本文選題：青光眼 + 濾過(guò)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(guò)術(shù)后濾過(guò)泡瘢痕化是手術(shù)失敗的最主要原因。失敗的濾過(guò)性手術(shù)的濾過(guò)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(guò)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(guò)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(guò)泡滲漏等并發(fā)癥,因此探索更加安全有效的治療方法對(duì)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(guò)程中重要的細(xì)胞學(xué)基礎(chǔ),而轉(zhuǎn)化生長(zhǎng)因子-β(TGF-β)是成纖維細(xì)胞表型轉(zhuǎn)化,進(jìn)一步遷移、增殖,合成細(xì)胞外基質(zhì),導(dǎo)致濾過(guò)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(guò)道瘢痕化的過(guò)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(dòng)傷口愈合反應(yīng)。MF在傷口愈合的不同時(shí)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(jìn)入凋亡的程序,若MF持續(xù)存在,則會(huì)導(dǎo)致細(xì)胞的過(guò)度增殖、細(xì)胞外基質(zhì)的合成增多,瘢痕形成,濾過(guò)道過(guò)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(lái)越多的研究選擇其作為研究靶點(diǎn),通過(guò)降低TGF-β表達(dá)來(lái)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(hào)通路非常復(fù)雜且相互形成交錯(cuò)網(wǎng)狀,所以太低位點(diǎn)的調(diào)節(jié)可能對(duì)其促進(jìn)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(hào)通路的較高的位點(diǎn)：TGF-β膜受體,來(lái)進(jìn)行干預(yù),借此來(lái)探究MF表型轉(zhuǎn)化的過(guò)程,以及纖維化的可能機(jī)制。 TGF-β的受體主要有3種,分別稱(chēng)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(hào)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(hào)通路。大多數(shù)的哺乳動(dòng)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(diǎn)。 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(duì)青光眼術(shù)后濾過(guò)道瘢痕化的作用,結(jié)果顯示濾過(guò)術(shù)后局部球結(jié)膜下注射SB-431542可以抑制濾過(guò)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(guò)建立細(xì)胞及動(dòng)物的瘢痕化模型,并人為調(diào)控其中ALK5的水平,觀察其對(duì)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(jī)制,為抑制青光眼濾過(guò)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(duì)HTF細(xì)胞及動(dòng)物青光眼術(shù)后濾過(guò)道瘢痕化模型中,活化素受體樣激酶5(activin receptor-like kinase5, ALK5)表達(dá)的調(diào)控作用,并通過(guò)慢病毒轉(zhuǎn)染技術(shù)建立ALK5基因高表達(dá)的細(xì)胞及動(dòng)物模型,人為調(diào)控ALK5表達(dá),研究ALK5水平對(duì)青光眼濾過(guò)術(shù)后濾過(guò)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(jìn)行細(xì)胞鑒定。取對(duì)數(shù)生長(zhǎng)期細(xì)胞,以10μg/μl的TGF-β1誘導(dǎo)HTF,在不同的時(shí)間,采用Western Blot檢測(cè)平滑肌肌動(dòng)蛋白(α-SM-actin)和纖連蛋白(fibronectin, FN)蛋白表達(dá),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(lái)鑒定ALK5蛋白的表達(dá),并以實(shí)時(shí)定量PCR檢測(cè)ALK5mRNA水平。 第二部分：采用Tronolab慢病毒載體系統(tǒng),構(gòu)建過(guò)表達(dá)ALK5基因的慢病毒載體,轉(zhuǎn)染HTF細(xì)胞,獲得過(guò)表達(dá)ALK5的HTF細(xì)胞。觀察細(xì)胞生長(zhǎng)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(zhǎng)特性,MTT試驗(yàn)檢測(cè)細(xì)胞增殖能力。并用10μg/μl的TGF-p1誘導(dǎo)HTF, Western Blot檢測(cè)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(dá)。 第三部分：1.應(yīng)用濾過(guò)性手術(shù)建立兔濾過(guò)道瘢痕化動(dòng)物模型,選用24只健康新西蘭白兔作為實(shí)驗(yàn)動(dòng)物,隨機(jī)分為3組：對(duì)照組、ALK5組、0.5mM SB-431542組。在全身麻醉下行左眼濾過(guò)性手術(shù)。術(shù)后觀察、測(cè)量眼壓(intraocularpressure,IOP)。術(shù)后第3、7、14、28天4個(gè)時(shí)間點(diǎn)各處死每組2只實(shí)驗(yàn)兔,摘除眼球,制作切片,免疫組化染色,光鏡下觀察濾過(guò)道α-SM-actin、ALK5及瘢痕化情況。2.對(duì)30只健康的新西蘭白兔施行右眼濾過(guò)性手術(shù)。隨機(jī)分為5組,對(duì)照組(NS+NS)、陰性對(duì)照組(NS+0.5mM SB-431542組)、高表達(dá)組(ALK5轉(zhuǎn)染+NS)、高表達(dá)+低劑量給藥組(ALK5轉(zhuǎn)染+0.5mM SB-431542組)和高表達(dá)+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(cè)量眼壓。術(shù)后第28d處死實(shí)驗(yàn)兔,摘除實(shí)驗(yàn)眼,制作切片,免疫組化染色,檢測(cè)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(jiàn)組織塊周?chē)莱鏊笮渭?xì)胞,繼續(xù)培養(yǎng)逐漸匯合成片,經(jīng)傳代后進(jìn)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(dá)有所上調(diào),于12-24h達(dá)到高峰值。Western Blot顯示蛋白表達(dá)水平也有所升高,于24-36h達(dá)到高峰值。細(xì)胞免疫熒光染色檢測(cè)也同樣顯示,ALK5表達(dá)水平升高。 第二部分：實(shí)驗(yàn)成功構(gòu)建了過(guò)表達(dá)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(zhǎng)速度及凋亡現(xiàn)象均有所逆轉(zhuǎn)。MTT實(shí)驗(yàn)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(wú)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(guò)表達(dá)ALK5的細(xì)胞,表達(dá)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(guò)程可被抑制。 第三部分：1.兔濾過(guò)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(shì),術(shù)后28d,眼壓相對(duì)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(guò)道周?chē)缙谝运[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(dá)在術(shù)后3天并不明顯,到第7天開(kāi)始逐漸增多,14天時(shí)結(jié)膜下有大量表達(dá),至28天,表達(dá)量有所下降,但仍高于術(shù)前。轉(zhuǎn)染ALK5的實(shí)驗(yàn)眼,可以見(jiàn)到結(jié)膜下陽(yáng)性的轉(zhuǎn)染細(xì)胞,周?chē)橛写罅康某衫w維細(xì)胞增生。2.各組在術(shù)后眼壓均有所下降,對(duì)照組IOP=7.83±2.14,P0.05；陰性對(duì)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(jì)學(xué)差異不明顯。組織學(xué)觀察顯示：對(duì)照組結(jié)膜下傷口周?chē)w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(tuán)狀結(jié)締組織；ALK5轉(zhuǎn)染后,傷口附近及結(jié)膜下可見(jiàn)大量增生的成纖維細(xì)胞,比對(duì)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(guò)道周?chē)?xì)胞增殖和瘢痕組織均有所減少,結(jié)膜下組織相對(duì)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(guò)術(shù)后瘢痕化的模型中,都可以觀察到TGF-βI型受體ALK5表達(dá)的增加,而通過(guò)慢病毒對(duì)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(dá)一方面能使成纖維細(xì)胞增殖加快,促進(jìn)兔濾過(guò)性手術(shù)后濾過(guò)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(shuō)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(hào)：1883358