CAJ | 학술논문

目的观察中药制剂明睛颗粒对激光诱导脉络膜新生血管(CNV)小鼠外周血骨髓来源细胞(BMCs)在CNV趋化和黏附的影响.方法将75只C57BL/6J小鼠按随机数字表方法随机分为3组:干预组、对照组、正常空白组.干预组和对照组各30只小鼠,正常空白组15只小鼠.干预组和对照组小鼠采用氪激光光凝诱导CNV发生.激光造模后0.5～1 h给予尾静脉注射绿色荧光蛋白(GFP)转基因小鼠的BMCs.造模后第1天干预组小鼠予以明睛颗粒溶剂每日灌胃直至处死取样,每只小鼠灌胃0.3 ml,相当于30 g/(kg·d)生药；对照组予以等体积蒸馏水灌胃；正常空白组小鼠常规喂养.分别在灌胃干预后第7、14、28天,采用脉络膜铺片观察CNV及BMCs在CNV处的趋化和黏附情况；光学显微镜观察小鼠视网膜脉络膜组织病理改变；酶联免疫吸附测定法(ELISA)检测小鼠外周血趋化因子受体4(CXCR4)的含量；免疫荧光法观察基质细胞衍生因子-1(SDF-1α)、血管细胞黏附分子(VCAM-1)、细胞间黏附分子(ICAM-1)在CNV区域的表达.结果脉络膜铺片观察显示,干预组和对照组均可见CNV生成,而正常空白组均未见CNV；明睛颗粒干预后第7、14天,干预组CNV中BMCs明显少于对照组,CNV面积亦较对照组小(t=10、9,P=0.007、0.024).光学显微镜观察显示,干预组视网膜脉络膜病变较对照组轻.ELISA检测结果显示,干预后7、14天干预组外周血CXCR4含量明显低于对照组和正常对照组(F=8.107、4.499,P＜0.05)；免疫荧光法检测结果显示,干预组CNV区域SDF-1α、ICAM-1、VCAM-1表达较对照组减弱.结论中药制剂明睛颗粒能在CNV形成早期抑制外周血BMCs在CNV的趋化和黏附及参与CNV形成作用.其机制可能与明睛颗粒减少外周血及CNV周围趋化因子和黏附因子的蛋白表达有关.
목적 관찰중약제제명정과립대격광유도맥락막신생혈관(CNV)소서외주혈골수래원세포(BMCs)재CNV추화화점부적영향.방법 장75지C57BL/6J소서안수궤수자표방법수궤분위3조:간예조、대조조、정상공백조.간예조화대조조각30지소서,정상공백조15지소서.간예조화대조조소서채용극격광광응유도CNV발생.격광조모후0.5～1 h급여미정맥주사록색형광단백(GFP)전기인소서적BMCs.조모후제1천간예조소서여이명정과립용제매일관위직지처사취양,매지소서관위0.3 ml,상당우30 g/(kg·d)생약；대조조여이등체적증류수관위；정상공백조소서상규위양.분별재관위간예후제7、14、28천,채용맥락막포편관찰CNV급BMCs재CNV처적추화화점부정황；광학현미경관찰소서시망막맥락막조직병리개변；매련면역흡부측정법(ELISA)검측소서외주혈추화인자수체4(CXCR4)적함량；면역형광법관찰기질세포연생인자-1(SDF-1α)、혈관세포점부분자(VCAM-1)、세포간점부분자(ICAM-1)재CNV구역적표체.결과 맥락막포편관찰현시,간예조화대조조균가견CNV생성,이정상공백조균미견CNV；명정과립간예후제7、14천,간예조CNV중BMCs명현소우대조조,CNV면적역교대조조소(t=10、9,P=0.007、0.024).광학현미경관찰현시,간예조시망막맥락막병변교대조조경.ELISA검측결과현시,간예후7、14천간예조외주혈CXCR4함량명현저우대조조화정상대조조(F=8.107、4.499,P＜0.05)；면역형광법검측결과현시,간예조CNV구역SDF-1α、ICAM-1、VCAM-1표체교대조조감약.결론 중약제제명정과립능재CNV형성조기억제외주혈BMCs재CNV적추화화점부급삼여CNV형성작용.기궤제가능여명정과립감소외주혈급CNV주위추화인자화점부인자적단백표체유관.
Objective To observe the effect of Chinese Herbal Preparation-Mingjing Granule on the recruitment and adhesion of bone-marrow derived cells (BMCs) to choroidal neovascu1arization (CNV) in mice induced by laser photocoagulation.Methods A total of 75 C57BL/6 mice were divided into treatment group (30 mice),control group (30 mice) and normal control group (15 mice).CNV was induced by krypton laser in the mice of treatment group and control group.BMCs from GFP-transgenic mice were injected through tail vein to those mice 0.5-1.0 hours after laser surgery.On day one after laser surgery,the mice of treatment group were gavaged with 0.3 ml Mingjing Granule solution every day[30 g/(kg · d)],while the control group mice were gavaged with distilled water.The mice of normal group fed with normal cereals.7,14,28 days after treatment,choroidal flatmount were prepared to measure the CNV severity and BMCs recruitment.The choroidal histopathological change was observed by optical microscope.CXCR4 level in peripheral blood was measured by enzyme-linked immuno sorbent assay (ELISA).Stromal cell derived factor-1α (SDF-1α),vascular cell adhesion molecule-1 (VCAM-1),intercellular adhesion molecule 1 (ICAM-1) were detected by immunofluorescent staining.Results Choroidal flatmount showed that CNV developed in treatment group and control group,but not in normal group.On day seven and 14,CNV lesions in the treatment group showed less incorporation of BMCs and smaller CNV in size compared with the control group (t=10,9; P=0.007,0.024),histologic sections also showed less severe lesions in the treatment group.CXCR4 level in peripheral blood in the treatment group was less than that in the control group and normal control group on day seven and day 14(t=8.107,2.747; P＜0.05).The expression of SDF-1α,ICAM-1 and VCAM-1 in the treatment group was lower compared with the control group.Conclusions Mingjing Granule could inhibit the recruitment and adhesion of BMCs in peripheral blood to CNV at the early stage.The mechanism may be related with the action of Mingjing Granule in lowing chemokines levels in peripheral blood and expression of adhesion molecules around CNV.