干細(xì)胞治療終末期肝病的進(jìn)展與挑戰(zhàn)

施明，劉振文，張政，王福生

肝移植是治療終末期肝病最有效的方法，但供肝來(lái)源短缺與肝移植需求之間的巨大差距促使人們努力尋求其他的替代療法。近來(lái)，干細(xì)胞治療終末期肝病越來(lái)越受到關(guān)注，特別是骨髓來(lái)源的干細(xì)胞[主要含造血干細(xì)胞(HSC)和間質(zhì)干細(xì)胞(MSC)]治療已有體外和動(dòng)物實(shí)驗(yàn)支持，自體回輸骨髓干細(xì)胞治療亦有相關(guān)臨床報(bào)道。此外，其他來(lái)源的MSC治療肝病亦取得了較好進(jìn)展。本文就此方面的研究進(jìn)展綜述如下。

1 研究背景

干細(xì)胞根據(jù)其發(fā)育階段可分為胚胎干細(xì)胞(ES)和成體干細(xì)胞，其重要特征是具有無(wú)限自我更新能力，可以分化成特定組織，在細(xì)胞發(fā)育過(guò)程中處于較原始階段。骨髓是成體干細(xì)胞的重要來(lái)源，主要含HSC和MSC。MSC在骨髓中含量最多，在其他組織器官如臍帶組織﹑臍帶血﹑胎盤(pán)﹑外周血﹑脂肪組織中的含量也較豐富?；诟杉?xì)胞強(qiáng)大的分化潛能，人們開(kāi)始嘗試應(yīng)用干細(xì)胞治療各種疾病。自1999年P(guān)etersen等[1]發(fā)現(xiàn)骨髓中某些干細(xì)胞具有向肝細(xì)胞分化的潛能以來(lái)，人們開(kāi)始研究應(yīng)用干細(xì)胞治療肝病的可行性，后來(lái)發(fā)現(xiàn)不同來(lái)源的干細(xì)胞如骨髓HSC﹑骨髓MSC等均可定向誘導(dǎo)分化為肝細(xì)胞樣細(xì)胞，具有正常肝細(xì)胞功能，如分泌尿素和白蛋白等[2]，且得到動(dòng)物實(shí)驗(yàn)的驗(yàn)證，臨床亦有自體回輸骨髓干細(xì)胞治療肝病的報(bào)道。目前開(kāi)展的肝病治療研究的干細(xì)胞類(lèi)型如圖1所示。

圖1 目前已經(jīng)開(kāi)展的治療肝病的干細(xì)胞類(lèi)型及其治療機(jī)制Fig.1 Types of stem cells on current liver disease therapy and its mechanism

2 臨床前研究

2.1 骨髓干細(xì)胞治療肝病 以前認(rèn)為只有具備HSC特性的細(xì)胞才能轉(zhuǎn)化為肝細(xì)胞，1999年P(guān)etersen等[1]發(fā)現(xiàn)骨髓干細(xì)胞或HSC能夠在鼠肝內(nèi)轉(zhuǎn)化為肝卵圓細(xì)胞甚至成熟的肝細(xì)胞和膽管細(xì)胞。2000年Theise等[3]在異性間骨髓移植或肝移植的受體中也發(fā)現(xiàn)了源于供體的肝細(xì)胞和膽管細(xì)胞。Lagasse等[4]用正常小鼠骨髓細(xì)胞移植治療延胡索酰乙酰乙酸鹽水解酶缺乏(FAH-/-)導(dǎo)致的遺傳性Ⅰ型酪氨酸血癥小鼠，發(fā)現(xiàn)骨髓中只有HSC能在體內(nèi)轉(zhuǎn)分化為肝細(xì)胞，并在受體內(nèi)形成供體源造血和肝細(xì)胞再生，受體小鼠血液生化指標(biāo)明顯改善。之后，人們嘗試應(yīng)用粒細(xì)胞集落刺激因子(G-CSF)直接動(dòng)員骨髓釋放干細(xì)胞來(lái)治療肝病。動(dòng)物部分肝切除后應(yīng)用G-CSF可以促進(jìn)肝再生，其機(jī)制可能與血液中CD133+或CD34+細(xì)胞動(dòng)員有關(guān)，且體外培養(yǎng)G-CSF動(dòng)員后CD133+細(xì)胞可表達(dá)肝細(xì)胞特異性標(biāo)志物[5]。為進(jìn)一步驗(yàn)證CD133+細(xì)胞對(duì)肝再生的促進(jìn)作用，am Esch等[6]將分選出的自體骨髓CD133+細(xì)胞回輸至選擇性門(mén)靜脈栓塞的中央巨型肝臟腫瘤患者中，發(fā)現(xiàn)CD133+細(xì)胞移植組非栓塞部分肝臟體積明顯增大，有利于患者接受進(jìn)一步手術(shù)。

Sakaida等[7]應(yīng)用四氯化碳(CCl4)建立的小鼠肝纖維化模型進(jìn)行研究發(fā)現(xiàn)，異體骨髓細(xì)胞移植能減輕肝纖維化程度，提高小鼠生存率。大鼠未受損的肝血竇內(nèi)皮細(xì)胞上有HSC表面標(biāo)志(CD45+和CD33+)及內(nèi)皮細(xì)胞的表面標(biāo)志(CD31+)，肝血竇內(nèi)皮細(xì)胞受損后，回輸骨髓來(lái)源的CD133+干細(xì)胞，在受體肝臟中可發(fā)現(xiàn)供體來(lái)源的肝血竇內(nèi)皮細(xì)胞，替代了原來(lái)的受損細(xì)胞[8]。

也有部分學(xué)者對(duì)HSC向肝細(xì)胞分化的潛能及治療意義提出了質(zhì)疑，他們應(yīng)用動(dòng)物的骨髓造血祖細(xì)胞在體外并不能誘導(dǎo)成功能性的肝細(xì)胞[9]，應(yīng)用轉(zhuǎn)基因的HSC研究發(fā)現(xiàn)，在體內(nèi)只有極少部分HSC來(lái)源的肝細(xì)胞與受體的肝細(xì)胞形成融合細(xì)胞，幾乎沒(méi)有任何治療意義[10]。

2.2 MSC治療肝病 由于骨髓細(xì)胞成分復(fù)雜，何種細(xì)胞更適合用于肝細(xì)胞再生治療尚不清楚，但越來(lái)越多的研究?jī)A向于骨髓MSC。Sato等[11]比較了人骨髓中的MSC﹑CD34+﹑非MSC/CD34–等3種細(xì)胞直接注射到大鼠肝內(nèi)后轉(zhuǎn)分化成肝細(xì)胞的能力，結(jié)果骨髓MSC顯示出最強(qiáng)的向肝細(xì)胞分化的潛能。

在適當(dāng)條件尤其是肝臟損傷的情況下，骨髓MSC可以分化為肝細(xì)胞樣細(xì)胞，這些分化細(xì)胞表現(xiàn)出成熟肝細(xì)胞的形態(tài)和特征，如表達(dá)肝細(xì)胞特異性基因，具有合成和分泌白蛋白﹑儲(chǔ)存糖原﹑代謝尿素及解毒功能等。Kuo等[12]應(yīng)用人骨髓來(lái)源的MSC進(jìn)行移植治療，使小鼠暴發(fā)型肝衰竭得到有效恢復(fù)，并促進(jìn)了肝臟的再生。脂肪組織來(lái)源的MSC與骨髓來(lái)源的MSC相似，在體外可誘導(dǎo)成肝細(xì)胞樣細(xì)胞，將這些肝細(xì)胞樣細(xì)胞移植到CCl4肝損傷模型的小鼠體內(nèi)，可整合入宿主的肝臟中，使其肝臟功能得到改善[13-14]。Van Poll等[15]應(yīng)用D-半乳糖胺建立小鼠急性肝損傷模型，再輸入MSC條件培養(yǎng)基(MSC-CM)，結(jié)果有效地減少了肝細(xì)胞壞死，促進(jìn)了肝細(xì)胞增殖，并抑制了肝損傷標(biāo)志物的釋放。Chamberlain等[16]將人MSC分別通過(guò)腹腔和肝內(nèi)注射至胎羊中，發(fā)現(xiàn)經(jīng)肝內(nèi)途徑注射MSC后，人來(lái)源的肝細(xì)胞廣泛分布于肝實(shí)質(zhì)中，而通過(guò)腹腔途徑注射MSC后，人來(lái)源的肝細(xì)胞主要分布于門(mén)靜脈周?chē)?。用人羊水?lái)源的MSC，以及由MSC誘導(dǎo)分化的肝祖細(xì)胞樣細(xì)胞和肝細(xì)胞樣細(xì)胞治療CCl4損傷的急性肝衰竭動(dòng)物模型，結(jié)果羊水來(lái)源的MSC和肝祖細(xì)胞樣細(xì)胞取得了很好的治療效果，且回輸肝祖細(xì)胞樣細(xì)胞條件培養(yǎng)基也能取得較好效果[17]。人肝干細(xì)胞及其條件培養(yǎng)基對(duì)暴發(fā)性肝衰竭也有很好的保護(hù)作用，可通過(guò)分泌細(xì)胞因子抑制肝細(xì)胞凋亡，促進(jìn)肝細(xì)胞再生[18]。最近國(guó)內(nèi)有學(xué)者研究發(fā)現(xiàn)，通過(guò)門(mén)靜脈輸注人骨髓來(lái)源的MSC可提高急性肝衰竭豬的生存率，促進(jìn)肝細(xì)胞再生[19]。

但同時(shí)也有研究表明，MSC不能在體外誘導(dǎo)成成熟的肝細(xì)胞，但可在體內(nèi)分化為肝細(xì)胞，且免疫原性很低，是MSC治療肝病的良好細(xì)胞來(lái)源[20]。將人骨髓來(lái)源的MSC直接注入免疫抑制的肝臟損傷大鼠中，其分布只限于注射部位周?chē)?，雖然可以分化成肝細(xì)胞，但分化效率很低，且供體的MSC并不與受體的肝細(xì)胞發(fā)生融合，MSC分化的肝細(xì)胞單純來(lái)源于供體MSC[11]。Arikura等[21]將先天性白蛋白缺乏癥大鼠行70%肝切除后植入正常大鼠骨髓MSC，4周后在受體肝臟中可檢測(cè)到表達(dá)白蛋白mRNA的肝細(xì)胞。

2.3 胚胎干細(xì)胞(ES)治療肝病 ES是指從囊胚期的內(nèi)細(xì)胞團(tuán)中分離出來(lái)的尚未分化的胚胎細(xì)胞，可分化形成各種類(lèi)型的組織[22]。ES具有在體外無(wú)限增殖并保持分化成所有細(xì)胞類(lèi)型的特性，理論上可以向3個(gè)胚層的任何類(lèi)型細(xì)胞分化，其中也包括肝細(xì)胞[23-25]。已有研究表明ES對(duì)大鼠肝衰竭[26]和小鼠肝硬化[27]均有顯著療效。目前，有關(guān)ES研究的限制主要集中在倫理學(xué)﹑組織相容性以及移植后畸胎瘤發(fā)生等方面。

2.4 誘導(dǎo)多能干細(xì)胞(iPS)治療肝病 已有研究顯示iPS細(xì)胞可以誘導(dǎo)分化為肝細(xì)胞[28]，也可將人原代肝細(xì)胞重編程為肝細(xì)胞來(lái)源的iPS細(xì)胞，此iPS細(xì)胞可定向誘導(dǎo)分化為內(nèi)胚層細(xì)胞﹑肝祖細(xì)胞和成熟的肝細(xì)胞[29]。有研究發(fā)現(xiàn)小鼠iPS細(xì)胞可以在四倍體囊胚中發(fā)育成完整的胎肝，同時(shí)人iPS細(xì)胞可在體外誘導(dǎo)分化為具備相關(guān)功能的肝細(xì)胞樣細(xì)胞，這些細(xì)胞可以在小鼠的肝臟內(nèi)增殖并整合到肝實(shí)質(zhì)中[30]。最近發(fā)現(xiàn)應(yīng)用三種轉(zhuǎn)錄因子(缺少轉(zhuǎn)錄因子c-Myc)的iPS細(xì)胞及其誘導(dǎo)的具肝細(xì)胞表型和功能的肝細(xì)胞樣細(xì)胞，均能減輕CCl4所致的急性肝損傷，提高動(dòng)物生存率[31]。有研究者建立了以代謝性疾病患者真皮成纖維細(xì)胞制備的人iPS細(xì)胞系平臺(tái)，此iPS細(xì)胞系能誘導(dǎo)分化成具有肝細(xì)胞表型﹑基因型和功能的肝細(xì)胞樣細(xì)胞，顯示出較強(qiáng)的治療潛力[32]。利用iPS細(xì)胞治療肝病的具體療效及其安全性尚需在動(dòng)物模型中進(jìn)一步驗(yàn)證。

3 臨床研究

3.1 HSC治療肝病的臨床研究 2000年Alison等[33]在移植了男性骨髓細(xì)胞的女性患者肝臟內(nèi)發(fā)現(xiàn)了Y染色體陽(yáng)性的肝細(xì)胞，2002年Korbling等[34]在移植了男性外周血干細(xì)胞的女性患者肝臟內(nèi)也發(fā)現(xiàn)了來(lái)自男性的肝細(xì)胞，證明骨髓和外周血干細(xì)胞在人體內(nèi)可以分化為肝細(xì)胞。骨髓干細(xì)胞治療可以通過(guò)G-CSF動(dòng)員或直接抽取骨髓的方式進(jìn)行。

3.1.1 G-CSF動(dòng)員的骨髓干細(xì)胞 Gaia等[35]應(yīng)用G-CSF動(dòng)員骨髓干細(xì)胞治療終末期肝病，患者有較好的耐受性，部分患者Child和終末期肝病模型(MELD)評(píng)分改善，臨床癥狀好轉(zhuǎn)。Garg等[36]應(yīng)用G-CSF動(dòng)員骨髓CD34+干細(xì)胞治療慢加急性肝衰竭，結(jié)果患者耐受性良好，Child﹑MELD和序貫器官衰竭估計(jì)(SOFA)評(píng)分均顯著改善，生存率顯著提高。Levicar等[37]應(yīng)用G-CSF動(dòng)員骨髓，通過(guò)免疫磁珠分選外周血中的CD34+干細(xì)胞，并通過(guò)門(mén)靜脈或肝動(dòng)脈回輸治療慢性肝衰竭，結(jié)果未發(fā)現(xiàn)短期和長(zhǎng)期不良反應(yīng)，患者的肝臟功能有一定程度改善。Salama等[38]應(yīng)用G-CSF動(dòng)員骨髓，而后通過(guò)收集外周血中的骨髓HSC治療終末期肝病，結(jié)果患者白蛋白升高，膽紅素和丙氨酸轉(zhuǎn)氨酶(ALT)下降，國(guó)際標(biāo)準(zhǔn)化比值(INR)恢復(fù)正常，所有患者均耐受。但也有報(bào)道通過(guò)門(mén)靜脈回輸自體骨髓CD133+細(xì)胞和單個(gè)核細(xì)胞治療失代償性肝硬化效果均不十分理想[39]。

3.1.2 未經(jīng)動(dòng)員的骨髓干細(xì)胞 Terai等[40]通過(guò)骨髓穿刺獲取骨髓干細(xì)胞后，經(jīng)外周靜脈回輸治療肝硬化(包括失代償)，在24周隨訪(fǎng)期內(nèi)，患者血漿白蛋白水平明顯升高，Child評(píng)分明顯改善，肝臟活檢組織中甲胎蛋白(AFP)及增殖細(xì)胞核抗原(PCNA)水平明顯升高。Lyra等[41-42]分離終末期肝病患者骨髓單個(gè)核細(xì)胞后經(jīng)肝動(dòng)脈回輸，結(jié)果無(wú)明顯不良反應(yīng)，患者Child﹑MELD評(píng)分明顯改善，血清總膽紅素下降，白蛋白升高，INR明顯下降，通過(guò)肝動(dòng)脈回輸骨髓干細(xì)胞治療終末期肝病安全可行。Mohamadnejad等[43]采用抽取自體骨髓體外培養(yǎng)的方法，將富含干細(xì)胞標(biāo)記的細(xì)胞亞群回輸至等待肝移植的患者，4例患者在等待期內(nèi)均存活，1年內(nèi)MELD評(píng)分有所改善。Kim等[44]應(yīng)用自體骨髓干細(xì)胞(包括造血干細(xì)胞和上皮細(xì)胞等單核細(xì)胞)治療慢性乙肝相關(guān)的肝硬化患者，結(jié)果患者生活質(zhì)量明顯改善，80%患者肝臟體積增大，腹水減少，Child評(píng)分改善，肝臟祖細(xì)胞(HPC)活性增強(qiáng)，可在6個(gè)月繼續(xù)分化成肝細(xì)胞，但肝硬化的臨床分級(jí)無(wú)明顯改善(表1)。

3.2 MSC治療肝病的臨床研究 Mohamadnejad等[43]最早報(bào)道應(yīng)用自體骨髓MSC治療終末期肝病患者，經(jīng)穿刺獲取骨髓后通過(guò)密度梯度離心獲得骨髓單個(gè)核細(xì)胞，在體外誘導(dǎo)成MSC后，經(jīng)外周靜脈回輸至患者，經(jīng)過(guò)12個(gè)月的隨訪(fǎng)，該方法安全無(wú)副作用，患者癥狀明顯改善，血漿白蛋白升高，腹水減少，肝體積增大，MELD評(píng)分改善。Khayaziha等[45]報(bào)道應(yīng)用自體骨髓MSC經(jīng)外周或門(mén)靜脈回輸治療由乙肝﹑丙肝﹑酒精性肝硬化及不明原因?qū)е碌慕K末期肝病患者，結(jié)果表明該方法安全無(wú)副作用，能提升血漿白蛋白，降低膽紅素水平，促進(jìn)凝血酶原時(shí)間(PT)復(fù)常，改善MELD評(píng)分。近兩年Amer等[46]用骨髓MSC體外誘導(dǎo)成肝細(xì)胞樣細(xì)胞后進(jìn)行自體回輸治療丙肝相關(guān)的終末期肝病，結(jié)果患者癥狀明顯改善，腹水減少，白蛋白顯著增加，Child和MELD評(píng)分改善。

國(guó)內(nèi)學(xué)者最近應(yīng)用MSC治療終末期肝病取得了較好進(jìn)展。Peng等[47]應(yīng)用自體骨髓MSC治療乙肝相關(guān)的肝衰竭患者，結(jié)果短期(4周)內(nèi)患者白蛋白﹑PT水平明顯增加，而總膽紅素水平和MELD評(píng)分明顯下降，無(wú)明顯不良反應(yīng)，但長(zhǎng)期效果不明顯。Zhang等[48]應(yīng)用臍帶MSC移植治療慢性乙肝相關(guān)失代償性肝硬化，結(jié)果患者腹水明顯減少，肝臟功能明顯改善，白蛋白增加，血清膽紅素水平和MELD評(píng)分明顯下降。Shi等[49]應(yīng)用臍帶MSC移植治療慢性乙肝相關(guān)的慢加急性肝衰竭患者，結(jié)果發(fā)現(xiàn)可顯著提高患者生存率，改善MELD評(píng)分，增加白蛋白﹑膽堿酯酶﹑凝血酶原活動(dòng)度水平，而總膽紅素和轉(zhuǎn)氨酶水平明顯下降。Wang等[50]采用臍帶MSC治療原發(fā)性膽汁性肝硬化患者，結(jié)果血清膽堿磷酸和γ-谷氨酰轉(zhuǎn)移酶水平明顯下降(表1)。

表1 干細(xì)胞治療終末期肝病的臨床應(yīng)用情況Tab. 1 Clinical application of stem cells on treatment of end-stage liver disease

3.3 干細(xì)胞回輸與移植途徑 目前，干細(xì)胞治療肝病的主要移植途徑有外周循環(huán)移植﹑門(mén)靜脈移植﹑肝動(dòng)脈移植等，現(xiàn)有的結(jié)果顯示安全性好，患者可耐受，無(wú)明顯不良反應(yīng)。但也有報(bào)道經(jīng)穿刺獲得骨髓后應(yīng)用免疫磁珠分選出CD34+干細(xì)胞，經(jīng)過(guò)肝動(dòng)脈回輸，結(jié)果發(fā)生顯影劑性腎病，最后發(fā)展成1型肝腎綜合征[51]。因此，通過(guò)肝動(dòng)脈回輸干細(xì)胞的安全性尚需進(jìn)一步觀察。

3.4 療效評(píng)價(jià)及影響療效的因素 移植前的肝功能狀況直接影響干細(xì)胞移植的結(jié)局。Barba等[52]報(bào)道HSC移植前高膽紅素和高谷酰轉(zhuǎn)肽酶(GGT)水平可影響移植后病死率和生存率?，F(xiàn)有肝功能改善的評(píng)價(jià)方法主要包括Child和MELD評(píng)分﹑血液生化﹑臨床表現(xiàn)﹑生活質(zhì)量﹑生存時(shí)間(率)，以及應(yīng)用甲胎蛋白和PCNA評(píng)價(jià)肝細(xì)胞再生，利用TNF﹑IL-6和IL-10等評(píng)價(jià)肝臟炎癥環(huán)境的改善等[53]。利用磁性標(biāo)記的大鼠MSC進(jìn)行肝臟移植，再行磁共振成像活體示蹤，可在肝實(shí)質(zhì)中發(fā)現(xiàn)標(biāo)記的移植細(xì)胞[54]。肝組織活檢對(duì)評(píng)價(jià)肝再生及移植細(xì)胞狀態(tài)有重要意義，但有創(chuàng)性使其對(duì)終末期肝病患者的應(yīng)用受到限制。一些無(wú)創(chuàng)或微創(chuàng)的活體內(nèi)移植細(xì)胞示蹤方法如Y染色體探查等，可應(yīng)用于不同性別間的細(xì)胞移植，放射性同位素標(biāo)記移植細(xì)胞后應(yīng)用核素顯像或PET-CT等也已有臨床報(bào)道[55]。這些方法均可在不同程度上說(shuō)明移植細(xì)胞的存活和增殖情況，為評(píng)價(jià)干細(xì)胞移植的作用提供依據(jù)。

4 干細(xì)胞治療肝病的機(jī)制

4.1 干細(xì)胞歸巢功能 肝臟微環(huán)境改變是MSC歸巢的始動(dòng)因素，肝組織損傷時(shí)存在炎癥反應(yīng)，局部可表達(dá)及分泌多種趨化因子﹑黏附因子﹑生長(zhǎng)因子﹑基質(zhì)金屬蛋白酶9(MMP-9)等，這些因子與其受體的相互作用可引導(dǎo)干細(xì)胞特異性遷移至病損部位[56-58]?；|(zhì)細(xì)胞衍生因子SDF-1及其受體CXCR4構(gòu)成的SDF-1/CXCR4軸是引導(dǎo)MSC向損傷組織遷移的重要生物軸[59]。此外，肝細(xì)胞生長(zhǎng)因子(HGF)及其受體c-met構(gòu)成的HGF/c-met軸﹑G-CSF﹑血管內(nèi)皮生長(zhǎng)因子(VEGF)﹑MMP等均與MSC的歸巢有關(guān)[58,60-61]。還有研究認(rèn)為，肝臟受損后肝臟環(huán)境中的鞘脂代謝物——鞘氨醇1-磷酸鹽(S1P)水平增高，且其與骨髓之間的濃度梯度差通過(guò)S1P3受體介導(dǎo)是骨髓MSC向肝臟歸巢的重要因素[62]。

4.2 干細(xì)胞分化功能 Petersen等[1]于1999年首先報(bào)道，在性別交叉骨髓細(xì)胞移植或全肝移植受體肝臟中發(fā)現(xiàn)來(lái)源于供體骨髓的肝細(xì)胞，隨后Alison等[33]和Theise等[3]相繼發(fā)現(xiàn)骨髓移植和肝移植患者的肝臟中也有來(lái)源于供體骨髓的肝細(xì)胞。Lagasse等[4]在延胡索酰乙酰乙酸水解酶(FAH)缺陷大鼠模型中發(fā)現(xiàn)骨髓HSC可在肝臟內(nèi)分化為具有功能的肝細(xì)胞，改善FAH缺陷大鼠的癥狀。Schwartz等[63]也報(bào)道，從大鼠﹑小鼠和人的骨髓中分離得到多能成體祖細(xì)胞，體外經(jīng)成纖維細(xì)胞生長(zhǎng)因子(FGF)和HGF誘導(dǎo)可分化為功能肝細(xì)胞。骨髓中存在可分化為肝細(xì)胞的干細(xì)胞，直接將其移植到肝臟，在肝臟微環(huán)境下可分化為肝細(xì)胞。因此骨髓干細(xì)胞移植為多種嚴(yán)重肝病的治療提供了新的策略(圖1)。

MSC具有跨胚層多向分化潛能，在合適的條件下，如HGF﹑FGF﹑表皮生長(zhǎng)因子(EGF)或制瘤素M(OSM)等誘導(dǎo)下，通過(guò)特定的細(xì)胞信號(hào)傳導(dǎo)途徑，可以跨胚層向內(nèi)胚層的肝細(xì)胞樣細(xì)胞﹑膽管細(xì)胞和血管內(nèi)皮樣細(xì)胞分化[64-66]，而Notch/Jagged信號(hào)通路在此過(guò)程中可能起重要作用[67-68]。在此分化系統(tǒng)中，肝臟局部微環(huán)境，如細(xì)胞因子﹑細(xì)胞外基質(zhì)(ECM)﹑激素﹑基質(zhì)細(xì)胞等，是誘導(dǎo)MSC定向分化的決定因素，其中細(xì)胞因子的類(lèi)型﹑濃度和添加次序是影響MSC分化的主要因素[69]。

4.3 干細(xì)胞旁分泌功能 以往認(rèn)為，干細(xì)胞移植可提供大量肝細(xì)胞樣細(xì)胞替代受損的肝細(xì)胞功能，然而急性肝損傷所造成的疾病進(jìn)展快，肝功能恢復(fù)不能依賴(lài)于干細(xì)胞分化為肝細(xì)胞過(guò)程，而更大程度上依賴(lài)其他機(jī)制，目前認(rèn)為干細(xì)胞旁分泌機(jī)制改變組織微環(huán)境比向肝細(xì)胞的轉(zhuǎn)分化更重要[70]。如MSC可以分泌多種細(xì)胞因子﹑生長(zhǎng)因子，發(fā)揮局部效應(yīng)，促進(jìn)受損肝臟增生及肝臟血管再生，抑制免疫細(xì)胞增殖及向肝臟遷移，調(diào)節(jié)肝臟及全身免疫炎癥反應(yīng)，從而減輕肝臟的急性損傷，提高生存率[12,71-72]。MSC的條件培養(yǎng)基能抑制肝細(xì)胞的抗凋亡，刺激肝臟再生[15]，機(jī)制可能與MSC的旁分泌功能一致，即為損傷的肝臟提供營(yíng)養(yǎng)和有利的生存環(huán)境，因?yàn)榈鞍踪|(zhì)組分析顯示條件性培養(yǎng)基中含有許多抗炎癥因子，如IL-10﹑IL-1ra﹑IL-13和IL-27等[17]，以及促進(jìn)肝細(xì)胞再生﹑抑制肝細(xì)胞凋亡的細(xì)胞因子，如HGF﹑IL-10﹑VEGF﹑IL-6和IL-8等[18]。此外，研究還發(fā)現(xiàn)移植的骨髓MSC可通過(guò)釋放促細(xì)胞增生因子和MMP-9刺激內(nèi)源性肝細(xì)胞再生[73-74]。

5 存在的問(wèn)題與展望

基于干細(xì)胞具有自我更新﹑無(wú)限增殖的能力以及多向分化潛能的特性，將其應(yīng)用于終末期肝病的臨床研究取得了令人鼓舞的進(jìn)展，但干細(xì)胞治療終末期肝病的長(zhǎng)期安全性(特別是致畸或癌變的風(fēng)險(xiǎn))和遠(yuǎn)期療效仍有待觀察，在療效評(píng)價(jià)方面如何檢測(cè)受體肝臟內(nèi)移植細(xì)胞的狀態(tài)和功能，如何減少和避免排斥反應(yīng)及其他不良反應(yīng)，特別是MSC具有形成肝細(xì)胞或促進(jìn)肝纖維化的雙刃劍作用[75]，仍需進(jìn)一步探討。此外，干細(xì)胞的來(lái)源﹑誘導(dǎo)培養(yǎng)條件﹑細(xì)胞質(zhì)量控制(包括表型﹑功能﹑微生物安全)﹑使用時(shí)機(jī)﹑途徑和劑量﹑臨床適應(yīng)證等都可能對(duì)治療的后果產(chǎn)生影響，這一系列與安全和療效密切相關(guān)的問(wèn)題還須進(jìn)一步深入研究。因此，要在闡明其機(jī)制的基礎(chǔ)上，不斷積累各方面的資料，明確其臨床應(yīng)用的安全性和有效性，保證患者利益。

總之，干細(xì)胞強(qiáng)大的治療潛力有可能成為終末期肝病患者的有效治療手段，從而給患者帶來(lái)新的希望。

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