席 銳 鄒 琛 陳楨玥 吳春芳 陸國(guó)平 徐志紅

·基礎(chǔ)研究·

同型半胱氨酸促進(jìn)血管平滑肌細(xì)胞增殖的機(jī)制及辛伐他汀的干預(yù)作用

席 銳 鄒 琛 陳楨玥 吳春芳 陸國(guó)平 徐志紅

目的：探討同型半胱氨酸促進(jìn)大鼠血管平滑肌細(xì)胞(VSMC)增殖的機(jī)制及辛伐他汀的干預(yù)作用。 方法：貼壁培養(yǎng)的大鼠原代VSMC隨機(jī)分為3組。(1)對(duì)照組：用含10%胎牛血清的DMEM培養(yǎng)基(完全培養(yǎng)基)； (2)同型半胱氨酸組：在完全培養(yǎng)基中加入同型半胱氨酸(0.05 mmol/L)； (3)辛伐他汀組：在完全培養(yǎng)基中加入同型半胱氨酸(0.05 mmol/L)和辛伐他汀(10 μmol/L)。培養(yǎng)24 h后，光學(xué)顯微鏡下觀(guān)察各組細(xì)胞的形態(tài)；采用半定量PCR法檢測(cè)c-myc 、P27kip1的mRNA水平；用Western blot檢測(cè)細(xì)胞周期蛋白(cyclin )D1、cyclin E、cyclin A和核增殖抗原(PCNA)的蛋白表達(dá)水平。 結(jié)果：與對(duì)照組相比，同型半胱氨酸組c-myc mRNA水平上調(diào)，P27kip1mRNA水平下調(diào)，下游cyclin D1、cyclin E、cyclin A和PCNA蛋白表達(dá)增加。與同型半胱氨酸組相比，辛伐他汀組上述檢測(cè)指標(biāo)均得到逆轉(zhuǎn)。 結(jié)論：同型半胱氨酸可促進(jìn)大鼠VSMC c-myc、cyclin D1、cyclin E、cyclin A等增殖相關(guān)基因的表達(dá)，抑制P27kip1表達(dá)。辛伐他汀可逆轉(zhuǎn)上述效應(yīng)。

同型半胱氨酸；動(dòng)脈粥樣硬化；血管平滑肌細(xì)胞

同型半胱氨酸是一種含巰基氨基酸，是體內(nèi)蛋氨酸分解過(guò)程中產(chǎn)生的重要中間產(chǎn)物。臨床研究顯示，同型半胱氨酸與動(dòng)脈粥樣硬化性疾病(如冠心病、腦血管疾病及外周血管栓塞性疾病等)密切相關(guān)，是動(dòng)脈粥樣硬化的獨(dú)立危險(xiǎn)因子[1-11]。同型半胱氨酸的致動(dòng)脈粥樣硬化作用可能與其促進(jìn)血管平滑肌細(xì)胞(VSMC)增殖、導(dǎo)致血管內(nèi)皮功能障礙以及致炎、促凝和促氧化等作用有關(guān)[12-15]。本課題組前期研究證實(shí)，同型半胱氨酸可促進(jìn)大鼠VSMC增殖[16]，但具體機(jī)制不明。本研究進(jìn)一步探討同型半胱氨酸促進(jìn)VSMC增殖的機(jī)制以及辛伐他汀的干預(yù)作用。

1 材料與方法

1.1 材料

SD大鼠購(gòu)自上海中科院動(dòng)物研究所；兔源細(xì)胞周期蛋白(cyclin) D1多克隆抗體以及鼠源核增殖抗原(PCNA)單克隆抗體購(gòu)自Cell Signaling公司；兔源cyclin A多克隆抗體以及鼠源cyclin E單克隆抗體購(gòu)自Santa Cruz公司。PCR引物采用Primer 3 軟件設(shè)計(jì)，序列見(jiàn)表1。

1.2 方法

采用組織塊貼壁法原代培養(yǎng)SD大鼠的VSMC。取生長(zhǎng)狀態(tài)良好的第4代細(xì)胞制成細(xì)胞懸液(4×107個(gè)/ml)，接種于6孔細(xì)胞培養(yǎng)板中，每孔1 ml，孵育24 h，換無(wú)血清培養(yǎng)24 h后，依次分為3組。(1)對(duì)照組：用含10%胎牛血清的DMEM培養(yǎng)基(完全培養(yǎng)基)培養(yǎng)，不加特殊處理；(2)同型半胱氨酸組：完全培養(yǎng)基中加入同型半胱氨酸(0.05 mmol/L)；(3)辛伐他汀組：在完全培養(yǎng)基中加入同型半胱氨酸(0.05 mmol/L)和辛伐他汀(10 μmol/L)。各組細(xì)胞均設(shè)立3個(gè)復(fù)孔，藥物處理時(shí)間均為24 h。

采用Trizol法提取細(xì)胞總RNA。按逆轉(zhuǎn)錄試劑盒說(shuō)明書(shū)合成cDNA。PCR反應(yīng)條件：94℃變性5 min, 94℃變性45 s，50℃退火1 min，72℃延伸1 min，35個(gè)循環(huán)，最后再72℃延伸10 min，4℃終止反應(yīng)。電泳后膠條在凝膠成像儀中攝影，Image Quant軟件進(jìn)行灰度掃描分析。

采用Western blot檢測(cè)cyclin D1、cyclin E、cyclin A和PCNA的蛋白表達(dá)水平。

1.3 統(tǒng)計(jì)學(xué)分析

計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差表示，多組間比較用q檢驗(yàn)；計(jì)數(shù)資料以百分?jǐn)?shù)(%)表示，組間比較用χ2檢驗(yàn)；所有數(shù)據(jù)用SPSS 18.0軟件進(jìn)行分析。P<0.05表示差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 VSMC形態(tài)學(xué)變化

對(duì)照組細(xì)胞排列有序；同型半胱氨酸組細(xì)胞排列雜亂，呈團(tuán)狀密集樣增生；經(jīng)辛伐他汀干預(yù)后細(xì)胞密度降低，細(xì)胞體積縮小，細(xì)胞間連接緊密(見(jiàn)圖1)。

注：A為對(duì)照組;B為同型半胱氨酸組;C為辛伐他汀組

2.2 細(xì)胞增殖相關(guān)基因的表達(dá)

與對(duì)照組相比，同型半胱氨酸組原癌基因c-myc mRNA水平明顯升高，細(xì)胞周期蛋白激酶抑制蛋白P27kip1mRNA水平明顯下調(diào)；與同型半胱氨酸組相比，辛伐他汀組c-myc水平顯著降低，P27kip1水平明顯上調(diào)(P<0.05，見(jiàn)表2)。

表2 各組c-myc、P27kip1的mRNA相對(duì)表達(dá)量

與對(duì)照組相比，同型半胱氨酸組cyclin D1、cyclin A、cyclin E及PCNA蛋白表達(dá)水平明顯升高；與同型半胱氨酸組相比，辛伐他汀組上述蛋白表達(dá)水平明顯降低(P<0.05，見(jiàn)圖2、表3)。

注：A為對(duì)照組；B為同型半胱氨酸組；C為辛伐他汀組

表3 各組cyclin和PCNA蛋白表達(dá)水平

3 討論

同型半胱氨酸促進(jìn)VSMC增殖的機(jī)制尚未闡明，可能與細(xì)胞外各種絲裂原激活不同信號(hào)傳導(dǎo)途徑有關(guān)。研究發(fā)現(xiàn)，同型半胱氨酸可通過(guò)興奮氨基酸受體或氧化還原受體，引起Ca2+內(nèi)流。一方面激活磷脂酶/三磷酸肌醇傳導(dǎo)途徑，激活蛋白激酶C，進(jìn)而激活絲裂素活化蛋白激酶；另一方面可通過(guò)鈣調(diào)蛋白激活鈣調(diào)蛋白激酶，促進(jìn)原癌基因c-myc、轉(zhuǎn)錄因子c-myb及c-fos的表達(dá)，經(jīng)過(guò)絲裂素活化蛋白激酶的催化，促進(jìn)基因轉(zhuǎn)錄，最終導(dǎo)致VSMC增殖[17]。

c-myc是控制細(xì)胞生長(zhǎng)的基因之一，為myc癌基因家族中關(guān)鍵的一員，其編碼產(chǎn)物c-myc蛋白作用于基因調(diào)控區(qū)，可啟動(dòng)一系列與增殖有關(guān)的基因，促進(jìn)細(xì)胞增殖、分化，參與腫瘤發(fā)生、轉(zhuǎn)移等病理過(guò)程[18-19]。本研究表明，同型半胱氨酸可誘導(dǎo)體外培養(yǎng)的大鼠VSMC c-myc轉(zhuǎn)錄增加，說(shuō)明同型半胱氨酸誘導(dǎo)的VSMC增殖可能與癌基因調(diào)控的分子生物學(xué)機(jī)制有關(guān)，但是同型半胱氨酸如何啟動(dòng)c-myc基因轉(zhuǎn)錄尚有待進(jìn)一步研究。

cyclin和細(xì)胞周期蛋白依賴(lài)性激酶(CDK)是細(xì)胞內(nèi)調(diào)節(jié)細(xì)胞周期的主要物質(zhì)，cyclin D、cyclin A、cyclin E能誘導(dǎo)細(xì)胞進(jìn)入有絲分裂S期，進(jìn)而促進(jìn)細(xì)胞增殖[20-21]。P27kip1是細(xì)胞周期蛋白依賴(lài)性激酶抑制蛋白(CKI)家族中的重要成員之一，可抑制不同的CDK復(fù)合物(主要包括cyclin E-CDK2、cyclin D-CDK4、cyclin A等G1期復(fù)合物)的活性，使細(xì)胞周期不能通過(guò)G1-S點(diǎn)而阻斷于G1期[22-24]。本研究顯示，同型半胱氨酸促進(jìn)VSMC中 cyclin D1、cyclin E、cyclin A的表達(dá)，抑制P27kip1表達(dá)，其誘導(dǎo)的VSMC增殖可能與下調(diào)P27kip1表達(dá)引起cyclin表達(dá)升高有關(guān)。

辛伐他汀的非調(diào)脂作用包括改善內(nèi)皮細(xì)胞功能、抑制VSMC增殖及維持斑塊穩(wěn)定性，長(zhǎng)期應(yīng)用可減少心血管事件的發(fā)生[25]。既往研究發(fā)現(xiàn)，辛伐他汀抑制同型半胱氨酸誘導(dǎo)的大鼠VSMC增殖主要是通過(guò)降低S期和G2/M期百分率、提高G0/G1期百分率，即誘導(dǎo)G0/G1期靜止[23]。本研究顯示，經(jīng)10 μmol/L辛伐他汀干預(yù)24 h后，P27kip1表達(dá)明顯上調(diào)，同時(shí)cyclin D1、cyclin E、cyclin A和細(xì)胞增殖的重要指標(biāo)PCNA也被辛伐他汀顯著抑制。P27kip1和cyclin上述協(xié)調(diào)性表達(dá)的結(jié)果，可解釋辛伐他汀作用于增殖的VSMC后，細(xì)胞周期主要停滯在G0/G1期，S期和G2/M期百分率下降，細(xì)胞接近靜息水平。

本研究初步探討了同型半胱氨酸誘導(dǎo)大鼠VSMC增殖的機(jī)制及辛伐他汀的作用。P27kip1蛋白表達(dá)的變化可能是決定VSMC增殖的關(guān)鍵[22-23]。因此，未來(lái)研究的重點(diǎn)是全面認(rèn)識(shí)P27kip1基因的作用，以用于控制細(xì)胞增殖的基因治療。

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(收稿：2014-07-21 修回：2014-12-03)

(本文編輯：丁媛媛)

Homocysteine on vascular smooth muscle cell proliferation and effects of simvastatin

XIRui1,ZOUChen1,CHENZhenyue1,WUChunfang1，LUGuoping1,XUZhihong2.

1DepartmentofCardiology; 2DepartmentofGeriatrics,RuijinHospital,ShanghaiJiaotongUniversity,Shanghai200025,China

Objective:To explore the mechanism of homocysteine promoting the proliferation of rat vascular smooth muscle cells (VSMC) and intervention effects of simvastatin. Methods:Primary rat VSMC were divided into 3 groups. Cells in control group were cultured in DMEM with 10% fetal bovine serum. Cells in homocysteine group were treated with 0.05 mmol/L homocysteine，and cells in simvastatin group were treated with 0.05 mmol/L homocysteine and 10 μmol/L simvastatin. Morphology of cells was observed under optical microscope. The mRNA levels of c-myc和P27kip1were analyzed by semi-quantitative PCR, and the protein levels of cyclin D1, cyclin E, cyclin A and PCNA were evaluated by Western blot. Results:Compared with control group, the mRNA level of c-myc and protein levels of cyclin D1, cyclin E, cyclin A and PCNA in homocysteine group were significantly increased, while the mRNA level of P27kip1was decreased. After treatment with simvastatin, P27kip1was upregulated compared with homocysteine group accompanied by the inhibition of c-myc, cyclin D1, cyclin E, cyclin A and PCNA. Conclusion:Homocysteine promotes the expression of proliferation associated genes such as c-myc, cyclin D1, cyclin E, cyclin A and PCNA and inhibits P27kip1expression, which can be reversed by simvastatin.

Homocysteine; Atherosclerosis; Smooth muscle cells

上海市科委重大基礎(chǔ)研究項(xiàng)目(054119634)

200025 上海交通大學(xué)附屬瑞金醫(yī)院心臟科(席 銳,鄒 琛,陳楨玥,吳春芳,陸國(guó)平)；老年病科(徐志紅)

徐志紅，Email:zhihxu@163.com

10.3969/j.issn.1673-6583.2015.01.014