脂肪間充質(zhì)干細(xì)胞治療急性肝衰竭機制的研究進(jìn)展

趙高遠(yuǎn)，譚雪瑩，王占春

(青島大學(xué)醫(yī)學(xué)院附屬青島市市立醫(yī)院，山東青島 266011)

急性肝衰竭最有效的治療方法為原位肝移植，行緊急肝移植的患者預(yù)期生存率約為75%[2]。但目前因存在肝臟供體嚴(yán)重不足、手術(shù)損傷、免疫排斥反應(yīng)及費用昂貴等諸多問題，使得急性肝衰竭的治療成為了一個國際性的難題[2，3]。肝細(xì)胞移植是將分離純化的肝細(xì)胞植入患者體內(nèi)暫時性地對遺傳性代謝性肝病和急性肝衰竭等患者提供代謝性支持，與原位肝移植比較，具有價廉、操作簡單、可重復(fù)進(jìn)行、移植細(xì)胞可體外培養(yǎng)增殖及冷凍保存，還可進(jìn)行基因修飾等優(yōu)點[4]，是目前的研究熱點。但肝細(xì)胞來源短缺、肝細(xì)胞移植后增殖能力差及免疫排斥等問題限制了其臨床應(yīng)用。目前細(xì)胞來源的研究主要集中于胚胎干細(xì)胞(ESC)與成體干細(xì)胞(ASC)上，二者雖各有優(yōu)點，但ESC的應(yīng)用仍有倫理、道德、法律等方面的爭議，向肝細(xì)胞系分化的報道局限于鼠體外研究。ASC可分化成為其他組織細(xì)胞，亦即具有可塑性。目前成體干細(xì)胞移植方面的研究主要是骨髓間充質(zhì)干細(xì)胞以及脂肪間充質(zhì)干細(xì)胞(ADSCs)，脂肪組織中的間充質(zhì)干細(xì)胞(MSC)可分化為神經(jīng)元、內(nèi)皮細(xì)胞、肌細(xì)胞、成骨細(xì)胞、成軟骨細(xì)胞等[5,6]，并可以橫向分化為肝前體細(xì)胞和肝細(xì)胞[7]，其具有取材容易，增殖能力強，體外培養(yǎng)、傳代及擴增容易，可以直接取材于患者本人，能避免移植后免疫排斥反應(yīng)的發(fā)生等特點。用ADSCs替代成熟肝細(xì)胞進(jìn)行移植治療肝病，有望使細(xì)胞移植在肝病治療方面取得重大突破。本文就脂肪間充質(zhì)干細(xì)胞(ADSCs)移植治療急性肝功能衰竭的機制作一綜述。

1 抑制肝細(xì)胞

大量研究表明急性肝衰竭與肝細(xì)胞凋亡的關(guān)系十分密切，肝細(xì)胞凋亡是肝衰竭發(fā)病的關(guān)鍵環(huán)節(jié)[8]。ADSCs可以分泌大量抗凋亡因子，如VEGF、Caspase、HGF等因子[9,10]。VEGF作為一種促進(jìn)血管再生的重要因子，在ADSCs治療急性肝衰竭時表達(dá)上升[11,12]。Caspase是一組含半胱氨酸的天冬氨酸蛋白水解酶，是一種存在于細(xì)胞質(zhì)中具有類似結(jié)構(gòu)的蛋白酶。Caspase與真核細(xì)胞凋亡密切相關(guān)，并參與細(xì)胞的生長、分化與凋亡調(diào)節(jié)，急性肝衰竭時其表達(dá)上升，鄧?yán)甑萚13]研究發(fā)現(xiàn)，應(yīng)用Caspase抑制劑后急性肝衰竭小鼠肝細(xì)胞凋亡率明顯下降。HGF是存在于急性肝損傷動物血漿中的蛋白因子，能刺激肝細(xì)胞的DNA合成，在肝再生過程中起重要作用。HGF不同于再生肝和胚胎肝細(xì)胞中的肝刺激物質(zhì)(HSS)，愈來愈多的報道表明，HGF不僅作用于肝再生，且對許多組織和細(xì)胞的生長、分化起重要調(diào)控作用[14]。Nishino等[15]研究發(fā)現(xiàn),HGF基因移植能夠在早期改善行部分肝切除術(shù)的肝纖維化大鼠生存,其機制可能與HGF上調(diào)Bcl-xl的表達(dá)從而抑制肝細(xì)胞凋亡有關(guān)。許多研究證實ADSCs治療急性肝衰竭小鼠后，較模型組相比治療組肝細(xì)胞凋亡率明顯下降[16]。以上研究說明ADSCs可以通過抑制肝細(xì)胞凋亡進(jìn)而治療急性肝衰竭。

2 抑制肝纖維化

急性肝衰竭時細(xì)胞大量凋亡伴隨而來的是肝內(nèi)星狀細(xì)胞(成纖維細(xì)胞)大量增生，肝臟纖維化形成。因此抑制肝臟纖維化可以延緩肝衰竭的進(jìn)程。如前所述，ADSCs移植治療急性肝衰竭小鼠時，VEGF和HGF分泌增加；而VEGF可通過減少膠原蛋白的沉積減輕肝臟纖維化[17]。Narmada等[18]研究表明，對肝纖維化小鼠靶向輸注HGF基因后其肝纖維化標(biāo)志物α-SMA和膠原蛋白的表達(dá)顯著下降，這可能與HGF抑制Ⅰ型膠原合成酶有關(guān)。Jin等[19]報道，經(jīng)CCl4處理后肝硬化小鼠移植轉(zhuǎn)染Bcl-2基因的ADSCs后HLCs、mRNA、ALB及CK18蛋白水平明顯上升，肝纖維化程度及肝功能較模型組改善明顯。肝星狀細(xì)胞(HSCs)在肝纖維化形成過程中起關(guān)鍵性作用[20],其活化可以通過自分泌和旁分泌信號通路，其中一條主要通路是PDGF-β通路。PDGF-β通路可以激活其他通路，如Ras/MAPK、PKC、Phosphoinositide 3-kinase-AKT通路，導(dǎo)致HSCs增殖。陳國忠等[21]將間充質(zhì)干細(xì)胞與HSCs共培養(yǎng)后研究發(fā)現(xiàn)，HSCs的增殖明顯受到抑制。目前對ADSCs治療肝纖維化的基因及通路研究較少，有待進(jìn)一步完善。

3 抑制炎癥反應(yīng)及免疫調(diào)節(jié)

炎性因子浸潤及免疫介導(dǎo)的肝損傷是肝衰竭發(fā)生中的核心環(huán)節(jié)之一。研究表明，免疫因子(如NK細(xì)胞、Kupffer細(xì)胞、DC細(xì)胞等)的活化在肝衰竭的發(fā)病中起到關(guān)鍵作用。ADSCs的免疫原性很低而能顯著的表現(xiàn)出一些免疫調(diào)節(jié)的特點，其免疫原性可以通過成軟骨分化和IFN-γ的處理后得到加強[22]。在急性肝衰竭早期，免疫細(xì)胞尤其是Kupffer細(xì)胞分泌大量炎性介質(zhì)。而He等[23]通過靜脈注射ADSCs治療急性胰腺炎小鼠，檢測發(fā)現(xiàn)TNF-α、IL-1β、IL-6、IFN-γ等炎性因子表達(dá)明顯下降，這表明ADSCs可以通過抑制炎癥因子的分泌起到抗炎的作用。Shen等[24]的研究發(fā)現(xiàn)間充質(zhì)干細(xì)胞可以促進(jìn)M2型巨噬細(xì)胞的增殖及VEGF的分泌，抑制炎癥反應(yīng)。以上研究表明ADSCs可以在抗炎及免疫調(diào)節(jié)方面治療急性肝衰竭。

4 肝樣細(xì)胞分化

目前國內(nèi)外有大量的實驗證明了ADSCs的多向分化潛能[25～30]，其肝樣分化治療肝衰竭的潛能同樣受到了廣泛的關(guān)注。Guan等[28]研究發(fā)現(xiàn)ADSCs具有肝向分化潛能，并發(fā)現(xiàn)這種肝向分化可能與Caveolin-1通過MAPK通路調(diào)節(jié)有關(guān)。肝衰竭的發(fā)生與病毒性肝炎密切相關(guān)，Wang等[29]的研究發(fā)現(xiàn)與骨髓間充質(zhì)干細(xì)胞相比ADSCs分化為肝細(xì)胞的潛能更大。研究發(fā)現(xiàn)，從慢性乙肝患者身上分離出的ADSCs在試管內(nèi)培養(yǎng)時不易感染HBV病毒；Lee等[30]從人大腿脂肪組織分離ADSCs，加入細(xì)胞因子等誘導(dǎo)其向肝細(xì)胞分化，發(fā)現(xiàn)肝細(xì)胞標(biāo)志mRNA表達(dá)上調(diào)，亦檢測到白蛋白表達(dá)，本實驗為得到ADSCs提供了新的選擇。目前國內(nèi)外研究骨髓間充質(zhì)干細(xì)胞肝樣分化較多，關(guān)于ADSCs分化為肝細(xì)胞的機制研究較少，有待進(jìn)一步完善。

目前對ADSCs移植治療急性肝衰竭的研究很多，但主要集中在細(xì)胞體外培養(yǎng)及誘導(dǎo)分化方面，體內(nèi)移植實驗較少，且移植后細(xì)胞存活率較低。目前已有研究者通過基因工程的方法對移植前ADSCs進(jìn)行靶基因轉(zhuǎn)入，以提高移植后細(xì)胞的存活率及目的基因的表達(dá)[31]。靶基因的轉(zhuǎn)染及移植前預(yù)處理ADSCs已成為新的研究熱點。

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