間充質(zhì)干細(xì)胞定向分化及其在肝臟疾病應(yīng)用中的啟示

王漢裕 劉擁軍

?述 評(píng)?

間充質(zhì)干細(xì)胞定向分化及其在肝臟疾病應(yīng)用中的啟示

王漢裕 劉擁軍★

間充質(zhì)干細(xì)胞向肝細(xì)胞誘導(dǎo)分化成功后，分化后的肝細(xì)胞樣細(xì)胞應(yīng)用于肝病成為了研究熱點(diǎn)。不僅未分化的間充質(zhì)干細(xì)胞能治療肝病，其來(lái)源的分化后的肝細(xì)胞樣細(xì)胞亦能有效治療肝病。因此，有必要對(duì)間充質(zhì)干細(xì)胞分化前后的細(xì)胞治療效果進(jìn)行比較評(píng)價(jià)。本文從誘導(dǎo)分化培養(yǎng)方案、鑒定指標(biāo)及相關(guān)細(xì)胞生物學(xué)功能進(jìn)行述評(píng)。

間充質(zhì)干細(xì)胞；定向分化；細(xì)胞治療；肝病

干細(xì)胞研究的發(fā)展促進(jìn)了應(yīng)用干細(xì)胞治療肝病的研究，豐富了肝病的治療手段，尤其是肝硬化及肝功能衰竭的治療?；诟杉?xì)胞的分化潛能，利用干細(xì)胞，尤其是間充質(zhì)干細(xì)胞（mesenchymal stem cells，MSCs），向肝細(xì)胞進(jìn)行誘導(dǎo)分化培養(yǎng)，可獲得肝細(xì)胞樣細(xì)胞（hepatocyte-like cells，HLCs），并應(yīng)用于肝病的治療。不少研究開(kāi)始比較MSCs向肝細(xì)胞分化前后用于肝病治療。我們發(fā)現(xiàn)MSCs經(jīng)過(guò)向肝細(xì)胞分化誘導(dǎo)培養(yǎng)后，丟失了高分泌基質(zhì)金屬蛋白酶（matrix metalloproteinases，MMPs）和肝細(xì)胞生長(zhǎng)因子（hepatocyte growth factor，HGF）的能力。我們及其他實(shí)驗(yàn)均發(fā)現(xiàn)分化后的HLCs治療肝病的效果不如未分化的MSCs。本文結(jié)合本實(shí)驗(yàn)的研究，綜合目前相關(guān)研究文獻(xiàn)，對(duì)MSCs向肝細(xì)胞定向分化及其在肝病中的應(yīng)用進(jìn)行簡(jiǎn)要述評(píng)。

1 分化誘導(dǎo)培養(yǎng)方案的選擇依據(jù)

2001年小鼠胚胎干細(xì)胞（embryonic stem cells，ESCs）[1]和2002年多潛能成體祖細(xì)胞（multipotent adult progenitor cells，MAPCs）[2]在體外向HLCs誘導(dǎo)分化培養(yǎng)獲得成功后，2004年MSCs在體外向HLCs分化誘導(dǎo)培養(yǎng)也獲得成功[3]。小鼠ESCs向HLCs的誘導(dǎo)分化方案中，ESCs在不含白血病抑制因子（leukemia inhibitory factor，LIF）的培養(yǎng)基中培養(yǎng)5天，繼而接種于I型膠原包被的培養(yǎng)皿中培養(yǎng)4天（基礎(chǔ)培養(yǎng)基為含20% 胎牛血清和300μM硫代甘油的IMDM，依次添加酸性成纖維細(xì)胞生長(zhǎng)因子（acidic fibroblast growth factor，aFGF，100 ng/mL）、HGF（20 ng/mL）；在誘導(dǎo)培養(yǎng)的第15天，添加抑瘤素M（oncostatin M，OSM，10 ng/mL）、地塞米松（dexamethasone，DXM，10-7M）、ITS混合物（5 mg/mL胰島素、5 mg/mL轉(zhuǎn)鐵蛋白、5μm/mL亞硒酸）培養(yǎng)3天。經(jīng)過(guò)18天的誘導(dǎo)分化培養(yǎng)，ESCs成功分化為具有部分肝細(xì)胞功能的HLCs?；贓SCs和MAPCs的誘導(dǎo)方案，改進(jìn)的MSCs誘導(dǎo)方案采用了兩步法，首先用含肝HGF（20 ng/mL）、堿性成纖維細(xì)胞生長(zhǎng)因子（basic fi broblast growth factor，bFGF，10 ng/mL）和尼克酰胺（0.61 g/L）的IMDM培養(yǎng)基培養(yǎng)1周；更換為誘導(dǎo)成熟培養(yǎng)基，即含OSM（20 ng/mL）、地塞米松（1×10-6mol/L）和 ITS 混合物（25 g/L胰島素、25 g/L轉(zhuǎn)鐵蛋白、25 mg/L亞硒酸鈉）的IMDM，繼續(xù)培養(yǎng)3周。經(jīng)過(guò)4周的誘導(dǎo)分化培養(yǎng)，細(xì)胞形態(tài)從梭型轉(zhuǎn)變?yōu)榈湫偷母渭?xì)胞樣橢圓形，并能檢測(cè)到細(xì)胞色素P450（cytochrome P450，CYP）的2B6（CYP 2B6）亞型。隨后文獻(xiàn)中出現(xiàn)的培養(yǎng)分化方案都在此方案上加于變化，比如預(yù)先在I型[4]或IV型膠原[5]包被的培養(yǎng)皿上培養(yǎng)2天，再繼續(xù)往下分化培養(yǎng)；把MSCs接種在Matrigel包被的納米聚酰胺纖維上進(jìn)行分化培養(yǎng)[6]。

在干細(xì)胞向HLCs分化培養(yǎng)過(guò)程中，除了HGF外，目前的分化培養(yǎng)方案中均需要添加bFGF[1～5]。我們簡(jiǎn)化了上述的分化方案，MSCs向HLCs分化誘導(dǎo)培養(yǎng)依然獲得成功。我們的誘導(dǎo)方案也是采取兩步法，在第一階段采用10-8mol/L的地塞米松、50 ng/mL的HGF、10 ng/mL的表皮生長(zhǎng)因子（epidermal growth factor，EGF）、1% ITS的DMEM-F12（高糖）培養(yǎng)基誘導(dǎo)培養(yǎng)2周；第二階段，使用含2%的FBS、20 ng/mL的OSM、10-6mol/L的DXM、1% ITS的DMEM-F12培養(yǎng)基誘導(dǎo)培養(yǎng)4周?？紤]到第二階段的培養(yǎng)時(shí)間延長(zhǎng)，添加低濃度的FBS以維持細(xì)胞的活性。經(jīng)過(guò)6周的誘導(dǎo)培養(yǎng)后，細(xì)胞逐漸變成圓形（圖1），并具有成熟肝細(xì)胞的功能，比如合成糖元（圖2）、分泌尿素（圖3）等。這說(shuō)明MSCs向HLCs分化的過(guò)程中，bFGF不是必需的。bFGF是胚胎干細(xì)胞培養(yǎng)基中維持ESCs未分化狀態(tài)的一個(gè)重要組成因子[7]。最近也有研究發(fā)現(xiàn)bFGF聯(lián)合骨形態(tài)發(fā)生蛋白4（bone morphogenetic protein-4，BMP-4）促進(jìn)了ESCs向成骨細(xì)胞和成軟骨細(xì)胞分化[8]。

圖1 誘導(dǎo)分化前后的細(xì)胞形態(tài)Figure 1 Cells morphology changes after hepatic differentiation

圖2 糖原染色（PAS法）Figure 2 Glycogen detection by PAS staining

圖3 隨著誘導(dǎo)分化培養(yǎng)時(shí)間的延長(zhǎng)，尿素分泌逐漸增多Figure 3 The differentiated cells synthesized urea in a timedependent manner

2 分化誘導(dǎo)的鑒定標(biāo)記及其意義

不管大鼠[9]、小鼠[10]，還是人類(lèi)[3]，各種來(lái)源的（骨髓[11]、臍帶、臍帶血[12]或脂肪[11,13,14]）MSCs均能分化為HLCs，為將來(lái)肝病的細(xì)胞治療解決了細(xì)胞來(lái)源的技術(shù)難題。但是，目前沒(méi)有提出一個(gè)特異的指標(biāo)來(lái)明確干細(xì)胞向HLCs分化成功與否，也沒(méi)有明確的鑒定指標(biāo)來(lái)評(píng)價(jià)分化效率。因此，在相關(guān)文獻(xiàn)中，均采用多指標(biāo)評(píng)價(jià)系統(tǒng)。常用的鑒定標(biāo)記有白蛋白（albumin）、甲胎蛋白（α-fetoprotein，AFP）、細(xì)胞角蛋白（cytokeratine，CK）、肝細(xì)胞核因子（hepatocyte nuclear factor，HNF）、細(xì)胞色素（cytochrome，CYP）P450、酪氨酸轉(zhuǎn)氨酶（tyrosine-aminotransferase）、色氨酸2，3雙加氧酶（tryptophan 2，3-dioxygenase）等，體現(xiàn)具有肝細(xì)胞功能的鑒定方法有過(guò)碘酸-希夫（Periodic Acid-Schiff，PAS）染色、Dil-Ac-LDL吸收、尿素合成等。另外，部分上述指標(biāo)還存在亞型，比如細(xì)胞角蛋白包括CK7[6]、CK18[5]、CK19[2]，HNF包括HNF-1α[2]、HNF-3β[2]、HNF-4α[5]。然而，尚未有某一篇文獻(xiàn)全部采用上述指標(biāo)。有研究發(fā)現(xiàn)誘導(dǎo)分化早期出現(xiàn)的指標(biāo)有HNF-3β、CK19、AFP，而CK18、albumin、HNF-1α、CYP則表達(dá)于誘導(dǎo)分化晚期[2]。經(jīng)過(guò)4周的誘導(dǎo)，HLCs表達(dá) CYP 2B6，而肝細(xì)胞核因子4（HNF-4）在誘導(dǎo)6周后才表達(dá)[3]。

不少文獻(xiàn)提到MSC在誘導(dǎo)分化之前沒(méi)有表達(dá)ALB、AFP、CK18[5,15,16]、CK19[15]。亦有文獻(xiàn)報(bào)道，經(jīng)過(guò)誘導(dǎo)分化培養(yǎng)后的細(xì)胞只有70%表達(dá)白蛋白[5]。BM-MSCs低表達(dá)白蛋白、CK18、色氨酸2，3雙加氧酶，但不表達(dá) AFP[3]。但是，我們發(fā)現(xiàn)未經(jīng)過(guò)誘導(dǎo)分化的UC-MSCs本身高表達(dá)白蛋白、CK19和AFP（圖4）。這些數(shù)據(jù)證明了白蛋白、色氨酸2，3雙加氧酶、CK18、CK19和AFP不能作為骨髓和臍帶來(lái)源的MSCs在向肝細(xì)胞誘導(dǎo)分化的鑒定指標(biāo)。分泌尿素是肝細(xì)胞活性的一個(gè)特性，但是腎小管上皮細(xì)胞也分泌尿素。CYP雖然首先在肝細(xì)胞中發(fā)現(xiàn)，但也在其他細(xì)胞中表達(dá)[18]。即使是肝細(xì)胞相對(duì)特異性指標(biāo)的CYP，其不同亞型的活性亦存在種屬特異性，如大鼠的CYP 2B1[19]、小鼠的CYP 2B9和CYP 2B13[20,21]、人的CYP 2B6[2]。盡管肝細(xì)胞具有LDL吸收的功能，但其他細(xì)胞也有這樣的特性[22]。綜合目前的文獻(xiàn)報(bào)道，只有肝細(xì)胞能合成和存儲(chǔ)糖原[2]。在包被了Matrigel上培養(yǎng)的MAPCs分化為HLCs（同時(shí)表達(dá)白蛋白、CK18、HNF-3β）的分化率能達(dá)到 91%[2]。經(jīng)過(guò)6周的誘導(dǎo)，大約 50%的分化細(xì)胞具有存儲(chǔ)糖原的功能[3]。

3 誘導(dǎo)前后的細(xì)胞生物學(xué)相關(guān)研究

自從建立了干細(xì)胞向HLCs分化誘導(dǎo)的成熟方案，不少研究者隨即對(duì)這些HLCs生物學(xué)功能和特性進(jìn)行深入的研究。由于ESCs具有高度的致瘤性，在體外進(jìn)行誘導(dǎo)分化時(shí)，很容易混雜未能充分分化的ESCs[23,24]，這就給臨床使用帶來(lái)高風(fēng)險(xiǎn)，限制其臨床應(yīng)用。和ESCs相比，成體干細(xì)胞不具有明顯潛在的成瘤性，沒(méi)有倫理問(wèn)題的困擾，非常適合于臨床的應(yīng)用[25]。

圖4 免疫熒光方法檢測(cè)間充質(zhì)干細(xì)胞表達(dá)的白蛋白、細(xì)胞角蛋白19、甲胎蛋白Figure 4 Expressions of albumin, CK19 and AFP from MSCs by immuno fl uorescence microscopy

MSCs向HLCs誘導(dǎo)分化的培養(yǎng)方案已經(jīng)得到很好的建立[3]。分化后的HLCs具有向損傷肝臟部位遷移的能力，而且能在體內(nèi)分泌白蛋白[26～28]。人脂肪來(lái)源的MSCs誘導(dǎo)分化為HLCs后，能減輕四氯化碳對(duì)裸鼠造成的急性肝損傷程度[29]。這些結(jié)果說(shuō)明來(lái)源于干細(xì)胞的HLCs合適于治療肝病，尤其是急性肝衰竭和終末期肝病[30]。分化后的HLCs和未經(jīng)分化誘導(dǎo)的MSC均有報(bào)道其對(duì)肝病的治療作用，例如肝纖維化[31～33]、肝損傷[34,35]、爆發(fā)性肝衰竭[27,29,36]和肝再生[26,37,38]。由于MSCs向肝細(xì)胞分化前后，對(duì)肝病的治療均有效果，因此，需要對(duì)分化前后的細(xì)胞進(jìn)行功能相關(guān)比較研究。

動(dòng)物實(shí)驗(yàn)證明，MSCs不僅能遷移到損傷的部位，修復(fù)損傷的組織，并使損傷的組織恢復(fù)一定的功能[27,39,40]；而且能減少細(xì)胞的死亡，并促進(jìn)內(nèi)源性再生[41,42]。另外，MSCs也易于體外培養(yǎng)擴(kuò)增。這些特性使得MSCs成為臨床細(xì)胞治療的最佳來(lái)源。MSCs修復(fù)組織的可能機(jī)制在于分泌可溶性細(xì)胞因子，這些細(xì)胞因子能抑制炎癥和免疫反應(yīng)，也能刺激內(nèi)源性干細(xì)胞的增殖和分化，從而起到組織修復(fù)作用[43,44]。體內(nèi)實(shí)驗(yàn)證明未分化的MSCs比分化后的HLCs治療急性肝衰竭的效果更好[27]，我們的數(shù)據(jù)也證明了這點(diǎn)。由于未分化的MSCs亦表達(dá)高水平的白蛋白，這有益于肝病的治療。這也說(shuō)明前期動(dòng)物實(shí)驗(yàn)發(fā)現(xiàn)HLCs在肝臟分泌白蛋白的數(shù)據(jù)是值得商榷的。需要注意的是，發(fā)現(xiàn)MSCs輸入治療后有向肝細(xì)胞分化的實(shí)驗(yàn)研究中，均采用的是免疫缺陷小鼠[15,26]，而MSCs在免疫系統(tǒng)正常小鼠體內(nèi)存留時(shí)間大概為23天[45]。在不同的動(dòng)物模型中，MSCs能改善肝臟功能，而沒(méi)有出現(xiàn)細(xì)胞融合[27,46]。

HGF是促進(jìn)肝細(xì)胞增殖[47,48]和肝再生[47,49]的最強(qiáng)效細(xì)胞因子。HGF刺激肝干/祖細(xì)胞的增殖來(lái)修復(fù)肝損傷[50]。HGF可引起B(yǎng)cl-xL的強(qiáng)烈表達(dá)，阻斷Fas及其配體激活后的信號(hào)傳導(dǎo)，抑制Fas介導(dǎo)的肝細(xì)胞凋亡[51]，從而治療Fas引起的爆發(fā)性肝衰竭[52]。HGF除了能誘導(dǎo)肝實(shí)質(zhì)細(xì)胞的迅速增殖外，還能刺激膽管上皮細(xì)胞增殖，因而影響整個(gè)器官的發(fā)育。由于HGF有促進(jìn)肝細(xì)胞增殖和抑制凋亡的特性，使得HGF可能是肝病細(xì)胞治療的一個(gè)關(guān)鍵因子。我們和其他的研究小組都發(fā)現(xiàn)MSCs分泌高水平的HGF[53]。而且，臍帶來(lái)源的MSCs分泌HGF的量比骨髓來(lái)源的MSCs高30倍[53]。 除了HGF，MSCs也分泌高水平的MMPs，這些分泌的MMPs有利于組織修復(fù)和肝纖維化的治療。MSCs分化為HLCs后，其分泌的HGF（圖5）和MMPs（表1）都顯著降低。也有數(shù)據(jù)證明MSCs向HLCs分化培養(yǎng)結(jié)束后，并不是處于分化終末期的穩(wěn)定狀態(tài)，這增加了成瘤性的風(fēng)險(xiǎn)[54]。

圖5 細(xì)胞分化前后肝細(xì)胞生長(zhǎng)因子的表達(dá)水平變化Figure 5 The change of hepatocyte growth factor expression after hepatic differentiation

表1 細(xì)胞分化前后基質(zhì)金屬蛋白酶的分泌水平變化Table 1 Matrix metalloproteinase changes after hepatic differentiation

綜上所述，由于沒(méi)有明確的鑒定標(biāo)準(zhǔn)，也難于評(píng)價(jià)分化效率。MSCs分化為HLCs后，丟失了分泌HGF和MMPs的能力，而HGF和MMPs均有利于相關(guān)肝病的治療。這些數(shù)據(jù)說(shuō)明直接用未分化的MSCs治療肝病效果好于經(jīng)過(guò)誘導(dǎo)分化培養(yǎng)后的細(xì)胞，而且還節(jié)約培養(yǎng)成本。

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Mesenchymal stem cells committed differentiation and its application in liver diseases therapy

WANG Hanyu, LIU Yongjun★
(Alliancells Institute of Stem Cells and Translational Regenerative Medicine, Tianjin 300300, China)

The protocols for differentiation of hepatocyte-like cells (HLCs) from mesenchymal stem cells (MSCs) have been well established. Previous datas have shown that MSCs and their derived-HLCs were able to engraft injured liver and alleviate injuries. It is necessary to elvaluate the cells therapeutic effects before and after MSCs differentiation. We will make a brief comment on the induction and differentiation culture protocol, hepatic markers and related functions of cell biology.

Mesenchymal stem cells; Committed differentiation; Cytotherapy; Liver diseases

國(guó)家自然科學(xué)基金（30872618）

和澤干細(xì)胞轉(zhuǎn)化再生醫(yī)學(xué)研究中心，天津 300300

★通訊作者：劉擁軍，E-mail: andyliuliu2001@yahoo.com.cn