自体骨髓单核细胞移植在肝纤维化兔肝脏内的定植能力

The Planting Ability of Autologous Bone-Marrow Mononuclear Cell Transplantation in Rabbit Hepatic Fibrosis Model

目的建立四氯化碳诱导的兔肝纤维化动物模型,观察体外分离标记的自体骨髓单核细胞(ABM-MNCs)经肠系膜上静脉自体移植至肝纤维化区及周边区后的存活、定植状况。方法将40只普通级日本大耳家兔随机分为细胞移植组和对照组各20只,实验组腹腔注射40%CCl4橄榄油溶液建立肝纤维化模型,对照组腹腔注射等量生理盐水。细胞移植组于模型稳定后自体髂骨处抽取骨髓,采用氯化氨红细胞溶解法分离得到单核细胞,以5溴-2脱氧尿嘧啶核苷(BrdU)标记体外ABM-MNCs及鉴定;分离培养ABM-MNCs,将3×109个ABM-MNCs经肠系膜上静脉回输体内,对照组回输等量生理盐水,移植前、移植后3、7、14、21 d分别取肝组织固定,进行免疫组织化学检测。结果BrdU体外标记ABM-MNCs的免疫组织化学表现示:20μmol/L BrdU孵育ABM-MNCs 72 h的阳性标记率达95%;肝组织20μmol/L BrdU免疫组化染色切片显示:自体骨髓单核细胞移植后第3天,肝小叶中央静脉周围BrdU染色阳性,随着时间的推移,阳性染色逐渐增强,并逐步向肝组织内部延伸。阳性染色主要分布于肝组织汇管区周围组织,而对照组BrdU染色则阴性。结论ABM-MNCs经肠系膜上静脉移植后,可在纤维化区及周边区存活,定植。

Objective To establish a rabbit model of CCl4-induced hepatic fibrosis,and to study the survival and field planting ability of autologous bone-marrow mononuclear cells（ABM-MNCs） in the liver of the rabbit model of hepatic fibrosis after transplanting through the superior mesenteric vein.Methods 40 conventional Japanese white rabbits were randomized into two groups.The rabbits of experimental group（bone marrow cell transplantation） were injected 40% CCl4 and olive oil suspension into the abdominal cavity.The rabbits of control group were injected equal weight of normal saline solution.Bone marrow cells were taken from the iliac bone in rabbits of the experimental group,and ABM-MNCs were obtained from bone marrow after using NH4Cl to destroy the erythrocytes.The ABM-MNCs were cultured and labeled by 5-bromo-2′-deoxyuridine（BrdU）,and 3×10^9 ABM-MNCs were transplanted into the superior mesenteric vein in rabbits of the experimental group.The rabbits of control group received normal saline solution injection.The distribution of ABM-MNCs in the liver was detected by immunohistochemical staining on days 3,7,14 and 21 after transplantation.Results The BrdU-labeled ABM-MNCs showed that the optimal concentration of cobalt catalyst is 20 μmol/L BrdU,and at 3 days after transplantation the ratio of BrdU labeling was 95%.The immunohistochemically stained 20 μmol/L BrdU-labeled hepatic tissue showed that at 3 days after transplantation BrdU-positive cells distributed around the central vein of hepatic lobules.The amount of BrdU-positive cells increased along with the passing of time,and concentrated into the portal area in the liver tissue.BrdU staining was negative in the control group.Conclusion ABM-MNCs can survive in the central area of hepatic fibrosis and its periphery after transplantation.