本文選題：青光眼 + 濾過性手術(shù)�。� 參考：《復(fù)旦大學(xué)》2012年博士論文

【摘要】：青光眼濾過術(shù)后濾過泡瘢痕化是手術(shù)失敗的最主要原因。失敗的濾過性手術(shù)的濾過泡結(jié)膜上皮下有異常增厚的致密膠原纖維結(jié)締組織,伴有成纖維細(xì)胞的活躍增生,阻塞濾過道,從而失去房水引流功能。目前手術(shù)中經(jīng)常聯(lián)合使用的抗代謝藥物,如五氟脲嘧啶(5-FU),絲裂霉素(mitomycin, MMC)等,雖然可以減少術(shù)后濾過道瘢痕形成,提高手術(shù)的成功率,但其抗代謝作用也可導(dǎo)致一系列術(shù)后低眼壓、濾過泡滲漏等并發(fā)癥,因此探索更加安全有效的治療方法對抗青光眼術(shù)后瘢痕化有著重要的意義。 細(xì)胞表型轉(zhuǎn)化是纖維化過程中重要的細(xì)胞學(xué)基礎(chǔ),而轉(zhuǎn)化生長因子-β(TGF-β)是成纖維細(xì)胞表型轉(zhuǎn)化,進一步遷移、增殖,合成細(xì)胞外基質(zhì),導(dǎo)致濾過泡瘢痕化的重要細(xì)胞因子。人結(jié)膜下Tenon's囊成纖維細(xì)胞(human tenon's capsule fibroblast, HTF)在青光眼術(shù)后濾過道瘢痕化的過程中扮演了主要的角色。在手術(shù)或損傷條件下,TGF-β水平增加,使局部的成纖維細(xì)胞(fibroblast,FB)激活并轉(zhuǎn)化成肌成纖維細(xì)胞(myofibroblast, MF)啟動傷口愈合反應(yīng)。MF在傷口愈合的不同時期均起到重要作用,一旦創(chuàng)傷愈合,MF一般迅速恢復(fù)為FB或進入凋亡的程序,若MF持續(xù)存在,則會導(dǎo)致細(xì)胞的過度增殖、細(xì)胞外基質(zhì)的合成增多,瘢痕形成,濾過道過早愈合,房水引流通道被阻,致使手術(shù)失敗。 由于TGF-β在細(xì)胞表型轉(zhuǎn)化中的關(guān)鍵作用,越來越多的研究選擇其作為研究靶點,通過降低TGF-β表達來抑制細(xì)胞表型轉(zhuǎn)化,從而降低瘢痕化的程度。但由于TGF-β的下游信號通路非常復(fù)雜且相互形成交錯網(wǎng)狀,所以太低位點的調(diào)節(jié)可能對其促進細(xì)胞增殖及表型轉(zhuǎn)化的作用不甚明顯。因此我們考慮在信號通路的較高的位點：TGF-β膜受體,來進行干預(yù),借此來探究MF表型轉(zhuǎn)化的過程,以及纖維化的可能機制。 TGF-β的受體主要有3種,分別稱為Ⅰ型(TGF-βR Ⅰ或活化素受體樣激酶,activin receptor-like kinase, ALK)、Ⅱ型受體(TGF-βRⅡ)和Ⅲ型受體(TGF-βRⅢ)。其中Ⅰ、Ⅱ型受體主要參與信號通路的轉(zhuǎn)導(dǎo),兩者的復(fù)合物與TGF-β相結(jié)合,導(dǎo)致Ⅰ型受體磷酸化,并激活下游的信號通路。大多數(shù)的哺乳動物細(xì)胞中,存在的RI是ALK5,所以人們把ALK5作為研究TGF-β受體的重點。 在前期的工作中,本研究組選擇一種ALK5抑制劑SB-431542作為工具,研究抑制ALK5對青光眼術(shù)后濾過道瘢痕化的作用,結(jié)果顯示濾過術(shù)后局部球結(jié)膜下注射SB-431542可以抑制濾過道瘢痕形成。由此,我們認(rèn)為,ALK5可能與調(diào)控TGF-p致瘢痕化有重要關(guān)系,我們希望通過建立細(xì)胞及動物的瘢痕化模型,并人為調(diào)控其中ALK5的水平,觀察其對細(xì)胞表型轉(zhuǎn)化及纖維化的影響作用,探索ALK5抑制劑在結(jié)膜損傷后局部組織重塑中的作用及其細(xì)胞學(xué)機制,為抑制青光眼濾過道瘢痕化的研究提供更多的理論依據(jù)。 目的：研究細(xì)胞因子TGF-β1對HTF細(xì)胞及動物青光眼術(shù)后濾過道瘢痕化模型中,活化素受體樣激酶5(activin receptor-like kinase5, ALK5)表達的調(diào)控作用,并通過慢病毒轉(zhuǎn)染技術(shù)建立ALK5基因高表達的細(xì)胞及動物模型,人為調(diào)控ALK5表達,研究ALK5水平對青光眼濾過術(shù)后濾過道瘢痕化的影響。 材料和方法： 第一部分：原代培養(yǎng)人結(jié)膜下Tenon's囊成纖維細(xì)胞,并進行細(xì)胞鑒定。取對數(shù)生長期細(xì)胞,以10μg/μl的TGF-β1誘導(dǎo)HTF,在不同的時間,采用Western Blot檢測平滑肌肌動蛋白(α-SM-actin)和纖連蛋白(fibronectin, FN)蛋白表達,確定細(xì)胞發(fā)生表型轉(zhuǎn)化及纖維化。再采用Western Blot和免疫細(xì)胞化學(xué)技術(shù)來鑒定ALK5蛋白的表達,并以實時定量PCR檢測ALK5mRNA水平。 第二部分：采用Tronolab慢病毒載體系統(tǒng),構(gòu)建過表達ALK5基因的慢病毒載體,轉(zhuǎn)染HTF細(xì)胞,獲得過表達ALK5的HTF細(xì)胞。觀察細(xì)胞生長特性,。采用普通培養(yǎng)基,以及加入ALK5抑制劑SB-431542的培養(yǎng)基分別培養(yǎng)轉(zhuǎn)染及未轉(zhuǎn)染的HTF細(xì)胞,觀察細(xì)胞生長特性,MTT試驗檢測細(xì)胞增殖能力。并用10μg/μl的TGF-p1誘導(dǎo)HTF, Western Blot檢測相關(guān)表型轉(zhuǎn)化(α-SMA)、纖維化指標(biāo)(FN)以及凋亡指標(biāo)(Caspase-3)的表達。 第三部分：1.應(yīng)用濾過性手術(shù)建立兔濾過道瘢痕化動物模型,選用24只健康新西蘭白兔作為實驗動物,隨機分為3組：對照組、ALK5組、0.5mM SB-431542組。在全身麻醉下行左眼濾過性手術(shù)。術(shù)后觀察、測量眼壓(intraocularpressure,IOP)。術(shù)后第3、7、14、28天4個時間點各處死每組2只實驗兔,摘除眼球,制作切片,免疫組化染色,光鏡下觀察濾過道α-SM-actin、ALK5及瘢痕化情況。2.對30只健康的新西蘭白兔施行右眼濾過性手術(shù)。隨機分為5組,對照組(NS+NS)、陰性對照組(NS+0.5mM SB-431542組)、高表達組(ALK5轉(zhuǎn)染+NS)、高表達+低劑量給藥組(ALK5轉(zhuǎn)染+0.5mM SB-431542組)和高表達+高劑量給藥組(ALK5轉(zhuǎn)染+2.0mM SB-431542組),每組6只兔(6只眼,n=6)。術(shù)后觀察并測量眼壓。術(shù)后第28d處死實驗兔,摘除實驗眼,制作切片,免疫組化染色,檢測a-SM-actin、ALK5及瘢痕化情況。 結(jié)果： 第一部分：貼壁法培養(yǎng)的人結(jié)膜下Tenon's囊組織,約3-5天可見組織塊周圍爬出梭形細(xì)胞,繼續(xù)培養(yǎng)逐漸匯合成片,經(jīng)傳代后進行波形蛋白(vimentin)染色,鑒定為成纖維細(xì)胞。TGF-β1誘導(dǎo)HTF后,ALK5的mRNA表達有所上調(diào),于12-24h達到高峰值。Western Blot顯示蛋白表達水平也有所升高,于24-36h達到高峰值。細(xì)胞免疫熒光染色檢測也同樣顯示,ALK5表達水平升高。 第二部分：實驗成功構(gòu)建了過表達ALK5基因的慢病毒載體,并獲得了較高滴度的病毒液。采用慢病毒轉(zhuǎn)染HTF細(xì)胞后,觀察發(fā)現(xiàn)細(xì)胞增殖速度明顯增加,并伴有凋亡現(xiàn)象,加入SB-431542后細(xì)胞生長速度及凋亡現(xiàn)象均有所逆轉(zhuǎn)。MTT實驗顯示,轉(zhuǎn)染GFP病毒的細(xì)胞與未轉(zhuǎn)染的HTF細(xì)胞增值速度無明顯變化,轉(zhuǎn)染ALK5病毒的HTF細(xì)胞,增殖速度明顯增加,而在培液中加入SB-431542后,細(xì)胞增殖速度明顯降低。轉(zhuǎn)染后,過表達ALK5的細(xì)胞,表達a-SMA以及FN蛋白水平有所下降,而加入ALK5抑制劑SB-431542后,該過程可被抑制。 第三部分：1.兔濾過性術(shù)后術(shù)眼的眼壓各組均有所下降,術(shù)后第1天,平均IOP為5.63±1.73mmHg,P0.05。之后術(shù)眼眼壓逐漸上升,到術(shù)后第10天,眼壓逐漸恢復(fù)到手術(shù)前水平,為11.07±1.72mmHg,P0.05。之后兔眼眼壓稍有上升趨勢,術(shù)后28d,眼壓相對術(shù)前稍有上升,為12.40±1.16mmHg,P0.05。組織學(xué)觀察顯示：術(shù)后濾過道周圍早期以水腫及炎癥細(xì)胞為主,之后逐漸出現(xiàn)較多增殖的成纖維細(xì)胞,晚期成纖維細(xì)胞數(shù)量減少,膠原纖維沉積增多,產(chǎn)生瘢痕組織。ALK5在結(jié)膜下成纖維細(xì)胞的表達在術(shù)后3天并不明顯,到第7天開始逐漸增多,14天時結(jié)膜下有大量表達,至28天,表達量有所下降,但仍高于術(shù)前。轉(zhuǎn)染ALK5的實驗眼,可以見到結(jié)膜下陽性的轉(zhuǎn)染細(xì)胞,周圍伴有大量的成纖維細(xì)胞增生。2.各組在術(shù)后眼壓均有所下降,對照組IOP=7.83±2.14,P0.05；陰性對照SB組：IOP=5.5±0.84mmHg,P0.05；轉(zhuǎn)染ALK5組：IOP=5.17±1.60mmHg,P0.05；轉(zhuǎn)染ALK5+SB0.5mM組：IOP=4.67±0.82mmHg,P0.05；轉(zhuǎn)染ALK5+SB2.0mM組：IOP=5±1.10mmHg,P0.05,到術(shù)后第7-10d,眼壓逐漸恢復(fù)到手術(shù)前水平,但各組間統(tǒng)計學(xué)差異不明顯。組織學(xué)觀察顯示：對照組結(jié)膜下傷口周圍纖維化明顯,充滿富含增生細(xì)胞的纖維化成分和明顯膠原化的團狀結(jié)締組織；ALK5轉(zhuǎn)染后,傷口附近及結(jié)膜下可見大量增生的成纖維細(xì)胞,比對照組和用藥組明顯增多,并伴有少量的膠原組織沉積。加入抑制劑后,術(shù)眼濾過道周圍細(xì)胞增殖和瘢痕組織均有所減少,結(jié)膜下組織相對疏松。 結(jié)論：在人Tenon's囊成纖維細(xì)胞(HTF)表型轉(zhuǎn)化以及兔眼濾過術(shù)后瘢痕化的模型中,都可以觀察到TGF-βI型受體ALK5表達的增加,而通過慢病毒對HTF細(xì)胞及兔眼結(jié)膜下組織轉(zhuǎn)染ALK5基因,并加入抑制劑SB-431542調(diào)控ALK5水平發(fā)現(xiàn),ALK5高表達一方面能使成纖維細(xì)胞增殖加快,促進兔濾過性手術(shù)后濾過道瘢痕的形成,另一方面也可能誘導(dǎo)細(xì)胞的凋亡從而使纖維化得到控制。加入ALK5抑制劑SB-431542以后,可以抑制由其產(chǎn)生的細(xì)胞增殖以及瘢痕化作用。這說明ALK5在TGF-p引發(fā)的瘢痕化中,可能有重要的調(diào)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錛，

本文編號：1883358