應(yīng)用現(xiàn)代生物技術(shù)和設(shè)備測定和評估肝臟的心肌保護(hù)作用

劉樹謙

美國西北大學(xué) 生物醫(yī)學(xué)工程系，美國伊利諾斯州 埃文斯頓

0 前言

冠狀動脈病變引起的心肌缺血可導(dǎo)致心肌細(xì)胞損傷和心功能衰退，但另一方面可激活心肌保護(hù)機(jī)制，抑制心肌死亡,改進(jìn)心肌功能。哺乳動物包括人類有兩種心肌保護(hù)機(jī)制：心源性的和非心源性的。心源性機(jī)制包括保護(hù)分子釋放及心肌干細(xì)胞激活。在分子水平, adenosine , bradykinin, and opioids等小分子可以快速地(在幾小時內(nèi))從損傷的心臟細(xì)胞釋放到細(xì)胞外間隙, 作用于缺血心肌，激活G-蛋白耦合受體信息通道，進(jìn)而激活細(xì)胞活性刺激分子PI3K和Akt1，引起細(xì)胞死亡分子BAD磷酸化，以此抑制心肌細(xì)胞死亡[1-6]。隨著小分子的釋放, 損傷心臟細(xì)胞及激活的白細(xì)胞也可在幾天之內(nèi)表達(dá)多種生長因子，包括成纖維細(xì)胞增長因子1和2，血管內(nèi)皮生長因子，和胰島素樣生長因子。這些因子可激活酪氨酸激酶耦合受體及PI3K-Akt1和MEK-ERK1/2信息通道，進(jìn)而刺激心肌細(xì)胞修復(fù)[7-20]。在細(xì)胞水平，心肌損傷可激活心源性干細(xì)胞。這些細(xì)胞可分化成心肌細(xì)胞, 進(jìn)而促進(jìn)心肌修復(fù)，改進(jìn)心肌功能[21-23]。

非心源性心肌保護(hù)機(jī)制主要涉及系統(tǒng)器官和組織。骨髓和肝臟是目前發(fā)現(xiàn)的主要心肌保護(hù)器官。隨著心肌缺血,骨髓可釋放造血干細(xì)胞和血管內(nèi)皮前體細(xì)胞于血液循環(huán)系統(tǒng), 部分游離骨髓細(xì)胞可以進(jìn)入缺血性心肌，促進(jìn)心肌修復(fù)[24-27]。與此同時，心肌缺血引起心肌與血液促炎因子如interlekin-6的水平增加。這些因子可作用于肝臟，引發(fā)肝細(xì)胞表達(dá)心肌保護(hù)因子，包括-1-acid glycoprotein 2 (AGP2),bone morphogenetic protein binding endothelial regulator (BMPER),fibroblast growth factor 21 (FGF21), neuregulin 4 (NRG4), 和 trefoil factor 3 (TFF3)[53]。這些保護(hù)因子可釋放于血液內(nèi)，作用于缺血心肌，進(jìn)而減少心肌損傷[28]。肝臟的另一個心肌保護(hù)機(jī)制是將肝細(xì)胞游離和釋放于循環(huán)系統(tǒng)[29]。部分游離肝細(xì)胞可隨血流進(jìn)入缺血心肌[30]。這些細(xì)胞可能釋放心肌保護(hù)因子，提升心肌保護(hù)因子的局部水平，因而到達(dá)迅速保護(hù)心肌的目的[28,30]。這里，作者將主要討論應(yīng)用現(xiàn)代生物技術(shù)和設(shè)備對肝臟的心肌保護(hù)作用的測定和評估。

1 肝源性心肌保護(hù)因子

心肌缺血可導(dǎo)致肝細(xì)胞表達(dá)心肌保護(hù)分泌蛋白因子。這些蛋白因子可以被釋放入血液循環(huán)系統(tǒng)，隨血流進(jìn)入缺血心肌，因而保護(hù)心肌，減少心肌損傷[28-29]。這里我們將討論三個問題: 這些蛋白因子是怎么發(fā)現(xiàn)的, 蛋白因子的長期心肌保護(hù)功能以及作用機(jī)制。

1.1 肝源性心肌保護(hù)因子的篩選

在小鼠冠狀動脈結(jié)扎誘發(fā)的心肌局部缺血模型，肝細(xì)胞可在一到三天內(nèi)增進(jìn)九個分泌蛋白基因的表達(dá)，包括AGP2, BMPER, chemokine (C-X-C motif)ligand 13 (CXCL13),FGF21, NRG4, PRG4, serum amyloid A1 (SAA1)和 A2 (SAA2)，及TFF3[5,28]。鑒于這些蛋白在心肌缺血時表達(dá)，它們可能包括心肌保護(hù)因子。為識別其中的心肌防護(hù)因子，我們進(jìn)行了功能篩選試驗。在冠狀動脈結(jié)扎之后，我們對心肌缺血小鼠立即進(jìn)行肝分泌蛋白靜脈注射(每組小鼠僅注射一種蛋白，50 ng/gm 體重, PBS為對照劑)。SAA1 和SAA2沒有進(jìn)行篩選試驗, 因為這兩種蛋白經(jīng)常引起組織淀粉樣病變和組織功能損傷[5,28]。在肝分泌蛋白注射后24 h，小鼠左心室被用于心肌梗塞測試。與PBS比較，注射AGP2,BMPER, FGF21, NRG4, 或TFF3導(dǎo)致顯著心肌梗塞減少。相比而言，注射CXCL13或PRG4沒有引起顯著心肌梗塞改變。這些觀察顯示，AGP2, BMPER, FGF21, NRG4和TFF3有心肌保護(hù)的作用。在心肌缺血后一到三天,這些蛋白在肝細(xì)胞和血液都顯著增加[28]。而心肌細(xì)胞死亡正好發(fā)生在這一時期[28]?？梢娺@些蛋白是為心肌保護(hù)而生。

在以前的研究中，上述五個分子是從不同的細(xì)胞類種被發(fā)現(xiàn)的，并有著不同的功用[31]。AGP2，也稱為 orosomucoid 2 (ORM2)，是肝細(xì)胞合成的急性炎癥反應(yīng)血液蛋白。其由201 氨基酸組成，分子量為23.6 kDa。AGP2的表達(dá)受多個促炎因子的調(diào)控，包括糖皮質(zhì)激素，IL1，IL6和TNF。AGP2的以知功能包括調(diào)控炎癥和免疫反應(yīng)[32]。比如，AGP2可以抑制T淋巴細(xì)胞發(fā)育和增長[32]；抑制嗜中性粒細(xì)胞活性和增長[33]；抑制血小板聚合[34]；刺激單核細(xì)胞和巨噬細(xì)胞表達(dá)和分泌細(xì)胞因子，包括IL1，IL6，IL12 和腫瘤壞死因子[35]；以及刺激成纖維細(xì)胞增殖和促進(jìn)傷口愈合[36]。

BMPER, 也稱為 crossveinless 2, 是由 685 氨基酸組成 , 其分子量為76 kDa。BMPER 最處發(fā)現(xiàn)于Flk-1陽性血管內(nèi)皮細(xì)胞。該分子可釋放于細(xì)胞外空間,通過與BMP4相互作用來影響細(xì)胞的活性[32,57]。 BMP4的作用主要是參與調(diào)控Smad信號傳導(dǎo)，控制中胚層發(fā)育,促進(jìn)血管內(nèi)皮細(xì)胞分化和血管形成,以及刺激成骨細(xì)胞和軟骨細(xì)胞增長。但是,BMP4對BMPER的影響是有爭議的。許多調(diào)查表明BMPER抑制BMP4的活性[38-40]，但也有人表明BMPER刺激BMP4的活性[41-43]。最近研究提供了解決這個爭議的證據(jù)。BMPER可刺激或抑制BMP4的活性，其影響依賴于BMPER的相對水平。當(dāng)BMPER的水平超過BMP4，BMPER可掩蓋BMP4受體，從而抑制BMP4的活性。反之,BMPER刺激BMP4的活性[44]。

FGF21是由209氨基酸組成,其分子量為22.3kDa,屬于成纖維細(xì)胞生長因子家族[45]。FGF21主要在肝臟表達(dá)。胸腺和脂肪組織也有表達(dá),但表達(dá)程度較低[6,46]。大多數(shù)成纖維細(xì)胞生長因子有調(diào)節(jié)細(xì)胞增殖和分化的作用，而FGF21已知參與調(diào)節(jié)血糖和血脂代謝[47-48]。據(jù)報，F(xiàn)GF21有以下具體的代謝功能：[1]刺激細(xì)胞的葡萄糖攝取和代謝從而減少血糖水平; [2]誘導(dǎo)胰島素表達(dá)和分泌；[3]調(diào)節(jié)脂肪細(xì)胞內(nèi)脂肪酸代謝；[4]減少血漿低密度脂蛋白和增加高密度脂蛋白水平[47,49-53]。FGF21現(xiàn)已用于治療糖尿病, 血脂紊亂和肥胖的研究。

NRG4, 也稱為 heregulin 4 (HRG4)，由115氨基酸組成,分子量為12.7kDa,主要在胰腺和骨骼肌表達(dá)[54]。NRG4是一個細(xì)胞膜蛋白，其細(xì)胞外領(lǐng)域含有一表皮增長因子的部分。當(dāng)合成后，NRG4既分布于細(xì)胞膜。其細(xì)胞膜外部分可由蛋白酶切割游離。游離NRG4可作用于酪氨酸激酶耦合受體HER4,進(jìn)而調(diào)控細(xì)胞活性。NRG4的主要功用是刺激ErbB4陽性細(xì)胞生長,促進(jìn)神經(jīng)元軸突或枝狀突起延伸,調(diào)控胰腺島細(xì)胞發(fā)育[54-55]。

TFF3由80氨基酸組成,分子量為8.6 kDa,主要在胃腸道的杯狀細(xì)胞表達(dá)。三環(huán)狀氨基酸排列為該分子的結(jié)構(gòu)特征。TFF3的主要功用是在生理條件下維持胃腸道黏膜完整性;促進(jìn)損傷黏膜愈合和修復(fù)[56-57]。體外測試證明，TFF3可增強(qiáng)腸道內(nèi)的黏蛋白聚合，以致形成穩(wěn)定的胃腸道黏膜層。該黏膜層對胃腸系統(tǒng)有保護(hù)作用。

1.2 肝源性心肌保護(hù)因子的長期功能

肝源性心肌保護(hù)因子的主功能是增加心肌對缺氧的耐受性，減少心肌死亡和改進(jìn)心肌收縮功能。我們使用組織化學(xué)和血液動力學(xué)方法評估肝因子對心肌的長期保護(hù)作用。當(dāng)五個肝因子，包括AGP2，BMPER，F(xiàn)GF21，NRG4，和TFF3，按心肌缺血時血液肝因子濃度比注入心肌缺血的老鼠(靜脈注射，每12小時一次，連續(xù)注射三天)，心肌梗塞范圍在五，十，和三十天有很大程度減少[28]。連續(xù)注射三天的原因是這一期間正是心肌細(xì)胞死亡的時間。三天以后開始注射沒有顯著療效。進(jìn)而，五肝因子注射顯著改進(jìn)了左心室dp/dt和收縮指數(shù)。這些結(jié)果支持肝因子的心肌保護(hù)作用[28]。有一點應(yīng)該強(qiáng)調(diào)，既然肝臟可產(chǎn)生心肌保護(hù)因子，為什么仍然需要注射這些因子來保護(hù)心肌。這是因為在心肌缺血以后這些蛋白因子的表達(dá)大約需要一天的時間。在這段時間，心肌在肝源性保護(hù)因子達(dá)到有效水平前已開始死亡。因此，注射心肌防護(hù)因子是必要的，而且是越快越好[28]。

1.3 肝源性心肌保護(hù)因子作用機(jī)制

一個重要的議題是肝蛋白因子如何保護(hù)缺血心肌。每一個蛋白因子都有一不同的作用機(jī)制。目前，我們已經(jīng)研究了其中有兩個因子：FGF21和TFF3[58-59]。FGF21可作用于心肌細(xì)胞酪氨酸激酶耦合FGF受體 1 (FGFR1)， 進(jìn)而激活細(xì)胞活性刺激分子PI3K和Akt1，引起細(xì)胞死亡分子BAD磷酸化[58]。當(dāng)BAD處于脫磷酸狀態(tài)，可與抗細(xì)胞凋亡分子Bcl-2和Bcl-XL結(jié)合，從而抑制Bcl-2和Bcl-XL的抗細(xì)胞凋亡作用，導(dǎo)致細(xì)胞死亡增加。相反，當(dāng)BAD處于磷酸化狀態(tài)，Bcl-2和Bcl-XL由BAD釋放出來，不再受BAD的抑制，導(dǎo)致細(xì)胞死亡減少，缺血心肌得以保護(hù)[30]。TFF3也可激活PI3K和Akt1，但其心肌細(xì)胞受體有待研究[59]。 可見，F(xiàn)GF21和TFF3激活細(xì)胞內(nèi)的共同信號傳導(dǎo)途徑。

2 游離肝細(xì)胞對缺血心肌的保護(hù)作用

心肌缺血可導(dǎo)致肝臟細(xì)胞游離并釋放于血液循環(huán)系統(tǒng)。部分游離肝細(xì)胞可隨血流進(jìn)入缺血心肌，進(jìn)而保護(hù)心肌[28,30]。這里我們將討論兩個問題：肝細(xì)胞是如何釋放的以及肝臟細(xì)胞如何保護(hù)缺血心肌。

2.1 肝細(xì)胞的釋放和調(diào)控

隨著心肌缺血的誘發(fā)，肝細(xì)胞可在三到天五內(nèi)出現(xiàn)于血液循環(huán)系統(tǒng)。一個基本問題是如何確認(rèn)循環(huán)肝細(xì)胞。我們建立了一個肝臟細(xì)胞特異黃熒光蛋白表達(dá)的小鼠模型[29]。循環(huán)肝細(xì)胞可根據(jù)黃色熒光蛋白來辨認(rèn)。循環(huán)肝細(xì)胞的數(shù)量在心肌缺血后5天內(nèi)可達(dá)到約白血球的1%，之后逐漸減少。部分游離肝細(xì)胞可隨血流進(jìn)入缺血心肌[30]。這些細(xì)胞在心肌缺血30天后基本消失。下面， 筆者將主要討論肝細(xì)胞是如何游離和釋放的。

缺血心肌以及在心肌病灶內(nèi)激活的白血球可釋放促炎因子interleukin 6 (IL6)。 該因子可進(jìn)一步激活循環(huán)白血球。這些白血球可粘附于肝臟血管內(nèi)皮細(xì)胞，遷移到肝組織， 表達(dá)并釋放基質(zhì)蛋白酶 2 (MMP2)。MMP2進(jìn)而分解肝細(xì)胞外基質(zhì)，導(dǎo)致肝細(xì)胞游離。游離肝細(xì)胞隨血流進(jìn)入肝中央靜脈及腔靜脈[29]。這些細(xì)胞可在靜脈， 肺循環(huán)， 左心房， 和左心室系統(tǒng)生存。但是，一但游離肝細(xì)胞進(jìn)入體動脈，這些細(xì)胞在動脈血流剪應(yīng)力的作用下分解。所以， 在周邊動脈和靜脈是很難發(fā)現(xiàn)游離肝細(xì)胞的[5]。由于冠狀動脈相對較短，進(jìn)入冠狀動脈的游離肝細(xì)胞不會全部被分解，部分細(xì)胞滯留于心肌。在心肌缺血誘發(fā)五至十天后，IL6和MMP2的表達(dá)和釋放減少，肝細(xì)胞游離程度下降。

2.2 游離肝細(xì)胞對缺血心肌的保護(hù)

進(jìn)入缺血心肌的肝細(xì)胞對心肌細(xì)胞有保護(hù)作用。雖然這些肝細(xì)胞如何保護(hù)心肌的機(jī)制仍未完全了解，但有一點比較明確，既進(jìn)入心肌的肝細(xì)胞可釋放心肌保護(hù)因子[28,30]。與釋放于血液比較，直接釋放于局部缺血心肌將迅速提升保護(hù)因子的濃度，有利于迅速有效地保護(hù)心肌。有一點應(yīng)該說明，游離肝細(xì)胞僅出現(xiàn)于心肌缺血部位，但不出現(xiàn)在正常心肌。對這一現(xiàn)象的機(jī)制仍待研究。

3 生物技術(shù)和設(shè)備的應(yīng)用

在該項研究中，我們采用了兩種主要生物技術(shù)和設(shè)備：cDNA microarray analysis 和 flow cytometry。cDNA microarray analysis可用于系統(tǒng)性監(jiān)測基因在某一細(xì)胞種類的表達(dá)。通過測定小鼠心肌缺血誘發(fā)的肝臟細(xì)胞全基因表達(dá)， 我們發(fā)現(xiàn)了如上所述的九個表達(dá)增強(qiáng)的蛋白， 其中包括五個心肌防護(hù)蛋白。Flow cytometry 可用于測定細(xì)胞種類和蛋白表達(dá).我們就是使用了這個方法發(fā)現(xiàn)了心肌缺血時循環(huán)的肝細(xì)胞。cDNA microarray analysis[60-61]和 flow cytometry[62-64]的技術(shù)，設(shè)備以及作用原理已在文獻(xiàn)中詳細(xì)討論，這里不再重復(fù)?？梢?，這些生物技術(shù)和設(shè)備在發(fā)現(xiàn)肝臟對局部缺血心肌的保護(hù)上起了至關(guān)重要的作用。

4 結(jié)束語

以上研究表明，心肌損傷可誘發(fā)系統(tǒng)性自體保護(hù)措施。肝臟活化是其中措施之一。肝臟已知有多種功能，如調(diào)控新陳代謝、產(chǎn)生膽汁、解毒、和產(chǎn)生血漿蛋白等。除這些功能之外， 肝臟也起心肌保護(hù)的作用。這一發(fā)現(xiàn)對理解心肌保護(hù)機(jī)制以及開發(fā)心肌保護(hù)措施有重要的意義。

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