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大鼠視網(wǎng)膜Müller細(xì)胞在高糖、氧化應(yīng)激、缺氧中的表現(xiàn)

2019-07-18 05:52李春花馮朝暉張雪田冰玉雷曉琴周婷潔馬為梅
關(guān)鍵詞:糖尿病視網(wǎng)膜病變

李春花 馮朝暉 張雪 田冰玉 雷曉琴 周婷潔 馬為梅

[摘要] 目的 觀察Sprague-Dawley(SD)大鼠視網(wǎng)膜Müller細(xì)胞在高糖、氧化應(yīng)激、缺氧三種病理環(huán)境下的特征,為糖尿病視網(wǎng)膜病變的防治提供新的研究依據(jù)。 方法 體外培養(yǎng)SD大鼠視網(wǎng)膜Müller細(xì)胞,并用谷氨酰胺合成酶(GS)鑒定。將傳2代的細(xì)胞隨機(jī)分為四組:對(duì)照組、高糖組、氧化應(yīng)激組、缺氧組。倒置顯微鏡觀察細(xì)胞形態(tài)學(xué)改變,免疫熒光染色法觀察細(xì)胞GS和α-平滑肌肌動(dòng)蛋白(α-SMA)的變化,Transwell小室觀察細(xì)胞遷移能力。 結(jié)果 體外成功培養(yǎng)并鑒定鼠Müller細(xì)胞,四組Müller細(xì)胞功能均受損,氧化應(yīng)激組以及缺氧組結(jié)構(gòu)受損明顯,失去正常形態(tài)。缺氧組中α-SMA的表達(dá)呈陽(yáng)性,并且在Transwell小室中發(fā)生遷移的細(xì)胞數(shù)明顯多于其他組,差異有統(tǒng)計(jì)學(xué)意義(P < 0.05)。 結(jié)論 高糖、氧化應(yīng)激、缺氧均可導(dǎo)致Müller細(xì)胞功能受損,缺氧可誘導(dǎo)Müller細(xì)胞轉(zhuǎn)化為具有遷移能力的肌纖維樣細(xì)胞,提示Müller細(xì)胞參與了增生性糖尿病視網(wǎng)膜病變的發(fā)病過(guò)程。

[關(guān)鍵詞] SD大鼠;視網(wǎng)膜Müller細(xì)胞;糖尿病視網(wǎng)膜病變;上皮細(xì)胞間質(zhì)轉(zhuǎn)化;Transwell小室

[中圖分類號(hào)] R587.2 ? ? ? ? ?[文獻(xiàn)標(biāo)識(shí)碼] A ? ? ? ? ?[文章編號(hào)] 1673-7210(2019)05(c)-0018-04

[Abstract] Objective To observe the characteristics of Sprague-Dawley (SD) rats retinal Müller cells under three pathological conditions including high glucose, oxidative stress and hypoxia, and to provide new research basis for the prevention and treatment of diabetic retinopathy. Methods SD rat retinal Müller cells were cultured in vitro and identified by glutathione synthetase (GS). The second generation of cells were divided into control group, high glucose group, oxidative stress group and hypoxia group randomly. The morphological changes of the cells were observed under inverted microscope, the changes of GS and α-smooth muscle actin (α-SMA) were observed by immunofluorescence staining, and the cell migration ability was observed by Transwell chamber. Results The Müller cells were successfully cultured and identified in vitro. In all groups, the function of Müller cells was impaired, and in oxidative stress group and hypoxia group, the structure was damaged, and the normal morphology was lost. In hypoxia group, the expression of α-SMA was positive, and the number of cells in the Transwell chamber was significantly higher than that in other groups, the differences were statistically significant (P < 0.05). Conclusion High glucose, oxidative stress and hypoxia can destroy the function of Müller cells. Hypoxia can induce transforming into myofibroblast cells with migratory ability, which indicates that Müller cells are involved in the pathogenesis of proliferative diabetic retinopathy.

[Key words] SD rat; Retinal Müller cells; Diabetic retinopathy; Epithelial cell interstitial transformation; Transwell chamber

糖尿病視網(wǎng)膜病變(diabetic retinopathy,DR)是糖尿病的主要致盲原因[1-3],其發(fā)病機(jī)制復(fù)雜,近年來(lái)研究表明DR的發(fā)生發(fā)展與視網(wǎng)膜Müller細(xì)胞有密切關(guān)系[4-8],其通過(guò)調(diào)節(jié)鈉鉀平衡、攝取神經(jīng)遞質(zhì)等來(lái)維持視網(wǎng)膜細(xì)胞外環(huán)境的穩(wěn)定性[9]。糖尿病時(shí)的高糖、氧化應(yīng)激、缺氧可以使Müller細(xì)胞功能受損、結(jié)構(gòu)破壞,關(guān)于上述3種病理環(huán)境中Müller細(xì)胞的損傷是否一致的報(bào)道甚少,因此本研究在體外模擬了DR的這3個(gè)階段,來(lái)研究Müller細(xì)胞參與DR的發(fā)病機(jī)制,為延緩或逆轉(zhuǎn)其結(jié)構(gòu)及功能異常提供探索性研究。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物

正常出生后7 d的SD大鼠20只,清潔級(jí)別,由西安交通大學(xué)醫(yī)學(xué)院動(dòng)物中心提供,許可證號(hào):SCXK(陜)2018-001,倫理號(hào):2018-191,飼養(yǎng)于無(wú)特定病原體級(jí)(SPF)動(dòng)物房。

1.2 儀器與試劑

倒置顯微鏡SZ51型(日本olympus公司);CO2培養(yǎng)箱PYC-16型(美國(guó)Scheldon公司);低溫高速離心機(jī)5810r型(德國(guó)Eppendorf公司,半徑15.4 cm);恒溫水浴箱HH-1型(上海森信研究?jī)x器有限公司)。DMEM培養(yǎng)基(Gibco公司,貨號(hào):C1199550BT);胎牛血清(杭州四季青生物工程公司,貨號(hào):11011-8611);兔抗鼠谷氨酰胺合成酶(GS)抗體(博奧森公司,貨號(hào):BS-1003R);兔抗鼠α-平滑肌肌動(dòng)蛋白(α-SMA)抗體(博奧森公司,貨號(hào):BS-11665R);熒光素偶聯(lián)的山羊抗兔IgG(Abbkine公司,貨號(hào):A22120)。

1.3 實(shí)驗(yàn)方法

1.3.1 大鼠視網(wǎng)膜Müller細(xì)胞分離、體外培養(yǎng)、鑒定及分組 ?將出生7 d的SD大乳鼠斷頸處死后浸泡于75%的酒精中,挖出眼球,顯微鏡下分離出視網(wǎng)膜組織,分別加入木瓜蛋白酶(27 U/mL)和0.25%的胰蛋白酶37℃進(jìn)行消化30 min,后加入含20% FBS的DMEM/F12培養(yǎng)基終止消化。放入37℃、體積分?jǐn)?shù)為5% CO2培養(yǎng)箱培養(yǎng),2~3 d換液1次。在原代培養(yǎng)5 d時(shí),按1∶3傳代,并使用GS鑒別。將傳2代細(xì)胞液隨機(jī)分為以下四組:①對(duì)照組,細(xì)胞未經(jīng)任何處理。②高糖組,經(jīng)50 mmol/L的葡萄糖(Glc)溶液處理。③氧化應(yīng)激組,經(jīng)100 μmol/L的過(guò)氧化氫(H2O2)溶液處理。④缺氧組,經(jīng)500 μmol/L的氯化鈷(CoCl2)溶液處理。

1.3.2 免疫熒光染色 ?隨機(jī)取爬片,20%山羊血清室溫孵育30 min,封閉內(nèi)源性IgG,加GS、α-SMA抗體工作液,加1∶200稀釋的熒光素標(biāo)記的二抗,37℃溫室避光孵育1 h,20%甘油封片劑封片,熒光顯微鏡觀察,照相。

1.3.3 細(xì)胞移行分析 ?將Müller細(xì)胞傳代到8 μm孔徑濾膜的Transwell小室中,待細(xì)胞貼壁達(dá)70%后,將細(xì)胞隨機(jī)分為對(duì)照組、高糖組、氧化應(yīng)激組、缺氧組。用預(yù)冷的丙酮固定20 min,用細(xì)胞刷刮除正面細(xì)胞,對(duì)透過(guò)濾膜小孔移行至濾膜反面的細(xì)胞進(jìn)行蘇木紫著染10 min,自來(lái)水沖洗5 min。顯微鏡觀察,照相,隨機(jī)取5個(gè)區(qū)域計(jì)數(shù)。

1.4 統(tǒng)計(jì)學(xué)方法

采用SPSS 18.0統(tǒng)計(jì)軟件對(duì)數(shù)據(jù)進(jìn)行分析,計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差(x±s)表示,多組間比較采用方差分析,組間兩兩比較采用LSD-t和SNK-q檢驗(yàn)。以P < 0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 Müller細(xì)胞的體外培養(yǎng)及鑒定

體外培養(yǎng)的Müller細(xì)胞呈扁平的多邊形融合狀生長(zhǎng),形態(tài)不規(guī)則并有較長(zhǎng)的突起。GS是主要鑒別酶,在胞核及胞質(zhì)中表達(dá)。見(jiàn)圖1(封三)。

2.2 各組細(xì)胞形態(tài)學(xué)以及功能觀察

對(duì)照組的Müller細(xì)胞描述同前,α-SMA表達(dá)陰性;高糖組Müller細(xì)胞形態(tài)基本同對(duì)照組,GS、α-SMA表達(dá)陰性;氧化應(yīng)激組Müller細(xì)胞數(shù)量減少,細(xì)胞崩解,GS、α-SMA表達(dá)陰性;缺氧組Müller細(xì)胞胞體變大,不規(guī)則樣突起增多,胞質(zhì)內(nèi)可見(jiàn)絲狀纖維,如箭頭所示,GS表達(dá)陰性,α-SMA表達(dá)陽(yáng)性。見(jiàn)圖2(封四)。

2.3 各組細(xì)胞移行能力觀察

Transwell小室細(xì)胞遷移試驗(yàn)結(jié)果顯示高糖組、氧化應(yīng)激組與對(duì)照組細(xì)胞遷移能力相近,各組間遷移細(xì)胞數(shù)比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P > 0.05);缺氧組遷移細(xì)胞數(shù)明顯多于其他組,差異有統(tǒng)計(jì)學(xué)意義(P < 0.05)。見(jiàn)圖3(封四)、表1。

3 討論

視網(wǎng)膜膠質(zhì)細(xì)胞以Müller細(xì)胞居多[10-12],因此體外培養(yǎng)Müller細(xì)胞成功率高。目前培養(yǎng)方法主要有組織塊貼壁培養(yǎng)法和酶解法。本實(shí)驗(yàn)改進(jìn)了酶解法,采用了木瓜蛋白酶(27 U/mL)和0.25%胰酶的分步消化,減少了離心所帶來(lái)的機(jī)械損傷。GS是一種表達(dá)于Müller細(xì)胞,能夠?qū)⒐劝彼徂D(zhuǎn)化為谷氨酰胺的關(guān)鍵酶,它可以作為Müller細(xì)胞的免疫標(biāo)志物[13-14]。本實(shí)驗(yàn)使用GS鑒別Müller細(xì)胞,其主要表達(dá)于胞核以及胞質(zhì)內(nèi),這與既往研究一致[15]。

Thompson等[16]的研究表明,糖基化終末產(chǎn)物可以引起Müller細(xì)胞功能下降。本研究主要研究了高糖早期(24 h)的變化。體外培養(yǎng)25~30 mmol/L的高糖水平,可近似地等同于糖尿病時(shí)體內(nèi)高糖狀態(tài)。因此,本研究選用50 mmol/L葡萄糖模擬體內(nèi)的絕對(duì)高糖條件。以50 mmol/L高糖作用Müller細(xì)胞24 h后發(fā)現(xiàn),高糖早期細(xì)胞形態(tài)未發(fā)生明顯改變,而GS表達(dá)陰性,分析可能在DR發(fā)病的早期,Müller細(xì)胞雖然結(jié)構(gòu)完整,但功能已受損。

目前已有研究證明糖尿病時(shí)氧化應(yīng)激的存在[17-18]。在多種細(xì)胞中,氧化應(yīng)激反應(yīng)都可導(dǎo)致細(xì)胞凋亡。Abrahan等[19]使用H2O2模擬體內(nèi)氧化應(yīng)激環(huán)境。H2O2是氧化應(yīng)激的產(chǎn)物,來(lái)源廣泛,所以本研究使用H2O2造成體外氧化應(yīng)激環(huán)境。本研究使用100 μmol/L的H2O2溶液,作用細(xì)胞24 h發(fā)現(xiàn)Müller細(xì)胞GS表達(dá)下降,細(xì)胞正常形態(tài)破壞,這與之前的研究一致[20]。

本研究選用CoCl2模擬體內(nèi)缺氧。鈷的一個(gè)用途是用于化學(xué)模擬低氧,常用化合物是CoCl2(100~250 μmol/L)。本研究選用500 μmol/L CoCl2溶液處理Müller細(xì)胞,24 h后發(fā)現(xiàn)GS表達(dá)減少甚至缺失,細(xì)胞體積變大,提示缺氧可損害Müller細(xì)胞正常形態(tài)和功能,而α-SMA染色陽(yáng)性,Transwell小室遷移研究提示細(xì)胞遷移能力增強(qiáng)。α-SMA是一組表達(dá)在多種類型細(xì)胞胞質(zhì)中的具有收縮功能的微管微絲結(jié)構(gòu),是細(xì)胞移行和具有收縮功能的基礎(chǔ)[21]。Transwell小室技術(shù)可觀察具有遷移能力的細(xì)胞穿過(guò)多孔濾膜的情況[22]。本研究使用α-SMA抗體及Transwell小室,提示缺氧可導(dǎo)致Müller細(xì)胞表型發(fā)生改變,使其轉(zhuǎn)化為具有遷移、收縮能力的肌纖維樣細(xì)胞。因而推測(cè)缺氧中的Müller細(xì)胞有可能移位于視網(wǎng)膜表面,參與增生性糖尿病視網(wǎng)膜病變的形成。

總之DR是多種因素相互作用的結(jié)果。本研究提示高糖、氧化應(yīng)激及缺氧早期均可造成Müller細(xì)胞功能破壞,后兩者對(duì)Müller細(xì)胞的損害更為嚴(yán)重。因此合理的抗氧化及抗缺氧治療可能成為DR治療的新靶點(diǎn)。

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[22] ?李冬平,原莉莉,張東昌.轉(zhuǎn)化生長(zhǎng)因子β2對(duì)人視網(wǎng)膜色素上皮細(xì)胞間質(zhì)轉(zhuǎn)化的影響[J].山西醫(yī)藥雜志,2017, 46(17):2035-2037.

(收稿日期:2018-11-09 ?本文編輯:張瑜杰)

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