Apoptosis of Human Trabecular Meshwork Cells Induced by Transforming Growth Factor-p2 in vitro

Summary: Whether transforming growth factor-β2 (TGF-β2) induces apoptosis of human trabecular meshwork cells was investigated in vitro. Cultured 3-5 passage human trabecular meshwork cells were treated with 0 (control), 0.32, 1, 3.2 ng/ml TGF-β2 for 48 h and divided into control group and experimental group. The apoptosis of human trabecular meshwork cells was examined by transmisson electron microscopy, TUNEL technique and flow cytometry. The results showed characteristic morphologic changes of apoptotic cells were observed under transmission electron microscopy.DNA fragmentation of human trabecular meshwork cells was found by TUNEL technique. Quantitative analysis of flow cytometry showed that percentages of apoptotic human trabecular meshwork cells were (2.79±0.44) %, (4.43±1.17) % and (9. 60±2.05) % respectively with different concentrations [1 ng/ml (P＜0. 05), 3.2 ng/ml (P＜0.01)] of TGF-β2 with the difference being significant between experimental group and control group[(1. 41±0.34) %]. It was concluded that TGF-β2 can induce apoptosis of human trabecular meshwork cells in vitro and may be involved in the decrease of trabecular meshwork cells in the patients with primary open angle glaucoma and aging of normal people.     

Apoptosis of human trabecular meshwork cells induced by transforming growth factor-beta2 in vitro.

Whether transforming growth factor-beta2 (TGF-beta2) induces apoptosis of human trabecular meshwork cells was investigated in vitro. Cultured 3-5 passage human trabecular meshwork cells were treated with 0 (control), 0.32, 1, 3.2 ng/ml TGF-beta2 for 48 h and divided into control group and experimental group. The apoptosis of human trabecular meshwork cells was examined by transmission electron microscopy, TUNEL technique and flow cytometry. The results showed characteristic morphologic changes of apoptotic cells were observed under transmission electron microscopy. DNA fragmentation of human trabecular meshwork cells was found by TUNEL technique. Quantitative analysis of flow cytometry showed that percentages of apoptotic human trabecular meshwork cells were (2.79 +/- 0.44)%, (4.43 +/- 1.17)% and (9.60 +/- 2.05)% respectively with different concentrations [1 ng/ml (P<0.05), 3.2 ng/ml (P<0.01)] of TGF-beta2 with the difference being significant between experimental group and control group [(1.41 +/- 0.34)%]. It was concluded that TGF-beta2 can induce apoptosis of human trabecular meshwork cells in vitro and may be involved in the decrease of trabecular meshwork cells in the patients with primary open angle glaucoma and aging of normal people.     

大白鼠前房内灌注正电荷聚乙烯亚胺后小梁的超微结构变化

目的：研究大白鼠前房内灌注不同浓度正电荷聚乙烯亚胺(PEI)后小梁的超微结构变化。方法：12只大白鼠分别用生理盐水稀释的O．05％，O．5％和5％PEI在正常眼压下经前房灌注，维持20min。组织制成标本后用电镜现察。结果：在O．05％PEI灌注组，仅在前房侧表层小梁内皮细胞膜上观察到细小的PEI颗粒。在O．5％PEI灌注组，在小梁的内皮细胞膜表面，细胞外成份和管旁组织，以及Schlemm's管的内皮细胞膜观察到大小较一致的PEI颗粒。在5％PEI灌注组，所见PEI颗粒范围与O．5％PEI组基本相同，但PEI颗粒较其大而多，且小梁部分结构被破坏。结论：PEI经小梁进入Schlemm’s管与浓度有关，高浓度PEI可使小梁部分组织结构被破坏。     

重组人骨形态发生蛋白7对原发性开角型青光眼小梁网细胞增殖的影响

目的 建立原发性开角型青光眼(POAG)小梁网细胞体外原代培养体系; 分析不同终浓度重组人骨形态发生蛋白7(rhBMP7)对POAG小梁网细胞增殖的影响; 探讨rhBMP7与POAG发生、进展的相关性。 方法 取术中切除的带小梁网组织块(未使用丝裂霉素C),进行体外原代及传代培养,取3代小梁网细胞,在CFDA SE标记后,分别加入终浓度为0(对照组),20,50,80,100,200 ng/mL的rhBMP7无血清培养基,采用CCK8、荧光显微镜、流式细胞仪等方法检测POAG小梁网细胞增殖情况。 结果 细胞经传代培养,经鉴定为POAG小梁网细胞; 采用CCK8法检测发现:经终浓度为0(对照组),20,50,80,100,200 ng/mL的rhBMP7处理后,POAG小梁网细胞吸光度(OD值)分别为:(0.561 2±0.026 9),(0.724 2±0.039 3),(1.416 0±0.016 2),(1.740 4±0.039 2),(1.853 8±0.014 5),(1.936 4±0.054 6); 实验组细胞增殖率分别为1.37%,2.96%,3.70%,3.96%,4.15%; 实验组与对照组及各实验组间增殖率比较,差别具有统计学意义(P<0.05); 荧光显微镜示:随着rhBMP7终浓度的增加,经CFDA SE标记的POAG小梁网细胞荧光染色变浅、细胞量及密度增加; 采用流式细胞仪检测经20,50,80,100,200 ng/mL终浓度rhBMP7干预的POAG患者小梁网细胞,分裂、增殖细胞所占的比例分别为17.85%,18.63%,20.10%,27.45%,72.41%。 结论 运用组织块培养法,可体外原代培养出POAG患者的小梁网细胞; rhBMP7在一定程度上可促进POAG小梁网细胞的增殖,且在一定范围内呈剂量依赖性。     

Apoptosis of human trabecular meshwork cells induced by transforming growth factor-β<Subscript>2</Subscript><Emphasis Type="Italic">in vitro</Emphasis><Emphasis Type="Italic">in vitro</Emphasis>

Summary Whether transforming growth factor-β₂ (TGF-β₂) induces apoptosis of human trabecular meshwork cells was investigatedin vitro. Cultured 3–5 passage human trabecular meshwork cells were treated with 0 (control). 0. 32. 1, 3. 2 ng/ml TGF-β₂ for 48 h and divided into control group and experimental group. The apoptosis of human trabecular meshwork cells was examined by transmisson electron microscopy. TUNEL technique and flow cytometry. The results showed character istic morphologic changes of apoptotic cells were observed under transmission electron microscopy. DNA fragmentation of human trabecular meshowork cells was found by TUNEL technique. Quantitative analysis of flow cytometry showed that percentages of apoptotic human trabecular meshwork cells were (2.79±0.44)%. (4.43±1.17)% and (9.60±2.05)% respectively with different concentrations [1 ng/ml (P<0.05), 3.2 ng/ml (P<0.01)] of TGF-β₂ with the difference being significant between experimental group and control group [(1.41±0.34)%]. It was concluded that TGF-β₂ can induce apoptosis of human trabecular meshwork, cellsin vitro and may be involved in the decrease of trabecular meshwork cells in the patients with primary open angle glaucoma and aging of normal people. CAO Yang, male, born in 1972, M. D., Ph. D., Associate Professor This project was supported by a grant from the National Natural Sciences Foundation of China (No. 38970758).     

Antagonistic Effects of Tranilast on Proliferation and Collagen Synthesis Induced by TGF-β2 in Cultured Human Trabecular Meshwork Cells

Whether tranilast had antagonistic effect on proliferation inhibition and collagen synthesis promotion induced by TGF-β2 in cultured human trabecular meshwork cells was investigated. Suspension of 1 × 104 cultured human trabecular meshwork cells of 3-5 passage was distributed in each well of a 96-well disk and divided into control group and experimental group. After 24 h, 0 μg/ml (control), 12.5 μg/ml, 25 μg/ml, 50 μg/ml tranilast with 3.2 ng/ml TGF-β2 were added into the incubation medium. Another 24 h later, proliferation and collagen synthesis in cultured human trabecular meshwork cells were examined respectively by using tetrazolium-based semiautomated colormetric (MTT) assay and 3 H-proline incorporation with liquid scintillation technique. The results showed absorbance (A) values of the experimental groups were 0. 9036 ± 0. 3017, 1.1361 ±0.1352, 1.2457 ±0.1524 according to the different concentrations of tranilast, and 0. 8956 ±0. 1903 of the control group. In comparison with the control group, 25 μg/ml (q′= 3. 23, P＜0.05), 50 μg/ml (q=4.70, P＜0.01) tranilast significantly antagonized the decrease of the A values induced by TGF-β2 in the cultured human trabecular meshwork cells. In comparison with the control group [817.37±124.21 cpm/104 cells], 12.5 μg/ml (620.33±80.46 cpm/104 cells, q′=4.26, P＜0.05),25 μg/ml (594. 58±88.13 cpm/104 cells, q′=4. 81, P＜0.01), 50 μg/ml (418. 64±67.90 cpm/104 cells, q′=8.62, P＜0.01) tranilast significantly inhibited the incorporation of 3 H-proline into the cultured human trabecular meshwork cells promoted by TGF-β2 in a dose-dependent manner. It was concluded that tranilast had the antagonistic effect on the proliferation inhibition and collagen synthesis promotion induced by TGF-β2 in the cultured human trabecular meshwork cells.     

Effect of transforming growth factor-β2 on phagocytosis in cultured bovine trabecular meshwork cells

Summary The effect of transforming growth factor-β₂ (TGF-β₂) on phagocytosis in bovine trabecular meshwork cellsin vitro was investigated. After the cultured bovine trabecular meshwork cells were treated with 0 ng/ml, 0.32 ng/ml, 1 ng/ml, 3. 2 ng/ml TGF-β₂ for 24 h, latex beads were added into the incubation medium, and the numbers of the latex beads in 20 adjacent cells were counted under a microscope 24 h later, after treatment with Wright’s stain. Our results showed that the average numbers of the latex beads in the trabecular meshwork cells treated with TGF-β₂ of different concentrations were 53. 1±1. 7 beads/cell, 56. 4±2. 9 beads/cell and 77. 9±6. 5 beads/cell respectinvely, in comparison with 45. 5 ±3. 3 beads/cell of the control group. TGF-β₂ significantly increased the number of the latex beads phagocytosed by cultured bovine trabecular meshwork cells in a dose-dependent manner. TGF-β₂ could promote the phagocytosis of bovine trabecular meshwork cellsin vitro. It may be involved in the cellularity decrease of the trabecular meshwork in the patients of primary open angle glaucoma through promoting the phagocytosis of trabecular meshwork cells. This project was supported by a grant from the National Natural Science Foundation of China (No. 38970758).     

压力对猪眼小梁细胞表达ELAM-1、MMPs/TIMPs的影响

目的 培养猪眼小梁细胞,研究压力对体外培养的猪眼小梁内皮细胞白细胞粘附分子-1 (ELAM-1)、基质金属蛋白酶MMP-2、MMP-3及基质金属蛋白酶组织抑制剂TIMP-2表达的影响。方法 培养猪眼小梁细胞并鉴定,建立细胞水平的青光眼模型,对传第3代猪眼小梁细胞分别施加20、40、60 、80mmHg压力作为实验组,0mmHg为对照组。培养6h后行ELAM-1免疫组织化学SP法染色,培养24h后行MMP-2、MMP-3和TIMP-2免疫组织化学SP法染色,并对染色结果进行统计学分析。结果 培养细胞为猪眼小梁细胞,正常小梁细胞不表达ELAM-1,压力为40、60、80mmHg时,ELAM-1的表达同0、20mmHg组相比表达明显增加。正常小梁细胞可以少量表达MMP-2、MMP-3及TIMP-2。压力为40、60mmHg时,MMP-2、TIMP-2的表达同0、20、80mmHg相比表达明显增加,压力不影响小梁细胞MMP-3的表达。结论 一定范围内压力的变化可以促进猪眼小梁细胞表达ELAM-1,可以改变MMPs／TIMPs之间的平衡状态,进而影响小梁细胞外基质(ECM)的代谢,改变房水外流阻力, ELAM-1、MMPs在青光眼的发病中可能发挥重要作用。     

A Study of Toxicity of 5-Fluorouracil on Bovine Trabecular Meshwork Cells in Vitro

5- fluorouracil( 5 - Fu) is widely used as an ad-junct in the surgery of ocular diseases such as glauco-ma[1-3 ] ,proliferative vitreoretinopathy[4 ] ,etc.Be-cause of its cytotoxicity,itmay cause corneal epithe-lial defects,conjunctival wound leak[5,6] in filteringsurgery or othercomplications.The trabecularmesh-work cells in the patients with glaucoma may alreadyhave sustained some previous damage,so it is crucialto make sure whether 5 - Fu would cause further in-jury of the cells.Therefore,t…     

Antagonistic effects of tranilast on proliferation and collagen synthesis induced by TGF-beta2 in cultured human trabecular meshwork cells.

Whether tranilast had antagonistic effect on proliferation inhibition and collagen synthesis promotion induced by TGF-beta2 in cultured human trabecular meshwork cells was investigated. Suspension of 1 x 10(4) cultured human trabecular meshwork cells of 3-5 passage was distributed in each well of a 96-well disk and divided into control group and experimental group. After 24 h, 0 microg/ml (control), 12.5 microg/ml, 25 microg/ml, 50 microg/ml tranilast with 3.2 ng/ml TGF-beta2 were added into the incubation medium. Another 24 h later, proliferation and collagen synthesis in cultured human trabecular meshwork cells were examined respectively by using tetrazolium-based semiautomated colormetric (MTT) assay and 3H-proline incorporation with liquid scintillation technique. The results showed absorbance (A) values of the experimental groups were 0.9036 +/- 0.3017, 1.1361 +/-0.1352, 1.2457 +/- 0.1524 according to the different concentrations of tranilast, and 0.8956 +/-0.1903 of the control group. In comparison with the control group, 25 microg/ml (q'= 3.23, P< 0.05), 50 microg/ml (q'=4.70, P<0.01) tranilast significantly antagonized the decrease of the A values induced by TGF-beta2 in the cultured human trabecular meshwork cells. In comparison with the control group [817.37+/-124.21 cpm/10(4) cells], 12.5 microg/ml (620.33+/-80.46 cpm/10(4) cells, q'= 4.26, P<0.05), 25 microg/ml (594.58+/-88.13 cpm/10(4) cells, q'=4.81, P<0.01), 50 microg/ml (418.64+/-67.90 cpm/10(4) cells, q'=8.62, P<0.01) tranilast significantly inhibited the incorporation of 3H-proline into the cultured human trabecular meshwork cells promoted by TGF-beta2 in a dose-dependent manner. It was concluded that tranilast had the antagonistic effect on the proliferation inhibition and collagen synthesis promotion induced by TGF-alpha2 in the cultured human trabecular meshwork cells.     

Transforming Growth Factor-β_2 Gene Cloning and Protein Expression in Human Trabecular Meshwork Cells

Primary open- angle glaucoma (POAG) is aleading cause of blindness,which involves optic neu-ropathy accompanied by characteristic visual field de-fects and is often associated with elevated intraocularpressure due to disturbance ofaqueoushumor outflowthrough the trabecularmeshwork (TM) .The patho-physiology of the TM in POAG has been character-ized by an increasein extracellularmatrix componentsand a decrease in the number of TM cells[1,2 ] .It hasbeen found that,compared with the non- P…     

氧化应激对猪眼小梁细胞内皮细胞白细胞黏附分子-1表达的影响

目的 研究氧化应激对猪眼小梁细胞内皮细胞白细胞黏附分子-1（ELAM-1）表达的影响,探讨氧化应激诱导的ELAM-1的表达与白细胞介素1α（IL-1α）的关系。方法原代培养的猪眼小梁细胞经过血清饥饿培养后,用不同浓度白细胞介素-1受体拈抗剂（IL-1rα）处理或直接用1mmol／L的H2O2刺激后,用免疫细胞化学法检测小梁细胞ELAM-1的表达。结果 H2O2处理的原代培养的猪眼小梁细胞中ELAM-1表达呈阳性,高浓度（180μg／ml和600μg／m1）拈抗剂IL-1rα预处理的猪眼小梁细胞ELAM-1表达呈阴性。结论 氧化应激可诱导猪眼小梁细胞表达ELAM-1。在体外细胞培养体系,高浓度的IL-1α可拮抗氧化应激对ELAM-1表达的作用。     

开放式压力控制培养系统对人眼小梁细胞超微结构及纤维连接蛋白合成的影响

目的采用开放式压力控制培养系统，研究压力对人眼小梁细胞（HTMCs）超微结构及纤维连接蛋白（FN）合成的影响。方法组织块法体外培养HTMCs，选取第5代细胞分为实验组和对照组。对照组不施加压力，实验组采用开放式压力控制培养系统分别施加20、40、608、0 mmHg（1 mmHg=0.133 kPa）的压力，压力作用时间分别为12、24、36 h。在光学显微镜、透射电镜下观察HTMCs形态及超微结构的变化。采用免疫组化方法和图像分析法检测FN的表达变化。结果透射电镜显示：20 mmHg压力作用12、24、36 h后与对照组比较，HTMCs超微结构未见明显改变；40mmHg压力作用12、24、36 h后HTMCs胞质内出现大小不等的空泡；60、80 mmHg压力作用12、24、36 h后，HTMCs出现线粒体嵴变得短而少或者丧失、溶酶体髓样结构增多等不可逆性改变。免疫组化结果显示体外培养的HTMCs均阳性表达FN；图像分析显示：20 mmHg压力作用12、24、36 h后HTMCs合成FN与对照组比较差异均无显著性意义（均P〉0.05）；40 mmHg压力作用12、24、36 h后HTMCs合成FN明显增加，差异均有显著性意义（均P〈0.01）；而60、80 mmHg压力作用12、243、6 h后HTMCs合成FN则逐渐下降（均P〈0.01），但与对照组比较仍有不同程度增高（均P〈0.01）。结论环境压力升高可以引起小梁细胞形态、超微结构以及FN的表达发生变化，从而可能在原发性开角型青光眼的发生和发展过程中发挥重要作用。     

PGF_(2α)类抗青光眼药Unoprostone对培养猴睫状肌细胞MMP-2表达的影响

目的：研究ＰＧＦ２α类抗青光眼药物Ｕｎｏｐｒｏｓｔｏｎｅ对培养猴睫状肌细胞及小梁网细胞中ＭＭＰ－２表达的影响。方法：猴睫状肌及小梁网细胞融合成单层以后,暴露于不同浓度的Ｕｎｏｐｒｏｓｔｏｎｅ代谢物Ｍ１、Ｍ２或ＰＧＦ２α中,采用Ｚｙｍｏｇｒａｐｈｙ技术比较其中ＭＭＰ－２活性。结果：Ｚｙｍｏｇｒａｐｈｙ技术对睫状肌细胞培养液中ＭＭＰ－２的定量分析表明：１０－８ｍｏｌ／Ｌ,１０－７ｍｏｌ／Ｌ,１０－６ｍｏｌ／Ｌ的Ｕｎｏｐｒｏｓｔｏｎｅ代谢物Ｍ１及Ｍ２增强了ＭＭＰ－２活性表达,并且成剂量增强关系。１０－７ｍｏｌ／Ｌ,１０－６ｍｏｌ／Ｌ的Ｍ１及Ｍ２对小梁网细胞中ＭＭＰ－２表达无明显影响。结论：在通过ＭＭＰ降解细胞外基质这方面作用机理上,Ｕｎｏｐｒｏｓｔｏｎｅ可能作用于猴葡萄膜—巩膜房水流出路而对传统通路无明显影响。     

青光眼患者小梁网中ONOO^-的表达及与眼压升高关系的研究

目的检测正常人和原发性急性闭角型青光眼(primary acute angle closure glaucoma,PAACG)及原发性慢性闭角型青光眼(primary chronic angle closure glaucoma,PCACG)患者小梁网组织标本中过氧亚硝基阴离子(peroxynitrite,ONOO-)的表达,探讨小梁网中ONOO-的变化、分布及与青光眼的关系。方法取正常眼6例及PAACG患者14例18只眼及PCACG患者12例15只眼小梁网组织标本,应用形态学观察,免疫组织化学方法检测小梁细胞ONOO-的标志物硝基酪氨酸(nitrotyrosine,NT)的表达,并采用计算机图像分析系统对检测结果进行分析。结果正常人的小梁网组织标本NT的表达呈弱阳性或不表达,PAACG组小梁网组织中NT呈强阳性表达,PCACG组小梁网组织中NT呈阳性表达,3组间的差异均具有统计学意义(F=25.32,P<0.01),眼压与小梁网NT着色强度呈相关关系(P<0.01)。结论正常人的小梁网细胞ONOO-微弱表达而PAACG与PCACG患者小梁网细胞ONOO-大量表达且其表达量随眼压的升高而增多,表明眼压升高可导致ONOO-过量生成,从而损伤小梁网细胞,进一步加重青光眼眼压升高。     

体外培养的人眼小梁细胞的观察

自1986年来，我们共培养99只人眼的小梁组织，原代培养成活率为18.2％，其中供体年龄小于2岁者成活率22.5％；在24h内取材者为27.3％。共传14～16代，细胞生长呈有限性。成活的小梁细胞在第3～7代生长最快，细胞形态与活体小梁细胞相似。研究这些细胞将有助于我们了解小梁细胞形态和功能变化的特点。     

人眼组织小梁网细胞的体外培养及鉴定的新方法

目的探讨人原代小梁网细胞的体外培养,以及利用其特性建立一种鉴定小梁网细胞的新方法。方法从眼球破裂伤病人的眼球以及角膜移植后剩余的角膜环分离出小梁网组织,利用组织块贴壁法以及消化法对人原代小梁网细胞进行体外培养。倒置显微镜下观察细胞生长状态并利用CCK8法检测其生长速率。利用细胞免疫荧光技术对所培养的细胞进行纤维连接蛋白、Ⅳ型胶原蛋白、层黏连蛋白、水通道蛋白-1等蛋白的染色鉴定。并利用100 nmol/L地塞米松对所培养的细胞诱导10 d,通过荧光定量PCR和western blotting方法检测myocilin的表达水平以确定所培养的细胞是否为小梁网细胞。结果小梁网组织块贴壁培养1~2周后,开始有细胞从组织块旁向外长出,并逐渐增多。传代后小梁网细胞在第1~4天生长较快,第5~7天生长速度有所减慢,但依然显著高于第4天（P < 0.01）。所培养的细胞纤维连接蛋白、Ⅳ型胶原蛋白、层黏连蛋白、水通道蛋白-1的免疫荧光染色均呈阳性。地塞米松诱导后,与对照组相比,小梁网细胞中myocilin mRNA和蛋白表达水平均明显上升（P < 0.01）。结论本实验中所培养的细胞通过对其特点进行检测,确定所培养的细胞为人原代小梁网细胞。     

电压门控性氯通道3在高眼压模型大鼠小梁网中的表达

目的: 检测电压门控性氯离子通道3(ClC-3)在大鼠小梁网中的表达情况,探讨其在青光眼发病机制中的可能作用。方法: 30只健康雄性大鼠。左眼作为正常对照,右眼前房内注射玻璃酸钠,每周1次,连续10周,制备高眼压模型,每次全麻后玻璃酸钠注射前采用Tono-Pen XL眼压计测量双眼眼压并记录。实验分为正常对照组、玻璃酸钠注射3周组和10周组。分别取3组大鼠的小梁网组织,采用RT-PCR法检测ClC-3mRNA表达水平,免疫组织化学法检测ClC-3蛋白的表达。结果: 玻璃酸钠注射后1、3和10周,大鼠右眼眼压均较注射前增高(P<0.01),注射后1周眼压值较注射前轻微增高,注射后3周眼压较注射前明显增高,但眼压值未达到高眼压成模标准,注射后10周眼压值较注射前明显增高,达到高眼压成模标准。RT-PCR法检测,与正常对照组比较,玻璃酸钠注射3周组大鼠小梁网组织中ClC-3 mRNA表达水平明显增高(t=7.88,P<0.05);玻璃酸钠注射10周组大鼠小梁网组织中ClC-3 mRNA表达水平明显降低(t=15.93,P<0.05)。免疫组织化学检测,3组大鼠小梁网组织中ClC-3蛋白表达均为阳性,玻璃酸钠注射3周组大鼠小梁网组织中ClC-3蛋白表达强度较正常对照组大鼠增强,玻璃酸钠注射10周组大鼠小梁网组织中ClC-3蛋白表达强度较正常对照组大鼠减弱。结论: 大鼠小梁组织中存在ClC-3的表达,ClC-3可能与小梁网的病理调节有关联,进而参与青光眼的发病。