中华眼视光学与视觉科学杂志
中華眼視光學與視覺科學雜誌
중화안시광학여시각과학잡지
CHINESE JOURNAL OF OPTOMETRY OPHTHALMOLOGY AND VISUAL SCIENCE
2011年
4期
263-268
,共6页
罗旭昇%吴星伟%顾青%张红梅%施宇华%仇亚婷
囉旭昇%吳星偉%顧青%張紅梅%施宇華%仇亞婷
라욱승%오성위%고청%장홍매%시우화%구아정
复方血栓通,中草药%血管生成抑制剂%脉络膜新生血管%基质金属蛋白酶类%血管内皮生长因子类%细胞增生%细胞移行
複方血栓通,中草藥%血管生成抑製劑%脈絡膜新生血管%基質金屬蛋白酶類%血管內皮生長因子類%細胞增生%細胞移行
복방혈전통,중초약%혈관생성억제제%맥락막신생혈관%기질금속단백매류%혈관내피생장인자류%세포증생%세포이행
Complex Xueshuantong,drugs,Chinese herbal%Angiogenesis inhibtors%Choroidal neovascularization%Matrix metailoproteinases%Vascular endothelial growth factors%Cell proliferation%Cell migration
目的 探讨复方血栓通干预猴脉络膜-视网膜内皮细胞(RF/6A)参与血管生成的作用及机理。方法 实验研究。本研究分为四部分：①取对数生长期RF/6A细胞进行培养，设立空白对照组、阳性对照组以及不同浓度的复方血栓通观察组，每组均加入终浓度为10 ng/ml的血管内皮生长因子(VEGF)，阳性对照组加入终浓度0.20 mg/ml的硫酸鱼精蛋白，复方血栓通观察组设立0.39～50.00 mg/ml的多个不同浓度组。培养24h后采用酶联免疫检测仪测定各组吸光度A值，观察不同浓度的复方血栓通对VEGF诱导的RF/6A细胞增殖的影响。②设立空白对照组和终浓度为3.13、6.25、12.50 mg/ml的复方血栓通组，培养24h后检测不同浓度复方血栓通对RF/6A细胞移行的影响。③设立空白对照组和终浓度为0.39、0.78、1.56 mg/ml的复方血栓通组，培养12 h后检测不同浓度的复方血栓通对RF/6A细胞管腔形成的影响；④设立空白对照组、终浓度为0.20 mg/ml的硫酸鱼精蛋白阳性对照组和终浓度为1.56、3.13、6.25 mg/ml的复方血栓通组，培养24h后，运用Western Blot和实时定量逆转录聚合酶链反应(RT-PCR)的方法，分别检测不同浓度的复方血栓通对RF/6A细胞表达VEGF、基质金属蛋白酶2(MMP-2)蛋白和mRNA的影响。对多组计量资料进行单因素方差分析。结果 ①各组间A值差异有统计学意义(F=158.669，P＜0.01)，同空白对照组比较，0.78～25.00 mg/ml浓度的复方血栓通对VEGF诱导的RF/6A细胞增殖具有明显抑制作用（P均＜0.01）；②复方血栓通浓度为0、3.13、6.25和12.50 mg/ml时，RF/6A细胞移行数分别为123.3±13.8、114.3±15.5、54.0±6.1和40.3±10.1，组间差异有统计学意义(F=36.918，P＜0.01)；与空白对照组比较，6.25和12.50 mg/ml浓度的复方血栓通对RF/6A细胞移行具有明显的抑制作用（P均＜0.01）；③复方血栓通浓度为0、0.39、0.78和1.56 mg/ml时RF/6A细胞内皮管腔形成数分别为20.3±2.5、12.7±2.1、9.7±1.2和0.7±0.6，组间差异有统计学意义(F=64.324，P＜0.01)；与空白对照组比较，0.39、0.78和1.56 mg/ml浓度的复方血栓通对RF/6A细胞内皮管腔形成均具有明显的抑制作用（P均＜0.01），且抑制作用随浓度增加而增强；④空白对照组、阳性对照组以及1.56、3.13和6.25 mg/ml浓度的复方血栓通组VEGF和MMP-2蛋白相对含量组间差异均有统计学意义(F=343.346、1670.505，P均＜0.01)；与空白对照组比较，其余各组均具有显著抑制RF/6A细胞VEGF和MMP-2蛋白表达的作用（P均＜0.01），复方血栓通的抑制作用随浓度增加而增强；各组VEGF和MMP-2 mRNA相对含量的差异也有统计学意义(F=228.130，208.579，P均＜0.01)，与空白对照组比较，其余各组均具有显著抑制RF/6A细胞VEGF mRNA表达的作用（P均＜0.01），复方血栓通的抑制作用随浓度增加而增强。结论 复方血栓通可能通过抑制RF/6A细胞增殖、移行以及管腔形成来抑制其参与新生血管生成，其机理可能与抑制RF/6A细胞VEGF、MMP-2的表达有关。
目的 探討複方血栓通榦預猴脈絡膜-視網膜內皮細胞(RF/6A)參與血管生成的作用及機理。方法 實驗研究。本研究分為四部分：①取對數生長期RF/6A細胞進行培養，設立空白對照組、暘性對照組以及不同濃度的複方血栓通觀察組，每組均加入終濃度為10 ng/ml的血管內皮生長因子(VEGF)，暘性對照組加入終濃度0.20 mg/ml的硫痠魚精蛋白，複方血栓通觀察組設立0.39～50.00 mg/ml的多箇不同濃度組。培養24h後採用酶聯免疫檢測儀測定各組吸光度A值，觀察不同濃度的複方血栓通對VEGF誘導的RF/6A細胞增殖的影響。②設立空白對照組和終濃度為3.13、6.25、12.50 mg/ml的複方血栓通組，培養24h後檢測不同濃度複方血栓通對RF/6A細胞移行的影響。③設立空白對照組和終濃度為0.39、0.78、1.56 mg/ml的複方血栓通組，培養12 h後檢測不同濃度的複方血栓通對RF/6A細胞管腔形成的影響；④設立空白對照組、終濃度為0.20 mg/ml的硫痠魚精蛋白暘性對照組和終濃度為1.56、3.13、6.25 mg/ml的複方血栓通組，培養24h後，運用Western Blot和實時定量逆轉錄聚閤酶鏈反應(RT-PCR)的方法，分彆檢測不同濃度的複方血栓通對RF/6A細胞錶達VEGF、基質金屬蛋白酶2(MMP-2)蛋白和mRNA的影響。對多組計量資料進行單因素方差分析。結果 ①各組間A值差異有統計學意義(F=158.669，P＜0.01)，同空白對照組比較，0.78～25.00 mg/ml濃度的複方血栓通對VEGF誘導的RF/6A細胞增殖具有明顯抑製作用（P均＜0.01）；②複方血栓通濃度為0、3.13、6.25和12.50 mg/ml時，RF/6A細胞移行數分彆為123.3±13.8、114.3±15.5、54.0±6.1和40.3±10.1，組間差異有統計學意義(F=36.918，P＜0.01)；與空白對照組比較，6.25和12.50 mg/ml濃度的複方血栓通對RF/6A細胞移行具有明顯的抑製作用（P均＜0.01）；③複方血栓通濃度為0、0.39、0.78和1.56 mg/ml時RF/6A細胞內皮管腔形成數分彆為20.3±2.5、12.7±2.1、9.7±1.2和0.7±0.6，組間差異有統計學意義(F=64.324，P＜0.01)；與空白對照組比較，0.39、0.78和1.56 mg/ml濃度的複方血栓通對RF/6A細胞內皮管腔形成均具有明顯的抑製作用（P均＜0.01），且抑製作用隨濃度增加而增彊；④空白對照組、暘性對照組以及1.56、3.13和6.25 mg/ml濃度的複方血栓通組VEGF和MMP-2蛋白相對含量組間差異均有統計學意義(F=343.346、1670.505，P均＜0.01)；與空白對照組比較，其餘各組均具有顯著抑製RF/6A細胞VEGF和MMP-2蛋白錶達的作用（P均＜0.01），複方血栓通的抑製作用隨濃度增加而增彊；各組VEGF和MMP-2 mRNA相對含量的差異也有統計學意義(F=228.130，208.579，P均＜0.01)，與空白對照組比較，其餘各組均具有顯著抑製RF/6A細胞VEGF mRNA錶達的作用（P均＜0.01），複方血栓通的抑製作用隨濃度增加而增彊。結論 複方血栓通可能通過抑製RF/6A細胞增殖、移行以及管腔形成來抑製其參與新生血管生成，其機理可能與抑製RF/6A細胞VEGF、MMP-2的錶達有關。
목적 탐토복방혈전통간예후맥락막-시망막내피세포(RF/6A)삼여혈관생성적작용급궤리。방법 실험연구。본연구분위사부분：①취대수생장기RF/6A세포진행배양，설립공백대조조、양성대조조이급불동농도적복방혈전통관찰조，매조균가입종농도위10 ng/ml적혈관내피생장인자(VEGF)，양성대조조가입종농도0.20 mg/ml적류산어정단백，복방혈전통관찰조설립0.39～50.00 mg/ml적다개불동농도조。배양24h후채용매련면역검측의측정각조흡광도A치，관찰불동농도적복방혈전통대VEGF유도적RF/6A세포증식적영향。②설립공백대조조화종농도위3.13、6.25、12.50 mg/ml적복방혈전통조，배양24h후검측불동농도복방혈전통대RF/6A세포이행적영향。③설립공백대조조화종농도위0.39、0.78、1.56 mg/ml적복방혈전통조，배양12 h후검측불동농도적복방혈전통대RF/6A세포관강형성적영향；④설립공백대조조、종농도위0.20 mg/ml적류산어정단백양성대조조화종농도위1.56、3.13、6.25 mg/ml적복방혈전통조，배양24h후，운용Western Blot화실시정량역전록취합매련반응(RT-PCR)적방법，분별검측불동농도적복방혈전통대RF/6A세포표체VEGF、기질금속단백매2(MMP-2)단백화mRNA적영향。대다조계량자료진행단인소방차분석。결과 ①각조간A치차이유통계학의의(F=158.669，P＜0.01)，동공백대조조비교，0.78～25.00 mg/ml농도적복방혈전통대VEGF유도적RF/6A세포증식구유명현억제작용（P균＜0.01）；②복방혈전통농도위0、3.13、6.25화12.50 mg/ml시，RF/6A세포이행수분별위123.3±13.8、114.3±15.5、54.0±6.1화40.3±10.1，조간차이유통계학의의(F=36.918，P＜0.01)；여공백대조조비교，6.25화12.50 mg/ml농도적복방혈전통대RF/6A세포이행구유명현적억제작용（P균＜0.01）；③복방혈전통농도위0、0.39、0.78화1.56 mg/ml시RF/6A세포내피관강형성수분별위20.3±2.5、12.7±2.1、9.7±1.2화0.7±0.6，조간차이유통계학의의(F=64.324，P＜0.01)；여공백대조조비교，0.39、0.78화1.56 mg/ml농도적복방혈전통대RF/6A세포내피관강형성균구유명현적억제작용（P균＜0.01），차억제작용수농도증가이증강；④공백대조조、양성대조조이급1.56、3.13화6.25 mg/ml농도적복방혈전통조VEGF화MMP-2단백상대함량조간차이균유통계학의의(F=343.346、1670.505，P균＜0.01)；여공백대조조비교，기여각조균구유현저억제RF/6A세포VEGF화MMP-2단백표체적작용（P균＜0.01），복방혈전통적억제작용수농도증가이증강；각조VEGF화MMP-2 mRNA상대함량적차이야유통계학의의(F=228.130，208.579，P균＜0.01)，여공백대조조비교，기여각조균구유현저억제RF/6A세포VEGF mRNA표체적작용（P균＜0.01），복방혈전통적억제작용수농도증가이증강。결론 복방혈전통가능통과억제RF/6A세포증식、이행이급관강형성래억제기삼여신생혈관생성，기궤리가능여억제RF/6A세포VEGF、MMP-2적표체유관。
Objective To investigate the effect and mechanism of Complex Xueshuantong on the angiogenesis of rhesus choroid-retina endothelial (RF/6A) cells. Methods This experimental research included 4 parts. ①RF/6A cells in logarithmic growth phase were cultured and divided into blank control group, positive control group (0.20 mg/ml protamine) and Complex Xueshuantong groups at different concentrations (0.39 to 50.00 mg/ml). Vascular endothelial growth factor (VEGF) at terminal concentration of 10 ng/ml was added into all groups. The optic absorptions (A values) of each group were measured 24 hours later. ②RF/6A cells in logarithmic growth phase were cultured in Transwell insert and divided into blank control group, Complex Xueshuantong groups at different concentrations of 3.13, 6.25, 12.50 mg/ml. The number of cells that migrated to under the Transwell membrane was counted after cultured for 24 hours. ③RF/6A cells were divided into blank control group, Complex Xueshuantong groups at different concentrations of 0.39, 0.78, 1.56 mg/ml. The number of full-formed tubes was counted after cultured for 12 hours. ④RF/6A cells were divided into blank control group, positive control group of protamine at terminal concentration of 0.20 mg/ml, and Complex Xueshuantong groups at different concentrations of 1.56, 3.13, 6.25 mg/ml. Western Blot and real-time quantitative RT-PCR were used to detect the expression of VEGF and matrix metalloproteinase 2 (MMP-2) protein and mRNA of RF/6A cells after cultured for 24 hours. Data were statistically analyzed by ANOVA method. Results ①The difference of the A values within groups was statistically significant (F=158.669, P＜0.01). Compared with that of blank control group,the A values of Complex Xueshuantong groups at different concentrations from 0.78 to 25.00 mg/ml were significantly reduced (all P＜0.01), the proliferation of RF/6A cells induced by VEGF was inhibited by Complex Xueshuantong at certain concentrations. ②When the concentrations of Complex Xueshuantong were 0, 3.13, 6.25, and 12.50 mg/ml, the number of RF/6A cells that migrated to under the Transwell membrane was 123.3±13.8, 114.3±15.5, 54.0±6.1, and 40.3±10.1, respectively.The difference within groups was statistically significant (F=36.918, P＜0.01). Compared with that of blank control group, the cells of Complex Xueshuantong groups at different concentrations of 6.25 and 12.50 mg/ml were significantly reduced (all P＜0.01), the migration of RF/6A cells was inhibited by Complex Xueshuantong at certain concentrations. ③When the concentrations of Complex Xueshuantong were 0, 0.39, 0.78, and 1.56 mg/ml, the number of tubes formed in Matrigel was 20.3±2.5, 12.7±2.1, 9.7±1.2, and 0.7±0.6, respectively. The difference within groups was statistically significant (F=64.324, P＜0.01). Compared with that of blank control group, the number of tubes in 0.39, 0.78,and 1.56 mg/ml groups were significantly reduced (P＜0.01), the tube formation of RF/6A cells was inhibited by Complex Xueshuantong at certain concentrations and with the concentration being increased, the inhibition was enhanced. ④In blank control group, positive control group, and Complex Xueshuantong groups at the concentrations of 1.56, 3.13, and 6.25 mg/ml, there were significant differences in the relative content of VEGF and MMP-2 protein expression level (F=343.346,1670.505, P＜0.01). Compared with that of blank control group, the other groups were significantly reduced (all P＜0.01), the VEGF and MMP-2 protein expression level of RF/6A cells was inhibited by Complex Xueshuantong at certain concentrations and with the concentration being increased, the inhibition was enhanced. There were significant differences in the relative content of VEGF and MMP-2 mRNA expression level (F=228.130, 208.579, P＜0.01). Compared with that of blank control group, the relative content of VEGF and MMP-2 mRNA expression level of all other groups were significantly reduced (all P＜0.01), the VEGF and MMP-2 mRNA expression level of RF/6A cells was also inhibited by Complex Xueshuantong at certain concentrations and with the concentration being increased, the inhibition was enhanced. Conclusion Complex Xueshuantong could inhibit the angiogenesis of RF/6A cells through the path of suppression of the proliferation, migration, and tube formation of RF/6A cells. The mechanism was likely to be related to the inhibition of the expression of proteins and mRNA of VEGF and MMP-2 in RF/6A cells.
