【摘要】目的了解转化生长因子-β₁(transforming growth factor-β₁,TGF-β₁)对培养的人眼小梁细胞微丝肌动蛋白的影响,探讨房水引流阻力在原发性开角型青光眼(primary&127;open angle glaucoma,POAG)的病因和发病机制中的作用。方法小梁网来源于除眼部疾患以外的各种原因死亡的6岁以下儿童,在24h内取材于34例68只正常眼球。用各相对质量浓度的TGF-β₁及中和抗体进行人眼小梁细胞原代和传代培养，于光镜和电镜下观察培养的人眼小梁细胞。结果根据培养细胞的生长特性和形态特征,以及细胞外基质染色特点等,确定培养的细胞为人眼小梁细胞。各浓度组与对照组比较,TGF-β₁使张力纤维束增多,F-肌动蛋白含量增加,β-肌动蛋白mRNA表达增强;其上调作用呈时间依赖性;TGF-β₁中和抗体可拮抗此作用。TGF-β₁在高浓度、作用时间延长时,使汇合前的小梁细胞生长增殖抑制、细胞膜破裂及细胞脱落。结论将逆转录-半定量多聚酶链式反应用于POAG研究领域,可加速、加强定量分析在基因水平上的POAG病因研究。TGF-β₁在一定范围内上调培养的人眼小梁细胞微丝肌动蛋白,可加强小梁细胞收缩能力,有利于降低小梁网房水引流阻力。

Influenceof transforming growth factor-β₁ on actin in cultured human trabecular cellsZHONG Lichun, LI Meiyu. Department of Ophthalmology, First School of Clinical Medicine, Beijing Medical University, Beijing 100034

【Abstract】ObjectiveTo understand transforming growth factor-β₁ (TFG-β₁) influencing microfilament actins in cultured human trabecular cells (HTCs) and approach to the effect of resistance of aqueous outflow in the pathogenesis and mechanism of primary open angle glaucoma (POAG).MethodsTrabecular meshwork was collected from 68 eyeballs of 34 human donors under 6 years old within 24 hours after death. HTCs were primarily cultured and subcultured. Cultured cells were observed by light and electron microscopes. Laminin (LN) and IVcollagen (IVC) in extracellular matrix (ECM) were immunohistochemically stained by S-P method. Various doses (0, 1, 5 ng/ml) of TFG-β₁ and 30 μg/ml neutralizing antibody of TFG-β₁ were respectively added to the third passage cells before confluence and acted for 6, 12, 24 hours. Stress fibers stained with fluorescein isocyanate (FITC)-phalloidin were observed under light and electron microscopes. Various doses (0, 0.5, 1, 5 ng/ml) of TFG-β₁ and 30 μg/ml neutralizing antibody of TFG-β₁ were respectively added to the third passage cells after confluence for one week and acted for 6, 12, 24, 48 hours. The positive numbers of labeled F-actins were quantitatively evaluated by flow cytometry. Various doses (0, 0.5, 1, 5 ng/ml) of TFG-β₁ and 30 μg/ml neutralizing antibody of TFG-β₁ were respectively added to the third passage cells after confluence for one week and acted for 6, 12, 24, 36, 48 hours. The amount of β-actin mRNA expression was semi-quantitatively evaluated by densitometry with reverse transcription-polymerase chain reaction (RT-PCR) using specific oligonucleotide primers for β-actin and glyceraldehyde-3-phophate dehydrogenase (G3PDH) as an internal standard. ResultsAccording to the growing characteristics and morphological features of the cultured cells, they were identified as HTCs. In comparison with the control group, TGF-β₁ could increase the stress fibrous bundle, the contents of F-actins and the expression of β-actin mRNA; the effects of TFG-β₁ appeared time dependent and were antagonized by neutralizing antibody of TFG-β₁. When TFG-β₁ was at higher concentration and longer acting time, cell proliferation was inhibited, cell membrane ruptured and cells peeled off in cultured HTCs before confluence.ConclusionsApplication of reverse transcription-semiquantitative PCR can promote the pathogenic research of POAG at genetic level, in a certain extent, TFG-β₁ can obviously upregulate microfilament actins in the confluent HTCs, enhance the contractility of trabecular cells and decrease the resistance of aqueous outflow in trabecular meshwork.

【Key words】Cells, trabecularGrowth factorActinGlaucoma, open angle

原发性开角型青光眼(primary open angle glaucoma,POAG)是致盲的主要疾病之一,POAG的视神经损害是由于小梁网房水引流阻力增加使眼压升高所致。受各种因素调节的小梁细胞形态与功能异常使小梁网清除和吞噬能力下降,细胞外基质堆积等,以致引起房水引流阻力增加。细胞骨架微丝肌动蛋白是小梁细胞功能活动的主要结构基础,其数量与质量直接影响到小梁细胞的形态与功能。转化生长因子-β₁(transforming growth factor-β₁,TGF-β₁)是活跃于眼部的多肽细胞生长因子,推测TGF-β₁可能通过调节小梁细胞微丝肌动蛋白而参与小梁网房水引流活动。

材料与方法

一、标本来源

小梁网来源于除眼部疾患以外的各种原因死亡的6岁以下儿童,在24h内取材于34例68只正常眼球。

二、人眼小梁细胞体外培养

在解剖显微镜下找到Schawlbe线和巩膜突的小梁网前、后界,用玻璃针插入Schlemm管内,在玻璃针与巩膜突之间环形切开Schlemm管,从Schwalbe线处分离下小梁网。小梁网组织块原代培养,胰蛋白酶消化传代培养,传3代细胞用于实验研究。

三、培养细胞光镜和电镜标本制备

传3代汇合前和汇合后的细胞铺片分别进行Wright-Giemsa染色、常规电镜标本制备及细胞外基质染色,包括层粘连蛋白(Laminin,LN)与Ⅳ型胶原(Ⅳ collagen,Ⅳ C)免疫组化S-P法染色;用相对质量浓度TGF-β₁为0、1、5ng/ml和TGF-β₁中和抗体为30μg/ml,作用于传3代汇合前的小梁细胞铺片6、12、24h,经异硫氰酸荧光素-鬼笔环肽(fluorescien isothiocyanate, FITC-phalloidin)染色;用光镜和电镜观察培养的细胞、细胞外基质及张力纤维束形态。

四、流式细胞计测定

相对质量浓度TGF-β₁为0、0.5、1、5ng/ml,TGF-β₁中和抗体为30μg/ml,分别作用于传3代汇合后1周的小梁细胞6、12、24、48h，共20个样品经FITC-phalloidin染色,流式细胞仪定量测定F-肌动蛋白的相对荧光强度。

五、细胞总RNA提取和逆转录-多聚酶链式反应(reverse transcription-polymerase chain,RT-PCR)

TGF-β₁相对质量浓度为0、0.5、1、5ng/ml,分别作用于传3代汇合后1周的小梁细胞3、6、12、24、36、48h,共24个样品,每个样品细胞计数后用异硫氢酸胍、酚、氯仿等一步法抽提细胞总RNA^［1］。每个样品在进行RT-PCR实验前RNA浓度调整为1μg/μl。人的β-肌动蛋白引物序列(460bp),上游:5′CAG AGC AAG AGA GGC ATC3′,下游:5′TGT CAC GCA CGA TTT CCC GC3′;内参照人的甘油醛-3-磷酸酯脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,G3PDH)引物序列(214bp),下游:5′CCA TCA CCA TCT TCC A3′,5′AGG CTG TTG TCA TAC TTC TC 3′。热循环RT-PCR仪条件设置:43℃，30min；94℃，1min；50℃，30s；72℃，1.5min；15～36个循环数。以PCR产物进行2%琼脂糖电泳,用可见紫外光检测仪观察与摄像,薄层光密度扫描仪半定量测定DNA电泳带光密度吸收值。

六、统计学处理方法

选用方差分析的F检验、q检验及直线相关分析。

结果

一、培养细胞的细胞鉴定

原代培养6～28天细胞从组织块向外生长,6～8周细胞围绕在组织块周围形成单细胞层。传代培养24h后多数细胞已贴壁,2或3周细胞汇合形成单细胞层(图1)。汇合后的细胞均匀一致且数量衡定,细胞可稳定1或2个月,2个月后开始降解,可传至8或9代。光镜下见汇合前细胞形态呈三角形或梭形,汇合后细胞形成一单细胞层,胞突重叠,但胞体不重叠。扫描电镜下细胞表面有均匀分布的短状微绒毛和包涵体小囊突起。透射电镜下均可见各种细胞器丰富、核膜不明显,有黑色峰带状楔形异染色质位于核膜内侧。培养细胞的细胞外基质免疫组化S-P法染色显示LN和ⅣC阳性。综上所述,从培养细胞的生长特性、形态特征和LN、ⅣC阳性S-P法染色特点确定培养的细胞为人眼小梁细胞。