The development of cell therapy for liver fibrolysis and prevention of carcinogenesis using bone marrow cell transplantaion

我们研究了骨髓细胞(BMC)移植对已建立的肝纤维化的影响。将绿色荧光蛋白(GFP)小鼠的骨髓细胞通过尾静脉移植到四氯化碳(CCl4)染毒的C57BL6小鼠体内，再用CCl4染毒4周(共8周)。天狼星红和GFP染色清楚地显示迁移的骨髓细胞与纤维一起存在，抗基质金属蛋白酶-9抗体和原位杂交显示基质金属蛋白酶-9呈强阳性表达。双荧光免疫组织化学显示GFP阳性细胞表面有基质金属蛋白酶9的表达。胶片原位酶谱分析显示门脉周围区域有很强的明胶溶解活性，这与基质金属蛋白酶-9阳性的骨髓细胞的位置相吻合。骨髓间充质干细胞移植四周后，小鼠的肝纤维化程度显著低于仅接受CCl_4治疗的小鼠，这是通过肝脏羟脯氨酸含量来评估的。Liv8阴性的骨髓细胞亚群与这种纤维溶解作用有关。结论：与单纯注射CCl_4的小鼠相比，连续注射CCl_4的BMC移植小鼠肝纤维化程度减轻，存活率显著提高。这一发现为肝纤维化的治疗引入了一个新的概念。此外，众所周知，预防纤维化可以减少癌变的发生。因此，这些结果可能表明，纤维溶解的骨髓细胞移植可以预防肝癌的发生。

We investigated the effect of bone marrow cell (BMC) transplantation on established liver fibrosis. BMCs of green fluorescent protein (GFP) mice were transplanted into 4-week carbon tetrachloride (CCl_4)-treated C57BL6 mice through the tail vein, and the mice were treated for 4 more weeks with CCl_4 (total, 8 weeks). Sirius red and GFP staining clearly indicated migrated BMCs existing along with fibers, with strong expression of matrix metalloproteinase (MMP)-9 shown by anti-MMP-9 antibodies and in situ hybridization. Double fluorescent immunohistochemistry showed the expression of MMP-9 on the GFP-positive cell surface. Film in situ zymographic analysis revealed strong gelatinolytic activity in the periportal area coinciding with the location of MMP-9-positive BMCs. Four weeks after BMC transplantation, mice had significantly reduced liver fibrosis, as assessed by hydroxyproline content of the livers, compared to that of mice treated with CCl_4 alone. Subpopulation of Liv8-negative BMCs was responsible for this fibrolytic effect. In conclusion, mice with BMC transplants with continuous CCl_4 injection had reduced liver fibrosis and a significantly improved survival rate after BMC transplantation compared with mice treated with CCl_4 alone. This finding introduces a new concept for the therapy of liver fibrosis. Also it has been known that prevention of fibrosis may reduce the carcinogenesis. Thus these results may suggest that fibrolysis bone marrow cell transplantation leads prevention of hepato-carcinogenesis.