高于空白對照組的(0.78±0.11)箇/視野,差異有統計學意義(t=2.24,P＜0.05);中劑量熊果痠組、高劑量熊果痠組小鼠視網膜突破內界膜的新生血管內皮細胞覈數分彆為(13.32±1.87)箇/視野和(8.93±1.09)箇/視野,明顯少于PBS對照組和低劑量熊果痠組的(18.65±3.24)箇/視野和(15.44±2.02)箇/視野,差異均有統計學意義(均P＜0.05),而高劑量熊果痠組小鼠視網膜突破內界膜的新生血管內皮細胞覈數與暘性對照組的(9.14±1.13)箇/視野接近,差異無統計學意義(t=1.17,P＞0.05).RT-PCR檢測錶明,PBS對照組小鼠視網膜組織中COX-2 mRNA、VEGF mRNA和MMP-2 mRNA相對錶達量均明顯高于空白對照組,差異均有統計學意義(t=13.45、12.49、14.32,均P＜0.05),且高劑量熊果痠組小鼠視網膜組織中COX-2mRNA、VEGF mRNA和MMP-2 mRNA相對錶達量均明顯低于PBS對照組和低劑量熊果痠組,差異均有統計學意義(均P＜0.05),而高劑量熊果痠組與暘性對照組間的差異均無統計學意義(均P＞0.05).結論 熊果痠以劑量依賴的方式下調氧誘導的缺血小鼠視網膜組織中VEGF、COX-2及MMP-2的錶達,從而抑製視網膜新生血管的產生.
배경 시망막신생혈관질병엄중영향시공능,목전수연유교다적항신생혈관약물,단다침대단일적간예파점,료효유한.연구증실,웅과산구유다충생물학효응,기중포괄항혈관신생작용,단기대안과혈관질병적료효상불청초. 목적 관찰웅과산대소서양유도시망막신생혈관형성적억제작용. 방법 채용수궤수자표법장7일령청길급C57BL/6J소서60지수궤분위6개조,즉공백대조조、PBS대조조、양성대조조화저、중、고제량웅과산조.공백대조조소서재정상배경중위양,기타각조소서여포유적모서치우체적분수(75±2)％적고양배경중련속사양5d,소서12일령시장모형소서급기포유모서반회정상공기배경중,이유도시망막신생혈관산생.조모성공후안조분조불동분별우소서파리체강주사무균PBS 3μl、곡안내덕주사액(1 ml∶40 mg)3μl혹1.5、3.0、6.0μg웅과산각3μl.소서17일령시과량마취법처사,적제쌍측안구제비시망막조직절편.채용조직병이학방법검사각조소서시망막혈관신생정황,계수돌파시망막내계막적신생혈관내피세포핵수목;채용역전록PCR(RT-PCR)법분별검측화비교각조소서시망막조직중혈관내피생장인자(VEGF) mRNA、배양합매-2(COX-2)mRNA급기질금속단백매-2(MMP-2)mRNA적상대표체량. 결과 시망막절편후소목정-이홍염색현시,PBS대조조소서시망막중돌파내계막적신생혈관내피세포핵수위(18.65±3.24)개/시야,명현고우공백대조조적(0.78±0.11)개/시야,차이유통계학의의(t=2.24,P＜0.05);중제량웅과산조、고제량웅과산조소서시망막돌파내계막적신생혈관내피세포핵수분별위(13.32±1.87)개/시야화(8.93±1.09)개/시야,명현소우PBS대조조화저제량웅과산조적(18.65±3.24)개/시야화(15.44±2.02)개/시야,차이균유통계학의의(균P＜0.05),이고제량웅과산조소서시망막돌파내계막적신생혈관내피세포핵수여양성대조조적(9.14±1.13)개/시야접근,차이무통계학의의(t=1.17,P＞0.05).RT-PCR검측표명,PBS대조조소서시망막조직중COX-2 mRNA、VEGF mRNA화MMP-2 mRNA상대표체량균명현고우공백대조조,차이균유통계학의의(t=13.45、12.49、14.32,균P＜0.05),차고제량웅과산조소서시망막조직중COX-2mRNA、VEGF mRNA화MMP-2 mRNA상대표체량균명현저우PBS대조조화저제량웅과산조,차이균유통계학의의(균P＜0.05),이고제량웅과산조여양성대조조간적차이균무통계학의의(균P＞0.05).결론 웅과산이제량의뢰적방식하조양유도적결혈소서시망막조직중VEGF、COX-2급MMP-2적표체,종이억제시망막신생혈관적산생.
Background Retinal neovascular diseases affect visual function.Although many drugs have been used to manage the visual diseases,their effectiveness is less than satisfactory.Studies showed that ursolic acid has multiple biological effects including anti-vascularization.However,the effect of ursolic acid on retinal neovascular diseases is unclear now.Objective This study was to observe the inhibitory effect of ursolic acid on the high oxygen-induced mouse retinal neovascularization after intravitreal injection.Methods Sixty clean 7-day-old C57BL/6J mice were divided into the blank control group,PBS control group,positive control group (triamcinolone) and low,moderate and high dose (1.5,3.0 and 6.0 μg) ursolic acid groups randomly.The blank control group mice were raised in normal environment,and the mice from other groups were fed in the environment with O2 concentration at (75±2)％ for 5 days together with the maternal mice.The mice then were back to the normal air environment to induce retinal neovascularization.Then,the drugs were intravitreally immediately injected in the mice of the different groups.The mice were sacrificed at the 17-day old for the preparation of retinal sections.Retinal new blood vessel was examined by haematoxylin and eosin stain under the light microscope,and the number of vascular endothelial cell nucleus breaking the inner limiting membrane was counted.The gene expressions of vascular endothelial growth factor (VEGF),cyclooxygenase-2 (COX-2) and matrix metalloproteinase-2 (MMP-2) in the mouse retinas were quantitatively assayed using reverse transcription PCR.Results The number of endothelial nuclei newly-generated vessel breaking internal limiting membrane in the mice of PBS control group was (18.65±3.24)/field,which was more than (0.78±0.11)/field of the blank control group obviously (t =2.24,P＜0.05).The number of endothelial nuclei newly-generated breaking internal limiting membrane in the moderate-or high-dose ursolic acid group was less than that of moderate group obviously,it was statistically significant(P＜0.05).The number of vascular endothelial cell nuclei breaking internal limiting membrane in high high-dose group was (13.32 ± 1.87)/field and (8.93 ± 1.09) /field,showing significant decreases in comparison with the PBS control group and low-dose ursolic acid group (18.65±3.24)/field (15.44±2.02)/field (all at P＜0.05).However,no significant difference were seen in the number of new vascular endothelial cell nucleus between the high-dose ursolic acid group and the positive control group(9.14±1.13)/field (t=1.17,P＞0.05).The relative expressions of COX-2 mRNA,VEGF mRNA and MMP-2 mRNA in the mouse retinas were higher in the PBS control group than those in the blank control group (t =13.45,12.49,14.32,all at P＜0.05),and those in the moderate-dose or high-dose ursolic acid group were lowed in comparison with the PBS control group and the low-dose ursolic acid group (all at P＜0.05),but there were no significant differences between the high-dose ursolic acid group and the positive control group (all at P＞0.05).Conclusions Ursolic acid can suppress retinal neovascularization by down-regulating the expressions of VEGF,COX-2 and MMP-2 in oxygen-induced retinopathy of mouse in dose-dependent manner.
