房水对培养的角膜基质细胞的影响

目的观察房水对角膜基质细胞生长的作用。方法将新西兰大白兔的角膜去除后弹力层、内皮层和上皮层后,得到基质层,用组织块培养法体外培养角膜基质细胞。在实验组的培养基中加入10％房水,对照组使用常规培养基培养。通过CCK8实验测得角膜基质细胞的吸光度（A）值以分析细胞增生的情况。分别在培养基中加入2．5％、5％、10％、15％、20％的房水,用明胶酶谱法检测基质金属蛋白酶（MMPs）的活性。结果倒置显微镜观察可见培养的角膜基质细胞呈多角形或树枝状,与对照组相比,实验组的细胞生长状态良好,培养的角膜基质细胞数量明显增加。明胶酶谱法显示实验组的条带比对照组清晰。实验第1～5天分别测角膜基质细胞的A值,实验组的检测条带明显强于对照组,〉10％的房水培养组检测的条带明显强于2．5％、5％房水培养组。CCK8检测表明,培养1～5d实验组的角膜基质细胞A值明显高于对照组,差异均有统计学意义（P〈0．05）。结论房水作用于角膜基质细胞后,可促进角膜基质细胞的生长,10％房水对体外培养角膜细胞有促细胞增生的作用。     

Verapamil对角膜基质细胞影响的研究

目的：观察Ｖｅｒａｐａｍｉｌ（异搏定）对体外培养角膜基质细胞的影响,为其临床应用提供基础。方法：进行兔角膜基质细胞的原代和早期传代培养,并用ＭＴＴ自动比色法检测Ｖｅｒａｐａｍｉｌ对兔角膜基质细胞增殖的影响。结果：Ｖｅｒａｐａｍｉｌ浓度１０～１０００μｇ／ｍｌ作用４８ｈ和７２ｈ对角膜基质细胞增殖有明显抑制作用（Ｐ＜０．０５）。作用７２ｈ抑制率高于作用４８ｈ。结论：Ｖｅｒａｐａｍｉｌ为剂量依赖型和时间依赖型药物,能有效地抑制角膜基质细胞增殖,可望成为调节角膜伤口愈合的新药物。     

tPA对晶体上皮细胞增殖和细胞外基质代谢的影响

观察了ｔＰＡ对体外培养的牛晶体上皮细胞的增殖、胶原和透明质酸合成的影响。结果表明ｔＰＡ对细胞增殖和胶原合成无影响；可使细胞透明质酸合成减少，以８０００ｕ／ｍｌ时效果较明显。提示（１）其抑制后囊混浊形成作用是通过减少纤维蛋白和其对晶体上皮细胞的作用来实现；（２）其用于眼部是安全的。     

手机电磁辐射对耳蜗超微结构及边缘细胞的影响

目的 探讨900 MHz手机电磁辐射对大鼠耳蜗超微结构及边缘细胞的影响。方法 选择成年SD大鼠40只,通过每日接受不同时长900 MHz手机电磁辐射暴露28 d建立手机电磁辐射大鼠模型,分为对照组、辐射6 h组、辐射12 h组和辐射24 h组。电磁辐射暴露后,测试大鼠的听性脑干反应(ABR)、40 Hz听觉事件相关电位和听觉稳态反应的反应阈以评价电磁辐射对大鼠听觉功能的影响;扫描电镜及苏木精-伊红染色后光镜下观察耳蜗超微结构;彗星实验检测耳蜗边缘细胞DNA损伤情况;流式细胞仪检测电磁辐射对边缘细胞凋亡的影响及活性氧水平。结果 手机电磁辐射后大鼠听觉功能各指标反应阈值均高于对照组(P值均<0.05);大鼠耳蜗超微结构随每日暴露时长增加而出现不同程度的外毛细胞纤毛排列及结构异常、柯蒂器形态改变和耳蜗内红细胞渗出等;大鼠耳蜗边缘细胞的DNA损伤情况及细胞凋亡率在各组间差异无统计学意义(P> 0.05);辐射24 h组的活性氧水平显著高于对照组(P <0.001),其余组与对照组间差异无统计学意义(P值均> 0.05)。结论 长时间手机电磁辐射可引起大鼠耳蜗超微结构损伤...     

高糖对牛眼小梁细胞形态、增殖及凋亡的影响

目的:建立牛眼小梁细胞体外培养体系,观察高浓度葡萄糖对小梁细胞的影响,研究高糖与开角型青光眼(primary open angle glaucoma,POAG)以及糖尿病与POAG之间的关系,探讨糖尿病开角型青光眼的发病机制,从而为指导开角型青光眼的临床用药提供重要依据。方法:以新鲜牛眼为材料,分离并培养小梁细胞,用光学镜、电镜观察正常小梁细胞的形态。将传至第3代的小梁细胞制作成细胞悬液、计数并接种于培养板(或培养瓶)中,待细胞贴壁后,将细胞分为两组:对照组(葡萄糖浓度为5.5mmol/L)和高糖组(葡萄糖浓度为35mmol/L),分别培养7d后收集细胞,用透射电子显微镜、MTT法、流式细胞仪(FCM)检测法,研究高糖对小梁细胞形态、增殖和凋亡的影响。结果:高糖组与对照组相比较,小梁细胞胞浆内细胞器减少,内质网、高尔基复合体、线粒体等细胞器肿胀,溶酶体及脂肪滴增多,核浆比减小,可见凋亡小体;高浓度葡萄糖对小梁细胞的增殖有明显的抑制作用,能够促进小梁细胞的凋亡,均有统计学意义(P<0.05)。结论:高糖可能通过使小梁细胞的增殖能力下降,凋亡率增加,使近管小梁网的网状结构改变,网孔变小,改变房水流出途径的阻力导致眼内压升高,可能是糖尿病患者PO-AG发病率增高的原因之一。     

基质细胞衍生因子-1α对体外培养人视网膜色素上皮细胞增殖的影响研究

目的:研究基质细胞衍生因子-1α(stromal cell-derived fac-tor-1α,SDF-1α)对体外培养的人类视网膜色素上皮(hu-man retinal pigment epithelium,hRPE)细胞增殖的影响。方法:应用MTT法研究SDF-1α对体外培养的人RPE细胞增殖的影响;用细胞化学染色法检测添加SDF-1α前后人视网膜色素上皮细胞内细胞增殖核抗原(proliferatingcell nucler antigen,PCNA)的表达情况。结果:MTT法表明SDF-1α可促进体外培养的hRPE细胞的增殖(P<0.05);细胞化学电镜下观察发现:添加SDF-1α前hRPE细胞未表达或低表达PCNA,而添加SDF-1α后细胞高度表达PCNA。结论:细胞因子SDF-1α可促进体外培养的hRPE细胞增殖。     

兔角膜内皮、上皮及基质细胞体外培养扩增的研究

目的 建立角膜上皮、基质及内皮细胞体外培养扩增的简单稳定的方法，为组织工程化角膜的构建提供种子细胞。方法 内皮细胞与后弹力层在培养基中孵育后消化法获原代细胞，胰酶消化去除表层上皮后取角膜缘，组织块法培养角膜缘上皮细胞，基质细胞应用胶原酶消化法获原代培养，各细胞融合后胰酶消化依次传代培养。结果 原代内皮细胞4—5d融合成单层细胞，可连续传6—7代。上皮细胞1周左右生长融合，连续传3—4代后细胞形态改变。基质细胞接种6—7d后近融合，传代后增殖明显，可连续传10代。结论依据角膜组织特征选择合适的方法体外分离、培养角膜3种细胞成分，可获连续传代扩增的角膜细胞。     

牛角膜基质细胞的两步酶消化法高效分离及体外培养观察

背景 高效、低成本分离出生物学功能活性高的角膜基质细胞是开展角膜基础研究的需要.目前的分离方法成本高、分离效率低,而通过培养达到扩增细胞数会导致细胞表型快速改变.应用成本较低的I型胶原酶,通过改良的两步酶消化法可能达到高效、快速、低成本分离牛角膜原代基质细胞的目的.目的 评价设计的I型胶原酶两步酶消化法分离原代牛角膜基...     

牛角膜基质细胞的两步酶消化法高效分离及体外培养观察

背景高效、低成本分离出生物学功能活性高的角膜基质细胞是开展角膜基础研究的需要。目前的分离方法成本高、分离效率低,而通过培养达到扩增细胞数会导致细胞表型快速改变。应用成本较低的Ⅰ型胶原酶,通过改良的两步酶消化法可能达到高效、快速、低成本分离牛角膜原代基质细胞的目的。目的评价设计的Ⅰ型胶原酶两步酶消化法分离原代牛角膜基质细胞的效果,并观察体外培养原代牛角膜基质细胞的形态学变化。方法分别用基础培养液配制的0．5g／L及1．0g／L Ⅰ型胶原酶以两步酶消化法顺序消化牛角膜组织,分离角膜基质细胞,以细胞计数板进行计数,检测基质细胞收获效率;锥虫蓝染色法检测收获细胞的存活率;分离的细胞进行原代培养,倒置显微镜下观察细胞形态和生长的变化;应用Alexa488标记的鬼笔环肽检测原代培养的牛角膜基质细胞中F—actin的分布。结果牛角膜经两步酶消化法基质逐步解离和降解,绝大多数细胞得以释放和分离,分离的牛角膜基质细胞呈圆形,透亮且大小均匀。每个角膜收获（2．109±0．142）×10。个基质细胞,细胞存活率（91．693±3．551）％,贴壁率（81．195±1．214）％。原代培养的牛角膜基质细胞贴壁呈树突样,铺伸至星状,融合时树突连接呈网状,其F—aetin局限性分布于细胞皮质。结论两步酶消化法可使牛角膜基质完全消化降解,具有高细胞收获率、高细胞存活率和操作简便等特点。原代培养的牛角膜基质细胞呈树突状,F—actin分布于细胞皮质。     

脱细胞猪角膜基质体外支持角膜上皮和基质细胞的生长

目的:探讨脱细胞猪角膜基质体外能否支持兔角膜细胞的生长。方法:体外培养兔角膜上皮细胞和基质细胞,并接种到制备的脱细胞猪角膜基质上,倒置相差显微镜和组织学观察细胞生长情况。结果:上皮细胞能在脱细胞猪角膜基质上贴附生长,10d时可形成2~3层的复层结构。基质细胞在脱细胞猪角膜基质上贴附生长后可向材料深层迁徙。结论:制备的脱细胞猪角膜基质体外可支持兔角膜上皮细胞和基质细胞的生长。     

全反式视黄酸对体外培养大鼠Müller细胞生长的影响

目的:观察全反式视黄酸(All-trans retinoic acid,RA)对体外培养大鼠Müller细胞生长的影响。方法:用不同浓度的RA作用于培养的Müller细胞,利用相差显微镜、MTT法、细胞计数法、流式细胞仪检测RA对Müller细胞形态、生长状况及其凋亡的影响。结果:RA在低浓度(<0.1μM)时对Müller细胞的抑制作用不明显。当RA在高浓度(1μM、10μM、100μM)时产生明显抑制作用,细胞形态出现显著变化,48h较24h更明显。细胞计数显示20μM的RA作用48h后引起细胞数目明显减少。流式细胞仪检测结果显示不同浓度的RA(5μM、10μM、20μM)均可诱导Müller细胞凋亡,20μM的RA作用48h时细胞凋亡率最高(P<0.01),凋亡指数为(35.87±7.40)%。结论:RA能抑制Müller细胞的增殖、促进其凋亡,并具有浓度和时间依赖性。     

金丝桃素抑制兔外伤性增生性玻璃体视网膜病变

目的:研究蛋白激酶C(protein kinase C,PKC)特异性抑制剂—金丝桃素对外伤性增生性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)的防治作用机制。方法:用自体富含血小板血浆玻璃体内注射制备免外伤性PVR模型,并同时将生理盐水、1μM、10μM和100μM金丝桃素0.1ml注入兔眼玻璃体内,在不同时间点观察金丝桃素对PVR形成的防治作用。结果:在对照组第10d就出现视网膜脱离,PVR随着时间延长发展至更严重等级。在金丝桃素组,PVR也发生,但其严重程度在每个时间点均低于对照组。在10μM和100μM金丝桃素组给药5d后,PVR的临床分级显著低于对照组(P<0.05)。组织学检查金丝桃素组未发现明显视网膜形态改变,视网膜电图检查也未观察到显著功能变化。结论:金丝桃素玻璃体腔内注射是安全的,并能有效抑制兔外伤性PVR的发生发展,为将来临床应用提供可靠的理论依据。眼科学报 2002;18:240-246.     

丹参对低气压噪声暴露豚鼠耳蜗一氧化氮合酶活性的影响

目的探讨丹参对低压及噪声环境下豚鼠耳蜗诱导型一氧化氮合酶(iNOS)活性的影响.方法豚鼠随机分为暴露1 d及7 d两大组,又各分为刘照、治疗及防治组.3组均置于模拟高原5 500 m低气压环境,110 db SPL白噪声刺激;防治组和治疗组每日肌注丹参注射液(1.3 ml·kg-1).采用免疫组织化学和图像分析半定量法.观察暴露后不同时间各组耳蜗iNOS的表达和阳性产物的平均灰度值.结果正常耳蜗未见iNOS阳性表达.低压噪声暴露后豚鼠耳蜗外毛细胞、内毛细胞、血管纹及螺旋神经节等部位均可见iNOS呈棕黄色颗粒的阳性反应.耳蜗iNOS阳性反应各组随暴露时间延长逐渐增强.随出舱后时间延长而逐渐减弱.除暴露1d组出舱1 d的对照组与治疗组的灰度值差异不显著(P＞0.05)外,其它各组间差别显著(P＜0.05).暴露7 d组的防治组、治疗组与对照组、防治组与治疗组间差异非常显著(P＜0.01).结论低压噪声暴露后,豚鼠耳蜗内iNOS阳性反应随暴露时间延长逐渐增强,随出舱后时间延长而逐渐减弱;丹参的抗氧化反应,可减轻iNOS对一氧化氮(NO)的生成,可能为丹参对低压噪声暴露后听器损伤具一定保护功能的机理之一.     

Expression of Pigment Epithelium-Derived Factor and Vascular Endothelial Growth Factor in Fibrovascular Membranes from Patients with Proliferative Diabetic Retinopathy

Purpose

The expression of pigment epithelium-derived factor (PEDF), a strong inhibitor of angiogenesis, has not been examined in human ocular fibrovascular membranes, to the best of our knowledge. The purpose of this study was to determine whether PEDF is expressed in the fibrovascular membranes in eyes of patients with proliferative diabetic retinopathy (PDR), and to compare the expression of PEDF with that of vascular endothelial growth factor (VEGF).

Methods

The expression of PEDF and VEGF in the fibrovascular membranes excised during vitreous surgery in eight cases of PDR was determined by immunohistochemistry.

Results

VEGF was strongly expressed in the endothelial cells of newly formed vessels in the fibrovascular membranes. In contrast, PEDF was weakly expressed in the endothelial cells and was prominently expressed in the extracellular matrix and fibrous tissue surrounding the new vessels.

Conclusions

Our results suggest that PEDF, along with VEGF, may modulate the formation of fibrovascular membranes in patients with PDR.?Jpn J Ophthalmol 2006;50:116–120 © Japanese Ophthalmological Society 2006     

Elevated Vascular Endothelial Growth Factor Levels in the Vitreous of Patients With Proliferative Diabetic Retinopath

Objective: To detect the levels of vascular endothelial growth factor (VEGF) in the vitreous of patients with proliferative diabetic retinopathy (PDR) and to investigate the possible role of VEGF in the development of neovascularization in PDR. Methods ; Undiluted vitreous samples and fasting venous blood samples were obtained from 27 patients with PDR and 14 subjects with idiopathic macular hole who underwent pars plana vitrectomy. The concentration of VEGF was determined by quantitative enzyme - linked immunosorbent assay (ELISA).Results: The level of vitreous VEGF in patients with PDR (median 0. 41ng/ml, range 0. 09- 11. 56ng/ml) was significantly elevated when compared with that in control subjects (median 0.017ng/ml, range 0.008-0.04ng/ml)(P<0. 001). The median of PDR patients' serum VEGF concentration was 0.19ng/ml (0. 090. 46ng/ ml) which was far lower than vitreous VEGF concentration (P<0. 05). Vitreous VEGF concentration was higher in PDR patients with retinal detachment than that in patient wi     

The Effects of Panretinal Photocoagulation on Dark Adaptation in Diabetics with Proliferative Retinopathy

The dark adaptation of 14 diabetic patients was measured before and after panretinal photocoagulation (PRP). The rod-cone break of diabetics prior to PRP was delayed 1.7 minutes compared to normal controls and the final rod threshold was elevated 0.7 log units. After PRP, the rod-cone break was delayed 2.9 minutes and the final rod threshold was elevated 1.1 log units.     

Proliferative response of corneal endothelial cells from young and older donors

PURPOSE: To compare the effect of epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor-BB (PDGF-BB), bovine pituitary extract, and fetal bovine serum (FBS), alone or in combination, on proliferation of human corneal endothelial cells (HCEC) cultured from young (<30 years old) and older donors (>50 years old). METHODS: Corneas from donors 2 to 79 years old were obtained from the National Disease Research Interchange. Descemet's membrane with intact endothelium was dissected. Cells were isolated by EDTA treatment and cultured to confluence. The HCEC marker, antibody 9.3.E, tested for pure endothelial populations. Antibody Ki67 and ZO-1 tested either before or after cultured cells reached confluence to indicate cell proliferation and cell-cell contact formation. Cell morphology was documented by inverted phase-contrast microscopy. Passages I through VII were used to test the effect of various factors on cell proliferation. For each study, equal numbers of cells were seeded, maintained overnight in 4% FBS to permit cell attachment, washed, and incubated for up to 3 weeks in one of the following: modified Eagle's Minimum Essential Medium (Opti-MEM-I) alone; Opti-MEM-I plus EGF, NGF, PDGF-BB, bovine pituitary extract, or FBS; or a combination of factors. At various times after seeding, cell numbers were determined by electronic cell counter. For each condition, three separate wells were tested and each sample was counted three times. Studies were repeated at least twice using cells from different donors and age groups. Within each study, a one-way ANOVA test was performed to analyze statistical significance. RESULTS: Cells stained positively with antibody 9.3.E, indicating isolation of HCEC and lack of contamination with epithelial cells or keratocytes. Positive staining of Ki67, indicating cycling cells, was found in subconfluent cultures. Plasma membrane-associated ZO-1 staining and lack of Ki67 staining indicated that cultured cells formed a contact-inhibited monolayer. Cultured cells decreased in density, increased in size, and became more heterogeneous depending on donor age and on the number of passages. Incubation in OptiMEM-I promoted attachment and induced a moderate proliferative response above that of MEM (P < 0.001). In general, proliferative responses to growth stimuli were relatively slow, with cell counts generally plateauing 10 to 14 days after exposure to growth-promoting agents. EGF yielded a broad, dose-dependent effect and, at 5-50 ng/mL, peak cell counts were significantly higher (P < 0.001) than basal levels. EGF consistently stimulated proliferation in cells from younger donors, but was less effective in stimulating growth of cells from older donors. NGF did not show a consistent significant stimulatory effect at any concentration tested. PDGF-BB (25 ng/mL) tended to stimulate growth to a greater extent than EGF (P < 0.05) in cultures from the same donor. Pituitary extract significantly increased counts at 1.0 (P < 0.05) to 100 ug/mL (P < 0.001). PDGF-BB plus pituitary extract demonstrated greater stimulation than pituitary extract (P < 0.01) or PDGF-BB alone (P < 0.01). FBS (1%-8%) increased cell numbers in a dose-dependent manner, and, at 4%-8%, yielded counts significantly higher (P < 0.001) than that of any single growth-promoting agent tested. CONCLUSIONS: HCEC from both young and older donors can proliferate in vitro in response to growth-promoting agents. Proliferation in the presence of multiple mitogens ceased when confluence was reached, indicating the formation of a contact-inhibited monolayer. In general, cells cultured from young donors were more responsive to the agents tested, but the relative response of HCEC to these agents was similar, regardless of donor age. The relative difference in the extent of the response of the same cell population to different mitogens suggests that these mitogens induce different downstream signals. The relatively robust proliferative response of HCEC to FBS may involve stimulation of multiple downstream signaling pathways may involve stimulation of multiple downstream signaling pathways and/or induce more sustained downstream signaling than the other growth-promoting agents tested.