【摘要】 目的：观察密蒙花提取物滴眼剂对去势所致干眼症雄鼠基础泪液分泌量、泪膜稳定性、泪腺凋亡相关基因蛋白Bax和Bcl2表达的影响。方法：将45只Wister雄性大鼠随机分为空白组(A1，A2，A3)、模型组(B1，B2，B3)、密蒙花提取物滴眼剂治疗组(C1，C2，C3)，共9组，1：饲养1mo，2：饲养2mo，3：饲养3mo，每组5只。B，C组行去势术建立动物模型，C组以密蒙花提取物滴眼剂连续滴眼治疗，对全部实验大鼠行Schirmer Ⅰ试验、测量泪膜破裂时间，采用免疫组织化学的方法，对大鼠泪腺组织中凋亡相关基因蛋白Bax和Bcl2进行检测。结果：C组Schirmer Ⅰ试验测量值明显高于B组(P<0.01)，泪膜破裂时间明显长于B组(P<0.01)。泪腺导管及腺泡上皮细胞中Bax阳性表达的细胞数均明显低于B组，而Bcl2阳性表达的细胞数均明显高于B组(P<0.01)。结论：密蒙花提取物滴眼剂可显著抑制雄激素水平降低后大鼠干眼症的发生，抑制泪腺细胞凋亡，维持泪腺基础分泌量和泪膜的稳定性。
【关键词】 去势;干眼症;泪腺;细胞凋亡;密蒙花提取物;滴眼剂
Effects of extract of buddleja officinalis eye drops on lacrimal gland cells apoptosis of castrated rabbits with dry eyeQingHua Peng ， XiaoLei Yao ，Jun Peng ，QuanLong Wu ，HanYu Tan ，JingRong Zhang Foundation items:General Project of National Natural Science Fund of China(No.30772824);Project of Doctor Fund of University of Ministry of Education,China(No.200805410004); Project of Hunan Provincial Natural Science Fund,China(No.07JJ3049); Project of Scientific Research Fund of Hunan Provincial Science and Technology Department of China(No.2009FJ3001); Project of Hunan Provincial Masters Innovation Fund in 2008 of China1 Key Discipline of Ophthalmology, the First Affiliated Hospital of Hunan University of Traditional Chinese Medicine, Changsha 410007, Hunan Province, China;2 Medical School of Nanhua University, Hengyang 421001, Hunan Province, ChinaAbstractAIM：To evaluate the effects of the extract of buddleja officinalis eye drops in basic tears secretory volume, tear film stability, expression of Bax and Bcl2 in castrated rats with dry eye, and to investigate the therapeutic effects of extract of buddleja officinalis on dry eye caused by gonadal hormones level imbalance. METHODS：Fortyfive Wister masculinity rats were divided randomly into 9 groups, including normal group(A1，A2 and A3)， model group(B1，B2 and B3)， therapy group with buddleja officinalis extract eye drops(C1，C2 and C3). The "1" stood for being fed for 1 mouth， and the "2" stood for being fed for 2 mouths， and the "3" stood for being fed for 3 mouths. The dry eye model was established with orchiectomy on group B， C. Group C were treated with buddleja officinalis extract eye drops. All rats were detected with SⅠt and BUT. Bax, Bcl2 were checked on immunohistochemistry. RESULTS：The SⅠt value of group C was significantly higher than that of group B(P<0.01) and the BUT value of group C was significantly longer than that of group B(P< 0.01), which indicated the eye drop could significantly keep basic tears secretory volume and tear film stability. The quantity of expression of Bax in acinar epithelial cell and glandular tube cell of group C were significantly higher than that of group B(P<0.01) and the expression of Bcl2 in acinar cell and glandular tube cell of group C were significantly higher than that of group B too (P<0.01). CONCLUSION：The main components of extract of buddleja officinalis is the flavonoids, which could significantly inhibit happening of dry eye of rat after androgen level lowered and lacrimal gland apoptosis and keep basic tears secretory volume and tear film stability
KEYWORDS: castrate;dry eye;lacrimal gland;apoptosis; extract of buddleja officinalis;eye drops0引言
干眼症可导致视力明显下降,发病率很高，已成为目前研究的热点问题。雄激素水平下降是干眼症的重要发病原因，雄激素替代疗法是治疗雄激素水平下降所致干眼症唯一的对因治疗方法，但是长期使用将会带来许多难以避免的副作用。现有其他治疗方法均存在着一定的缺陷和局限性。密蒙花有效部位为黄酮类物质。雄激素和黄酮类化合物均为杂环多酚类化合物，利用其化学结构的相似性，发挥其拟内源性雄激素作用，可治疗雄激素水平下降所致的干眼症。本实验观察密蒙花提取物滴眼剂去势大鼠干眼症模型泪腺组织细胞凋亡的影响。
1材料和方法
1.1材料
取健康1月龄Wistar雄性大鼠45只，体质量0.2kg，由湖南中医药大学动物实验中心提供。裂隙灯显微镜和眼底镜检查眼前节及眼底无异常，地卡因眼液表面麻醉后Schirmer Ⅰ试验(SⅠt)≥10mm/5min,泪膜破裂时间(BUT)≥10s。密蒙花提取物滴眼剂由湖南中医药大学第一附属医院药剂科制备。50g/L BSA封闭液，鼠抗鼠Bax和Bcl2多抗，生物素化山羊抗兔IgG(FITCIgG)，过氧化物酶标记链酶白卵素(strept avidinbiotin complex，SABC)免疫组织化学试剂盒，3，3二氨基联苯胺(3，3diaminobenzidine，DAB)显色剂购自武汉博士德生物工程有限公司。Waters15252996高效液相色谱仪(美国Waters公司，含2996二极管阵列检测器、1525泵、717自动进样器);Empower中文色谱工作站(美国Waters公司);双光束紫外可见分光光度计(北京普新通用仪器有限责任公司);LEICA DM LB2型双目显微镜(德国LEICA公司产);Shandon325型石蜡切片机(英国Shandon公司生产);DNP9162 型电热恒温培养箱(上海精宏实验设备有限公司生产);Motic B5 显微摄像系统(麦克奥迪实业集团公司生产)。
1.2方法
将Wistar雄性大鼠45只，随机分为A1，B1，C1，A2，B2，C2，A3，B3，C3，共9组，每组5只。A：假手术空白组;B：手术对照组;C：密蒙花提取物滴眼剂治疗组;1：饲养1mo;2：饲养2mo;3：饲养3mo。雄激素水平下降导致干眼症动物模型的制作参照马轶群等[1]的方法，行双侧睾丸切除术(ORX)，将B1，B2，B3，C1，C2，C3组实验鼠术前12h禁食水，后肢大腿外侧肌肉碘伏消毒，盐酸氯胺酮注射液30mg/kg ip麻醉，雄鼠取仰卧位，四肢张开并固定，下腹部褪毛剂褪毛，阴囊皮下加用20g/L利多卡因局部麻醉，碘伏消毒，铺无菌巾单，将一侧睾丸由腹腔挤入阴囊并捏紧，勿使其滑动。用消毒刀片将阴囊切一小口，用力挤出睾丸，结扎精索静脉及输精管，切除睾丸及附睾，连续缝合阴囊皮肤后局部涂碘酊预防感染。另侧睾丸及附睾同法切除。A1,A2,A3组只切开阴囊，不切除睾丸，术后连续缝合阴囊皮肤。术前im青霉素针20万U，术后连续3d im青霉素针20万U，预防感染。A1,B1组每日以生理盐水滴眼，ou，tid;C1组每日以密蒙花滴眼剂滴眼，1滴/次，ou，tid;每组均滴眼1mo。以常规饲料喂养1mo后处死。A2,B2组每日以生理盐水滴眼，ou，tid;C2组每日以密蒙花滴眼剂滴眼，1滴/次，ou，tid;每组均滴眼2mo。以常规饲料喂养2mo后处死。A3,B3组每日以生理盐水滴眼，ou，tid;C3组每日以密蒙花滴眼剂滴眼，1滴/次，ou，tid;每组均滴眼3mo。分别于分组前和常规饲料喂养末次给药完成后进行Schirmer Ⅰ试验(SⅠt)和泪膜破裂时间(BUT)检查，检测标准参照SⅠt与BUT的诊断标准。检查完后立即以断头法处死动物，即刻摘除泪腺。予以40g/L多聚甲醛固定，常规石蜡包埋，每组石蜡块予以Shandon325型石蜡切片机连续切片，常规脱蜡至水，蒸馏水洗涤2min×2次。免疫组化检测严格按照说明书操作。木素轻度复染，脱水，透明，封片，显微镜观察，Motic B5显微摄像系统进行摄像。
统计学分析:用SPSS 13.0软件进行统计学分析，双侧检验，以P<0.05为差异有统计性意义。
[1] [2] [3] 下一页 |
