兔眼晶状体上皮细胞的体外培养

目的 建立兔晶状体上皮细胞体外培养的模型。方法 采用组织块培养法,对兔眼晶状体前囊膜进行培养,并利用形态学检查方法和免疫组化技术鉴定。结果 组织块接种２４ｈ后即可见细胞生长,且保持上皮细胞形态,１ｗｋ左右细胞融合,在体外可传至７代,５代以后细胞呈成纤维细胞状,α－晶状体蛋白间接免疫荧光呈阳性反应。结论 成功地建立晶状体上皮细胞体外培养模型,可用于后发性白内障发病机制的研究。     

兔晶体上皮细胞培养的技术改进

对兔晶体上皮细胞培养方法作了改进，为控制后发性白内障的预防提供实验手段，取新西兰家兔晶体前囊膜作细胞培养，在原代培养中，以吸管转移囊膜，细胞融合后，用胰酶消化转代，结果：原代培养４８～７２小时后，可见晶体上皮细胞长出，以后细胞呈贴壁单层，铺砌型向外生长，传代后６～８小时细胞贴壁生长。方法经济简便，可为各种实验提供兔晶体上皮细胞。     

去整合素抑制体外培养人晶状体上皮细胞增殖的研究

目的探讨去整合素Kistrin(disintegrin kistrin)对体外培养的人眼晶状体上皮细胞(human lensepithelial cells,HLECs)增殖的抑制作用.方法对先天性白内障患者术中环行撕前囊膜,进行原代培养并传代,取第二代细胞用于实验.增殖抑制实验将悬浮的活细胞接种于涂有鼠尾胶原的96孔培养板,16 h后,去掉旧培养液,换用不同浓度的去整合素(5 ng/ml、10 ng/ml、20ng/ml、40 ng/ml、80 ng/ml)37℃、5%CO2条件下共同孵育,7 d后,MTT法测定晶状体上皮细胞的数目.结果20ng/ml、40 ng/ml、80 ng/ml的去整合素对体外培养的晶状体上皮细胞的增殖进行干预后,OD值分别为0.254±0.008、0.224±0.009,0.202±0.010,与空白对照组相比,差异有统计学意义(P＜0.05).结论去整合素可抑制体外培养的人眼晶状体上皮细胞的增殖.     

不同术式晶状体摘除术后兔眼后发性白内障形成的组织病理学分析

目的：探讨可有效预防后发生白内障发生的内白障手术式方式。方法：将18例实验家兔随机分为A、B、C3个组,分别进行超声乳化晶状体吸附术、超声乳化晶状体吸附联合后囊膜连续环形撕囊术及超声乳化晶状体吸除联合的囊膜连续环形撕囊及前部玻璃体切除术。术后3个月对3个组术眼进行裂隙灯、眼底、组织病理学及电镜检查。结果：术后3个月3组术眼周边囊膜的混浊情况相似;A、B组术眼中央视区混浊情况相似,C组多数术眼中央视区清亮。3个组均无玻璃体疝、视网膜脱离及黄斑囊样水肿等并发症发生。3个组周边后囊膜均可见形态改变的晶状体上皮细胞及程度相似的Elschnig小体和晶体状纤维。A组后囊膜中央可见Elschnig小体及晶体纤维。B组可见与后囊膜撕囊孔相连续的纤维增生,C组后囊膜撕囊孔缘未见明显病理改变,无纤维增生。结论：超声乳化白内障吸除联合后囊膜连续环形前部玻璃体切除术可有效预防中央视区后发性白内障的发生;远期疗效尚需进一步观察。     

白内障手术中后囊硬性斑的处理

目的：探讨白内障患者后囊“硬性斑”的诊断及处理方法。方法：根据术前裂隙灯检查和手术中证实硬性斑的不同情况，选择后囊处理方式；首选“oerHi”环形撕囊仪环形撕囊，次之选用刺开撕囊，特殊的硬性斑用不同的环形切囊法。结果：oerHi环形撕囊仪5眼无1眼出现玻璃体溢出晶状体囊袋内，而刺开撕囊2眼有1眼玻璃体溢出晶状体囊袋内、环形切囊2眼均出现玻璃体溢出晶状体囊袋内，给予前段玻璃体切除，本组9眼人工晶状体均植入晶状体囊袋内。结论：用oerHi环形撕囊仪环形撕后囊“硬性斑”，安全、简便。刺开撕囊时，勿伤及玻璃体前膜，深度掌握有难度，环形撕囊时需做前段玻璃体切除准备。     

核因子κB在年龄相关性白内障晶状体前囊膜的表达

目的研究核因子κB（NF-κB）在正常人、年龄相关性白内障患者晶状体前囊膜上皮细胞中表达的差异,探讨NF-κB在年龄相关性白内障发病机制中的作用。方法收集白内障术中60例60眼年龄相关性白内障的前囊膜组织（白内障组）,选取正常供体5例10眼的透明晶状体前囊膜组织作为正常对照组。取白内障组30眼标本和正常对照组5眼标本用于免疫组织化学法检测NF-κBp65蛋白在晶状体前囊膜上皮细胞中的表达;另外白内障组30例标本及正常对照组的5例标本采用实时荧光定量聚合酶链反应（PCR）方法检测NF-κBp65mRNA在晶状体前囊膜上皮细胞中的表达。结果正常晶状体前囊膜上皮细胞细胞质和细胞核中可见NF-κBp65蛋白的弱阳性表达,以细胞质为主;年龄相关性白内障晶状体前囊膜上皮细胞的细胞质和细胞核中NF-κBp65蛋白呈强阳性表达。白内障组NF-κBp65吸光度（A）值为0.1658±0.022,正常对照组为0.2889±0.043,差异有统计学意义（t=6.2109,P〈0.05）。白内障组NF-κBp65mRNA表达量为1.454±0.081,正常对照组为0.951±0.075,差异有统计学意义（t=12.9683,P〈0.05）。结论 NF-κB表达水平的升高与年龄相关性白内障的发病密切相关,其表达异常可能参与年龄相关性白内障的发生发展。     

硅油眼并发性白内障晶状体上皮细胞超微结构观察

目的白内障是眼内硅油填充术后最常见的并发症,其具体的发病机制目前尚未完全明确。应用扫描电子显微镜及透射电子显微镜对硅油眼并发性白内障晶状体前囊膜及上皮细胞的超微结构进行观察分析,为其发病机制及病理改变的研究提供新的线索及依据。观察硅油填充眼并发性白内障晶状体前囊膜及晶状体上皮细胞(lens epithelial cells,LECs)的超微结构改变,探讨硅油在并发性白内障的发生、发展中的作用。方法选取我科2006年5月至2007年10月行硅油取出术患者12例(12只眼),行白内障囊外(ECCE)或超声乳化(Phaco)联合硅油取出术,术中取晶状体前囊膜,浸入3%戊二醛固定液中固定后制作病理标本,分别在扫描电子显微镜(SEM)、透射电子显微镜(TEM)下观察其超微结构改变。结果硅油眼并发性白内障在SEM下可观察到可见部分上皮细胞及ECM脱离前囊膜,晶状体前囊膜LECs游离面呈舌状或瓦片状,覆盖于相邻细胞顶部,并大片状缺失或脱离前囊膜。上皮细胞呈扁平立方形,细胞核呈圆形或椭圆形。TEM下观察晶状体前囊膜为均质的基底膜,前囊下LECs形态极不规则,呈长梭形或扁平状,相邻细胞结合也较疏松,可见大量大小不等空泡存在。LECs间以指状突紧密镶嵌或缝隙连接,线粒体肿胀,嵴消失。细胞核呈椭圆形,密度加深,核内染色质浓缩、边集、固缩呈典型的细胞凋亡表现。结论硅油可促进并发性白内障的发生、发展,硅油眼并发性白内障最终以LECs细胞凋亡为主要形式。     

选择性后房型人工晶状体植入前的后环形撕囊术

目的：探讨后囊膜增殖钙化的白内障摘出术后的后囊膜处理技术。方法：对42例(49只眼)后囊膜增殖钙化的白内障患者，于摘除白内障后在后囊膜旁中心处用破囊针头划出起始瓣，沿起始瓣边缘撕开一直径约3～4mm的类圆孔，再植入人工晶状体。结果：全部术眼后囊膜中央均有一透明类圆孔，术后无人工晶状体偏位或眼底异常改变。结论：后房型人工晶状体植入前的后环形撕囊术可使后囊膜增殖钙化的白内障获得视轴透明区，是安全有效地处置后囊膜混浊方法之一。     

晶状体前囊膜下上皮细胞增殖性病变的临床病理分析

目的：观察晶状体前囊膜下上皮细胞增殖的组织病理学变化,并探讨其发生原因。方法：取不同病因致手术摘除的伴晶状体前囊膜下上皮细胞增殖的眼球标本64例,其中眼外伤36例,绝对期青光眼10例,角巩膜葡萄肿6例,视网膜母细胞瘤5例,眼内炎4例,白内障手术后2例,糖尿病性白内障1例,在光镜下进行组织病理学观察研究。结果：增殖的晶状体前囊膜下上皮细胞形态不规则,排列不整齐、向后囊膜下延伸;囊膜破裂或部分缺如 前囊膜下纤维结缔组织化生,纤维组织形成;晶状体纤维崩解、液化和钙化,结论：眼内多种病状态,如眼外伤、眼内炎、青光眼,以及糖尿病等全身疾患均可命名晶状体前囊膜下上皮细胞增殖,导致晶状体发生病变,眼内炎和眼外伤后晶状体发生增殖的程度最重。     

儿童先天性白内障不同术式后发性白内障形成的病理学分析

目的 探讨预防儿童后发性白内障的手术方式.方法 回顾性研究先天性白内障患儿50例69眼,按不同手术方式(超声乳化晶状体吸出术、超声乳化晶状体吸出联合后囊膜连续环形撕囊术及超声乳化晶状体吸出联合后囊膜连续环形撕囊及前部玻璃体切割术)分为Ⅰ、Ⅱ、Ⅲ 3组,术后随访3～48个月,对各组术眼进行眼底镜、裂隙灯、组织病理学及电镜检查.结果 3组术眼晶状体后囊膜周边部均可见残留晶状体上皮细胞(lens epithelial cells,LECs)向梭形成纤维细胞转化,并可见残留的LECs形成半透明的球形Elschnig 珍珠小体;晶状体前囊膜亦可见少量残留的LECs及Elschnig珍珠小体.Ⅰ组后囊膜中央部可见多量增生的成纤维细胞及LECs;Ⅱ组后囊膜撕囊孔缘处可见成纤维细胞及排列紊乱的LECs,术眼视区中央部的增生膜内可见多量的羽毛状胶原原纤维;Ⅲ组视区中央无增生膜形成,后囊膜撕囊孔缘仅见少量残留LECs.3组中央视区后发性白内障发生率分别是:Ⅰ组 43.48%,Ⅱ组17.39%,Ⅲ组0,Ⅲ组多数术眼中央视区清亮,后囊孔缘处未见明显病理学改变.结论 儿童先天性白内障摘出术中,行后囊连续环形撕囊 前部玻璃体切割术可有效预防儿童中央视区后发性白内障的发生.     

Human anterior lens capsule as a biologic substrate for the ex vivo expansion of limbal stem cells in ocular surface reconstruction

PURPOSE: To study the potential use of human anterior lens capsule as a scaffold for stem cell transplantation in treatment of limbal cell deficiency. METHODS: Limbal biopsies and anterior lens capsules were obtained (same eye) from 30 patients during cataract surgery. Biopsies were suspended in Dulbecco modified Eagle medium under sterile conditions and stored at 4 degrees C. Capsules were treated in distilled water under strict asepsis for 2 hours to eliminate the crystalline epithelium and stored at 4 degrees C. After initial processing, the limbal biopsy was plated epithelial-side down (48 hours) on the capsular specimen in a 35-mm culture dish. Samples were sorted into 4 groups. Group 1 was made up of 10 samples in which limbal biopsies were allowed to grow on corresponding capsules from the same eye (autologous). Group 2 was 10 limbal biopsies that were allowed to grow on capsules of different eye (allogeneic). The remaining specimens were randomized into 2 groups. Group 3 included 10 capsules on which an ex vivo expanded cell line was allowed to grow. Group 4 harbored 10 limbal biopsies that were allowed to grow on polystyrene culture plates. All specimens were incubated for 2 weeks at 37 degrees C and 5% CO2. Cell density, viability, morphology, and adherence of the cell-capsule complex were evaluated at 1, 3, 7, and 14 days. RESULTS: Rate of cell growth and density in groups 1 and 2 were comparable to the control groups. Cell viability was 95% or superior in all groups, and desmosomes developed between growing cells. CONCLUSIONS: Human anterior lens capsule is a potential scaffold for ex vivo expansion of limbal epithelial cells, possibly providing a substrate for ocular surface reconstruction.     

鼠晶状体上皮细胞的体外培养

目的:建立鼠晶状体上皮细胞体外培养的模型。方法:应用组织块贴片法和酶逐步消化法对10~14d的SD大鼠晶状体上皮细胞进行体外培养,在相差显微镜下观察其生长规律。结果:组织块贴片法在加入培养基4~5d后见细胞生长,2wk细胞融合。而酶逐步消化法在加入培养基后7d左右见细胞贴壁,2wk左右见细胞融合。结论:鼠晶状体上皮细胞体外培养较困难,本试验采用酶逐步消化方法和组织块贴片法。成功地建立了鼠晶状体上皮细胞体外培养的模型,为研究后发性白内障发病机制提供了基础。     

Lens epithelial cell death after cataract surgery

PURPOSE: To determine whether lens epithelial cells (LECs) undergo apoptosis during healing after cataract surgery to further characterize the healing process of the postoperative lens capsule. SETTING: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. METHODS: Apoptotic cells were detected in human postoperative lens capsules using the terminal deoxynucleotidyl transferase deoxy-UTP nick-end labeling (TUNEL) method. The effect of exogenous transforming growth factor-beta2 (TGF-beta2) on mouse LEC apoptosis was also examined in an organ-culture system. RESULTS: Three of 17 postoperative specimens contained TUNEL-positive cells. In 2 lens capsules obtained earlier than 10 days, many TUNEL-positive cells, presumably apoptotic LECs, were observed beneath the residual anterior capsule. In cell multilayers in capsule opacification extracted later than 10 days, a few TUNEL-positive cells were seen in 1 specimen; most cells remained negative. In mouse lenses organ-cultured with 1.0 ng/mL TGF-beta2 for 48 hours, TUNEL-positive cells were detected beneath the lens capsule. CONCLUSIONS: Lens epithelial cells undergo apoptosis during healing after cataract surgery, especially in the early phase. Transforming growth factor-beta2 may be a factor inducing apoptosis in in vivo LECs.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases of fibrous humans lens capsules with intraocular lenses

PURPOSE: We located immunohistochemically the matrix metalloproteinases (MMP) -1, -2, -3 and -9 and the tissue inhibitors of matrix metalloproteinases (TIMP) -1 and -2 in the fibrous capsule of patients with intraocular lenses (IOLs). METHODS: During vitreoretinal surgery in 10 patients we obtained post-cataract surgery lens capsules with or without an IOL. The mean interval between the previous cataract operation and the extraction of the specimens was 35.2 months (range: 2-120 months). Circular sections of the anterior capsule with lens epithelial cells (LECs) were also obtained during cataract surgery. Specimens were processed for immunohistochemical identification of MMPs and TIMPs by light microscopy. RESULTS: While all the members of MMPs and TIMPs were not detected in the normal anterior capsules, they were detected in the ECM and/or LECs on the lens capsules extracted within 18 months after IOL implantations in all of the 4 patients, but were not observed in specimens obtained 18 months or longer postoperatively. In LECs of 1 capsule specimen 10 years postoperatively, MMP-1, but not other MMPs and TIMPs, was detected. CONCLUSIONS: MMPs and TIMPs were detected in the ECM and/or LECs on post-cataract surgery capsules. These proteins may be remodeling the newly deposited ECM and regulating LEC behavior on residual lens capsules in the early phase of healing after cataract surgery.     

老年性白内障与透明晶状体上皮内蛋白酶体活性的比较

目的:比较老年性白内障晶状体上皮与透明晶状体上皮内蛋白酶体的糜蛋白酶样、胰蛋白酶样酶活性是否存在差异,进而探讨蛋白酶体在老年性白内障发生发展中的作用机制。方法:白内障晶状体囊膜取自在我院行白内障超声乳化及人工晶状体植入的患者,透明晶状体囊膜取自广东省眼库,年龄与白内障病人相匹配。反复冻融囊膜使细胞脱落,将囊膜从溶液中除去。各组样品中分别加入LLVY或VGR,在不同时间测定荧光强度。结果:透明晶状体上皮的蛋白酶体活性明显高于白内障组(P<0.01);皮质型与非皮质型白内障晶状体上皮的糜蛋白酶样酶活性无明显区别(P>0.05);MG-132明显抑制各组蛋白酶体的活性。结论:与透明晶状体相比,白内障晶状体上皮内蛋白酶体的糜蛋白酶样、胰蛋白酶样酶活性明显降低,蛋白酶体活性降低可能在老年性白内障的形成中起重要的调节作用。     

Human lens epithelial cell apoptosis and epithelial to mesenchymal transition in femtosecond laser-assisted cataract surgery

AIM: To evaluate human lens epithelium cell apoptosis and epithelial to mesenchymal transition (EMT) induced by femtosecond laser in femtosecond laser assisted cataract surgery (FLACS). METHODS: Sixty cataract patients with N2 to N3 stage according to the LOCS III were enrolled in this study and divided into three groups randomly: FLACS1 group (cataract surgery by FLACS with LenSx), FLACS2 group (cataract surgery by FLACS with LensAR) and manual group (cataract surgery by phacoemulsification). Patients in two FLACS groups performed anterior capsulotomy by LenSx or LensAR laser system. Patients in the manual group were performed continuous curvilinear capsulorrhexis (CCC) manually. The anterior capsules were fixed right after moved out of eye. Hematoxylin-eosine staining, immunofluorescence staining and real-time PCR were performed in order to observe human lens epithelium cells changes after cataract surgery. RESULTS: The capsule cutting edge was shown irregularity and roughness in two FLACS groups and smooth edge in manual capsulotomy by pathologic staining. Irregularities of the cell configuration with partly swollen and destroyed nuclei were observed in two FLACS groups. Femtosecond laser could induce a significantly higher cell apoptosis in human lens epithelium cell than manually performed CCC (P<0.05). Lens epithelium cells apoptosis were correlated with femtosecond laser duration according to Pearson correlation analysis. Decreased N-cadherin expression, alpha-SMA and FSP-1 level in two FLACS groups showed the inhibition of cell EMT. CONCLUSION: Femtosecond laser may affect the apoptosis and EMT of lens epithelium cells which are under the peeled central lens capsule.     

miR-181调控人晶状体上皮细胞凋亡的机制研究

目的：探讨 miR-181在白内障晶状体组织中的表达情况,及其对人晶状体上皮细胞凋亡的调控机制。
　　方法：利用Real time q-PCR方法,检测年龄相关性白内障患者晶状体前囊膜和人晶状体上皮细胞凋亡模型中miR-181的表达情况;利用Lipofectamine 2000瞬时转染miR-181 mimic 和 inhibitor 调节人晶状体上皮细胞中miR-181的表达,利用Real time q-PCR方法验证转染效率,利用流式细胞仪检测细胞凋亡率的变化。
　　结果：与对照组相比,年龄相关性白内障患者晶状体前囊膜组和人晶状体上皮细胞凋亡模型组,miR-181的表达显著升高;miR-181 mimic转染组,miR-181的表达显著升高,细胞凋亡率显著升高;miR-181 inhibitor转染组, miR-181的表达显著降低,细胞凋亡率显著降低,差异均有统计学意义(P<0.01)。
　　结论：miR-181在年龄相关性白内障晶状体组织中呈高表达,miR-181能够促进人晶状体上皮细胞凋亡,从而可能在年龄相关性白内障的发病过程中发挥一定作用,miR-181可能成为白内障非手术治疗的一种新途径,但具体机制有待进一步研究。     

Lens epithelial inhibition by PMMA optic: implications for lens design

It has been a clinical impression that posterior chamber lens implants in some way inhibit opacification of the posterior lens capsule after extracapsular cataract extraction. The mechanism of this inhibition is unclear; it may be related to mechanical contact or blockage of migration of lens epithelial cells, or possibly to the leeching of toxic factors from the lens itself. A better understanding of the exact mechanism of opacification inhibition may have important clinical implications for intraocular lens design. For example, some lens designs that facilitate Nd:YAG capsulotomy by physically separating the posterior chamber lens and the posterior capsule may result in less inhibition and in fact more opacification of posterior capsules. We performed in vitro tissue culture studies of the effect of the polymethylmethacrylate (PMMA) optic of a planoconvex intraocular lens on cultured rabbit lens epithelium. These studies demonstrated both inhibition of lens epithelial migration beneath the PMMA optic (plano side down) as well as metaplasia and necrosis of lens cells growing directly beneath the optic. The clinical implications of these studies for intraocular lens design are discussed.     

Influence of a lymphocyte-conditioned medium on the expression of smooth-muscle α-aktin in lens epithelial cells in situ

PURPOSE: Contraction of the capsule of the ocular lens is based upon proliferation and contraction of transformed lens epithelial cells. It is assumed that these processes can be assisted by postoperative intraocular inflammation. Previously, we reported that lens epithelial cell proliferation is enhanced by lymphocyte-conditioned medium (LCM). In this study we investigated the effect of LCM as well as of a culture medium conditioned by pigmented ciliary epitheilal cells (CBCM) on the expression of the smooth-muscle alpha-actin of the contractile cytoskeletal elements. METHODS: Explants of the anterior lens capsule of freshly enucleated bovine eyes were cultured in serum-free LCM and CBCM for 3 days, followed by fixation. Smooth-muscle alpha-actin was identified by indirect immunoflorescence. Explants cultured in serum-free bFGF-containing and TGF-beta containing medium served as control. RESULTS: Lens epithelial cells expressed smooth-muscle alpha-actin under the influence of LCM or TGF-beta. No smooth muscle alpha-actin could be detected under the influence of CBCM or bFGF. CONCLUSION: Our results demonstrate that secreted molecules of activated lymphocytes are able to induce the transformation of lens epithelial cells into contractile myofibroblasts and may be involved in the post-operative contraction of lens capsules.     

Expression of matrix metalloproteinases of human lens epithelial cells in the cultured lens capsule bag

AIM OF PURPOSE: To observe the different expression of matrix metalloproteinases (MMPs) between pre- and postoperation of sham cataract surgery in vitrohuman lens capsule bag model from the same donor eye in order to investigate a possible role of MMPs in posterior capsule opacification (PCO).Methods: Sham cataract surgeries were performed in six human donor eyes. Immunohistochemical staining was used to detect the expression of MMP-2 and -9 of human lens epithelial cells (LECs) on the anterior capsulorhexis. LEC migration on posterior capsule of human lens from the same donor eye was observed in a modified capsule bag model without pin. Total MMP-2 and -9 protein production were determined by enzyme-linked immunosorbent assay at days 2, 10, 20, and 30 postoperation, respectively. RESULTS: MMP-2 and -9 could not be detected immunohistochemically on the anterior capsulorhexis preoperation of cataract. Lens epithelia cells at the equator began to migrate by day 4. A confluent monolayer of lens epithelia cells was present on the posterior capsule at day 20. Total MMP-2 and -9 protein production increased with time with maximum levels reached on day 30. CONCLUSION: MMP-2 and -9 were showed to be upregulated following sham cataract surgery. MMP expression could play an important role in PCO.