修回日期: 2011-05-19
接受日期: 2011-05-24
在线出版日期: 2011-06-08
人们对使用细胞移植疗法恢复肝脏功能的前景寄予厚望, 期待其最终能够代替肝脏移植手术. 众多学者对成人成熟肝细胞, 不同来源干细胞的再生能力方面进行了大量研究, 特别是间充质干细胞来源的肝细胞移植疗法在恢复肝脏再生方面的效果在动物模型实验中取得令人鼓舞的结果. 本文将对近年来细胞移植治疗各种肝病的机制研究、动物实验以及临床试验等方面的最新进展作一综述.
引文著录: 杨大伟, 姚鹏. 细胞移植疗法在肝病中的研究与应用. 世界华人消化杂志 2011; 19(16): 1720-1725
Revised: May 19, 2011
Accepted: May 24, 2011
Published online: June 8, 2011
Cell transplantation is a promising way to restore liver function. Treatment of end-stage liver disease with stem cells, especially bone marrow stem cells, has attracted wild attention. There is ongoing research to use mature hepatocytes, liver progenitor cells, bone marrow stem cells and embryonic stem cells to restore liver function in patient with hepatic disease. Here we review the current research status of cell transplantation for hepatic disease in terms of cell biology, animal models and clinical trials.
- Citation: Yang DW, Yao P. Cell transplantation for hepatic disease: current research status. Shijie Huaren Xiaohua Zazhi 2011; 19(16): 1720-1725
- URL: https://www.wjgnet.com/1009-3079/full/v19/i16/1720.htm
- DOI: https://dx.doi.org/10.11569/wcjd.v19.i16.1720
由于肝脏疾病的高患病率, 肝脏供体的极度缺乏, 促使科研人员探索多种细胞的作用与潜能, 期待将其移植入肝脏中后能使其重新恢复功能. 人们关注的这些细胞, 特别是一些干细胞已经证明可以在某些特定体外条件下或者动物模型上表达出同肝细胞相同的细胞表型. 无论是急慢性肝损伤还是肝脏代谢紊乱, 都有可能通过细胞移植疗法治愈. 借助少量的相关干细胞移植来使肝脏修复无疑是一种非常具有吸引力的途径. 本综述关注并讨论肝细胞, 特别是肝脏相关干细胞治疗肝脏疾病的前景, 动物实验成果以及临床应用情况.
我们知道人类成熟肝细胞具有显著的再生潜能[1], 那么能否将健康的肝细胞移植进受损肝脏内来治疗疾病呢? 在啮齿类动物模型实验中科研人员证实了这一可能, 动物实验取得的效果也不错[2,3], 然而临床应用效果却并不理想, 因为在许多疾病情况下, 成熟肝细胞会端粒缩短而迅速衰老; 某些疾病如脂肪肝时的高氧化应激反应也可以抑制肝细胞的增殖[4-6]. 由于成体肝细胞难于培养, 在体外几乎不能增殖, 会发生去分化, 且难以大量获得与冻存, 这些特性促使我们去寻找其他的细胞来替代.
肝祖细胞(liver progenitor cell, LPC), 也被称为肝卵圆细胞(这是因为他的细胞核在啮齿类动物中呈椭圆形). 肝祖细胞的核质比较大, 位于终末胆小管的最末端. LPC的起源仍未肯定, 由于LPC与造血干细胞共有一定数量的细胞表面抗原, 所以猜测造血干细胞有可能是LPC的前身. LPC的再生潜力是很有吸引力的, 但是他能否有能力在注入人类肝脏之后安全地重建肝脏, 对此目前还没有详细的研究[7]. LPC的出现率与肝脏慢性疾病的严重度相关[8]. 如果人体出现成熟肝细胞的数目过低、肝细胞的复制能力衰竭或是毒素阻止了肝细胞的增殖等情况, LPC以及肝细胞就会开始复制. 与此同时某些炎性细胞因子会通过不同的方式刺激成熟的肝细胞和LPC的复制. 已知的一些对于LPC再生能力有影响的蛋白质有TNF-α、IL-6、淋巴毒素-β(lymphotoxin-β, LTB)、干扰素γ(interferon γ, IFN-γ)、IL-15、干细胞因子(stem cell factor, SCF)、基质源因子-1(stromal derived factor -1, SDF-1)、TGF-α、肝细胞生长因子(hepatocyte growth factor, HGF)等, 他们对LPC再生的作用主要有: TNF-α基因敲除的小鼠LPC的再生能力缺失[9], 其在体外引起已分化的LPC凋亡[10], 在体外刺激未分化的LPC增殖[11]; IL-6基因缺失小鼠的LPC再生能力降低[12]; 急性肝损伤时LTB生成受抑制[12], 慢性丙型肝炎时LTB过度表达[13], LTB基因敲除小鼠LPC的再生能力缺失[13]; 急性肝损伤时IFN-γ生成受到抑制[12], 在TNF-α或脂多糖存在时IFN-γ可诱导LPC增生, 但对肝细胞有抑制作用[12], IFN-γ基因敲除小鼠LPC的再生能力缺失[13]; IL-15与IL-2作用相同, 引导Th1应答和细胞毒T淋巴细胞汇聚[14], 主要导致LPC数量增加, 炎性细胞浸润和肝细胞增生[15]; 肝损伤时SCF可调节LPC应答[16]; 封闭SDF-1表达基因会使慢性肝损伤小鼠体内的LPC减少, 但对急性肝损伤的小鼠没有影响[17], LPC会产生SDF-1受体, 进行自分泌和旁分泌活动[17]; TGF-α主要是促进LPC分裂[18]; HGF可诱导LPC增生并刺激其分化为肝细胞[19].
Swenson等[20]对慢性肝损伤时对LPC的再生影响和SCF的分泌情况进行了研究. 在使用酪氨酸诱导致肝损伤后, 观察到LPC增殖现象缺失和SCF血浆水平的下降. 在二乙基二硫代氨基甲酯诱导所致的肝损害时, 可观察到LPC的增殖和血浆中SCF水平的略微下降. 当慢性肝病由局部病灶扩散以后, 持续性的肝损伤会使SCF的血浆水平升高, 但对于LPC的数量没有影响. 在一项实验中, 对Long-Evans棕色大鼠(Wilson disease模型)中分离出的LPC加以检测, 研究人员发现此类细胞的甲胎蛋白呈阳性而白蛋白呈阴性, 将其移植到白蛋白缺陷的大鼠体内, 在后来的10 wk时间里观察到白蛋白缺陷大鼠的血清白蛋白持续增长, 证明LPC有能力分化为肝细胞[21]. 因为直接从小鼠体内分离LPC更加困难, 所以Li等[22]首先用甲状旁腺激素诱发LPC增殖, 进而向肝内注射倒千里光碱, 最后分离出LPC. 这些细胞被分离出后进行单细胞克隆, 而且证明他们还可以在体外被诱导分化. 研究人员将这些LPC移植入由CCl4诱导慢性肝损伤的小鼠体内. 移植手术是成功的, 然而值得注意的是1 mo后, 移植的肝干细胞只剩原来的2%, 6 mo后只剩0.4%.
骨髓干细胞包括造血干细胞及间充质干细胞, 2004年首次证明人间充质干细胞在体外具有分化为肝祖细胞和肝细胞的能力[26]. 间充质干细胞广泛存在于骨髓、脐带血、脂肪组织、唾液腺、胰腺等组织, 这些细胞在体外具有高度的增生能力, 易于转染且可低温保存, 无论在体外还是体内都表现出高度的分化潜力. 他的分化需要EGF、HGF、bFGF、烟酰胺、制癌素、胰岛素、转铁蛋白、硒、地塞米松等物质组成的混合液的诱导[26]. 由于干细胞在特定环境下可定向分化为功能细胞, 在肝损伤后肝细胞的再生过程中, 骨髓干细胞是肝细胞的重要肝外来源, 其可在肝脏分化为肝祖细胞及肝细胞, 从而参与肝功能的修复和重构, 同时采取方便, 体外培养技术成熟以及具备自体移植潜能, 可作为治疗肝病的理想种子, 目前在临床上应用最多.
在动物实验方面, 有研究者将骨髓干细胞注入被CCl4损伤肝脏的裸鼠脾内, 可使裸鼠免于肝功能衰竭[27]. 将骨髓干细胞置于含有HGF的培养皿中培养3 wk后, 干细胞已能表达白蛋白mRNA, 此时将其移植入肝脏被CCl4破坏的小鼠体内, 短时间后小鼠的血清白蛋白水平得以恢复, 转氨酶类水平下降, 且小鼠的肝纤维化程度也得以好转[28]. 将过度表达HGF的骨髓干细胞移植入患肝病的发育不良小鼠体内, 小鼠的肝功能有所改善, 总体的死亡率也得以降低[29]. 以被D-半乳糖胺损伤肝脏的小鼠为实验模型, 当van Poll等[30]将含有骨髓干细胞的条件培养液注射入小鼠体内, 发现小鼠肝细胞凋亡现象减少了90%, 而且肝细胞再生的数量相对对照鼠增加了3倍之多.
但是自应用骨髓干细胞治疗患遗传性酪氨酸血症Ⅰ型的小鼠模型开始, 骨髓干细胞的作用出现了一定争议[3,31]. 将捐献者骨髓干细胞移植入这些小鼠肝内后, 尽管植入的细胞数量比率相对来说很少(相对每百万肝细胞只有1个骨髓干细胞), 但最终克隆扩充的肝细胞数量可以占到原有组织的一半并且使肝功能有所恢复; 但是新生的健康肝细胞中同时含有受者和捐献者的染色体, 健康的造血细胞的细胞核与不正常的肝细胞细胞核融合, 致使其原本的细胞核被改编, 创造出新的功能性肝细胞. 这一结果显示骨髓干细胞可能并不是在肝内分化为肝细胞从而发挥功能, 而是通过细胞融合形成嵌合细胞, 继而减数分裂转化为含有正常数目染色体的肝实质细胞, 促进肝脏的自我修复从而改善肝功能. 在使用骨髓干细胞或者脐带干细胞治疗其他由其他疾病造成的肝损伤小鼠时, 可能也存在这种情形[3,23,32]. Alvarez-Dolado等[33]在成熟肝实质细胞中发现有来源于骨髓供体的基因组分; Quintana-Bustamante等[34]利用GFP+BMSCs进行雌雄小鼠交叉移植, 在受体肝脏内鉴定出双核/Y染色体+/GFP+功能性肝细胞. 目前细胞融合机制被普遍认为是BMSCs转化为成熟肝细胞的主要方式和首要机制, 由于嵌合细胞染色体呈多倍性且不稳定, 所以BMSCs治疗存在致癌风险, 但目前尚无报道.
骨髓干细胞具有定向分化增生的能力. 在动物实验中证明移植后可以明显改善肝功能, 并且与原位肝脏移植手术相比具有一定优势. 因此短时间内在肝病治疗领域内, 肝动脉自体骨髓干细胞移植得到广泛开展. 我国是最早开展此项技术的国家, 姚鹏等在使用此技术治疗失代偿期肝硬化后, 患者的相关检验结果以及临床症状都得到了改善, 且未发现严重不良反应[35]. 几乎同时, 国外医疗人员也进行了相似尝试. 在3例患巨块型肝癌的患者身上先行门静脉栓塞术, 使肿瘤萎缩变小, 继而行自体骨髓干细胞移植, 从而使其余肝叶要比未经治疗的相同肝叶增大至少25%[36]. Gordon等[37]、Levicar等[38]报道5例肝硬化患者接受CD34+移植(3例门静脉, 2例肝动脉)并随访6-18 mo, 其中有4例肝功能好转; Yannaki等[39]观察接受CD34+移植的2例失代偿肝硬化患者, 30 mo后Child-Pugh、MELD分值分别平均下降1和8; Terai等[40]的研究也发现了肝功能好转, 并在移植后4 wk行肝活检, 发现AFP、增殖细胞核抗原表达增加.
然而由于干细胞移植的适应证未统一, Mohamadnejad等[41,42]先后进行了造血干细胞、间充质干细胞移植的临床研究, 发现造血干细胞移植疗效令人遗憾, 6 mo后仅有1例MELD分值下降, 并有1例死亡, 不过他们认为这与肝动脉造影导致放射性肾病有关. 最近, Kharaziha等[43]对8例终末期肝病患者进行间充质干细胞移植, 6 mo后MELD分值由17.9±5.6下降至10.7±6.3(P<0.05), 腹水明显减少, 且随访期间未发作肝性脑病. 以上结果都给我们展示了骨髓干细胞移植治疗肝病的巨大潜能.
胚胎干细胞(embryonic stem cells, ESCs)由人类胚胎的内细胞群产生, 他具有全方位分化的潜能. 因此, 对于肝脏再生而言比其他任何一种干细胞都更有可行性. 目前3组这类的细胞系已在仁济医院的干细胞研究实验室建立成功[44]. 多种针对ESCs分化为肝细胞谱系中一系列细胞的研究已经开始实施. 多种生长因子被用来促进胚胎干细胞的分化, 诸如: 激活蛋白-A、成纤维生长因子-2、骨形态发生蛋白-2、HGF等[3,45,46]. 另外, ESCs可以与其他一些相关细胞如肝非实质细胞一起培养来促进胚胎干细胞的分化[3,4,47,48]. 由非本体组织衍生的ESCs可能会存在排斥的危险, 现在也还不能生产供移植所用的足够的ESCs, 而且应用ESCs治疗疾病还要受到相关伦理道德的制约.
将ESCs诱导分化而来的小鼠肝细胞移植入CCl4诱导的肝损伤小鼠模型和二甲基亚硝胺诱导的肝硬化小鼠模型体内, 研究人员观察到小鼠的肝衰竭发生率得以下降[2,19,49,50]. 将在体外由ESCs分化培养来的一系列肝脏谱系中的细胞移植入患有严重尿蛋白和尿激酶型纤溶酶原激活物联合免疫缺陷的裸鼠肝内, 可以减轻CCl4对小鼠肝细胞的损害, 但是28 d后, 移植入的ESCs数量仅剩余30%[2,51]. 由于ESCs的特性, 其也会在某些动物模型体内形成畸胎瘤. Ishii等[52]报道他们将ESCs体外培养分化得到了可以表达甲胎蛋白的肝细胞. 将这些肝细胞移植入白喉毒素诱导的肝损伤小鼠体内, 在距移植手术1 wk时, 移植肝细胞占肝质量的3.4%, 距手术1 mo时, 移植肝细胞占到肝质量的32.8%, 1 mo后, 移植肝细胞的数量渐渐减少. 距手术2 mo后, 绝大多数小鼠都患了脾脏畸胎瘤, 警示我们胚胎干细胞移植的风险不容忽视. 目前人类ESCs主要应用于生物人工肝技术. 这是一种代替人体肝脏功能的体外装置, 用于维持肝病患者的生命从而等待接受整体肝脏移植手术. 实验证明切除小鼠90%的肝脏之后, 生物人工肝依然可以使小鼠免于急性肝衰竭, 同时还可以大大提高半乳糖胺诱导的肝衰竭小鼠的存活率[2,3,53,54].
利用各种干细胞进行的研究和临床应用方兴未艾. 骨髓干细胞移植治疗各种肝病的效果十分显著, 从而在临床得到迅速开展. 但是干细胞移植依然有较多未知待以研究: 第一: 经肝动脉或者门静脉移植的干细胞最终能有多少定植于肝脏? 第二: 移植的干细胞在肝脏中都转化为哪些细胞？受哪些条件影响? 第三: 干细胞是通过转化为肝细胞还是分泌细胞因子来改善肝功能? 其分子机制是怎样的? 第四: 具体哪种干细胞的作用最好? 第五: 干细胞移植治疗肝病的适应证及操作规范依然无共同标准. 尽管如此, 随着对干细胞移植疗法的不断研究和完善, 对某些患急性肝病和终末期肝病的患者来说, 干细胞疗法将会是一个较好的选择.
干细胞科学作为目前自然科学中最为引人注目的领域之一, 其理论的日臻完善和技术的迅猛发展对医学的基础研究和临床应用同样影响巨大.
倪润洲, 教授, 南通大学附属医院消化内科
van Poll等将含有骨髓干细胞的条件培养液注射入小鼠体内, 发现小鼠肝细胞凋亡现象减少了90%, 而且肝细胞再生的数量相对对照鼠增加了3倍之多.
本综述对近年来肝细胞, 特别是肝脏相关干细胞治疗肝脏疾病的基础研究, 动物实验成果以及临床应用情况的相关进展做一回顾和总结, 并提出目前干细胞移植研究和临床应用面临的难点与热点, 希望对初次接触或者正在进行肝脏相关干细胞研究的医学同仁有所帮助.
本文内容新颖, 对读者了解细胞移植在肝病治疗中的作用有较大的帮助.
编辑:李军亮 电编:何基才
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