葛茸茸，沈 煒

·基礎(chǔ)醫(yī)學(xué)· ·論著·

雷帕霉素靶蛋白信號(hào)通路介導(dǎo)轉(zhuǎn)化生長(zhǎng)因子-β2誘導(dǎo)的后發(fā)性白內(nèi)障的分子機(jī)制研究

葛茸茸，沈 煒

目的 探討雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)信號(hào)通路與轉(zhuǎn)化生長(zhǎng)因子(transforming growth factor，TGF)-β2誘導(dǎo)的晶狀體上皮細(xì)胞間質(zhì)化的相關(guān)機(jī)制。方法 顯微鏡觀察TGF-β2誘導(dǎo)的晶狀體上皮細(xì)胞的表型變化，Western blot進(jìn)一步驗(yàn)證TGF-β2誘導(dǎo)的HLEB-3上皮間質(zhì)轉(zhuǎn)化模型。MTT檢測(cè)TGF-β2誘導(dǎo)上皮間質(zhì)化后以及雷帕霉素對(duì)細(xì)胞增殖的影響。Western blot在分子水平檢測(cè)上皮間質(zhì)化和mTOR信號(hào)通路之間的機(jī)制關(guān)聯(lián)。結(jié)果 加入TGF-β2誘導(dǎo)處理24 h后，用顯微鏡觀察HLEB-3的細(xì)胞形態(tài)由橢圓形變?yōu)樾切位蚣忓N形，細(xì)胞連接減少。Western blot檢測(cè)發(fā)現(xiàn)TGF-β2誘導(dǎo)后的HLEB-3中上皮細(xì)胞標(biāo)志蛋白E-cadherin表達(dá)水平明顯降低，而間質(zhì)細(xì)胞標(biāo)記蛋白α-SMA的表達(dá)水平顯著降低，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。MTT檢測(cè)發(fā)現(xiàn)雷帕霉素預(yù)處理可以抑制TGF-β2對(duì)上皮細(xì)胞增殖的促進(jìn)作用。Western blot顯示當(dāng)用雷帕霉素抑制mTOR信號(hào)通路的激活后，可以逆轉(zhuǎn)TGF-β2對(duì)α-SMA的表達(dá)的上調(diào)作用，促進(jìn)上皮細(xì)胞標(biāo)志蛋白E-cadherin的表達(dá)。結(jié)論 mTOR信號(hào)通路介導(dǎo)TGF-β2誘導(dǎo)的上皮細(xì)胞間質(zhì)化作用，促進(jìn)后發(fā)性白內(nèi)障的發(fā)生。

轉(zhuǎn)化生長(zhǎng)因子-β2；雷帕霉素靶蛋白信號(hào)通路；后發(fā)性白內(nèi)障

后發(fā)性白內(nèi)障(posterior capsular opacification，PCO)是白內(nèi)障術(shù)后常見(jiàn)的主要并發(fā)癥之一，是目前白內(nèi)障術(shù)后視力下降的主要原因，其分子病理基礎(chǔ)為手術(shù)殘留的晶狀體上皮細(xì)胞的異常遷移、增殖及上皮間質(zhì)轉(zhuǎn)化[1]。雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)信號(hào)通路是調(diào)控細(xì)胞生長(zhǎng)與增殖、分化的關(guān)鍵通路，不僅可參與調(diào)節(jié)細(xì)胞周期的進(jìn)程，調(diào)控細(xì)胞生長(zhǎng)增殖[2]，而且促進(jìn)細(xì)胞的黏附和遷移，調(diào)節(jié)上皮間質(zhì)轉(zhuǎn)化[3]。雖然晶狀體上皮細(xì)胞發(fā)生上皮間質(zhì)轉(zhuǎn)化是后發(fā)性白內(nèi)障的病理基礎(chǔ)，然而，mTOR信號(hào)通路在晶狀體上皮細(xì)胞上皮間質(zhì)轉(zhuǎn)化過(guò)程中的作用及作用機(jī)制目前知之甚少。本研究擬通過(guò)轉(zhuǎn)化生長(zhǎng)因子(transforming growth factor，TGF)-β2誘導(dǎo)晶狀體上皮細(xì)胞構(gòu)建上皮間質(zhì)轉(zhuǎn)化模型，檢測(cè)mTOR信號(hào)通路在上皮間質(zhì)轉(zhuǎn)化過(guò)程中的表達(dá)，并利用mTOR 抑制劑雷帕霉素誘導(dǎo)晶狀體上皮細(xì)胞，探討其對(duì)晶狀體上皮細(xì)胞間質(zhì)轉(zhuǎn)化的影響，明確mTOR信號(hào)通路在晶狀體上皮細(xì)胞上皮間質(zhì)轉(zhuǎn)化過(guò)程中的作用及機(jī)制，從中尋找后發(fā)性白內(nèi)障防治的新靶點(diǎn)。

1 材料與方法

1.1 材料和試劑 TGF-β2購(gòu)買(mǎi)自PeproTech公司，人晶狀體上皮細(xì)胞HLEB-3購(gòu)買(mǎi)自上海生命科學(xué)院細(xì)胞所。DMEM培養(yǎng)液和胎牛血清(AusGeneX)購(gòu)買(mǎi)自上海銀海圣生物有限公司，青霉素、鏈霉素、胰酶、雷帕霉素購(gòu)買(mǎi)自上海聚仕隆有限公司，E-cadherin抗體，α-SMA抗體、mTOR抗體、p-mTOR抗體、cyclinD1抗體、BAX抗體購(gòu)買(mǎi)自上海煊翎有限公司。MTT試劑盒和Annexinv-FITC/PI雙染試劑盒購(gòu)買(mǎi)自南京建成生物有限公司。

1.2 方法 細(xì)胞培養(yǎng)與處理人晶狀體上皮細(xì)胞HLEB-3進(jìn)行常規(guī)傳代培養(yǎng)，DMEM培養(yǎng)液和10%胎牛血清在37 ℃ 5%二氧化碳的培養(yǎng)箱中培養(yǎng)。當(dāng)細(xì)胞約80%融合時(shí)，換用無(wú)血清DMEM培養(yǎng)液，加入終濃度為1 ng/ml TGF-β2誘導(dǎo)處理24 h，模擬人晶狀體上皮細(xì)胞HLEB-3上皮間質(zhì)轉(zhuǎn)化模型。為檢測(cè)mTOR信號(hào)通路對(duì)上皮間質(zhì)化的影響，當(dāng)人晶狀體上皮細(xì)胞HLEB-3約80%融合時(shí)，換用無(wú)血清DMEM培養(yǎng)液，先加入終濃度為100 μmol/L的雷帕霉素預(yù)處理2 h，然后用終濃度為1 μg/L TGF-β2誘導(dǎo)處理24 h，檢測(cè)mTOR信號(hào)通路抑制后對(duì)TGF-β2誘導(dǎo)的上皮間質(zhì)轉(zhuǎn)化的影響。

1.3 觀察及檢測(cè)指標(biāo) (1)顯微鏡觀察細(xì)胞形態(tài)。在培養(yǎng)室的倒置顯微鏡下觀察人晶狀體上皮細(xì)胞HLEB-3的形態(tài)以及加入TGF-β2和雷帕霉素的形態(tài)變化，拍照并對(duì)比。(2)MTT檢測(cè)細(xì)胞增殖。人晶狀體上皮細(xì)胞HLEB-3進(jìn)行常規(guī)傳代培養(yǎng)，收集對(duì)數(shù)生長(zhǎng)期細(xì)胞制成單細(xì)胞懸液，接種于96孔板，細(xì)胞約80%融合時(shí)，換用無(wú)血清DMEM培養(yǎng)液，先加入終濃度為100 μmol/L的雷帕霉素預(yù)處理2 h，然后用終濃度為1 μg/L TGF-β2誘導(dǎo)處理24 h，加入MTT溶液，孵育4 h后酶標(biāo)儀檢測(cè)570 nm處的光密度(OD值)。(3)Western blot檢測(cè)上皮間質(zhì)轉(zhuǎn)化相關(guān)分子的表達(dá)以及mTOR信號(hào)通路的激活情況。先加入終濃度為100 μmol/L的雷帕霉素預(yù)處理2 h，然后用終濃度為1 μg/L TGF-β2誘導(dǎo)處理24 h, Western blot檢測(cè)HLEB-3細(xì)胞中檢測(cè)mTOR信號(hào)通路的激活水平以及上皮細(xì)胞標(biāo)志蛋白E-cadherin以及間質(zhì)細(xì)胞標(biāo)志蛋白α-SMA的表達(dá)水平，探究mTOR信號(hào)通路的激活與上皮間質(zhì)轉(zhuǎn)化的關(guān)系。

1.4 統(tǒng)計(jì)學(xué)處理 采用 SPSS 14.55統(tǒng)計(jì)軟件處理。實(shí)驗(yàn)數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(x±s)表示，各組間樣本均數(shù)比較采用One-way ANOVA分析，兩樣本間成對(duì)比較用t檢驗(yàn)。P<0.05為差異具有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 顯微鏡觀察TGF-β2誘導(dǎo)的晶狀體上皮細(xì)胞上皮間質(zhì)轉(zhuǎn)化模型 顯微鏡觀察發(fā)現(xiàn)TGF-β2處理后細(xì)胞形態(tài)明顯變得細(xì)長(zhǎng)，形態(tài)變?yōu)樾切位蚣忓N形，細(xì)胞之間連接減少，具有成纖維細(xì)胞的雛形。這表明TGF-β2可能參與誘導(dǎo)的晶狀體上皮細(xì)胞上皮間質(zhì)轉(zhuǎn)化。見(jiàn)圖1。

2.2 Western blot驗(yàn)證TGF-β2誘導(dǎo)的晶狀體上皮細(xì)胞HLEB-3上皮間質(zhì)轉(zhuǎn)化模型 利用Western blot檢測(cè)結(jié)果發(fā)現(xiàn)，與對(duì)照組比較，TGF-β2誘導(dǎo)的上皮細(xì)胞標(biāo)志蛋白E-cadherin表達(dá)水平明顯降低，而間質(zhì)細(xì)胞標(biāo)記蛋白α-SMA的表達(dá)水平顯著升高，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。見(jiàn)圖2。

2.3 MTT檢測(cè)TGF-β2和雷帕霉素對(duì)晶狀體上皮細(xì)胞HLEB-3增殖的影響 TGF-β2可以成功誘導(dǎo)HLEB-3細(xì)胞間質(zhì)化，那么對(duì)細(xì)胞的增殖具有怎么樣的影響，當(dāng)用雷帕霉素預(yù)處理抑制mTOR信號(hào)通路后增殖的變化如何，尚不明確。因此，筆者利用MTT方法檢測(cè)了細(xì)胞間質(zhì)化后細(xì)胞增殖的變化，發(fā)現(xiàn)TGF-β2誘導(dǎo)細(xì)胞間質(zhì)化后細(xì)胞數(shù)量明顯增加。見(jiàn)圖3。

注：A表示對(duì)照組(加入PBS)，B表示TGF-β2處理組。TGF為轉(zhuǎn)化生長(zhǎng)因子圖1 顯微鏡觀察TGF-β2誘導(dǎo)的人晶狀體上皮細(xì)胞HLEB-3形態(tài)變化(×200)

注：與對(duì)照組比較aP<0.05。TGF為轉(zhuǎn)化生長(zhǎng)因子。A為2組蛋白電泳圖，B為2組蛋白的表達(dá)水平圖2 TGF-β2對(duì)人晶狀體上皮細(xì)胞HLEB-3中上皮細(xì)胞標(biāo)志蛋白和間質(zhì)細(xì)胞標(biāo)志蛋白表達(dá)的影響

注：與TGF-β2處理組比較aP<0.05。TGF為轉(zhuǎn)化生長(zhǎng)因子圖3 TGF-β2和雷帕霉素對(duì)晶狀體上皮細(xì)胞HLEB-3增殖的影響

2.5 TGF-β2和雷帕霉素對(duì)上皮間質(zhì)化和mTOR信號(hào)通路的影響 為了探究mTOR信號(hào)通路和TGF-β2誘導(dǎo)的HLEB-3細(xì)胞間質(zhì)化的關(guān)系，利用Western blot檢測(cè)了TGF-β2和雷帕霉素處理后，上皮間質(zhì)化標(biāo)志蛋白、間質(zhì)化標(biāo)志性蛋白和mTOR信號(hào)通路的激活情況，發(fā)現(xiàn)TGF-β2可以顯著激活mTOR信號(hào)通路，促進(jìn)HLEB-3細(xì)胞中間質(zhì)化標(biāo)志性蛋白α-SMA的表達(dá)，促進(jìn)上皮細(xì)胞間質(zhì)化，當(dāng)用雷帕霉素抑制mTOR信號(hào)通路的激活后，可以逆轉(zhuǎn)TGF-β2對(duì)α-SMA的表達(dá)的上調(diào)作用，逆轉(zhuǎn)TGF-β2對(duì)上皮細(xì)胞間質(zhì)化的作用。見(jiàn)圖4。

注：與TGF-β2處理組比較aP<0.05。TGF為轉(zhuǎn)化生長(zhǎng)因子。A為各組蛋白電泳圖，B為各組蛋白的表達(dá)水平圖4 TGF-β2和雷帕霉素對(duì)上皮間質(zhì)化和mTOR信號(hào)通路的影響

3 討論

白內(nèi)障術(shù)后晶狀體后囊膜混濁又名PCO，是現(xiàn)代白內(nèi)障術(shù)后最常見(jiàn)的主要并發(fā)癥之一[4]，盡管手術(shù)技術(shù)和人工晶狀體材料的進(jìn)步已經(jīng)在很大程度上降低了后發(fā)性白內(nèi)障的發(fā)生率，但后發(fā)性白內(nèi)障仍是術(shù)后視力下降的主要原因[5]，目前認(rèn)為手術(shù)殘留的晶狀體上皮細(xì)胞的異常遷移、增殖及上皮間質(zhì)轉(zhuǎn)化是后發(fā)性白內(nèi)障的主要發(fā)病機(jī)制[6]，積極預(yù)防后發(fā)性白內(nèi)障的發(fā)生是保障白內(nèi)障手術(shù)遠(yuǎn)期療效的關(guān)鍵途徑[7]。因此，應(yīng)積極探究PCO發(fā)病的調(diào)控機(jī)制，為PCO的治療尋找新的分子靶位點(diǎn)。

近年來(lái)，研究發(fā)現(xiàn)肝細(xì)胞生長(zhǎng)因子，成纖維細(xì)胞生長(zhǎng)因子[8]、轉(zhuǎn)化生長(zhǎng)因子、表皮生長(zhǎng)因子[9]等均參與后發(fā)性白內(nèi)障的發(fā)生，其中，TGF-β2參與調(diào)節(jié)晶體狀上皮細(xì)胞的增殖、遷移、上皮間質(zhì)轉(zhuǎn)化等多種活動(dòng)，是公認(rèn)的誘發(fā)PCO發(fā)生的重要因子[10]。目前TGF-β誘導(dǎo)晶狀體上皮細(xì)胞上皮間質(zhì)轉(zhuǎn)化細(xì)胞模型已成為眾多學(xué)者認(rèn)可的研究白內(nèi)障形成的細(xì)胞模型[11]。

目前在PCO發(fā)生發(fā)展過(guò)程中研究比較明確的信號(hào)傳導(dǎo)途徑有TGF-β/Smad信號(hào)通路[12]、PI3K/AKT/mTOR信號(hào)通路[13]、Rho/ROCK信號(hào)通路[14]等。目前認(rèn)為mTOR信號(hào)通路不僅在調(diào)節(jié)細(xì)胞周期進(jìn)程和細(xì)胞生長(zhǎng)增殖過(guò)程中發(fā)揮中心樞紐的作用[15]，而促進(jìn)細(xì)胞的黏附和遷移，并參與調(diào)節(jié)上皮間質(zhì)轉(zhuǎn)化[16]。研究證實(shí)，mTOR信號(hào)通路介導(dǎo)TGF-β誘導(dǎo)的小鼠乳腺上皮細(xì)胞上皮間質(zhì)轉(zhuǎn)化[17]。近期有研究[18]表明，在上皮細(xì)胞的上皮間質(zhì)轉(zhuǎn)化過(guò)程中，TGF-β通過(guò)激活PI3K/Akt信號(hào)通路進(jìn)而激活mTOR信號(hào)通路，然而，mTOR途徑在晶狀體上皮細(xì)胞上皮間質(zhì)轉(zhuǎn)化過(guò)程中的作用目前研究甚少。

本課題擬通過(guò)TGF-β誘導(dǎo)的晶狀體上皮細(xì)胞上皮間質(zhì)轉(zhuǎn)化模型，檢測(cè)mTOR途徑在此過(guò)程中的活化情況，并利用mTOR抑制劑誘導(dǎo)晶狀體上皮細(xì)胞，探討其對(duì)細(xì)胞形態(tài)、增殖、凋亡以及間質(zhì)轉(zhuǎn)化的影響，初步明確mTOR途徑在晶狀體上皮細(xì)胞上皮間質(zhì)轉(zhuǎn)化過(guò)程中的作用及機(jī)制，力求從中尋找PCO防治的新靶點(diǎn)。結(jié)果發(fā)現(xiàn)TGF-β能夠活化mTOR信號(hào)通路，促進(jìn)人晶狀體上皮細(xì)胞的遷移能力以及上皮細(xì)胞向間質(zhì)的轉(zhuǎn)化，表明TGF-β能夠通過(guò)mTOR信號(hào)通路調(diào)控PCO的發(fā)生發(fā)展。這提示了抑制mTOR信號(hào)通路的活化，可能會(huì)減弱TGF-β對(duì)人晶狀體上皮細(xì)胞增殖及遷移能力的增強(qiáng)作用，mTOR信號(hào)通路可能為改善上皮間質(zhì)轉(zhuǎn)化的靶位點(diǎn)[19]。筆者利用mTOR 抑制劑雷帕霉素誘導(dǎo)晶狀體上皮細(xì)胞，發(fā)現(xiàn)雷帕霉素可以顯著抑制晶狀體上皮細(xì)胞間質(zhì)轉(zhuǎn)化，進(jìn)一步驗(yàn)證了mTOR信號(hào)通路介導(dǎo)TGF-β2誘導(dǎo)的上皮細(xì)胞間質(zhì)化作用，促進(jìn)PCO的發(fā)生這一結(jié)論，為PCO的治療及藥物研發(fā)提供了一定的分子理論依據(jù)。

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(本文編輯：莫琳芳)

Study on the molecular mechanism of after-cataract induced by mTOR signal pathway and mediated by TGF-β2

Ge Rongrong, Shen Wei

(DepartmentofOphthalmology,ChanghaiHospital,SecondMilitaryMedicalUniversity,Shanghai200433,China)

Objective To investigate the mechanism of the association between mammalian target of rapamycin(mTOR) signal pathway and TGF-β2-induced lens epithelial mesenchymal transition.MethodsPhenotypic changes in lens epithelial cells induced by TGF-β2 were closely observed by microscopy. Western blotting was used to further verify the HLEB3 epithelial mesenchymal transition model induced by TGF-β2. MTT was applied to detect the effects of TGF-β2 after induction of epithelial mesenchymal and Rapamycin on cell proliferation. Western blotting was used to detect the mechanism of the relationship between epithelial mesenchymal transition and mTOR signal pathway at the molecular level.ResultsAfter TGF-β2 treatment for 24 hours, microscopic observation revealed that HLEB-3 cells were transformed from the oval shape into the star or fusiform shape, and cell connection was also reduced. Western blotting revealed that the expression levels of epithelial protein marker E-cadherin in HLEB-3, following induction of TGF-β2, was obviously decreased, while the expression levels of the stromal cell protein marker α-SMA were significantly decreased, and statistical significance could be noticed when comparisons were made between them(P<0.05). MTT test also indicated that Rapamycin pretreatment could inhibit the enhancing effect of TGF-β2 on epithelial cell proliferation. Western blotting showed that Rapamycin could reverse the up-regulation of α-SMA by TGF-β2 and promote the expression of epithelial protein marker E-cadherin, following the activation of mTOR signal pathway inhibited by Rapamycin.ConclusionEpithelial mesenchymal transition induced by mTOR signal pathway and mediated by TGF-β2 could promote the onset of after-cataract.

TGF -β2; mTOR signal pathway; Post capsular opacification

虹口區(qū)衛(wèi)生和計(jì)劃生育委員會(huì)醫(yī)學(xué)科研課題(虹衛(wèi)1603-27)；上海市科學(xué)技術(shù)委員會(huì)科研計(jì)劃項(xiàng)目(15ZR1413200)

200433 上海，第二軍醫(yī)大學(xué)附屬長(zhǎng)海醫(yī)院眼科[葛茸茸(現(xiàn)工作單位解放軍第四一一醫(yī)院眼科)、沈煒]

沈煒，電子信箱：shenwzz@163.com

R776.1

A

10.3969/j.issn.1009-0754.2016.06.006

2016-10-11)