毛果芸香碱凝胶的研究

与毛果芸香碱眼水比较，毛果芸香碱凝胶经动物实验证实其眼内房水及睫状体药物浓度较高，持续时间较长，ＰＧ对瞳孔的作用时间与其粘度和浓度有关，对角膜上皮可造成损害；临床研究表明：ＰＧ每晚一次维持１８－２４小时，药效与ＰＳ每日４次相当。     

角膜胶原膜载释毛果芸香碱的研究

目的研究自制的胶原膜以不同方式载释毛果芸香碱的功能。方法将新西兰白兔４０只（８０只眼）随机分为胶原膜浸药组、胶原膜载药后滴药组及合成胶原药膜组，测定用药后０．５、１、３及６小时前房水中毛果芸香碱浓度及缩瞳效应。结果胶原膜三种不同载药方式均能在短时间内达到较高房水药物浓度并持续６小时以上，起到“临时药库”作用。胶原膜载释毛果芸香碱的房水药物代谢过程符合一级动力学模式，并由缩瞳效应得到进一步证实。结论国产胶原膜具有良好的载释药物功能。     

毛果芸香碱新剂型对高眼压兔的降眼压作用

目的　评价毛果芸香碱 (pilocarpine,PC)原位胶化滴眼剂、PC微乳滴眼剂、PC增效滴眼剂及PC滴眼剂对卡波姆诱导的兔高眼压模型的降眼压作用。方法　人工诱导兔高眼压模型 ,取高眼压兔 2 8只 (43只眼 ) ,随机分成Ⅰ、Ⅱ、Ⅲ、Ⅳ及Ⅴ组 ,结膜囊内分别滴入 2 %PC原位胶化滴眼剂、4 %PC原位胶化滴眼剂、2 %PC微乳滴眼剂、2 %PC增效滴眼剂及 2 %PC滴眼剂 ,于用药前及用药后 1、2、3、4、5、7、10、12、2 2及 2 4h测眼压。结果　 2 %PC滴眼剂的降眼压作用持续 7h ,最大降眼压幅度为(12 2 5± 5 2 8)mmHg(1mmHg =0 133kPa) ;2 %PC原位胶化的降眼压作用持续 12h ,最大降眼压幅度为 (13 6 7± 4 6 1)mmHg ;4 %PC原位胶化的降眼压作用持续 2 4h ,最大降眼压幅度为 (13 6 3± 3 5 8)mmHg;2 %PC微乳滴眼剂的降眼压作用持续 12h ,最大降眼压幅度为 (13 5 5± 3 90 )mmHg ;2 %PC增效滴眼剂的降眼压作用持续 10h ,最大降眼压幅度为 (10 89± 4 0 4 )mmHg ,几种PC新剂型的降眼压作用持续时间均比 2 %PC滴眼剂延长 ,按作用持续时间排序 :4 %PC原位胶化滴眼剂 >2 %PC原位胶化滴眼剂 =2 %PC微乳滴眼剂 >2 %PC增效滴眼剂 >2 %PC滴眼剂。各组最大降眼压幅度差异无显著意义 (P >0 0 5 )。实验眼滴药后除轻度结膜充血外均     

兔泪腺上皮细胞的体外分离和培养

分离和培养泪腺上皮细胞，是研究泪腺功能的基础，也是研究干眼症发病机制的重要方法之一。本实验参考国内外文献，探索出稳定和经济的分离方法，报告如下。     

4％毛果芸香碱凝胶单次应用治疗青光眼的临床观察

目的：比较４％毛果芸香碱凝胶（ｐｉｌｏｅａｒｐｉｎｅ ｇｅｌ,ＰＧ）和１％毛果芸香碱溶液（ｐｉｌｏｅａｒｐｉｎｅ ｓｏｌｕｔｉｏｎ,ＰＳ）的降眼压效果及安全性。方法：２０例（４０只眼）高眼压症及早期原发性开角型青光眼患者,采用自身对照,分别比较ＰＧ（每晚一次）和ＰＳ（每天４次）对日间多次眼压的降眼压效应,分别比较二者连续４周用药对某一时点（１０ＡＭ）的眼压、瞳孔及屈光度的影响,以及眼部副作用。结果     

前房内应用毛果芸香碱对角膜内皮细胞的影响

对白内障囊外摘除后房型人工晶体植入术中前房内用０．１％毛果芸香碱缩瞳的２３只眼，术后３～６个月使用非接触型内皮显微镜观察内皮细胞变化，内皮损失率为１７．３５％。术中不用毛果芸香碱缩瞳的对照组２８只眼术后内皮损失率为１６．４３％，两组经卡方检验Ｐ＞０．０５，差异无显著性。我们认为在后房型人工晶体植入术中，前房内应用０．１％毛果芸香碱缩瞳是安全、可靠的。     

催醒安与毛果芸香碱降眼压和缩瞳作用的比较

催醒安是我国首创的新药,由中国军事医学科学院自行设计并人工合成.催醒安的化学结构与毒扁豆碱相似,药理作用均为可逆性胆碱脂酶抑制剂.经过动物实验和正常志愿者点眼观察,发现均有一定程度的降眼压和缩瞳作用.在此基础上,北京市眼科研究所又作了催醒安在开     

联合毛果芸香碱滴眼液治疗眼外伤房角后退继发性青光眼

目的探讨联合毛果芸香碱滴眼液治疗外伤引起房角后退继发性青光眼的临床疗效。方法回顾性分析山东省眼科医院2007年8至2012年7收治12例(12只眼)眼外伤房角后退继发性青光眼患者的临床资料,患者均有眼球钝挫伤史且超生生物显微镜(UBM)和前房角镜检查可见外伤眼有不同程度的房角后退,入院眼压为(48.50±3.10)mm Hg,瞳孔直径为(5.15±0.70)mm。治疗:积极治疗原发性损伤,控制炎症,口服及静脉给予降眼压药物联合局部应用0.5%马来酸噻吗洛尔滴眼液、1%布林佐胺滴眼液、酒石酸溴莫尼定滴眼液降眼压治疗3～5 d,眼压不再下降,记录眼压为(35.00±3.55)mm Hg,此时加用0.5%毛果芸香碱滴眼液每日4次。详细记录患者治疗转归,观察眼压变化。结果加用毛果芸香碱眼滴眼液24 h后眼压为(21.90±3.87)mm Hg,平均下降(13.10±1.10)mm Hg。治疗前后瞳孔直径缩小(2.10±0.31)mm。4例患者最终逐渐减量药物致停,眼压正常;8例患者眼压降低后行小梁切除术控制眼压。结论毛果芸香碱滴眼液可以应用于眼外伤房角后退继发性青光眼患者,在减轻炎症的的基础上,联合应用毛果芸香碱滴眼液能够有效的降低眼压。     

毛果芸香碱微乳滴眼剂及滴眼液在兔眼房水中的药代动力学研究

目的 对２％毛果芸香碱微乳滴眼剂与２％毛果芸香碱滴眼液兔房水中的药代动力学进行对比研究。方法 健康白兔６０只,随机分成２０组,１０个实验组,１０个对照组,每组３只兔（６只眼）。对各实验组双眼结膜囊内准确滴入２％毛果芸香碱微乳滴眼剂５０μｌ,对各对照组双眼同法滴入２％毛果芸香碱滴眼液５０μｌ,分别于给药后５、１０、３０、４０、６０、９０、１２０、１８０、２４０及３６０ｍｉｎ时抽取房水。用于氯甲烷提取     

毛果芸香碱透明质酸钠药膜药代动力学研究

目的 探讨2%毛果芸香碱透明质酸钠药膜在兔眼的药代动力学特征,评价其相对生物利用度.设计实验性研究.研究对象 48只健康白兔.方法 48只兔随机分成实验组和对照组,每组24只兔.分别给兔眼结膜囊内嵌入2%毛果芸香碱透明质酸钠药膜和滴入4%毛果芸香碱凝胶滴眼液.评价用药后兔眼刺激症状得分、瞳孔大小,并于给药后0.5、1、3、6、12、24、48及72h时抽取房水,每组各时间点随机选取3只兔(6眼),用反相高效液相色谱法(HPLC)测定房水中的毛果芸香碱浓度.主要指标兔眼刺激症状,瞳孔大小,房水药物浓度.结果 实验组与对照组比较兔结膜充血症状无显著性差异(P＞0.05);瞳孔缩小持续时间分别为(54.78±4.52)h、(21.33±2.28)h(P＜0.01);药物房水达峰时间分别为30min和3h,达峰浓度分别为(18.44 9.56)μg/ml、(16.61±4.92)μg/ml.结论 2%毛果芸香碱透明质酸钠药膜可明显提高毛果芸香碱的生物利用度,减少用药频率,并具有临床开发应用的前景.(眼科,2008,17:283-285)     

Characterization of beta-hexosaminidase secretion in rabbit lacrimal gland

The present study was aimed at validating the use of the lysosomal enzyme beta-hexosaminidase as a marker of secretory function in cultured rabbit lacrimal gland acinar cells. The secretory response and morphological characteristics of isolated acinar cells cultured in a serum-free medium supplemented with an extracellular matrix extract were monitored over time as part of optimization of our culturing protocol. Secreted beta-hexosaminidase activity was analyzed and compared with that of another lysosomal enzyme, cathepsin B, as well as protein secreted into the media, w or w/o the presence of secretagogues or protein kinase C activators and inhibitors. Lacrimal gland fluid was obtained from pilocarpine stimulated rabbits, and the activities of beta-hexosaminidase and cathepsin B were measured. A membrane fraction and a soluble fraction were obtained from isolated acinar cells and used for kinetic studies of beta-hexosaminidase in comparison with that released from cultured cells, in the lacrimal gland fluid and in serum. Optimal secretory response was obtained when the cells had been in culture for 2-3 days, coinciding with the formation of acinus-like structures. Stimulation of the cultured cells by carbachol or phorbol esters resulted in a more than 3-fold increase of beta-hexosaminidase release over basal, whereas no effect on cathepsin B release could be detected. Treatment with the protein kinase C inhibitor, chelerythrine chloride, significantly decreased the carbachol and phorbol ester-stimulated secretion. Cathepsin B could not be detected in rabbit lacrimal fluid, but beta-hexosaminidase was easily measured in quantities corresponding to as low as 0.4 microl of tear fluid. Using 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide as a substrate for beta-hexosaminidase, the K(m) in lacrimal gland fluid (1.22+/-0.15 mM) was not significantly different from that of the membrane-associated fraction, the soluble fraction, rabbit serum or activity secreted from cultured cells. Beta-hexosaminidase is secreted by rabbit lacrimal gland, in vivo, and by acinar cells in primary culture, whereas cathepsin B is not secreted under the conditions described. Beta-hexosaminidase therefore provides a versatile marker for secretion in studies of tear production utilizing the rabbit as a model. Our results also indicate that PKC is an important regulator of rabbit lacrimal gland secretion.     

丙酸睾酮对泪腺上皮细胞保护作用的实验研究

目的 研究雄激素对泪腺上皮细胞的保护作用.方法 酶消化法培养的兔泪腺上皮细胞,经不同剂量丙酸睾酮（10×10^-9mol·L^-1、100×10^-9mol·L^-1、1×10^-6mol·L^-1、10×10^-6mol·L^-1）作用24 h以及过氧化氢作用60 min后,分别采用MTT法检测细胞活性和TUNEL法检测细胞凋亡.结果 加入不同剂量的丙酸睾酮（10×10^-9 mol·L^-1、100×10^-9 mol·L^-1、1×10^-6 mol·L^-1、10×10^-6 mol·L^-1）,泪腺上皮细胞的活性分别为0.376±0.013、0.398±0.018、0.418±0.023和0.439±0.015;泪腺上皮细胞的凋亡率分别为（13.94±0.73）%、（12.51±0.91）%、（10.63±0.78）%和（8.19±0.85）%,与H2O2组[（0.354±0.022）%和（15.15±1.05）%]比较差异有显著统计学意义（P＜0.01）,并在一定浓度范围内表现出剂量依赖性.结论 丙酸睾酮可以抑制泪腺上皮细胞的凋亡,保护泪腺上皮细胞.     

Effect of Buddleia flavonoids drug-containing plasma on the expression of STAT1 phosphoprotein in lacrimal gland epithelial cells in vitro

AIM: To explore the effect of Buddleia flavonoids drug-containing plasma and androgen receptor (AR) blocker on the expression of STAT1 phosphoprotein. · METHODS: In vitro lacrimal gland epithelial cells were cultivated with H2O2 to establish the dry eye apoptosis state. Blank plasma group, Buddleia officinalis plasma total flavonoids interfere with drug-containing group, and the intervention group of testosterone propionate were set. The expressions of STAT1 phosphoprotein of each group were observed by Western blot. AR blocker flutamide was used to explore the intended androgen effect of Buddleia flavonoids. · RESULTS: After the intervention of drug-containing plasma, the expression of STAT1 phosphoprotein in Buddleja officinalis drug-containing plasma intervention group (0.353±0.494) and testosterone propionate intervention group (0.502±0.036) were enhanced and the differences between the two groups were significant (P <0.01). After using AR blocker, the expression of STAT1 phosphoprotein in each group (0.268±0.061, 0.283±0.106, 0.213±0.071) had no difference. · CONCLUSION: Buddleja officinalis drug-containing plasma total flavonoids can promote the expression of STAT1 phosphorylation.     

密蒙花总黄酮含药血浆干预干眼症细胞凋亡模型STAT1磷酸化蛋白表达

目的:探讨密蒙花总黄酮含药血浆和雄激素受体阻滞剂对泪腺上皮细胞中STAT1的磷酸化蛋白表达的影响。方法:体外分离及培养泪腺上皮细胞。以H2O2诱导大鼠泪腺上皮细胞凋亡,建立雄激素水平下降所致干眼症的细胞凋亡状态。设立空白血浆组、密蒙花总黄酮含药血浆干预组、丙酸睾酮干预组,分别观察各组STAT1的磷酸化蛋白表达情况;并应用雄激素受体阻滞剂氟他胺,考察密蒙花总黄酮的拟雄激素效应。结果:免疫印迹结果表明,含药血浆干预后,密蒙花总黄酮含药血浆干预组中(0.353±0.494)与丙酸睾酮干预组中STAT1的磷酸化蛋白(0.502±0.036)的表达增强,两组间的差异有统计学意义(P<0.01)。各组加入雄激素受体阻滞剂后,各组间(分别是0.268±0.061,0.283±0.106,0.213±0.071)的STAT1的磷酸化蛋白表达间无差异。结论:密蒙花总黄酮含药血浆可促进STAT1的磷酸化表达,并激活STAT1细胞信号传导通路,而产生与丙酸睾酮相同的雄激素效应。     

角膜缘上皮细胞体外培养、冻存和复苏的实验研究

目的 建立兔角膜缘上皮细胞体外培养、冻存和复苏的方法。方法 兔角膜缘上皮细胞组织块接种培养。取第三代细胞进行冻存。于冻存后第2周，3、6个月复苏细胞。用MTT法测细胞生长曲线。结果 兔角膜缘上皮细胞体外生长良好。培养细胞AE1单克隆抗体染色阳性，PAS染色阴性。冻存细胞复苏成功。冻存细胞复苏后生长曲线良好。结论 兔角膜缘上皮细胞可以在体外培养、冻存和复苏。     

巨噬细胞对体外培养兔晶状体上皮细胞增殖的影响

目的探讨白内障术后后囊混浊的发病机制。方法观察巨噬细胞对兔晶状体上皮细胞（rabbitlensepithelialcells，RLEC）增生的影响。结果实验组加有巨噬细胞，BLEC增殖活跃。巨噬细胞上清液48及72小时能明显促进BLEC的生长。结论巨噬细胞促进晶体上皮细胞的增生，可能是引起白内障术后后囊混浊的原因之一。     

兔角膜缘上皮细胞培养后自体移植修复

目的：运用培养角膜缘上皮细胞联合人羊膜行自体移植的方法，观察植片修复兔眼角膜上皮的疗效。方法：选用健康新西兰白兔20只，制成右眼角膜缘干细胞缺乏的兔眼模型，其中12只兔行角膜缘上皮细胞培养联合羊膜自体移植，另外8只兔只进行单纯羊膜移植。术后每周对眼表情况进行评分，术后1mo眼角膜进行苏木素-伊红(HE)染色和透射电镜观察。结果：移植了含有自体角膜上皮细胞的兔眼，术后早期都形成了角膜上皮化并明显抑制了新生血管的再生，HE染色和电镜观察表明培养并移植的角膜上皮与正常的角膜上皮无明显差异；而只接受羊膜移植的兔眼，术后又出现角膜混浊和明显的新生血管，表明角膜表面被结膜上皮覆盖。结论：该方法术后早期可以恢复角膜上皮化，重建正常眼表，疗效明显优于单纯羊膜移植。     

晶状体上皮细胞培养及Ⅳ型胶原表达

目的体外培养小牛晶状体上皮细胞，免疫组织化学SABC法检测1V型胶原表达。方法小牛晶状体前囊组织块培养，晶状体上皮细胞原代和传代培养。传代2次后免疫组织化学法检测细胞内1V型胶原表达。结果原代培养2-3周后细胞成单层后传代培养，传代细胞易贴壁，9～10d后可再传代，免疫组织化学检测发现细胞质内有大量Ⅳ型胶原表达，形成细胞的骨架成分。结论晶状体上皮细胞在增生时有大量Ⅳ型胶原表达，其与晶状体后囊混浊有关。     

Traffic of endogenous, transduced, and endocytosed prolactin in rabbit lacrimal acinar cells

The rabbit lacrimal gland undergoes an immunophysiological transformation during pregnancy, reminiscent of that of the mammary gland as it prepares to deliver secretory IgA into the nascent fluid product. The contents of TGF-beta and prolactin (PRL) within ductal epithelial cells increase, and their primary localizations shift from the apical to the basal cytoplasm, suggesting a transformation from exocrine to paracrine secretion. Studies with ex vivo acinar cell models demonstrated that elevated PRL suppresses traffic of secretory proteins into the regulated exocrine apparatus and directs them into a novel, induced, regulated paracrine apparatus [Wang, Y., Chiu, C.T., Nakamura, T., Walker, A.M., Petridou, B., Trousdale M.D., Hamm-Alvarez S.F., Schechter J.E., Mircheff A.K., 2007. Elevated prolactin redirects secretory vesicle traffic in rabbit lacrimal acinar cells. Am. J. Physiol. Endocrinol. Metab. 292, E1122-E1134]. However, it was not clear whether PRL itself entered the induced paracrine apparatus. In the present study, confocal immunofluorescence microscopy revealed that natively expressed PRL and over-expressed PRL co-localized with PRL receptors (PRLR); rab11, a marker for the recycling endosome; gamma-adaptin, a marker for the Golgi complex and trans-Golgi network; and rab7, a marker for the autophagic lysosomal apparatus. Natively expressed, over-expressed, and endocytosed PRL also co-localized with rab4 and rab5A, markers for the early endosome, and with rab3D, a marker for regulated exocrine secretory vesicles. Endocytosed PRL was stored in intact form and released in response to stimulation with carbachol. Subcellular fractionation analysis detected relative excesses of PRL over PRLR in fractions that contained fragments of the recycling endosome and fractions that contained both secretory vesicle fragments and prelysosomal and autolysosomal fragments. EM-gold microscopy demonstrated PRL within small vesicles, consistent with endosomes or secondary lysosomes, and in large vesicles, consistent with regulated secretory vesicles. The secretory vesicles were preponderantly localized in the apical cytoplasm of control cells, and in the basal cytoplasm of PRL over-expressing cells. These results indicate that when lacrimal epithelial cells synthesize PRL, and when they endocytose it from their ambient medium, they traffic it both into the endosomes that constitute the constitutive transcytotic paracrine apparatus and also into regulated secretory vesicles, which are associated with the exocrine apparatus at low PRL levels and with the induced paracrine apparatus at high PRL levels.