人角膜内皮细胞压力调节培养的初步研究

Objective To investigate the effect of pressure bionic culture on the morphology and function of rabbit corneal endothelial cells. Methods Corneal endothelial cells were separated and purified by tearing apart the descemet and digesting with trypsin and EDTA, then cultured in the plate. The cells were divided into two groups: group A were cultured under atmosphere; cells exposed to 2 kPa( 14. 66 mm Hg) pressure in vitro was group B; the morphology and growth pattern of cells were observed by inverted microscope; cells origin were identified by neuron-specific enolase immunoassay. Cellular changes in the structure were observed by HE staining and scanning and transmission electron microscopy (SEM and TEM) analysis. Cells activity was detected by flow cytometry. Results NSE antibody of the primary corneal endothelial cells was positive without corneal epithelial cells and corneal stroma cells. Two groups of cells were cultured for 120-144 h respectively, the morphology was flat, polygon, most of cells were hexagon and abundant cytoplasms in group B (pressure bionic culture), but in group A, the cells size was not uniform and there were much granules in the cytoplasm. There was no difference in the time of formation of monolayer in two groups. SEM showed that cells exposed to pressure connected tightly and the surface was rich in microvilli, extended foot processes and attached to the substrate tightly, while cells cultured under atmosphere with more off-chip. In group B, Annexiv-FITC/PI detection of apoptosis showed cell survival rate was 98.2%, early apoptosis rate was 0.7%, late apoptosis rate was 1.0%, death rate was 0. 1%; the corresponding data were 92.2%, 5.2%, 2.3%, and 0.3% in group A, respectively; There was statistically significant difference between the two groups (x2 =594. 0,P ＜0. 01 ). After cultured for 96 h,the expression of ZO-1 protein in cells exposed to pressure was higher than those in control. Conclusions The biological activity of endothelial cells is regulated positively by bionic pressure. The establishment of a new biomimetic pressure model will help to investigate the physiological function and injury repair of corneal endothelial cells in vitro.     

高压力培养下角膜内皮细胞凋亡的启动机制

目的 探讨高压力培养下角膜内皮细胞凋亡的启动机制.方法 原代兔角膜内皮细胞经免疫组织化学鉴定后,在50mmHg(1 kPa =7.5 mmHg)压力条件下,培养lh、2h、24 h,另设正常压力(15 mmHg)作为正常压力组,培养24 h.第一代兔角膜内皮细胞融合达70％～80％后,细胞在加压前lh分别使用浓度均为10-6 mol·L-l抗Caspase-8和抗Caspase-9预处理,然后置于压力装置中,压力设定为50 mmHg,培养24h后检测蛋白Bcl-2和P53表达,并且以无抑制剂处理的50 mmHg压力培养的细胞为对照组.各组培养的细胞均经Western blot检测Bcl-2和P53的表达.免疫荧光染色检测兔角膜内皮细胞胞浆细胞色素C的含量.结果 50 mmHg压力组角膜内皮细胞,加压lh、2h、24 h P53的表达量分别为0.651±0.007、0.805±0.006、0.839±0.011,较正常压力组(0.033±0.004)升高,差异均有统计学意义(均为P＜0.01);50 mmHg压力组随着加压时间的延长,角膜内皮细胞中P53蛋白表达量逐渐增加,各时间两两比较,差异均有统计学意义(均为P＜0.01).50 mmHg压力组中加压lh、2h、24 h Bcl-2的表达量分别为0.590±0.009、0.724±0.005、0.314±0.016,较正常压力组(0.081±0.013)反应性升高,差异均有统计学意义(均为P＜0.01);50 mmHg压力组各时间两两比较差异均有统计学意义(均为P＜0.01).实验发现正常压力组培养24h后,细胞核呈蓝色,胞浆中无细胞色素C释放;50 mmHg压力组培养lh可见部分细胞胞浆呈红色,提示细胞色素C释放进入到胞浆内,随着加压时间延长荧光强度增加,加压24 h见胞浆内出现广泛红色强荧光.抗Caspase-9和抗Caspase-8预处理组的角膜内皮细胞P53表达量分别为0.535±0.007、0.703±0.010,较对照组(0.727±0.021)下调,差异均有统计学意义(均为P＜0.0I);抗Caspase-9和抗Caspase-8预处理组中Bcl-2的表达量呈抑制性下降,分别为0.312±0.003、0.442±0.011,较对照组(0.501±0.011)下降,差异均有统计学意义(均为P＜0.01).抗Caspase-9预处理组的角膜内皮细胞P53和Bcl-2表达量较抗Caspase-8预处理组下降,差异亦均有统计学意义(均为P＜0.01),表明抗Caspase-9对高压下角膜内皮细胞凋亡的抑制作用更显著.结论 Caspase-9抑制剂可有效阻断高压力对角膜内皮细胞的促凋亡作用,高压力对角膜内皮细胞的损伤主要是触发了线粒体细胞色素C的释放,激活了Caspase-9参与的内源性酶联反应性凋亡途径.     

人角膜内皮细胞压力调节培养的初步研究

Objective To investigate the effect of pressure bionic culture on the morphology and function of rabbit corneal endothelial cells. Methods Corneal endothelial cells were separated and purified by tearing apart the descemet and digesting with trypsin and EDTA, then cultured in the plate. The cells were divided into two groups: group A were cultured under atmosphere; cells exposed to 2 kPa( 14. 66 mm Hg) pressure in vitro was group B; the morphology and growth pattern of cells were observed by inverted microscope; cells origin were identified by neuron-specific enolase immunoassay. Cellular changes in the structure were observed by HE staining and scanning and transmission electron microscopy (SEM and TEM) analysis. Cells activity was detected by flow cytometry. Results NSE antibody of the primary corneal endothelial cells was positive without corneal epithelial cells and corneal stroma cells. Two groups of cells were cultured for 120-144 h respectively, the morphology was flat, polygon, most of cells were hexagon and abundant cytoplasms in group B (pressure bionic culture), but in group A, the cells size was not uniform and there were much granules in the cytoplasm. There was no difference in the time of formation of monolayer in two groups. SEM showed that cells exposed to pressure connected tightly and the surface was rich in microvilli, extended foot processes and attached to the substrate tightly, while cells cultured under atmosphere with more off-chip. In group B, Annexiv-FITC/PI detection of apoptosis showed cell survival rate was 98.2%, early apoptosis rate was 0.7%, late apoptosis rate was 1.0%, death rate was 0. 1%; the corresponding data were 92.2%, 5.2%, 2.3%, and 0.3% in group A, respectively; There was statistically significant difference between the two groups (x2 =594. 0,P ＜0. 01 ). After cultured for 96 h,the expression of ZO-1 protein in cells exposed to pressure was higher than those in control. Conclusions The biological activity of endothelial cells is regulated positively by bionic pressure. The establishment of a new biomimetic pressure model will help to investigate the physiological function and injury repair of corneal endothelial cells in vitro.     

青少年近视屈光度数与眼压的相关性研究

目的探讨青少年近视屈光度数与眼压之间的相关性。方法对在我院就诊患有近视的青少年75例进行眼压测量,并进行检影验光测试屈光度数。按等效球镜的屈光度数分为轻、中、重度3组,非近视眼组为无近视组,对各组数据进行t检验,分析近视屈光度数与眼压之间的关系。结果轻、中度组与非近视组之间存在统计学差异,重度组与非近视组之间无统计学差异。结论青少年近视患者的眼压并不随屈光度的变化而改变,两者之间无明确的相关性。     

单眼上转缺陷行水平直肌移植优化术的远期疗效分析

目的 探讨水平直肌移植术在单眼上转缺陷中的远期疗效。方法 回顾性分析在本院确诊并手术的23例患者,观察术前、术后患者假性上睑下垂、代偿头位的改善情况、评估患眼术后运动功能的变化和眼位的情况。采用手术方法：水平直肌移植优化术。随访时间不少于12个月。结果 术后所有患眼上转功能及代偿头位均明显好转,假性上睑下垂消失,23例患者术前、术后6个月、术后12个月的第一眼位垂直斜视度分别是（50.23±5.07）PD、（1.77±2.23）PD,(1.81±2.35)PD,统计学分析显示术前与术后6个月垂直斜视度比较,有显著性差异（P<0.05）,术后6个月与术后12个月垂直斜视度比较,差异无统计学意义。结论 单眼上转缺陷患者采用水平直肌移植优化术可有效改善患眼上转功能且扩大双眼单视野,远期疗效稳定确切,值得推广。     

角膜碱烧伤的免疫学机制研究进展

角膜碱烧伤是眼科临床常见的致盲性眼病,一旦发生,治疗相当棘手,预后极差,其损伤机制的研究一直受到众多学者的重视。许多研究表明,角膜碱烧伤后免疫系统的参与起着非常重要的作用。角膜碱烧伤后角膜免疫微环境的变化致使各种效应细胞和炎性细胞出现规律性变化,另外各种炎性因子也参与角膜碱烧伤的病理损伤过程。就角膜碱烧伤的免疫学机制进行综述。     

头孢呋辛钠前房给药对白内障患者术后预防眼内感染的疗效及其对不良反应发生的影响

目的:探究头孢呋辛钠前房给药对白内障患者术后预防眼内感染的疗效及其对不良反应发生的影响。方法:选取2017年1月—2018年10月间本院收治的行防盲白内障手术治疗患者106例资料,按治疗方法的不同将其分为对照组和观察组,每组53例;对照组患者给予常规预防感染,观察组患者在对照组基础上加用头孢呋辛钠前房给药预防感染;比较两组患者用药后术后视力复明率、眼内感染发生率及用药期间不良反应发生率差异。结果:观察组患者用药术后脱盲率(94.34%)和脱残率(83.02%)均高于对照组(71.70%)和(56.60%()P<0.05),眼内感染发生率(0.00%)低于对照组(13.21%()P<0.05),不良反应发生率低于对照组(P<0.05)。结论:采用头孢呋辛钠前房给药预防白内障患者术后感染,有效提高了患者的复明率,减少了术后眼内感染,且安全性较高。     

先天性特发性眼球震颤合并水平面转型代偿头位的手术疗效的观察

目的 观察单纯水平面转型代偿头位的先天性眼球震颤的手术效果.方法 回顾性分析在我院收治的13例伴有单纯水平代偿头位但不合并斜视的先天性眼球震颤患儿,术前分别检查代偿头位、头位扭转角和三棱镜耐受试验,手术方式根据头位扭转角为15°-25°者采用改良Anderson法,头位扭转角为25°-40°者采用Parks法.结果 13例患儿术后第一眼位双眼矫正视力较术前提高两行或两行以上,正前方眼球震颤明显减轻甚至消失,均恢复了不同程度的双眼视功能,代偿头位明显改善,头位扭转角均小于10°,无斜视和反向代偿头位出现.结论 根据不同的头位扭转角选择合适的中间带转位术可以有效地改善眼球震颤患者的水平面转型代偿头位,同时减轻正前方眼球震颤和提高双眼视力,改善双眼视功能.     

直肌移植优化术在难治性单眼上转不足中的应用

目的：初步探讨直肌移植优化术在伴有下直肌限制且原在位垂直斜视度较大由双上转肌麻痹导致的难治性单眼上转不足中的应用。

方法：回顾性分析本院2015-01/2019-01收治的双上转肌麻痹病例6例6眼,经保守治疗无效后采用直肌移植优化术,观察术前与术后眼位、垂直斜视度、麻痹眼上下转的运动功能以及假性上睑下垂和代偿头位的改善情况。

结果：患者6例解除下直肌限制后行直肌移植优化术,术前,术后1、6mo的原在位垂直斜视度分别是49.83±6.55、2.67±2.07、1.83±2.40PD,术前斜视度与术后1、6mo比较均有差异(P<0.001),术后1mo与6mo垂直斜视度比较无差异(P=0.899)。所有患者术后原在位眼位均正位,麻痹眼上转功能显著改善; 术前4例假性上睑下垂和2例有代偿头位者,术后假性上睑下垂和代偿头位基本消失。

结论：直肌移植优化术适合伴有下直肌限制的单眼上转不足,可有效改善患眼的上转功能且疗效稳定。