摘要
研究背景
生物人工肝和肝细胞移植面临一个共同的问题：高质量、来源丰富的人源性细胞来源的问题。干细胞以及人源性肝细胞系的研究进展为获取肝细胞提供了新的思路。
成体肝干细胞中，骨髓源性干细胞具有可以自体获取、易于获取、来源丰富和免疫原性小等特点，是比较理想的肝干细胞。骨髓MSCs可以分化为肝细胞，然而，目前难以通过骨髓源性MSCs获取足够临床应用数量的肝细胞，因为人源性骨髓源性干细胞的培养条件非常严格，难以大量扩增或大规模诱导分化。通常骨髓源性MSCs均在体外诱导分化为肝细胞，通过动物肝脏人肝细胞化获取人肝细胞的方法尚未见报道。
目前，人们已建立了许多人源性肝细胞系，迄今为止，只有C_3A细胞系开始应用于生物人工肝的临床研究，其他细胞系均没有进入临床试验，更没有一个细胞系用于肝细胞移植的临床研究。其中一个很重要的问题是这些细胞系应用的安全性，其中致瘤性是人肝细胞系应用安全性的一个重要指标。CL—1细胞系具有很好的生物学功能，其致瘤性的值得探讨。
第一部分 人源性细胞大鼠肝内移植的实验研究
材料与方法
建立SD大鼠2—AAF+CCL_4+CP大鼠肝再生模型，经门静脉移植入骨髓源性MSCs和CL—1细胞。移植后每日持续给予环孢素A皮下注射(3.5mg／kg)抑制排斥反应。比较大鼠在处理过程中肝功能(ALT，TBiL)的变化，通过免疫组化、PCR、实时荧光定量RT—PCR的方法检测大鼠肝脏中人肝细胞的存在以及计算所占的比例。
结果与讨论
移植人骨髓源性MSCs和CL—1细胞的大鼠肝功能明显好转(P＜
0.01二，但在观察期内未达到正常水平(P<0.01=，未移植大鼠全部死亡。
免疫组化以及PCR检测显示移植后大鼠肝脏中存在人源性细胞;实时荧
光定量RT一PCR显示移植人骨髓源性MSCs的大鼠肝脏中人肝细胞的比
例分别为9.79%(14天)和9.09%(28天)，移植CL一1细胞的大鼠肝
脏中人肝细胞的比例分别为9.27%(14天)和8.86%(28天)
结论
人骨髓源性MSCs和CL一1细胞可以在2一AAF+CCL4+CP大鼠
肝脏内存活;人骨髓源性MSCs可以在2一AAF+CCL4+CP选择性肝再
生大鼠肝脏内诱导分化为肝样细胞并实现部分替代(9.79%);人CL一1
细胞可以在大鼠肝脏内存活并能部分替代(9.27%);肝内移植人骨髓
MSCs和CL一1细胞均有助于2一AAF+CCL4+CP大鼠肝损伤后肝功能
的恢复;在观察期限内，人CL一1细胞在免疫抑制的2一AAF+CCL4+
CP大鼠肝脏内未发现肿瘤形成。
第二部分人cL一1细胞裸鼠体内致瘤性的实验研究
材料与方法
将体外培养的CL一1细胞接种到裸鼠皮下(n二12)，观察其致瘤性，
4W后切取种植瘤、肝肺脑进行病理检查，接种1 640作阴性对照。
结果与讨论
前3周瘤体生长缓慢，后生长加快;接种后4周，总致瘤率为58%
(7/12)，对照组裸鼠皮下未见种植瘤生长;种植瘤大体标本示:瘤体呈
光滑的圆球型，界限清楚，有完整包膜，瘤体直径为(8 .26士1.49)~。。
镜下CL一1种植瘤显示:细胞形态大小比较均一，细胞核深染，核仁不
明显，核分裂像较多，细胞呈双梁状排列，有少量的侵犯肌层和脂肪组
织;肝、肺、脑组织切片染色未发现有转移瘤。
结论
人CL一1细胞在裸鼠体内有一定的致瘤性(7/12)。
Background
Bioartificial liver (BAL) and hepatocyte transplantation (HTx) confront a common problem: abundant source of high quality human-derived hepatocyte. The progress of the study on stem cells and human-derived hepatocyte cell line provides a new way to obtain human-derived hepatocyte.
Bone marrow-derived stem cells are abundant in cell source, low-immunogenicity, can be obtained easily and from oneself, are ideal hepatic stem cell. Bone marrow-derived mesenchymal stem cell (MSCs) can differentiate into hepatocyte, nevertheless, it is difficult to obtain sufficient hepatocyte for clinic application by the differentiation of bone marrow-derived MSCs because the cultivation condition of bone marrow-derived MSCs is very strict in vitro so that they cannot repopulate and differentiate on a large scale. Generally, bone marrow-derived MSCs differentiate into hepatocyte in vitro. It is not reported to obtain human hepatocyte by the way of human hepatocytized of animal liver.
Nowadays, there are many human hepatocyte cell line (hHCL) but only C3A hHCL are used in BAL. The other hHCL are not used in BAL. No hepatocyte cell lines are used in HTx mainly because of the problem of security. The oncogenicity of hHCL is an important aspect of security. CL-1 hHCL has good biological function while its oncogenicity should be discussed.
Part I The experimental study on the transplantation on human-derived cells into the rat liver
Materials and Methods
The human MSCs from bone marrow and human CL-1 cells(5 X104) were transplanted in to the liver of SD rats which 2-AAF+CCL4+CP rat models were set up. After transplantation, rats were maintained on a daily dose of CsA (3.5mg/kg). The hepatic function (ALT, TBiL) of the rats were checked, and then the human-derived cells of the transplanted rats were detected by Immunohistochemistry staining and PCR detection; lastly, the human-derived cells of the transplanted rats were detected by the real time RT-PCR
Result and Discussion
The liver function of the rats which had been transplanted human MSCs from bone marrow and human CL-1 cells were ameliorated but were not normal while those who had not been transplanted cells all die in a short time; the results of Immunohistochemistry staining and PCR detection indicate there were human-derived cells in the liver of the transplanted rats; the proportion of human hepatocyte in the rat which transplanted with human MSCs was 9.79% and 9.09% respectively and the proportion of human hepatocyte in the rat which transplanted with human MSCs was 9.27% and 8.86% respectively.
Conclusion
The human MSCs from bone marrow and human CL-1 cells can survive in the liver of 2-AAF+CCL4+CP rat model; the human MSCs can diffenentiate to functional hepatocyte-like cells in the 2-AAF/CCLF4/CP rat
model and replace 9.79%(14d) and 9.09% (28d) ; the CL-1 cells can replace 9.27%(14d) and8.86% (28d) ; the transplantation of the human MSCs and CL-1 cells can ameliorate the recovery of the 2-AAF+CCL4+CP rats; within 28 days, the CL-1 cells failed to formed planted tumor in the 2 -AAF+CCL4+CP rats.
Part II The experimental study on the oncogenicity of human hepatocyte line CL-1 cells in nude mice
Materials and Methods
CL-1 cells(2X106) were cultured in vitro and transplanted into the subcutaneous tissue of nude mice The planted tumor, liver, lung and brain of the nude mice were removed for pathological examination
Results
The planted tumor grew slowly initially while grew fast in the last
week. 7/12 of the planted place formed planted tumor, the proportion of
oncogenicity is 58%. The planted tumors were round which diameter is
( 8.26+ 1.49 ) mm. There are no planted tumors formed in the liver, lung and
brain.
Conclusion
The CL-1 cells have a little oncogenicity in immunodeficiency mice.
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