ALK5在青光眼滤过术后滤过道瘢痕化中的调控作用

发布时间：2018-05-13 13:34

  本文选题：青光眼 + 滤过性手术　； 参考：《复旦大学》2012年博士论文

【摘要】：青光眼滤过术后滤过泡瘢痕化是手术失败的最主要原因。失败的滤过性手术的滤过泡结膜上皮下有异常增厚的致密胶原纤维结缔组织,伴有成纤维细胞的活跃增生,阻塞滤过道,从而失去房水引流功能。目前手术中经常联合使用的抗代谢药物,如五氟脲嘧啶(5-FU),丝裂霉素(mitomycin, MMC)等,虽然可以减少术后滤过道瘢痕形成,提高手术的成功率,但其抗代谢作用也可导致一系列术后低眼压、滤过泡渗漏等并发症,因此探索更加安全有效的治疗方法对抗青光眼术后瘢痕化有着重要的意义。 细胞表型转化是纤维化过程中重要的细胞学基础,而转化生长因子-β(TGF-β)是成纤维细胞表型转化,进一步迁移、增殖,合成细胞外基质,导致滤过泡瘢痕化的重要细胞因子。人结膜下Tenon's囊成纤维细胞(human tenon's capsule fibroblast, HTF)在青光眼术后滤过道瘢痕化的过程中扮演了主要的角色。在手术或损伤条件下,TGF-β水平增加,使局部的成纤维细胞(fibroblast,FB)激活并转化成肌成纤维细胞(myofibroblast, MF)启动伤口愈合反应。MF在伤口愈合的不同时期均起到重要作用,一旦创伤愈合,MF一般迅速恢复为FB或进入凋亡的程序,若MF持续存在,则会导致细胞的过度增殖、细胞外基质的合成增多,瘢痕形成,滤过道过早愈合,房水引流通道被阻,致使手术失败。 由于TGF-β在细胞表型转化中的关键作用,越来越多的研究选择其作为研究靶点,通过降低TGF-β表达来抑制细胞表型转化,从而降低瘢痕化的程度。但由于TGF-β的下游信号通路非常复杂且相互形成交错网状,所以太低位点的调节可能对其促进细胞增殖及表型转化的作用不甚明显。因此我们考虑在信号通路的较高的位点：TGF-β膜受体,来进行干预,借此来探究MF表型转化的过程,以及纤维化的可能机制。 TGF-β的受体主要有3种,分别称为Ⅰ型(TGF-βR Ⅰ或活化素受体样激酶,activin receptor-like kinase, ALK)、Ⅱ型受体(TGF-βRⅡ)和Ⅲ型受体(TGF-βRⅢ)。其中Ⅰ、Ⅱ型受体主要参与信号通路的转导,两者的复合物与TGF-β相结合,导致Ⅰ型受体磷酸化,并激活下游的信号通路。大多数的哺乳动物细胞中,存在的RI是ALK5,所以人们把ALK5作为研究TGF-β受体的重点。 在前期的工作中,本研究组选择一种ALK5抑制剂SB-431542作为工具,研究抑制ALK5对青光眼术后滤过道瘢痕化的作用,结果显示滤过术后局部球结膜下注射SB-431542可以抑制滤过道瘢痕形成。由此,我们认为,ALK5可能与调控TGF-p致瘢痕化有重要关系,我们希望通过建立细胞及动物的瘢痕化模型,并人为调控其中ALK5的水平,观察其对细胞表型转化及纤维化的影响作用,探索ALK5抑制剂在结膜损伤后局部组织重塑中的作用及其细胞学机制,为抑制青光眼滤过道瘢痕化的研究提供更多的理论依据。 目的：研究细胞因子TGF-β1对HTF细胞及动物青光眼术后滤过道瘢痕化模型中,活化素受体样激酶5(activin receptor-like kinase5, ALK5)表达的调控作用,并通过慢病毒转染技术建立ALK5基因高表达的细胞及动物模型,人为调控ALK5表达,研究ALK5水平对青光眼滤过术后滤过道瘢痕化的影响。 材料和方法： 第一部分：原代培养人结膜下Tenon's囊成纤维细胞,并进行细胞鉴定。取对数生长期细胞,以10μg/μl的TGF-β1诱导HTF,在不同的时间,采用Western Blot检测平滑肌肌动蛋白(α-SM-actin)和纤连蛋白(fibronectin, FN)蛋白表达,确定细胞发生表型转化及纤维化。再采用Western Blot和免疫细胞化学技术来鉴定ALK5蛋白的表达,并以实时定量PCR检测ALK5mRNA水平。 第二部分：采用Tronolab慢病毒载体系统,构建过表达ALK5基因的慢病毒载体,转染HTF细胞,获得过表达ALK5的HTF细胞。观察细胞生长特性,。采用普通培养基,以及加入ALK5抑制剂SB-431542的培养基分别培养转染及未转染的HTF细胞,观察细胞生长特性,MTT试验检测细胞增殖能力。并用10μg/μl的TGF-p1诱导HTF, Western Blot检测相关表型转化(α-SMA)、纤维化指标(FN)以及凋亡指标(Caspase-3)的表达。 第三部分：1.应用滤过性手术建立兔滤过道瘢痕化动物模型,选用24只健康新西兰白兔作为实验动物,随机分为3组：对照组、ALK5组、0.5mM SB-431542组。在全身麻醉下行左眼滤过性手术。术后观察、测量眼压(intraocularpressure,IOP)。术后第3、7、14、28天4个时间点各处死每组2只实验兔,摘除眼球,制作切片,免疫组化染色,光镜下观察滤过道α-SM-actin、ALK5及瘢痕化情况。2.对30只健康的新西兰白兔施行右眼滤过性手术。随机分为5组,对照组(NS+NS)、阴性对照组(NS+0.5mM SB-431542组)、高表达组(ALK5转染+NS)、高表达+低剂量给药组(ALK5转染+0.5mM SB-431542组)和高表达+高剂量给药组(ALK5转染+2.0mM SB-431542组),每组6只兔(6只眼,n=6)。术后观察并测量眼压。术后第28d处死实验兔,摘除实验眼,制作切片,免疫组化染色,检测a-SM-actin、ALK5及瘢痕化情况。 结果： 第一部分：贴壁法培养的人结膜下Tenon's囊组织,约3-5天可见组织块周围爬出梭形细胞,继续培养逐渐汇合成片,经传代后进行波形蛋白(vimentin)染色,鉴定为成纤维细胞。TGF-β1诱导HTF后,ALK5的mRNA表达有所上调,于12-24h达到高峰值。Western Blot显示蛋白表达水平也有所升高,于24-36h达到高峰值。细胞免疫荧光染色检测也同样显示,ALK5表达水平升高。 第二部分：实验成功构建了过表达ALK5基因的慢病毒载体,并获得了较高滴度的病毒液。采用慢病毒转染HTF细胞后,观察发现细胞增殖速度明显增加,并伴有凋亡现象,加入SB-431542后细胞生长速度及凋亡现象均有所逆转。MTT实验显示,转染GFP病毒的细胞与未转染的HTF细胞增值速度无明显变化,转染ALK5病毒的HTF细胞,增殖速度明显增加,而在培液中加入SB-431542后,细胞增殖速度明显降低。转染后,过表达ALK5的细胞,表达a-SMA以及FN蛋白水平有所下降,而加入ALK5抑制剂SB-431542后,该过程可被抑制。 第三部分：1.兔滤过性术后术眼的眼压各组均有所下降,术后第1天,平均IOP为5.63±1.73mmHg,P0.05。之后术眼眼压逐渐上升,到术后第10天,眼压逐渐恢复到手术前水平,为11.07±1.72mmHg,P0.05。之后兔眼眼压稍有上升趋势,术后28d,眼压相对术前稍有上升,为12.40±1.16mmHg,P0.05。组织学观察显示：术后滤过道周围早期以水肿及炎症细胞为主,之后逐渐出现较多增殖的成纤维细胞,晚期成纤维细胞数量减少,胶原纤维沉积增多,产生瘢痕组织。ALK5在结膜下成纤维细胞的表达在术后3天并不明显,到第7天开始逐渐增多,14天时结膜下有大量表达,至28天,表达量有所下降,但仍高于术前。转染ALK5的实验眼,可以见到结膜下阳性的转染细胞,周围伴有大量的成纤维细胞增生。2.各组在术后眼压均有所下降,对照组IOP=7.83±2.14,P0.05；阴性对照SB组：IOP=5.5±0.84mmHg,P0.05；转染ALK5组：IOP=5.17±1.60mmHg,P0.05；转染ALK5+SB0.5mM组：IOP=4.67±0.82mmHg,P0.05；转染ALK5+SB2.0mM组：IOP=5±1.10mmHg,P0.05,到术后第7-10d,眼压逐渐恢复到手术前水平,但各组间统计学差异不明显。组织学观察显示：对照组结膜下伤口周围纤维化明显,充满富含增生细胞的纤维化成分和明显胶原化的团状结缔组织；ALK5转染后,伤口附近及结膜下可见大量增生的成纤维细胞,比对照组和用药组明显增多,并伴有少量的胶原组织沉积。加入抑制剂后,术眼滤过道周围细胞增殖和瘢痕组织均有所减少,结膜下组织相对疏松。 结论：在人Tenon's囊成纤维细胞(HTF)表型转化以及兔眼滤过术后瘢痕化的模型中,都可以观察到TGF-βI型受体ALK5表达的增加,而通过慢病毒对HTF细胞及兔眼结膜下组织转染ALK5基因,并加入抑制剂SB-431542调控ALK5水平发现,ALK5高表达一方面能使成纤维细胞增殖加快,促进兔滤过性手术后滤过道瘢痕的形成,另一方面也可能诱导细胞的凋亡从而使纤维化得到控制。加入ALK5抑制剂SB-431542以后,可以抑制由其产生的细胞增殖以及瘢痕化作用。这说明ALK5在TGF-p引发的瘢痕化中,可能有重要的调控作用。
[Abstract]:Filtering bleb scar after glaucoma filtration is the main reason for the failure of the operation. Drugs such as five fluorouracil (5-FU) and Mitomycin (mitomycin, MMC) can reduce the postoperative scar formation and improve the success rate of the surgery, but their anti metabolic effects can also lead to a series of postoperative complications such as low intraocular pressure and filtration bleb leakage. Therefore, a safer and more effective treatment is explored to combat scar formation after glaucoma surgery. It is of great significance.
Cell phenotype transformation is an important cytological basis in the process of fibrosis, and transforming growth factor - beta (TGF- beta) is an important cytokine in the phenotype transformation of fibroblasts, which further migrate, proliferate and synthesize extracellular matrix, leading to the scarring of filtration vesicles. The subconjunctival Tenon's capsule fibroblast (HTF) is in the human conjunctival conjunctiva. (HTF) in the human conjunctiva of human conjunctiva (HTF) in the human conjunctiva In the process of glaucoma surgery, the filter plays a major role in the process of scar formation. Under the conditions of surgery or injury, the level of TGF- beta is increased, and the local fibroblasts (fibroblast, FB) are activated and converted into myofibroblast (myofibroblast, MF) to start the wound healing reaction.MF to play an important role in the different period of wound healing. After wound healing, MF generally quickly recovered to FB or into a program of apoptosis. If MF persisted, it could lead to excessive proliferation of cells, increase of extracellular matrix synthesis, scar formation, premature healing of filter channel and obstruction of aqueous drainage channel, which resulted in the failure of the operation.
Because of the key role of TGF- beta in cell phenotype transformation, more and more studies have chosen it as a target, reducing the phenotype transformation by reducing the expression of TGF- beta, thus reducing the degree of scar formation. But the downstream signal pathway of TGF- beta is very complex and interlaced with each other, so the regulation of the low point may be against it The role of promoting cell proliferation and phenotypic transformation is not very obvious. Therefore, we consider the higher locus of the signal pathway, TGF- beta membrane receptor, to intervene in order to explore the process of MF phenotypic transformation and the possible mechanism of fibrosis.
There are 3 main receptors for TGF- beta, which are called type I (TGF- beta R I or activin receptor like kinase, activin receptor-like kinase, ALK), type II receptor (TGF- beta R II) and type III receptor (TGF- beta R III). In which type I, type II receptors are mainly involved in signal transduction, and the complex of both is combined with TGF- beta, leading to the phosphorylation of type I receptors. Activation of downstream signaling pathways. In most mammalian cells, the presence of RI is ALK5, so ALK5 is used as the focus of TGF- beta receptors.
In the previous work, the study group chose a ALK5 inhibitor SB-431542 as a tool to study the effect of inhibition of ALK5 on the scar formation after glaucoma surgery. The results showed that the local subconjunctival injection of SB-431542 could inhibit the formation of the filter scar. Therefore, we think that ALK5 may be important for the regulation of TGF-p scar formation. We hope to explore the effect of ALK5 on cell phenotypic transformation and fibrosis by establishing the scar model of cell and animal, and observing its effect on cell phenotypic transformation and fibrosis, and explore the role of ALK5 inhibitors in the local tissue remodeling after conjunctival injury and the mechanism of cytology, providing a study on the inhibition of the scar formation of glaucoma filters. More theoretical basis.
Objective: To study the regulatory effect of cytokine TGF- beta 1 on the expression of activin receptor like kinase 5 (activin receptor-like kinase5, ALK5) in HTF cells and the model of filter hypertrophic scar after glaucoma surgery, and to establish a fine cell and animal model of high expression of ALK5 gene by lentivirus transfection technology, and to regulate the expression of ALK5 and study ALK5 water artificially. The effect of Ping on the scarring of filtering passages after glaucoma filtering surgery.
Materials and methods:
The first part: primary cultured human conjunctival Tenon's fibroblasts and cell identification. The logarithmic growth phase cells were taken to induce HTF with TGF- beta 1 of 10 mu L. Western Blot was used to detect the expression of smooth muscle actin (alpha -SM-actin) and fibronectin (fibronectin, FN) protein by Western Blot to determine the phenotype transformation of cells. Fibrosis. Western Blot and immunocytochemistry were used to identify the expression of ALK5 protein and the ALK5mRNA level was detected by real-time quantitative PCR.
The second part: using the Tronolab lentivirus vector system to construct the lentivirus vector expressing ALK5 gene, transfect the HTF cells and obtain the HTF cells expressing ALK5. Observe the cell growth characteristics. The transfected and untransfected HTF cells are cultured with the medium of ALK5 inhibitor SB-431542 and the cell growth is observed, and the cell growth is observed. Properties, MTT test was used to detect cell proliferation, and HTF was induced with TGF-p1 of 10 mu g/ L, Western Blot was used to detect related phenotypic transformation (alpha -SMA), fibrosis index (FN) and expression of apoptosis index (Caspase-3).
The third part: 1. the animal model of the rabbit filter cicatricial pathway was established with filtration surgery. 24 healthy New Zealand white rabbits were selected as experimental animals, and they were randomly divided into 3 groups: the control group, the ALK5 group, the 0.5mM SB-431542 group. The left eye surgery was performed under the general anesthesia. The postoperative observation, the measurement of intraocular pressure (intraocularpressure, IOP). 3,7,14,28 after operation. 2 rabbits in each group were killed at 4 time points at 4 days. The eyeball was removed, the immunohistochemical staining was made, the filter channel alpha -SM-actin, ALK5 and cicatricial situation.2. were performed on 30 healthy New Zealand white rabbits. They were randomly divided into 5 groups, the control group (NS+ NS), the negative control group (NS+0.5mM SB-431542 group), the high expression group (AL). K5 transfected +NS), high expression + low dose group (ALK5 transfected +0.5mM SB-431542 group) and high expression + high dose group (ALK5 transfected +2.0mM SB-431542 group), 6 rabbits in each group (6 eyes, n=6). Observation and measurement of intraocular pressure after operation. After operation, the experimental rabbits were executed, the experimental rabbits were executed, the experiment eyes were removed, the immunohistochemical staining was made, a-SM-actin, ALK5, and cicatricicization was detected. Situation.
Result锛，

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